Pathology and Pathogenesis of Human Viral Disease

r-®i

Endothelium

c. Cell recruitment and activation Binding to epithelium

d. Resolution

( ^ ^ ^

VlNFaJL-l If' IFNa.y

Neurogenic & cellular inflammation

FIGURE 5.1 Hypothetical schema illustrating likely mechanism of immune-mediated disease in the RSV-infected human. (A) Respiratory viral infections are initiated when a virus enters a host epithelial cell, and viral replication causes the release of new infectious particles and activates secretion of cytokines, chemokines, and mediators by the epithelial cells. (B) Viral particles released into the airway cause activation of resident cells in the airway, including macrophages, lymphocytes, and granulocytes. Cytokines from epithelial cells and other resident airway cells increase adhesion molecule expression and increase airway responsiveness. (C) The combination of chemokine secretion and increased adhesion molecule expression causes recruitment and activation of additional leukocytes, which further add to airway inflammation and increased responsiveness. (D) Cytotoxic T cells kill virus-infected cells, and cytokines such as TGF-p and IL-10 are likely to play a role in downregulation of airway inflammation after viral infection. Increased numbers of airway lymphocytes and eosinophils may persist for weeks after the viral infection. The steps illustrated in this diagram can occur sequentially and/or in parallel, and the timing depends on factors related to both the host and virus. Abbreviations: EOS = eosinophil; PMN = polymorphonuclear leukocyte; mac = macrophage; lymph = lymphocyte; CTL = cytotoxic lymphocyte; 0«2~ = superoxide; AHR = airway hyperresponsiveness. Reprinted with permission from Folkerts et al. (1998).

55

Respiratory Syncytial Virus

of the viral antigens in the vaccine elicited a response in the children. Recent experimental observations argue for a cellmediated disease process. In RSV-infected mice, circulating antibody and both CD4+ (helper/suppressor) cells and CD8+ (cytolytic) cells appear to participate in virus clearance. When these lymphoid cells are eliminated from the circulation experimentally, respiratory disease fails to develop (Anderson and Heilman, 1995). Studies in humans indicate that cell-mediated immunity is greatest in children younger than 6 months of age (Scott et al, 1978). The intensity of this response correlates with the occurrence of wheezing in infected infants and degree of hypoxia (Welliver et al, 1981). The clinical manifestations may depend upon the specific T cell subset involved in the immune response and the capacity of the cells to elaborate pathogenic cytokines (Anderson et al, 1994; Tang and Graham, 1997; Roman et al., 1997). The severity of respiratory illness in RSV-infected infants correlates with the amounts of the proinflammatory mediators histamine, leukotriene C4, and eosinophilic cationic protein in the nasopharyngeal secretions (Saito et ah, 1997). The so-called cc cytokines and RANTEs are believed to be elaborated by the infected epithelial cells and most probably play a role in the accumulation of eosinophils in lung tissue (Saito et al, 1997) (Figure 5.1). Additional work relating immune factors to the pathogenesis of bronchiolitis focuses on the role of IgE antibody (Welliver et al, 1980). These allergenic immunoglobulins are bound to infected exfoliated respiratory cells during the acute stages of infection. Their presence is associated with wheezing by the child and the release of histamine into the respiratory tract. Overproduction of IgE in persons with atopy could be the result of an imbalance in regulatory populations of T cells. Airway hyperresponsiveness and enhanced airway sensitization to allergens can be documented in

mice and guinea pigs experimentally infected with RSV (Schwarze et al, 1997; van Schaik et al, 1998; Reidel et al, 1997). Despite an enormous amount of experimental study, we lack key insights into the pathogenesis of the bronchiolitis associated with RSV infection and the means for inducing effective immunity. Infants and children with immune deficiency disorders and recipients of corticosteroid agents and chemotherapy are at increased risk of contracting RSV infections and death due to giant cell pneumonia (Hall et al, 1986; Milder et al, 1979; Delage et al, 1984). In these conditions, the virus replicates with relative abandon and for extended periods of time in the lower respiratory tract (Table 5.1) (Hall et al, 1976). Recipients of bone marrow cell transplants are particularly susceptible to RSV infections. As documented by Hertz et al (1989), the majority of patients with severe lower respiratory tract disease develop infections during or shortly after periods of intense irradiation and chemotherapy, but before engraftment of the transplanted bone marrow occurs. At this time, graft recipients are profoundly neutropenic and the respiratory epithelium exhibits the cytolytic effects of irradiation and chemotherapy. A lethal outbreak among bone marrow transplant recipients of various ages was reported by others (Harrington et al, 1992). More than % of the RSV-infected pre-engraftment patients developed pneumonia, whereas fewer than half of those with functioning grafts were so affected. Seventy-eight percent of those with pneumonia died after an extended period of infection. A predisposition to lower respiratory tract disease has also been reported in childhood liver transplant recipients (Pohl et al, 1992). At autopsy, the lungs of patients with severe pulmonary insufficiency exhibit the pathologic features associated with intense mechanical respiratory support, in addition to giant cell pneumonia. These observations in transplant

TABLE 5.1 Shedding Patterns of RSV in Children with Compromised Immune Function as Compared with Those in Children with Normal Immune Function Matched for Type of Illness (Pneumonia) and Age*^

Mean peak titer (logioTCIDso/ml) Duration of shedding in days — mean Percent shedding >20 days

Cancer

Controls

Steroid therapy

Controls

Immunodeficiency

Controls

4.7

2.8

3.6

2.2

5.2

3.1

16

6

9

4

26

7

55

2.5

14

5

80

0

Adapted with permission from Hall et al. (1986). "Not significant. All other differences significant at the level of 0.02 or greater.

56

Pathology and Pathogenesis of Human Viral Disease

B "I ^<'/^^''-

^%'V' ,,.# -%-;

f^. i ^ 1

^ W' '{m,"'0^

%• 4

FIGURE 5.2 Confluent multinucleate cells lining airways of an immunocompromised bone marrow recipient infected with RSV. Note the absence of intranuclear inclusions, permitting a differentiation of the giant cell from a similar multinucleate cell in measles virus pneumonia. In the latter, prominent intranuclear and indistinct cytoplasmic inclusions are usually found.

recipients emphasize the important role that cell-mediated immune mechanisms play in the control of pulmonary parenchymal infections. Pathological descriptions of the lungs in those who have died with RSV infection are limited and incomplete. The histopathologic picture is often confused by the occurrence of secondary bacterial infections and the effects of prolonged therapeutic respiratory support. At autopsy, the mucosa of the bronchi and bronchioles exhibit widespread necrosis, and the submucosa is infiltrated by mononuclear cells (Aherne et al, 1970). The lumen of these small airways are often plugged by debris and secretions. Studies of experimentally infected mice have shown that the degree of respiratory insufficiency inversely correlates with the number of neutrophils and lymphocytes in bronchiolar lavage specimens (van Schaik et al, 1998). In the immunologically intact patient, characteristic multinucleate epithelial cells with eosinophilic intracytoplasmic inclusions are only rarely observed in the respiratory tract, whereas giant cell pneumonia is regularly described in autopsy reports of patients with compromised immune systems (Figure 5.2). Cells of the respiratory mucosa and alveolar macrophages invariably contain viral antigens at these times (as demonstrated by immunological labeling studies). In some studies, the amount in the bronchiolar mucosa is limited; it proves to be much greater in the diseased lung parenchyma (Gardner et ah, 1970). Infected cells lining the airways often exhibit distinct eosinophilic inclusions that represent concretions of viral nucleocapsid (Parham et al, 1993) (Figures 5.3 and 5.4). Studies in Cebus monkeys experimentally infected with high concentrations of virus provide insights into the features of the disease (Richardson ef al, 1978). Four

B

FIGURE 5.3 Specimens from an immunocompromised bone marrow recipient with RSV pneumonia. (A) RSV-infected cells in a bronchoalveolar lavage specimen stained with May-Brunwald-Giemsa dyes. Note the distinct cytoplasmic inclusion. (B) Bronchial epithelial lining cell from autopsy specimen exhibiting complex accumulations of eosinophilic inclusions. Reprinted with permission from Parham et al (1993) through the courtesy of D. Parham, MD.

Respiratory Syncytial Virus

57

FIGURE 5.4 (A) Ultrastructural features of epithelial cell in autopsy specimen (N = nucleus; I = cytoplasmic inclusion; 5,000x). (B) Cytoplasmic inclusion (left) comprised of dense bundles of filamentous nucleocapsids typical of paramyxoviruses (17,000x). (C) RSV nucleocapsid in RSV cytoplasmic inclusion (115,000x). Reprinted with permission from Parham et al. (1993) through the courtesy of D. Parham, MD.

days after intratracheal inoculation, the lungs of these primates exhibit pneumonia characterized by extensive interstitial infiltrates of neutrophils and mononuclear cells. Two days later, prominent numbers of multinucleate mucosal epithelial cells are evident in the lungs, and by the 8th day after inoculation the lungs are consolidated with extensive interstitial infiltrates and intra-airspace exudates of proteinaceous fluid and cells. Studies using organ cultures of fetal human trachea have demonstrated the formation of syncytial giant cells from infected ciliated respiratory epithelial cells (Henderson et al, 1978). Symptomatic RSV infections develop in adults of all ages. In a study of almost 1200 adult patients in Ohio, USA, who were hospitalized with lower respiratory tract illness, 4.4% were infected with RSV during the winter months and 1% during the summer (Dowell et

al, 1996). Reports from Europe have yielded similar findings (Fransen et al, 1967; Morales et al, 1983; Guidry et al, 1991). The risk of acquiring a clinically significant RSV by the elderly correlates with waning of immunity, as documented by low serum antibody concentrations (Falsey and Walsh, 1998). Although customarily mild, the illness is characterized by nasal congestion and rhinorrhea accompanied by pharyngitis and persistent cough and fever. Examination of the chest often reveals rhonchi and wheezes. Clinically, these features help differentiate RSV pulmonary disease from other forms of pneumonia in the older adult. Symptoms often persist for protracted periods of time, and physiological monitoring of these patients has documented increases in airway resistance over a period of weeks (Hall et al, 1978). At times, the virus can be recovered from nasopharyngeal secretions for long

58

Pathology and Pathogenesis of Human Viral D i s e a s e

periods of time. Although asthma occasionally accompanies RSV infections in children, exacerbations of bronchitis and the symptoms of chronic obstructive pulmonary disease do not seem to occur excessively in infected adults. Several RSV outbreaks have been reported in residents of extended care facilities for the elderly (Agius et ah, 1990). Bacterial superinfections occasionally develop in these aged patients, and deaths have resulted (Vikerfors et al, 1987). The pathological features of RSV infection in these adult patients have not been described.

References Agius, G., Dindinaud, G., Biggar, R., Peyre, R., Vaillant, V., Ranger, S., Poupet, J., Cisse, M., and Castets, M. (1990). An epidemic of respiratory syncytial virus in elderly people: Clinical and serological findings. /. Med. Virol 30,117-127. Aheme, W., Bird, T., Court, S., Gardner, P, and McQuillin, J. (1970). Pathological changes in virus infections of the lower respiratory tract in children. /. Clin. Pathol. 23, 7-18. Anderson, L., and Heilman, C. (1995). Protective and disease-enhancing immune responses to respiratory syncytial virus. /. Infect. Dis. 171,1-7. Anderson, L., Tsou, C , Potter, C , Keyserling, H., Smith, T., Ananaba, G., and Bangham, C. (1994). Cytokine response to respiratory • syncytial virus stimulation of human peripheral blood mononuclear cells. /. Infect. Dis. 170,1201-1208. Brandt, C , ICim, H., Arrobio, J., Jeffries, B., Wood, S., Chanock, R., and Parrott, R. (1973). Epidemiology of respiratory syncytial virus infection in Washington, DC, III: Composite analysis of eleven consecutive yearly epidemics. Am. ]. Epidemiol. 98, 355-364. Craighead, J. (1975). Report of a workshop: Disease accentuation after immunization with inactivated microbial vaccines. /. Infect. Dis. 131, 749-753. Delage, G., Brochu, P., Robillard, L., Jasmin, G., Joncas, J., and Lapointe, N. (1984). Giant cell pneumonia due to respiratory syncytial virus: Occurrence in severe combined immunodeficiency syndrome. Arch. Pathol. Lab. Med. 108, 623-625. Dowell, S., Anderson, L., Gary Jr., H., Erdman, D., Plouffe, J., File Jr., T., Marston, B., and Breiman, R. (1996). Respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults. /. Infect. Dis. 174,456462. Falsey, A., and Walsh, E. (1998). Relationship of serum antibody to risk of respiratory syncytial virus infection in elderly adults. /. Infect. Dis. Ill, 463^66. Folkerts, G., Busse, W, Nijkamp, F., Sorkness, R., and Gem, J. (1998). Virus-induced airway hyperresponsiveness and asthma. Am. J. Respir. Crit. Care Med. 157,1708-1720. Fransen, H., Sterner, G., Forsgren, M., et al. (1967). Acute lower respiratory illness in elderly patients with respiratory syncytial virus infection. Acta Med. Scand. 182, 323-330. Gardner, P., McQuilli, J., and Court, S. (1970). Speculation on pathogenesis in death from respiratory syncytial virus infection. Br. Med. ]. 1, 327-330. Gilchrist, S., Torok, T., Gary Jr., H., Alexander, J., and Anderson, L. (1994). National surveillance for respiratory syncytial virus. United States, 1985-1990. /. Infect. Dis. 170, 986-990.

Giles, T., and Gohd, R. (1976). Respiratory syncytial virus and heart disease: A report of two cases. JAMA 236,1128-1130. Groothuis, J., Gutierrez, K., and Lauer, B. (1988). Respiratory syncytial virus infection in children with bronchopulmonary dysplasia. Pediatrics 82,199-203. Guidry, G., Black-Payne, C , Payne, D., Jamison, R., George, R., and Bocchini, J. (1991). Respiratory syncytial virus infection among intubated adults in a university medical intensive care unit. Chest 100,1377-1384. Hall, C , and Douglas Jr., R. (1976). Respiratory syncytial virus and influenza: Practical community surveillance. Am. J. Dis. Child. 130, 615-620. Hall, C , Douglas Jr., R., and Geiman, J. (1976). Respiratory syncytial virus infections in infants: Quantitation and duration of shedding. /. Pediatr. 89,11-15. Hall, C , Kopelman, A., Douglas Jr., G., Geiman, J., and Meagher, M. (1979). Neonatal respiratory syncytial virus infection. New Engl. J. Med. 300, 393-396. Hall, C , Powell, K., MacDonald, N., Gala, C , Menegus, M., Suffin, S., and Cohen, H. (1986). Respiratory syncytial viral infection in children with compromised immune function. New Engl. ]. Med. 315, 77-81. Hall, W, Hall, C , and Speers, D. (1978). Respiratory syncytial virus infection in adults: Clinical, virologic, and serial pulmonary function studies. Ann. Intern. Med. 88, 203-205. Harrington, R., Hooton, T., Hackman, R., Storch, G., Osborne, B., Cleaves, C , Benson, A., and Meyers, J. (1992). An outbreak of respiratory syncytial virus in a bone marrow transplant center. /. Infect. Dis. 165, 987-993. Heilman, C. (1990). Respiratory syncytial and parainfluenza viruses. /. Infect. Dis. 161, 402-406. Henderson, R, Hu, S.-C, and Collier, A. (1978). Pathogenesis of respiratory syncytial virus infection in ferret and fetal human tracheas in organ culture. Am. Rev. Respir. Dis. 118, 29-37. Hertz, M., Englund, J., Snover, D., Bitterman, P., and McGlave, P. (1989). Respiratory syncytial virus-induced acute lung injury in adult patients with bone marrow transplants: A clinical approach and review of the literature. Medicine 68, 269-281. Kurlandsky L., French, G., Webb, P, and Porter, D. (1988). Fatal respiratory syncytial virus pneumonitis in a previously healthy child. Am. Rev. Respir. Dis. 138, 468-472. MacDonald, N., Hall, C , Suffin, S., Alexson, C , Harris, P, and Manning, J. (1982). Respiratory syncytial viral infection in infants with congenital heart disease. New Engl. J. Med. 307, 397-400. Milder, J., McDearmon, S., and Walzer, P. (1979). Presumed respiratory syncytial virus pneumonia in an adolescent compromised host. South. Med. J. 72,1195-1198. Morales, R, Calder, M., Inglis, J., Murdoch, P., and Williamson, J. (1983). A study of respiratory infections in the elderly to assess the role of respiratory syncytial virus. /, Infect. 7, 236-247. Parham, D., Bozeman, P., Killian, C , Murti, G., Brenner, M., and Hanif, I. (1993). Cytologic diagnosis of respiratory syncytial virus infection in a bronchoalveolar lavage specimen from a bone marrow transplant recipient. Am. J. Clin. Pathol. 99, 588-592. Parrott, R., Kim, H., Brandt, C , and Chanock, R. (1974). Respiratory syncytial virus in infants and children. Prev. Med. 3, 473-480. Pohl, C , Green, M., Wald, E., and Ledesma-Medina, J. (1992). Respiratory syncytial virus infections in pediatric liver transplant recipients. /. Infect. Dis. 165,166-169. Reidel, R, Oberdieck, B., Streckert, H., Philippou, S., Krusat, T., and Marek, W (1997). Persistence of airway hyperresponsiveness and viral antigen following respiratory syncytial virus bronchiolitis in young guinea-pigs. Eur Respir J. 10, 639-645.

Respiratory Syncytial Virus Richardson, L., Belshe, R., Sly, L., London, W., Prevar, D., Camargo, E., and Chanock, R. (1978). Experimental respiratory syncytial virus pneumonia in Cebus monkeys. /. Med. Virol 2, 45-59. Roman, M., Calhoun, W., Hinton, K., Avendano, L., Simon, V., Escobar, A., Gaggero, A., and Diaz, R (1997). Respiratory syncytial virus infection in infants is associated with predominant Th-2-like response. Am. ]. Respir. Crit. Care Med. 156,190-195. Saito, T., Deskin, R., Casola, A., Haeberle, H., Olszewska, B., Ernst, R, Alam, R., Ogra, R, and Garofalo, R. (1997). Respiratory syncytial virus induces selective production of the chemokine RANTES by upper airway epithelial cells. /. Infect. Dis. 175, 497-504. Schwarze, J., Hamelmarm, E., Bradley, K., Takeda, K., and Gelfand, E. (1997). Respiratory syncytial virus infection results in airway hyperresponsiveness and enhanced airway sensitization to allergen. /. Clin. Invest. 100, 226-233. Scott, R., Kaul, A., Scott, M., Chiba, Y, and Ogra, R (1978). Development of in vitro correlates of cell-mediated immunity to respiratory syncytial virus infection in humans. /. Infect. Dis. 137, 810817. Simpson, W., Hacking, R, Court, S., and Gardner, R (1974). The radiological findings in respiratory syncytial virus infection in children, II: The correlation of radiological categories with clinical and virological findings. Pediatr. Radiol. 2,155-160.

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Tang, Y, and Graham, B. (1997). T cell source of type 1 cytokines determines illness patterns in respiratory syncytial virus-infected mice. /. Clin. Invest. 99, 2183-2191. van Schaik, S., Enhoming, G., Vargas, L, and Welliver, R. (1998). Respiratory syncytial virus affects pulmonary function in BALB/c mice. /. Infect. Dis. 177, 269-276. Vikerfors, T, Grandien, M., and Olcen, P. (1987). Respiratory syncytial virus infections in adults. Am. Rev. Respir. Dis. 136,561-564. Walsh, E. (1994). Humoral, mucosal, and cellular immune response to topical immunization with a subunit respiratory syncytial virus vaccine. /. Infect. Dis. 170, 345-350. Walsh, E., McConnochie, K., Long, C , and Hall, C. (1997). Severity of respiratory syncytial virus infection is related to virus strain. /, Infect. Dis. 175, 814-820. Welliver, R., and Ogra, P. (1981). Respiratory syncytial virus. Comp. Then 7, 3 4 ^ 0 . Welliver, R., Kaul, T, and Ogra, P. (1980). The appearance of cellbound IgE in respiratory-tract epithelium after respiratorysyncytial-virus infection. New Engl. J. Med. 303,1198-1202. Welliver, R., Wong, D., Sun, M., Middleton, E. J., Vaughan, R., and Ogra, P. (1981). The development of respiratory syncytial virusspecific IgE and the release of histamine in nasopharyngeal secretions after infection. New Engl. ]. Med. 305, 841-846.

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C H A P T E R

6 Herpesviruses: General Principles INTRODUCTION 61 CELLULAR VIRUS REPLICATION 61 HERPESVIRUS LATENCY 62 HERPESVIRUS CYTOPATHOLOGY (ALPHA AND BETA) REFERENCES 63

mon Epstein-Barr virus (EBV) and three recently discovered herpesviruses (nos. 6, 7, and 8). These lymphotropic agents have not been shown to replicate in animals and in cultured epithelial and mesenchymal cells. Members of the herpesvirus family exhibit a common structure but vary in size, with the alphaherpesviruses being the smallest and cytomegalovirus the largest. The viruses have a DNA core in the form of a torus that is comprised of a linear double-stranded DNA chain ranging in size from 150 (HSV) to 180 (CMV) kbp. A capsid made up of 162 pentameric protein capsomeres, arranged in an icosahedral symmetry, invests the double-stranded DNA core. The DNA of HSV-1 is comprised of about 70 open reading frames, these being the site where the genes that characterize the virus are located. The capsid is surrounded by a tegument of poorly described structure and function. In turn, the tegument is invested by an irregular trilaminar envelope that is derived from modified host cell membrane. The DNA of HSV-1 is comprised of about 70 open reading frames. These are sites where the genes that characterize the virus are found. HSV-1 and 2 are believed to encode at least 84 different polypeptides (Whitley et al, 1998). Other members of the family are similarly endowed. Accordingly, the herpesviruses possess an enormous resource of genetic material for the purposes of carrying out their mission. The products of some genes are essential for viral replication, where other genes encode "luxury" functions that provide a virus strain with its unique characteristics.

63

INTRODUCTION The clinical manifestations of herpes simplex viruses (HSV 1 and 2) and varicella-zoster virus (VSV) were recognized by physicians of antiquity. Intense medical and scientific interest in these agents and the "newer" members of the herpesvirus group continues to date because of their enormous clinical importance and their intriguing but incompletely understood biology. Humans are infected with eight different herpesviruses of clinical importance. These agents are somewhat artificially classified into three subfamilies: the alpha, beta, and gamma herpesviruses. Herpes simplex (1 and 2) viruses are the prototype agents of the family and are categorized as alphaherpesviruses, as are VSV and herpes "B" virus, a common pathogen of Old World subhuman primates that occasionally infects humans. Members of the alphaherpesviruses replicate rapidly in vitro and in vivo and in the process destroy cells. Under experimental circumstances, these viruses have the capacity to infect a variety of animal species and cultured cells. Cytomegalovirus (CMV) is the only human pathogen classified as a betaherpesvirus because of its limited host range and characteristic "slow" pattern of replication in both cultured cells and in humans. Thus, CMV recovered from humans only infects human cells in vitro and does not replicate in subhuman primates and lower animals. CMV has the capacity to destroy cells in tissues, but typically it causes a chronic nonlytic cellular infection in humans, thus the common presence of histologically detectable cytomegalic cells with both intranuclear and cytoplasmic inclusions in infected human tissues. The gammaherpesviruses of humans are represented by the comPATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

CELLULAR VIRUS REPLICATION Infectivity under natural circumstances requires an intact virion. Cellular infection is initiated by fusion of the virus with the plasma membrane at specific receptor sites. Uncoating of the DNA core occurs in the cytoplasm, after which the DNA is transported to the nu61

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62

Pathology and Pathogenesis of Human Viral Disease

clear membrane where it enters into the nucleus through membrane pores. The linear DNA is circularized in the nucleus, and both nuclear and cytoplasmic viral protein are transcribed and synthesis of new viral proteins is initiated. Nuclear chromatin is degraded at this point and viral DNA replication begins. Additional protein synthesis results in formation of the empty capsid, which then receives its core of newly synthesized DNA. Viral and tegument proteins are then synthesized and accumulate as patchy layers on the inner aspect of the cell's internal or plasma membranes, where the virus is enveloped. These complex events are accompanied by profound changes in cell structure and function that depend upon the specific characteristics of the herpesvirus type. The biologic diversity and unique pathogenicity of herpesviruses reflect their endowment with a rich but incompletely characterized supply of enzyme systems and proteins. Studies with deletion mutants are beginning to define the complexities and factors that contribute to pathogenicity, but the diverse biological effects and characteristics of these agents no doubt reflect the heterogeneous composition of viral genetic material. It is not surprising that various strains of herpesviruses differ in their pathogenicity for humans. A detailed discussion of the molecular biology of the herpesviruses can be found in numerous contemporary reference sources.

HERPESVIRUS LATENCY Latency is a prototypical biologic characteristic of the herpesvirus. Its molecular and cellular bases have been the subject of countless investigations, but at present our understanding is incomplete. Shortly after the turn of the century, the observations of the surgeon Sir Harvey Gushing on patients with trigeminal neuralgia provided insights into the role of the trigeminal nerve trunk in the occurrence of facial herpetic lesions (Gushing, 1904). When these nerves were severed surgically, recurrent herpetic lesions failed to develop in the distribution of the nerve. Plausibly, Gushing concluded that the nerves served as a viral conduit from the ganglia. Some 20 years later, Goodpasture and Teague (1923) provided experimental confirmation of Gushing's fundamental observations and documented centripetal movement of virus from the skin along the nerves of experimentally infected animals to the ganglion. These findings provided a basis for understanding the detailed studies of Head and Gampbell (see Ghapter 10), who documented for the first time the inflammatory and necrotizing lesions of the dorsal and

cranial sensory nerve ganglia associated with herpes zoster skin lesions (Head and Gampbell, 1900). Later studies by several investigators showed that both HSV and VZV can be recovered from ganglia when active skin lesions exist in the appropriate related dermatomes as well as during periods of apparent dormancy (Baringer, 1974; Bastian et ah, 1972). In the absence of recoverable virus, transcripts of viral RNA can often be demonstrated (Stevens et al, 1988; Hass, 1935; Galloway et al, 1982). Gurrent research has addressed the biology of latency from a molecular perspective (Rock, 1993). During this state, viral DNA exists as a covalent closed circle in a nonintegrated form within the nucleus of roughly 10 to 20% of neurons in the sensory ganglia. Approximately 10 to 20 copies are estimated to be present in each of these cells. However, the mRNAs that characterize cells undergoing a lytic infection are not demonstrable in these cells, although a viral-coded unique latency-associated transcript (LAT) mRNA can be found in the cells of the ganglia. This transcript may regulate a specific locus in the viral DNA that codes for the early replicative protein (IE) involved in initiation of a productive herpesvirus infection (Ho, 1992; Stevens, 1994). Glearly, much work remains to be completed before we possess a full understanding of the phenomenon of latency in HSV and VZV infections. It is estimated that most adults with serologic evidence of past HSV and VZV infections carry latent viral genomic DNA in sensory ganglia of the nervous system, but systematic studies to confirm this claim are lacking. We know very little regarding the distribution of latent viral DNA in individuals. A number of environmental (UV light, trauma) and physiological (fever, menses) influences provoke activation of latent infections. Studies in experimentally infected animals have shown that viral replication begins in the ganglia 48 to 72 hours after a hyperthermic insult or the administration of a single intravenous dose of corticosteroids (Rock and Reed, 1982; Rock et al, 1992; Sawtell and Thompson, 1992; Sheffy and Davies, 1972). The molecular mechanisms that serve as a basis for these events have not been defined. Latency among gammaherpesvirus such as EBV is less clearly defined, for typical cytolytic infections do not occur with this virus in human tissues. Moreover, the viral latent state appears to differ in the various diseases associated with EBV infection. While the viral DNA exists as a nonintegrated circular episome in lymphoid cells, various encoded proteins can be found in either the nucleus or cytoplasm. In Burkitt's lymphoma, a specific intranuclear antigen (Epstein-Barr nuclear antigen [EBNA]) is the only transcript identi-

63

Herpesviruses: General Principles

fied. In tumor cells from nasopharyngeal carcinomas, EBNA and two latent membrane proteins (LMPl and LMP2) are found. And, finally, in B-cell lymphomas and in cultured transformed cells, a panoply of six nuclear antigens and three plasma membrane antigens can be detected (Stevens, 1994). Alas, at this time, the biologic significance of these intriguing observations is uncertain. Attempts to isolate actively replicating virus from cells exhibiting these antigens have not been carried out. Thus, definition of viral latency in these conditions is not critically defined.

HERPESVIRUS CYTOPATHOLOGY (ALPHA A N D BETA) The hallmark of herpesvirus infections in human tissues (but not gamma viruses) is the intranuclear inclusion originally classified as type A by Cowdry (1934). This inclusion was contrasted with the type B cytoplasmic inclusion observed by Cowdry in cells infected with a variety of other viruses. As described in the initial publication, "the nuclear reaction is total and proceeds to complete degeneration. The inclusions are amorphous and particulate, but may be condensed in rounded masses. The ground substance of the entire nucleus is profoundly disturbed, and all of the basophilic chromatin eventually marginates on the nuclear membrane except in the salivary gland inclusions ... which are more basophilic than acidophilic." Subsequent pathologists have documented the spectrum of nuclear changes that fall short of a true inclusion with a clear zone "halo" surrounding it. In these cells, the chromatin structure is lost and the nucleoplasm acquires a granular, homogeneous, amphophilic, smoky, or ground-glass appearance, and the nucleolus is obscured to a variable extent. In humans, these latter cellular features predominate and only occasional cells exhibit typical inclusions. Indeed, inclusions with "halos" appear to be a fixation artefact that is accentuated by fixtures that contain heavy metals (such as Zenker's). This conclusion is borne out by electron microscopic studies of herpesvirus-infected cells that demonstrate the altered chromatin associated with nonenveloped virions but no evidence of a halo. Multinucleate cells — variously termed polykaryocytes, polykaryome, syncytia, and giant cells — are a second feature of herpesvirus-infected human tissue (Poste, 1970). As with inclusions, they vary in number in a lesion and contain within them variable numbers of nuclei. These cells were initially described before the turn of the century in VZV-infected tissues and have

been the subject of considerable research and speculation since that time. Debate has centered on the mechanism of giant cell formation. While some have advocated the concept of amitotic division, the bulk of the evidence supports the view that polykaryosomes result from fusion of the membranes of adjacent cells. The plasma membranes of herpesvirus-infected cells undergo profound changes in structure and antigenicity during infection, but it is currently uncertain whether these effects are responsible for giant cell formation. There is also evidence supporting the notion that interferons may play a key role in cell fusion (Pais et ah, 1994). Information is currently lacking regarding the expression of plasma membrane adhesion factors in herpesvirus-infected cells. "Ballooning" of the cytoplasm is a third characteristic change in the alphaherpesvirus-infected cell. It is prominent in the infected epithelial cells of the vesicular lesions of HSV and VZV. While a mechanism for cellular alterations has not been defined, experimental studies have demonstrated alterations in intracellular electrolyte balance within the infected cells. Thus, it is plausible to suggest that water accumulation in the cytoplasm is at least one mechanism accounting for the typical cytological features of these cells. As its name indicates, CMV infections result in dramatic cellular enlargement in the absence of balloon degeneration (Smith and Vellios, 1950). Since these cells survive for indefinite, but undefined, periods of time in vivo, it has been assumed that the cells enlarge because of continued growth but fail to divide because of viral effects on mitotic spindle formation and molecular signaling mechanisms. It should be amply clear from this brief discussion that our understanding of the mechanisms of cytological changes in the herpesvirus-infected cells is currently limited. References Baringer, J. (1974). Recovery of herpes simplex virus from human sacral ganglions. New Engl. J. Med. 291, 828-830. Bastian, R, Rabson, A., Yee, C , and Tralka, T. (1972). Herpesvirus hominis: Isolation from human trigeminal ganglion. Science 178, 306-307. Cowdry, E. (1934). The problem of intranuclear inclusions in virus diseases. Arch. Pathol. 1, 527-542. Gushing, H. (1904). Perineal zoster, with notes upon cutaneous segmentation postaxial to the lower limb. Am. J. Med. Sci. 127, 375391. Pais, S., Burgio, V., Silvestri, M., Capobianchi, M., Pacchiarotti, A., and Pallone, F. (1994). Multinucleated giant cells generation induced by interferon-y. Lab. Invest. 71, 737-744. Galloway, D., Fenoglio, G., and McDougall, J. (1982). Limited transcription of the herpes simplex virus genome when latent in human sensory ganglia. /. Virol. 41, 686-691.

64

Pathology and Pathogenesis of Human Viral Disease

Goodpasture, E., and league, O. (1923). The transmission of the virus of herpes febrilis along sensory nerves with resulting unilateral lesions in the central nervous system in the rabbit. Proc. Soc. Exp. Biol Med. 20, 545-547. Hass, M. (1935). Hepato-adrenal necrosis with intranuclear inclusion bodies: Report of a case. Am. }. Pathol. 11, 127-142. Head, H., and Campbell, A. (1900). The pathology of herpes zoster and its bearing on sensory localization. Brain 23, 353-523. Ho, D. (1992). Herpes simplex virus latency: Molecular aspects. In ''Progress in Medical Virology'' (J. Melnick, ed.). Vol. 39, pp. 76115. Karger, Basel. Poste, G. (1970). Virus-induced polykaryocytosis and the mechanism of cell fusion. Adv. Virus Res. 16, 303-356. Rock, D. (1993). The molecular basis of latent infections by alphaherpesviruses. Sem. Virol. 4, 157-165. Rock, D., and Reed, D. (1982). Persistent infection with bovine herpesvirus type 1: Rabbit model. Infect. Immunol. 35, 371-373.

Rock, D., Lokensgard, J., Lewis, T., and Kutish, G. (1992). Characterization of dexamethasone-induced reactivation of latent bovine herpesvirus 1. /. Virol. 66, 2484-2490. Sawtell, N., and Thompson, R. (1992). Rapid in vivo reactivation of herpes simplex virus in latently infected murine ganglionic neurons after transient hyperthermia. /. Virol. 66, 2150-2156. Sheffy, B., and Davies, D. (1972). Reactivation of a bovine herpesvirus after corticosteroid treatment. Proc. Soc. Exp. Biol. Med. 140,974-976. Smith, M., and Vellios, F. (1950). Inclusion disease or generalized salivary gland virus infection. Arch. Pathol. 50, 862-884. Stevens, J. (1994). Overview of herpesvirus latency. Sem. Virol. 5, 191-196. Stevens, J., Haarr, L., Porter, D., Cook, M., and Wagner, E. (1988). Prominence of the herpes simplex virus latency-associated transcript in trigeminal ganglia from seropositive humans. /. Infect. Dis. 158, 117-123. Whitley, R., Kimberlin, D., and Roizman, B. (1998). Herpes simplex viruses. Clin. Infect. Dis. 26, 541-555.

C H A P T E R

7 Herpes Simplex Virus (HSV) Types 1 and 2 INTRODUCTION

UROGENITAL TRACT DISEASE

69

CENTRAL NERVOUS SYSTEM DISEASE RESPIRATORY TRACT DISEASE DIGESTIVE TRACT DISEASE

65

66

GENERALIZED SYSTEMIC DISEASE

LIVER DISEASE

evidence of a causative association was lacking. Now the concept has been discarded, as the papillomaviruses receive increasing attention as the cause, or major contributor, to this disease. As an intern, it was my lot to admit to the hospital a young nurse with fever, dementia, and unilateral neurological signs. Craniotomy revealed a destructive and hemorrhagic encephalopathy involving a temporal lobe. HSV was recovered from the tissue. This patient had no known risk factors, and the pathogenesis of the lesion then, as it would now, remained obscure. As the present chapter relates, the story of HSV infections is complex since the virus lingers in most of us in a latent, unexpressed form. Yet, on occasion, it can cause devastating disease. This chapter summarizes our current understanding of these conditions.

65

PRIMARY AND RECURRENT ORAL AND SKIN INFECTIONS

71

75

76

17

LYMPH NODE DISEASE EYE DISEASE

78

REFERENCES

81

78

INTRODUCTION A colleague has herpes lip vesicles rather often. They are triggered by menses, sunlight, and emotional stress, as usually is the case in adults. She can predict their appearance. There is a day or two of irritability and depression, followed by a tingling sensation and lancing pain at the site where the eruption develops. Then, as the vesicles burst forth, the nervous tension subsides and the inevitable one week wait for the eruption to resolve begins. Her husband has never had herpes, yet he takes no special precautions to avoid contact with his wife when a lip vesicle is present. Obviously, he is either resistant or immune — most probably the latter. It is likely he has a latent unexpressed infection. The risk factors for cervical cancer are well known to every medical student, that is, sexual experiences early in life with multiple partners. This association leads to the obvious conclusion that the disease has a venereal etiology, just as does herpes genitalis. Studies in recent years have made it clear that herpes infections of the male and female genital tract are far more common than previously thought. And, to our surprise, a new herpesvirus (HSV-2) was found to be the etiology in most cases (Fleming ei al., 1997). In the 1960s, it was generally concluded that cervical cancer was linked with this infection. A foregone conclusion as numerous papers in my files attest, but, in hindsight, clear-cut

PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

PRIMARY A N D RECURRENT ORAL A N D SKIN INFECTIONS (Bader ei al, 1978; Buddingh et al, 1953; Higgins et al, 1993; Spruance et al, 1977)

Gingivostomatitis is the typical primary lesion of HSV in children 1 to 3 years of age. Almost invariably, it is due to type 1 virus acquired from others by intimate contact or respiratory droplet transmission. The infection is complicated by generalized symptoms of fever, malaise, and regional lymphadenopathy. Resolution of these lesions occurs over a period of 7 to 10 days. Presumably, infants under 1 year of age are protected by maternally acquired antibodies, but exposure to the virus is limited because social contacts are usually few during this time of life. Inoculation herpetic lesions of the skin can occur as primary infections in immunologically susceptible persons due to abrasive interactions (herpes gladiatorum and herpetic whitlow), or as a complication of a chronic dermatological condition (Eiferman et al, 1979; Hambrick et al, 1962; Selling and Kibrick, 1964) (Figure 7.1). Thus, the virus is a hazard for hospitalized burn victims (Foley et al, 1970) and for those with an active 65

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

66

Pathology and Pathogenesis of Human Viral Disease

F I G U R E 7.1 Typical skin lesions in a healthy 25-year-old laboratory technologist with chronic acneiform dermatitis autoinfected by HSV. The eruption was accompanied by systemic symptoms, including fever, but resolved without event, and without scarring.

F I G U R E 7.2 A herpetic skin lesion at autopsy in an immunosuppressed renal allograft recipient. It was only one of several extensive confluent lesions found over the skin of this patient at autopsy.

dermatitis, such as eczema (Kaposi varicelliform eruption) (Indgin et ah, 1970). Skin lesions also develop in immunosuppressed patients, despite the presence of preexisting naturally acquired immunity. Often, these lesions are extensive, confluent, and hemorrhagic (Figure 7.2). The facial lesions of recurrent herpes simplex are a reflection of trigeminal nerve latency They characteristically occur on the mucocutaneous vermillion margin of the lip or occasionally in the nasal folds in the sensory nerve branch distribution of this nerve. HSV lesions rarely occur in the ophthalmic distribution. Approximately 20 to 40% of healthy men and women in developed countries regularly experience these eruptions. Both the primary and recurrent lesions exhibit comparable morphological features,, but almost no biopsy material is available for critical analysis. More frequently, diagnosis is established by the isolation of virus from the bullous fluid or by the cytological demonstration of multinucleate inclusion-bearing cells in the cellular sediments of the fluid (Figure 7.3) (Spruance et al, 1977; Solomon et al, 1984). The fluid contains a mixture of acute inflammatory cells, and inflammation is seen at the base of the ulcers. Infected keratinocytes and the cells of the inflammatory infiltrate elaborate a variety of cytokines that accumulate in the vesicular fluid. Undoubtedly, these cytokines contribute to the pathological picture, but the role of specific cytokines remains to be defined (Mikloska et al, 1998). To the extent justified, molecular diagnostic studies would be useful. HSV infections in the immunocompromised patient, particularly those with AIDS, are atypical in distribu-

tion and appearance. The buccal mucosa, floor of the mouth, palate, and tongue may be involved by extensive ulcerating lesions that can mimic bacterial glossitis or candidiasis. Longitudinal, linear, or branching fissures with a geometric pattern can form on the papillated surface of the tongue. The lesions of HSV in a patient with AIDS are customarily deeper and more painful than those occurring in an immunologically intact patient (Bustamante and Wade, 1991; Grossman et al, 1993).

UROGENITAL TRACT DISEASE (Nahmias et al, 1967; Nahmias and Roizman, 1973; Kaufman et al, 1973; Naib et al, 1973; Stern and Longo, 1963; Cone et al, 1994)

In the Journal of Cutaneous and Venereal Diseases, Unna (1883) provided the first clinical description of herpes progenitalis. Since that time, interest in this common venereal infection has grown as its common occurrence among the sexually active has become more fully appreciated. Concern is also justifiably focused on the contribution of genital herpes to disseminated herpes simplex infections of the newborn (Amstey, 1971; Nahmias et al, 1971; Kibrick, 1980; Arvin et al, 1986; Binkin et al, 1984; Randolph et al, 1993; Kulhanjian et al, 1992; Prober et al, 1987; 1992; Cone et al, 1994; Hain et al, 1980). Roughly 5% of the population of the United States has clinical histories suggestive of an HSV genital infection, although a substantially greater proportion exhibits serological evidence of an inapparent (i.e..

Herpesviruses: General Principles

67

'i^.'

F I G U R E 7.3 Material scraped from the floor of a vesicle smeared on a slide (Wright's stain). The multinucleate cell with nuclear molding and intranuclear inclusions is diagnostic. Inclusions in this example are represented by homogeneous alterations of the nucleoplasm. This procedure is the so-called Tzanck's test, which can be used effectively as an alternate to virological studies to establish the etiology of vesicular lesions rapidly. Reprinted with permission from Cohen et ah (1977).

subclinical) or latent, inactive infection. The majority of these infections are acquired venereally and are due to type 2 HSV virus. Overall, the prevalence of HSV-2 has increased by roughly 30% during the past two decades, a possible reflection of changes in sexual practices among adolescents and young adults (Fleming et ah, 1997). Women are more commonly affected than males, and in some studies blacks have a prevalence threefold greater than whites. Clinicians have long suspected that pregnant women are unusually susceptible to infection or exhibit reactivated latent infections more often; however, this clinical impression has not been established in a formal study. Disseminated HSV infections, although rare, occur with unusual frequency in pregnant women. In the female, primary HSV genital infections become symptomatic 3 to 7 days after sexual contact (Bryson et ah, 1993). The papules and vesiculations are frequently extensive, involving both the labia majora

and labia minora, as well as the vestibule of the vulva, the anus, and the perianal skin. To a variable extent, the lesions ulcerate with the passage of time, before resolving over a 3- to 6-week period (Guinan et ah, 1981) (Figure 7.4). Tissue edema and both local and generalized symptoms are common. Ulcerative changes occasionally occur on the exocervix and at the squamocolumnar junction. The histological features of these genital lesions are typical of HSV vesicles elsewhere and exhibit cellular features that are easily recognized by cytopathologists. The lesions of recurrent herpes develop in a distribution similar to those of the primary infection, but they usually are less prominent and often are inconspicuous. The pattern and frequency is somewhat dependent on virus type. In one study, genital recurrences were documented in almost 90% of those infected with HSV type 2 virus, but in only 25% of patients infected with type 1 virus (Lafferty et ah, 1987). In some patients, both virus types are recovered

68

Pathology and Pathogenesis of Human Viral Disease

TABLE 7.1 Comparative Results of Virological and Cytological Studies of 97 Women Who Had Previously Been Found to Yield Cells, Cytologically Positive for HSV Infection

Pain Index

Cellular changes Erythema 500/J Papule

50%|

Vesicle

50%

Ulcer

50%|

Crust

50%|

Virologic findings

Positive

Negative

Total

Positive Negative Total

13 3 16

16 65 81

29 68 97

Adapted with permission from Naib et al. (1966).

Completely Healed '"''^

%of Patients Culture Positive

Lesion Viral Titer Mean Log

3

3

T 4

6 or 7

8 or 9 10 or 11

Day of Illness

FIGURE 7.4 Natural history of primary HSV female genital infections. The evolution of lesions on the vermillion border of the lip is temporally comparable to the process on the vulva. Adapted with permission from Guinan (1981).

(Sucato et ah, 1998). Overall, the frequency of these events was 0.33 eruptions per month in women with latent infections. In a detailed study of young women with a history of recurrent genital herpes, Guinan et al. (1981) noted that 78% had only a single lesion while 4% had a maximum of four. Clinically important shedding of HSV during lesion-free periods is common (Wald et al, 1995). In cytologic survey studies of several hundred thousand indigent women, Naib (1966) detected fewer than 0.3% HSV positive preparations. Virological investigations of the survey-positive cases yielded HSV in over 80% of these women, but additional work showed that 20% of those lacking cytological evidence of infection had detectible virus in genital tract specimens (Table 7.1). Thus, both cytological and virological studies are required in order to identify all cases of infection. These investigators characterized the detailed sequence of changes in exfoliated cells typical of HSV infection as follows:

1. The alteration appears in younger cells, parabasal of squamous epithelium, or the young cuboidal cells of the endocervical glandular epithelium. 2. The first visible structural change is a true hypertrophy of the cytoplasm and the nucleus. 3. An irregular perinuclear halo is also often seen early in this stage, but this change is probably a fixation artefact and is common to most of the enlarged exfoliated epithelial cells. 4. The nucleolus, if present, enlarges for a short period, then becomes edematous, distorted, and finally disappears by lysis. 5. Marked disturbance of the cytoplasmic ground substance appears early on. From its normal granular composition, it becomes hyalin in appearance, denser, and more basophilic. 6. Multinucleation most probably resulting from amitotic division is associated with formation of the diagnostic giant cells. The cells differ from the multinucleated foreign body giant cells by their characteristic nuclear molding without overlapping and by variation in their size and shape. (Considerable questions remain today as to the mechanism of multinucleated cell formation in HSV infection.) 7. The nuclear chromatin loses its granularity, clumps, and marginates to the periphery, leaving in the center a bland amorphous eosinophilic zone. The nuclear basophilia gradually decreases, except for the chromatin clumps adherent to inner surface of nuclear membrane. 8. Particulate, acidophilic, single, and centrally positioned intranuclear inclusion bodies then develop, soon to be surrounded by a more or less prominent halo. 9. Ballooning-type cytoplasmic or nuclear degeneration with multiple vacuolization usually follows, indicating an irreversible cellular injury.

Herpesviruses: General Principles

Published documentation of genital herpes in males is less detailed, but the natural history of the cutaneous infections are similar to those in females (Powers et al, 1982; Barile et al, 1962). Studies of the internal male genital organs in men lacking a history of genital herpes have yielded virological evidence of infections of the prostate, seminal vesicle, or epididymis in approximately 15% of immunocompetent adults of all ages (Centifanto et ah, 1972). This astonishing prevalence of infection is particularly remarkable, because no pathological evidence of HSV infection has been described in these organs. The male may serve as a reservoir of infection. HSV may be the etiologic agent of prostatitis and epididymitis in a few men (Morrisseau et al, 1970). HSV hemorrhagic cystitis with viral antigens or inclusion-bearing cells in the bladder mucosa has been described (Nguyen et al, 1992), but it is difficult to assess how frequently this lesion occurs. HSV has also been recovered from prostatic secretions (Morrisseau et al, 1970), but there is no evidence to suggest that HSV is transmitted commonly by semen. On occasion, urogenital infections result in neuropathies of the sacral plexus (Figure 7.5).

69

\i^ fjf

GENERALIZED SYSTEMIC DISEASE (Miller et al, 1970; Wheeler and Huffines, 1965; Monif et al, 1985; Hanshaw, 1973)

Disseminated neonatal HIV infection was first recognized at autopsy by Hass (1935). Since that time, its pathogenesis has intrigued the curious. With the increasing prevalence of herpes progenitalis among sexually active young people, it has become an increasingly important cause of perinatal mortality. Although still rare, the incidence approximates 28 per 100,000 births per year in the United States today (SuUivanBolyai et al, 1983; Whitley 1993). Neonatal HSV infections are almost invariably due to exposure of the child to active ulcerating maternal genital lesions resulting from a primary infection acquired from a sex partner during pregnancy. The risk to the fetus has been estimated to be approximately 50%. In contrast, the risk to infants born of mothers with recurrent herpes is substantially less (approximately 2 to 5% of the maternal cases) (Prober et al, 1987). While primary infections are usually fulminant, there are several possible explanations for the low rate of viral transmission to infants by mothers with recurrent disease. First, in contrast to primary infections, the concentration of virus in genital secretions of women with active lesions is relatively low (Prober et al, 1987).

F I G U R E 7.5 A patient with HSV involvement of the sensory sacral nerve distribution. Note the distribution of the skin lesions along the course of the sensory nerves. The patient is incontinent and receiving catheter drainage.

Second, the cervix is rarely affected by herpes ulcerations. And, finally, the infant has acquired specific HSV type 2 antibody transplacentally and thus possesses a level of immune resistance prior to exposure in the birth canal. About 2 to 3% of all pregnant women shed infectious HSV in the absence of clinically apparent genital lesions. This is most probably a minimal number, since sensitive molecular techniques detect viral DNA in genital secretions of about 25% of pregnant women at the time of labor. Because of the relatively infrequent occurrence of primary infection in full-term women, most cases of generalized herpes in neonates result from recurrent HSV disease in the mother. Although the majority of neonatal infections are acquired in the birth canal, fragmentary information suggests that HSV infection is occasionally contracted transplacentally (Mitchell and McCall, 1963). Case

70

Pathology and Pathogenesis of Human Viral Disease

F I G U R E 7.6 HSV infection of the placenta. (A) Subchorionic cells of the placenta from an 18-year-old woman with a history of genital herpes but no evidence of an active infection at the time of parturition. The 2700-g offspring did not develop an HSV infection. Virus-positive cells are stained using a biotinylated DNA probe for HSV. (B) Virus-infected maternal cells of the decidua. Reprinted with permission from Schwartz and Caldwell (1991).

reports have documented viral antigens (Robb et ah, 1986a,b), intranuclear inclusions (Goldman, 1970), and viral particles in the endometrium (Abraham, 1978), decidua (Schwartz and Caldwell, 1991), and the placenta associated with villositis (Witzleben and Driscoll, 1965; Nakamura et ah, 1985) (Figure 7.6) and necrotizing funisitis (Heifetz and Bauman, 1994). The actual prevalence of transmission by this route is not documented. The clinical features of neonatal HSV infection are variable, as is the distribution and severity of the internal lesions at autopsy. In about 40% of cases, the disease is limited to the skin, eyes, and mouth. The lesions in the form of vesicles and keratoconjunctivitis develop shortly after birth, and resolve with the passage of

time, to reoccur sporadically. When the infection is disseminated, necrotizing hepatic and adrenal lesions are the most striking constant postmortem finding (Hass, 1935; Patrizi et al, 1968) and the prototype lesions of this disease. To differing extents, inclusion-bearing cells can be identified at the periphery of the necrotic foci. The lungs often exhibit necrotizing lesions of the airways and parenchyma. Meningoencephalitis (see below) occurs in about 30% of cases (Schlesinger and Storch, 1994). About 1 in 7 of these cases manifest an ocular infection that ranges in severity from a mild conjunctivitis to a destructive lesion of the retina or optic nerve (Gammon and Nahmias, 1985). Virus is readily recovered from these lesions. Disseminated intravascular coagulation occurs frequently in these

71

Herpesviruses: General Principles

cases (Nakamura et al, 1985; Miller et al, 1970). It has been suggested but not proven that the route of infection influences the distribution of lesions. Aspiration results in involvement of the aerodigestive system, whereas hepatoadrenal disease is due to transplacental hematogenous transmission. Neonatally infected infants experience a viremia; this is the likely source of the lesions in the liver and adrenals (Stanberry et al, 1994). Disseminated infections occur on rare occasions in pregnant women, particularly during the 2nd and 3rd trimesters (Kulhanjian et al, 1992; Young et al, 1996). Over half of these women die with encephalitis, pneumonitis, hepatitis, and a coagulopathy. Almost 60% have visible oropharyngeal, genital, or skin lesions, and in two-thirds of these patients type 2 HSV is recovered from the tissues. Pregnancy seems to create physiological conditions conducive to HSV dissemination. Experimentally infected pregnant mice experience a high mortality rate when inoculated intravaginally with type 2 HSV (Young and Gomez, 1979). Susceptibility of animals is also increased by progesterone treatment (Baker and Plotkin, 1978). Interestingly enough, progesterone has a decided immunosuppressive effect, particularly impacting cell-mediated immunity. However, disseminated HSV in patients with AIDS and recipients of immunosuppressive drugs is not reported, even though these patients often develop severe localized disease (Montgomerie et al, 1969; Hook et al, 1992).

CENTRAL NERVOUS SYSTEM DISEASE Dr. Margaret Smith, who contributed so much to our understanding of CMV, initially described HSV encephalitis in the newborn as a pathologic entity (Smith et al, 1941). A classical study of the neuropathology of the disease in adults followed shortly thereafter (Adams et al, 1949). In developed countries, HSV encephalitis is now recognized as the most common form of sporadically occurring necrotizing hemorrhagic encephalitis in both children and adults. The attach rate has been calculated at 2 to 4 cases per million (Whitley and Schlitt, 1991). Numerous reports document the clinical features of HSV encephalitis, and considerable virological information has accumulated (Drachman and Adams, 1962; Haymaker et al, 1958). The disease has a biphasic age distribution, with approximately 30% of cases occurring before the age of 20 and a sizable proportion of the remainder developing in later life (Figure 7.7). It is seen less often in the third and


1-5

5-10

10-15

15-20

20-30

>30

Age Group (Years)

F I G U R E 7,7 Age at onset of HSV encephalitis in patients admitted to civilian and military general hospitals. Reprinted with permission from Olson et al (1967).

fourth decades of life. Ten to 20% of patients develop disease subtly. liowever, in most adults, the illness evolves as a fulminating febrile condition with personality changes and bizarre behavior, seizures, and an altered state of consciousness. Radiological studies often reveal the classical picture of a "tumor-like" temporal lobe swelling. A small proportion (10-15%) of patients display herpetic pharyngeal or skin vesicular lesions (Whitley and Lakeman, 1995), and approximately 70% of patients possess serum antibodies against HSV type 1 at the onset of illness, an indication of a previous and presumptively latent infection. In the absence of effective treatment, the mortality rate is 50%, but many survivors exhibit significant residual neuropsychiatric problems. Studies by Nahmias et al (1982) have shown that the outcome (in the absence of antiviral treatment) correlates with the relative concentration of virus recovered from brain biopsies obtained during the acute stages of disease. Because of the availability of effective antiviral therapy, early diagnosis is a necessity. Among patients believed to have HSV encephalitis on clinical grounds, Nahmias et al (1982) detected infection in biopsies of brain tissue in the form of intranuclear cellular inclusions in 56% of patients, and by immunohistochemistry of the brain tissue in 70% (Whitley et al, 1989; Esiri, 1982; Merkel and Zimmer, 1981) (Figure 7.8). Electron microscopy is a less effective means of diagnosis (Swanson et al, 1966). Thus, it has been debated as to whether or not biopsy diagnosis should be established before antiviral treatment is begun (Hanley et al, 1987; Fishman, 1987). Antiviral drug treatment (currently, acyclovir) is often initiated based on diagnostic suspicion before the results of a brain biopsy are available.

72

Pathology and Pathogenesis of Human Viral D i s e a s e

FIGURE 7.8 Temporal lobe biopsy specimen from a young adult woman with clinically typical encephalitis. Note the prominent intranuclear eosinophilic inclusions surrounded by a halo. The nucleoplasm is marginated. The background brain substance shows degenerative vacuolar changes.

The common appearance of chemotherapy-resistant strains of HSV is an increasing concern (Swetter et ah, 1998; Read et al, 1998). Modern molecular approaches prove to be highly sensitive diagnostic tools (TroendleAtkins et al, 1993). In a multi-institutional study 98% of patients with biopsy-proven herpes encephalitis had PCR-positive cerebrospinal fluid. Interestingly enough, the fluid of 20% of patients was positive by PCR for longer than 15 days of treatment with antiviral drugs (Whitley and Lakeman, 1995). PCR evaluation of cerebrospinal fluid now appears to be sufficient to establish the diagnosis (Puchhammer-Stockl et al, 1990). Despite the characteristic clinical picture in the typical case, many encephalopathies mimic HSV encephalitis. Thus, a specific virological diagnosis is highly desirable whenever feasible (Whitley et al, 1989). While in most childhood and adult cases the temporal lobe is most extensively affected (see Figure 7.9), the orbital frontal cortex, occipital lobe, and hippocampus can be involved to a variable extent. In infants, the infection is often more widely disseminated in the brain (Figure 7.10). Histologically, during the acute stages of encephalitis, the brain tissue exhibits variable degrees of necrosis and hemorrhage accompanied by meningeal and perivascular mononuclear cell infiltrates (Figures 7.11 and 7.12). A vasculitis is commonly seen (Figure 7.13). The typical intranuclear inclusions are found in both neurons and neuralgia but are variable in number (see Figure 7.8). Often, cells with a smudgy homogeneous eosinophilic nucleoplasm provide evidence of HSV infection in the absence of characteristic inclusions. Patients die throughout the course of the acute process and later in convalescence. However, the availability of effective chemotherapy has dramatically altered the outcome of HSV encephalitis.

FIGURE 7.9 The brain at autopsy from the case illustrated histologically in Figure 7.8. Note the prominent edema and hemorrhage in the temporal lobes bilaterally. The diffuse swelling and distortion of the brain consequent to the infection is apparent. There is a meningeal reaction particularly prominent at the base of the brain. The tip of the right lobe had been removed for laboratory study.

FIGURE 7.10 Coronal sections of the brain of an infant with HSV encephalitis. The tissue exhibits an extreme degree of congestion, edema, and focal hemorrhages.

Herpesviruses: General Principles

F I G U R E 7.11 Acute HSV encephalitis demonstrating the marked perivascular accumulation of inflammatory cells and the diffuse astrocytosis and inflammation of the brain substance with necrosis of scattered neurons.

F I G U R E 7.12 Thrombosis of a small blood vessel in the edematous inflamed brain of a patient v^ith HSV encephalitis.

Isolated aseptic meningitis due to HSV sometimes occurs with a clinical picture that cannot be distinguished from meningitis caused by other agents, such as enteroviruses and mumps virus. Although diagnosis can often be accomplished by viral isolation and serological studies, as noted above, modern molecular approaches provide a sensitive means for identifying the virus or its fingerprints in brain tissue (Schiff and Rosenblum, 1998; Saldanha et ah, 1986; Sequiera et ah, 1979) and in cerebrospinal fluid (Schlesinger et ah, 1995). The unique but consistent development of lesions in the limbic regions of the brain in HSV infections of the central nervous system has intrigued many investigators, since no obvious routes of access for virus to the affected areas of the brain are evident. No other viral encephalitis exhibits such a remarkable localization to this region of the brain (Figure 7.14). Some investiga-

73

F I G U R E 7.13 Vasculitis in a vessel of the meninges adjacent to the temporal lobe areas of extensive necrosis. Lesions of this type are commonly found in herpes encephalitis.

F I G U R E 7.14 Bilateral destructive lesions in temporal lobes and cingulate gyri bilaterally in the brain of a middle-aged man dying late during convalescence from virologically documented HSV encephalitis. Note the prominent meningeal thickening representing chronic inflammatory changes.

tors have argued that the ophthalmic bulb serves as a means of access for the virus, allowing transmission along nerves to the rhinencephalon and limbic systems of the brain (Tomlinson and Esiri, 1983). Virus has been recovered from the ophthalmic nerve in a patient with typical HSV encephalitis (Ojeda, 1980). However, lesions of the ophthalmic bulb are rarely observed in cases of encephalitis, and, as noted above, most cases are believed to result from activation of a latent virus. Some workers have championed the notion that a latent infection in the trigeminal nerve ganglion serves as a source of infection, perhaps by shunting virus to the central nervous system through the nervous tentoria (Barnett et ah, 1994). Experimental studies in small animals have shown that the central nervous system can be infected by introducing the virus through the cornea and directly by injection into the central nervous system. Clearly, these animal studies do not replicate the

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situation occurring in humans. Neurovirulence of HSV strains may be an acquired genetic characteristic, but the evidence supporting the notion that strains of virus having neuropathic properties are responsible for disease of the central nervous system thus far is not compelling (Whitley and Lakeman, 1995). As indicated above, central nervous system diseases in infants are customarily a reflection of a systemic infection acquired in the birth canal. We have discussed above the various factors that influence the occurrence of infections in the neonate. In the adult, no plausible explanation has been advanced that would readily account for the sporadic occurrence of HSV encephalitis. Since most patients exhibit serological evidence of prior infection, one must assume that the disease reflects a manifestation of a recurrent infection in a substantial proportion of patients. However, the infrequent occurrence of herpetic lesions on the skin and in the oropharynx during the acute stages of herpes encephalitis argues that the disease of the central nervous system is not a reflection of an activated infection elsewhere in the body. Surprisingly, HSV encephalitis is not a common complication of immunosuppression or AIDS, although a case in an immunosuppressed renal transplant recipient has been reported (Linnemann ei ah, 1976). This is particularly perplexing since rampant skin infections (see Figure 7.2) occur commonly in immunosuppressed patients. Recently, Schiff and Rosenblum (1998) described three cases of herpes encephalitis in immunocompromised adults who had fever and changes in mental status, but no evidence of lesions in the brain by computer axial tomography. Cerebrospinal fluid changes were also minimal. Autopsy established the diagnosis by immunocytochemistry and electron microscopy demonstrated virions, but histologically the temporal lobes demonstrated none of the inflammatory hemorrhagic and destructive changes customarily seen in typical herpes encephalitis (Figure 7.15). Earlier reports previously documented somewhat similar findings in individual cases (Price et al, 1973; Martin et al, 1988). The evidence, although incomplete, suggests that immune mechanisms and possibly cytokines generated by infected cells (Mikloska et al, 1998) play an important role in the pathogenesis of the brain lesions of herpes encephalitis (Johnson and Valyi-Nagy, 1998). Jay and his associates (1995) documented HSV-1 infections by PCR in the brains of two young children undergoing treatment for refractory seizures. Pathological evidence of chronic encephalitis was established. The finding is consistent with the notion that HSV may at times account for subtle and as of yet unexplained chronic neurological syndromes (Rennick et al, 1973; Raskin and Frank, 1974). For example.

.f •, ->ii! F I G U R E 7.15 Schiff and Rosenblum (1998) recently described the unique clinical pattern of HSV encephalitis in patients with HIV-1 infections. The disease process evolves over a 2- to 3-week period with fever and a variety of neuropsychiatric findings. Radiological studies do not show localized disease. The case illustrated here is from a 35-year-old woman who had a subtotal resection of a left frontal lobe brain to treat a B cell immunoblastic lymphoma. Over the next 3 weeks, her general status deteriorated with the development of mucocutaneous herpetic lesion and a septicemia. Repeated contrast-enhancing cranial computer axial tomography scans disclosed no changes in the brain indicative of encephalitis. (A) The coronal section of the brain at the anterior commissure exhibits diffuse swelling and loss of the grey-white matter demarcation in the insular area. The temporal lobe changes were considered to be artefactual. (B) There is a noninflammatory "ischemic" appearance to the neurons with shrinkage and sponginess as well as perineural and perivascular vacuolization is depicted. (C) Immunocytochemical localization of HSV antigens. In this patient there is a chronic encephalopathy that does not have the dramatic clinical features of the typical HSV encephalitis occurring in immunologically intact persons. Reprinted with permission from Schiff and Rosenblum (1998) through the courtesy of M. Rosenblum, MD.

Herpesviruses: General Principles

75

FIGURE 7.16 Herpetic infection involving the (A) tracheal mucosa and (B) a submucosal gland. Intranuclear inclusions are seen (straight arrows). In A, cells with homogenous HSV inclusions are depicted by curved arrows. In B a possible herpetic giant cell (curved arrow) is displayed. Reprinted with permission from Nash (1972) through the courtesy of G. Nash, MD.

Shearer and Finch (1964) described a patient with recurrent episodes of severe organic psychosis temporally associated with the appearance of herpetic lesions of the lip.

RESPIRATORY TRACT DISEASE HSV infections of the airways and lungs occur sporadically in adults with preexisting traumatic lesions of the tracheal bronchial tree and under conditions of immunosuppression. Autopsy studies by Nash and Foley (1970) showed that herpetic complications occur commonly in persons who have died from widespread thermal burns. In a subsequent retrospective evaluation of 1000 consecutive autopsies in a large metropolitan general hospital, Nash (1972) identified pulmonary herpetic infections in 1% of patients. Interestingly enough, in this study the lung lesions had routinely been overlooked by both the clinicians and the pathologists. In two cases, the patients had undergone tracheal intubation, whereas the remaining eight cases in the series were victims of extensive body burns. The pulmonary complications in a few patients were believed to be secondary to HSV viremia resulting from herpetic lesions in the damaged skin. In these and other immunologically competent patients, upper airway HSV infections ojften develop in respiratory tissues that were traumatically or thermally damaged. As an end result, focal necrotizing viral lesions ultimately are located in the tracheobronchial tree (Figure 7.16) and lung parenchyma (Figure 7.17). Typically, these pulmonary lesions are superimposed upon one of a variety of acute inflammatory pulmonary conditions such as bacterial pneumonia and adult respiratory distress syndrome

(Tuxen et ah, 1982). Cytopathology has been shown to be an effective approach to the diagnosis of herpes respiratory disease (Frable et al, 1977). However, herpesvirus infections of the lung may be more common than initially perceived. Using immunohistochemistry, Oda et al. (1994) found infected cells in 52% of lungs obtained at autopsy from patients with a diversity of diseases. Diffuse HSV pulmonary disease also develops in the lungs of congenitally infected neonates (Andersen, 1987) and immunosuppressed adults (Douglas et al., 1969). HSV lung disease can occur as a manifestation of

FIGURE 7.17 HSV bronchopneumonia. The infection destroyed a small airway and spread to the surrounding lung parenchyma. Note the massive accumulations of nuclear debris, and the fragmented muscle remnants of the airway wall. Reprinted with permission from Nash (1972) through the courtesy of G. Nash, MD.

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either a primary or recurrent infection. These lesions can be extensive and often are life-threatening. Clinically, they occasionally exhibit the radiological picture of diffuse interstitial pneumonia. In a series of 16 immunosuppressed bone marrow recipients, Ramsey et al. (1982) found that HSV disease of the upper airways and lungs developed within 2 months of the time of transplantation. Orofacial herpetic lesions were identified in the majority of these patients, but two had herpes progenitalis. The isolates exhibited the virological type typical of either oropharynx or genital tissues, an indication that both types 1 and 2 can infect the respiratory tract. The lesions of the lung parenchyma typically are relatively circumscribed and exhibit hemorrhagic coagulative necrosis. Inflammation usually is not prominent. In the studies of Ramsey et al. (1982), typical HSV inclusions were found in 75% of the lesions and immunohistological studies to identify HSV antigens were positive in half of the cases. Cytology supplemented by immunohistochemistry of tracheal and bronchial aspirates is a useful diagnostic approach (Jordan et al, 1975; Vernon, 1982).

autopsy in about 2% of immunologically intact adults (Pearce and Dagradi, 1943; Moses and Cheatham, 1963; Nash and Ross, 1974; Matsumoto and Sumiyoshi, 1985; Galbraith and Shafran, 1992). Although they occur somewhat more often in patients with malignant disease, the esophageal lesions do not develop with unusual frequency and severity in the immunocompromised patient (Matsumoto and Sumiyoshi, 1985; Buss and Scharyj, 1979). For example, in a systematic study of 580 renal transplant recipients, only 5 biopsy or autopsy-proven cases of herpes esophagitis were recognized (Komorowski et al, 1986) (Figures 7.19 and 7.20). For unknown reasons, herpetic lesions of the digestive tract are found on rare occasions in immunologically intact and otherwise healthy children and adolescents (Ashenburg et al, 1986; Stillman, 1986; DeGaeta et al, 1985).

DIGESTIVE TRACT DISEASE Severe ulcerative disease of the oropharynx is common in immunocompromised patients (Figure 7.18). Interestingly enough, ulcerative lesions of the mucosa of the distal esophagus due to HSV type 1 are found at

FIGURE 7.18 Margin of ulcerative lesion of the tongue in a patient with AIDS. Note the inflammatory infiltrate and granulation tissue at the margin of the regenerating squamous epithelium. The typical cytopathological changes are evident with inclusion bodies surrounded by halos in the basal cells, and homogeneous granular basophilic changes in the nucleoplasm of the more superficial squamous cells. Vacuolar degeneration of these cells is evident.

FIGURE 7.19 Typical appearance of HSV esophagitis with discrete ulcers in the upper and middle thirds of the esophagus. Confluent lesions are located in the lower third. Herpetic involvement of the esophagus does not advance beyond the cardioesophageal junction. The gastric mucosa of the cardia exhibits petechial hemorrhages, a common nonspecific finding at autopsy. Reprinted with permission from Nash and Ross (1974).

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lesions in the course of sacral nerves is common in these patients (Samarasinghe et al, 1979).

LIVER DISEASE

F I G U R E 7.20 HSV lesions of the esophagus in an immunocompromised renal allograft recipient. In the mid-esophagus, an irregularly outlined proliferative lesion is located. At the cardia, there is a longitudinally oriented deep ulcer with granulation tissue in the crater. Evidence of cytomegalovirus infection of the endothelial cells and fibroblasts in the granulation tissue was found microscopically. The squamous mucosal cells on the margin of the ulcer exhibited the typical intranuclear inclusions of HSV. This is a common finding in lesions of this type.

Patients with herpes esophagitis typically experience odynophagia, retrosternal pain, and fever. In my review of the literature, only two cases of alleged herpes involvement of the gastric mucosa were found (Howiler and Goldberg, 1976; Buss and Scharyj, 1979), but it is not clear whether the stomach mucosal lesions were HSV ulcers. Esophageal herpetic ulceration raises perplexing questions of pathogenesis. These lesions are not found with increased frequency in patients with other herpes simplex conditions such as herpes labialis. There is no history of recurrent disease, but there is also no evidence to argue that the lesion represents a primary infection. Possible precursor conditions such as reflux esophagitis or indwelling catheters are not recognized to be predisposing factors, and immunosuppression does not appear to be a trigger. Anal and perianal ulceration due to herpes simplex accompanied by urinary dysfunction is a well-established syndrome in male homosexuals. The innervation of the urinary tract and perineum no doubt are involved, since sacral radiculomyelopathy with skin

HSV frequently involves the liver in the course of disseminated infections in newborns (Figure 7.21), older children with kwashiorkor and marasmus (McKenzie, 1961), and adult recipients of immunosuppressive drugs (Johnson et al, 1992; Chase et al, 1987) and those with AIDS (Figure 7.22). In addition, pregnant women exhibit an unusual susceptibility to the virus in the absence of therapeutic immunosuppression. In a literature review of published reports, pregnancy was found to be the only identifiable risk factor in 23% of adult cases (Chase et al, 1987). HSV hepatitis was found in roughly 1% of some 1000 recipients of bone marrow transplants who died while being administered immunosuppression (Johnson ^f al, 1992; Rosen and Hajdu, 1971). However, HSV hepatitis occasionally occurs in seemingly immunocompetent patients (Lee and Fortuny 1972; Goodman et al, 1986). Type 2 HSV usually is responsible for the disease in infants, since the infection is acquired in the birth canal or transplacentally In adults, both virus types appear to be involved. While the liver can be involved in the absence of disease in other major organs, not infrequently, herpetic lesions of the aerodigestive system and adrenal glands are also identified. Primary lesions are found in either the oropharynx or the genital tract.

F I G U R E 7.21 HSV infection of the liver of a neonate demonstrating extensive destructive necrosis and a multinucleate cell exhibiting the typical nuclear features of an infected cell. Note the homogeneous pattern of the nucleoplasm.

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FIGURE 7.22 HSV hepatitis in an immunocompromised adult. Note the involvement of the epitheliuni of a bile duct in the portal radical, and a localized lesion in the parenchyma comprised of a few clusters of mononuclear inflammatory cells. Hepatic lesions in adults are generally localized in comparison to the diffuse necrosis customarily seen in congenitally acquired infections.

However, HSV hepatitis can be manifest as a fulminant anicteric hepatitis, with death being the usual outcome. Eighty-six percent of the adult cases reviewed by Chase et al (1987) died. Liver biopsies and autopsy specimens typically show hemorrhagic coagulation necrosis in foci of variable size, surrounded by a zone of congestion. Inflammatory infiltrates are customarily not prominent. Reactive and regenerative changes characterize the involved parenchyma, and accumulations of fat in hepatocytes are a variable feature. The viral nature of the liver lesions is apparent when typical intranuclear inclusions with a halo are present, but frequently, cells with inclusions are not a prominent feature. In these cases, the nucleoplasm of affected cells often exhibits a typical homogenous "ground glass'' appearance.

LYMPH N O D E DISEASE

cortical expansion with accumulation of prominent numbers of immunoblasts and a variable representation of plasma cells and eosinophils. In contrast to the lesions observed with other herpesviruses, the lymph nodes in HSV infections exhibit focal circumscribed areas of necrosis in which occasional cells at the periphery of the lesion have intranuclear inclusion bodies (Figure 7.23). In early lesions, intact and karyolytic neutrophils are apparent in focal lesions of the lymph nodes, whereas in more advanced lesions coagulation necrosis is evident (Figure 7.24). The presence of inclusion-bearing cells permits differentiation of a herpesvirus lesion from other forms of necrotizing lymphadenitis, but, as discussed above, infected cells often exhibit viral-associated alterations of the nuclei in the absence of typical inclusions. In these situations, there is a need for immunologic or molecular diagnostic studies to establish the identity of the etiologic agent.

(Epstein et al, 1986; Gaffey et al, 1991; Tamaru et al, 1990; Miettinen and Ukkonen, 1982)

EYE DISEASE Lymphadenitis sporadically occurs among patients with CMV, EBV, and VZV infections. HSV is a rare cause of lymphadenitis. It occurs in those who are immunologically intact as well as in patients with lymphoma and leukemia. Microscopically, the lymph nodes in these infections exhibit either a diffuse or peri-

As noted above, HSV involves the eyes in about 16% of cases of neonatal infection (Gammon and Nahmias, 1985). In particular, these lesions commonly accompany overt neonatal encephalitis. In one study, uncomplicated keratoconjunctivitis was associated with both

Herpesviruses: General Principles

79

FIGURE 7.23 HSV lymphadenitis. Enlarged lymph node exhibiting well-circumscribed areas of necrosis. The insert documents the presence of inclusion-bearing cells in the node. Reprinted with permission from Epstein et at. (1986) and through the courtesy of J. Epstein, MD.

FIGURE 7.24 HSV lymphadenitis. Nuclear debris and coagulation necrosis of the lymph node. The nodal architecture is lost. Reprinted with permission from Gaffey et at. (1991) and through the courtesy of M. Gaffey, MD.

HSV-1 and HSV-2 infections, but more severe forms of the ocular disease proved almost invariably to be due to the type 2 virus (Reersted and Hansen, 1979). DukeElder and Perkins (1966) noted three categories of intraocular disease in the infected neonate: (1) iridocyclitis secondary to herpetic keratitis; (2) iridocyclitis unassociated with keratitis, but resulting from herpetic

uveitis; and (3) chorioretinitis secondary to a generalized herpetic infection, often with encephalitis. A variety of less easily defined but complicated lesions have also been described (Johnson and Wisotzkey, 1977). In diagrammatic form. Figure 7.25 depicts the various lesions that have been described in the eyes of congenitally infected newborn.

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Ocular Findings in Herpes Infections of the Newborn

e-^'^'^PSSsCj^ Anterior 1. 2. 3. 4. 5. 6. 7. 8.

Conjunctivitis Punctate Keratitis Dendrite Geographic Corneal Ulcer Stromal Keratitis Uveitis (Keratitic Precipitates) Posterior Synechiae Cataract

Posterior 1. Optic Neuritis (Disc Edema) 2. Intraretinal Hemorrhages (Flame, Dot and Blot) 3. Vasculitis 4. Vitritis 5. Yellowish-White Retinitis 6. Retinal Detachment 7. Retinal Pigmentary Hyperplasia 8. Macular Scars

FIGURE 7.25 The spectrum of ocular HSV lesions in the eye of the newborn in the anterior and posterior anatomic locations. Reprinted with permission from Gammon and Nahmias (1985).

In the adult, keratitis due to HSV is a leading cause of blindness. An estimated 3-5 x 10^ adults suffer from this recurrent disease of the external eye. First described over a century ago, its destructive manifestations were vividly portrayed in Picasso's early 1900 painting "La Celestine" (Figure 7.26). Grut (1886) defined keratotic dendritica as a "superficial ulceration (of the cornea) with a chronic course and tendency to become serpiginous; propagating by means of buds or excrescences so that the lines of demarcation of the very superficial ulcers become extremely irregular; the surrounding parts are clear, and the cornea is not vascularized." The etiology was established by transmission of the virus to the cornea of rabbits in 1912, with subsequent induction of keratitis in the eye of a blind man using corneal exudates from an infected rabbit. Thus, the basic tenets of Koch's postulates were fulfilled long before the virus was recovered and grown in vitro (Gruter, 1920). Herpetic keratitis begins as a primary infection with the typical geographic dendritic features of the lesion developing after the acute onset of conjunctivitis (Figure 7.27). At this time, the eye is inflamed and smears of the exudates show polymorphonuclear leukocytes.

but inclusion-bearing cells are customarily not seen. Subsequently, the virus becomes latent to reoccur erratically, as do the more common lesions of the vermillion margin of the lips. Usually, the eye lesion is unilateral (Openshaw et ah, 1995), and in adolescents and adults the disease occurs twice as often in males as in females (Howard and Kaufman, 1962). With the passage of time, the lesions evolve to develop deep in the cornea and assume a disciform appearance. Similar changes have been described in the eyes of herpes zoster-infected persons. The chronic inflammatory disease of the stroma of the cornea results in neovascularization and scarring, with glaucoma and cataracts occurring as secondary complications. The major inflammatory and destructive manifestations of herpes keratitis appear to be attributable to an immunopathologic process in which CD4+ T cells are a major actor. While evidence in humans is lacking, studies using a murine model of herpes keratitis show that specific clones of corneal reactive CD4+ T cells cross-react with an HSV virion-associated protein (UL6) (Zhao et al., 1998). Additional work by other investigators (Jayaraman et al, 1993) implicates T cells sensitized to glycoproteins in the envelope of HSV virions. Molecu-

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Herpesviruses: General Principles

FIGURE 7.26 The late effects of unilateral HSV keratitis illustrated in 'Ta Celestine/' a classical Blue Period painting by Pablo Picasso. Reprinted with the permission of the Estate of Pablo Picasso and the Artists Rights Society (ARS), New York.

lar mimicry may play an important role in elaboration of these pathogenic T cells. Conflicting evidence implicates both the Thl and Th2 CD4+ lymphocytes (Jayaraman et al, 1993; Verjans et ah, 1998). Some studies suggest a contribution of natural killer cells, macrophages, and polymorphonuclear leukocytes in the pathogenesis of the keratitis (Tamesis et al, 1994; Berra et al, 1994). The role of CD8+ cells in development of the lesion remains to be defined, although these lymphocytes may contribute to clearance of virus from the infected eye. In AIDS, inflammatory and destructive lesions of the retina and the optic nerve have been associated with HSV infections in some cases. In murine models, depletion of either CD4+ or CD8+ lymphocytes results in development of optic nerve lesions (Azumi and Atherton, 1994; Zhao et al, 1995). Unfortunately, the bulk of experimental work undertaken to evaluate the immunopathology of herpes keratitis has been conducted by necessity in inbred highly susceptible strains of mice. These models prove to be imperfect examples of herpes keratitis in humans.

FIGURE 7.27 HSV corneal dendrite is revealed by slit-lamp examination with fluorescein (upper panel) and fluorescein plus rose bengal (lower panel, high power) in a 70-year-old man who reported 2 days of pain and redness in his right eye. His visual acuity was minimally diminished. The lids (A) are normal. The conjunctiva (B) is injected. The diagnostic dendrite (C) results from the branching pattern of desquamation of infected corneal epithelial cells. The desquamated cornea stains with fluorescein, whereas the remaining infected epithelial cells at the margin of the dendrite stain brilliantly with rose bengal. This epithelial infection differs from a corneal ulcer, which involves infection and erosion of the corneal stroma. The iris (D) and pupil (E) are normal. The leading edge of the slit-lamp beam (F) reveals the sloping contour of the corneal surface and suggests that the depth of the anterior chamber is normal. The patient was successfully treated with a 14-day course of topical trifluridine, an antiviral agent, and had no permanent visual impairment. Reprinted with permission from Cicella and Johnson (1997).

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Douglas, R., Anderson, M., Weg, J., Williams, T, Jenkins, D., Knight, v., and Beall Jr., A. (1969). Herpes simplex virus pneumonia: Occurrence in an allotransplanted lung. JAMA 210, 902-904. Drachman, D., and Adams, R. (1962). Herpes simplex and acute inclusion-body encephalitis. Arch. Neurol. 7, 45-79. Duke-Elder, S., and Perkins, E. (1966). Diseases of the uveal tract. In ''System of Ophthalmology" (S. Duke-Elder, ed.). Vol. 9, pp. 336343. Heruy Kimpton, London. Eiferman, R., Adams, G., Stover, B., and Wilkins, T (1979). Herpetic whitlow and keratitis. Arch. Ophthalmol. 97,1079-1081. Epstein, J., Ambinder, R., Kuhajda, F, Pearlman, S., Reuter, V, and Mann, R. (1986). Localized herpes simplex lymphadenitis. Am. J. Clin. Pathol. 86, 444-448. Esiri, M. (1982). Herpes simplex encephalitis: An immunohistological study of the distribution of viral antigen within the brain. /. Neurol. Sci. 54, 209-226. Fishman, R. (1987). No, brain biopsy need not be done in every patient suspected of having herpes simplex encephalitis. Arch. Neurol. 44,1291-1292. Fleming, D., McQuillan, G., Johnson, R., Nahmias, A., Aral, S., Lee, F., and St. Louis, M. (1997). Herpes simplex virus type 2 in the United States, 1976 to 1994. New Engl. J. Med. 337,1105-1111. Foley R, Greenawald, K., Nash, G., and Pruitt Jr., B. (1970). Herpesvirus infection in burned patients. New Engl. J. Med. 282, 652-656. Frable, W, Frable, M., and Seney Jr., R (1977). Virus infections of the respiratory tract: Cytopathologic and clinical analysis. Acta Cytol. 21, 32-36. Gaffey, M., Ben-Ezra, J., and Weiss, L. (1991). Herpes simplex lymphadenitis. Am. J. Clin. Pathol. 95, 709-714. Galbraith, J., and Shafran, S. (1992). Herpes simplex esophagitis in the immunocompetent patient: Report of four cases and review. Clin. Infect. Dis. 14, 894-901. Gammon, J., and Nahmias, A. (1985). Herpes simplex ocular infections in the newborn. In "Viral Diseases of the Eye" (R. Darrell, ed.), pp. 46-58. Lea & Febiger, Philadelphia. Goldman, R. (1970). Herpetic inclusions in the endometrium. Obstet. Gynecol. 36, 603-605. Goodman, Z., Ishak, K., and Sesterhenn, I. (1986). Herpes simplex hepatitis in apparently immunocompetent adults. Am. J. Clin. Pathol. 85, 694-699. Grossman, M., Stevens, A., and Cohen, P. (1993). Brief report: Herpetic geometric glossitis. New Engl. J. Med. 329, 1859-1860. Grut, E. (1886). Sur deux Formes typiques de keratite. In "Proceedings of the Congres periodique international des sciences medicales (8th Session, 1884)." Copenhagen. Gruter, W (1920). Experimentelle und klinische Untersuchungen uber den sogenannten Herpes Comeae. Klin. Mbl. Augenheilk. 65, 398. Guinan, M., MacCalman, J., Kern, E., Overall Jr., J., and Spruance, S. (1981). The course of untreated recurrent genital herpes simplex infection in 27 women. New Engl. J. Med. 304, 759-763. Hain, J., Doshi, N., and Harger, J. (1980). Ascending transcervical herpes simplex infection with intact fetal membranes. Obstet. Gynecol. 56,166-169. Hambrick Jr., G., Cox, R., and Senior, J. (1962). Primary herpes simplex infection of fingers of medical personnel. Arch. Dermatol. 85, 583-589. Hanley, D., Johnson, R., and Whitley, R. (1987). Controversies in neurology: yes, brain biopsy should be a prerequisite for herpes simplex encephalitis treatment. Arch. Neurol. 44,1289-1290. Hanshaw, J. (1973). Herpesvirus hominis infections in the fetus and the newborn. Am. J. Dis. Child. 126, 546-555.

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8 Cytomegalovirus HISTORICAL OVERVIEW

due to this same virus were obscure. Several years later, I had the opportunity to explore some of these influences when the same virus, now known as cytomegalovirus (CMV), was discovered in the lung tissue of a renal allograft transplant recipient with a fatal pneumonia at the Peter Bent Brigham Hospital in Boston (Hedley-Whyte and Craighead, 1965). The distinctive cellular inclusions of CMV have intrigued generations of pathologists. In 1881, Rivert noted "protozoan-like" cells in the kidney epithelium of a stillborn fetus thought to have died from congenital syphilis. His observations were published in 1904, as were those of Jesionek and Kiolemenoglou (1904), who observed similar cells in the lungs, kidneys, and liver of an 8-month-old fetus with the clinical picture of congenital lues. From this time to the present, pathologists have documented the seemingly paradoxical occurrence of these same cytomegalic cells with intranuclear inclusions in a variety of epithelial organs of children lacking evidence of a systemic virus infection. The striking resemblance of the inclusions in these cells to amoeba resulted in the designation of what was believed to be a new species of amoebae. Goodpasture and Talbot (1921) noted the similarity of the intranuclear bodies in the enlarged epithelial cells to similar structures in the squamous epithelial blisters of chicken pox. They coined the term "cytomeglia" and described the disease of newborn infants we now know as Cytomegalic Inclusion Disease. Accumulating evidence soon led to the conclusion that the bodies were not in fact protozoa, but represented the cytopathology of a virally infected cell. The evidence was based on the finding of similar enlarged inclusion body-bearing cells in the salivary gland tissue of several species of lower mammals. The modern history of CMV begins with the near simultaneous isolation of a cytopathic virus from congenitally infected newborns by Smith and Vellios (1950) (Figure 8.1) and Weller et al (1957), as well as the recovery of CMV by Rowe et al. (1956) from cultured tonsillar and adenoid tissue of older children. Clinical and epidemiological studies in several labora-

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MONONUCLEOSIS AND THE POSTTRANSFUSION SYNDROME

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NERVOUS SYSTEM INFECTION AND DISEASE PULMONARY INFECTION AND DISEASE

DIGESTIVE TRACT INFECTION AND DISEASE LIVER INFECTION AND DISEASE

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GENITOURINARY TRACT INFECTION AND DISEASE MYOCARDIAL INFECTION AND DISEASE EYE DISEASE

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POSSIBLE ROLE OF CMV IN ATHEROSCLEROSIS REFERENCES

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HISTORICAL OVERVIEW In 1957, the author began work in the virology laboratory of Wallace Rowe at the National Institutes of Health. It had been just a year since Rowe (Rowe et ah, 1956) and Margaret Smith of Washington University independently isolated from infants and children an infectious agent they termed ''salivary gland virus" (Smith, 1956). In tissue culture, the virus produced large intranuclear inclusions that strikingly resembled structures seen microscopically in the salivary gland epithelial cells of some humans and many lesser mammalian species. I was responsible for collecting urine and oropharyngeal secretions from seemingly healthy young children at a District of Columbia orphanage in an effort to recover the virus and thus better understand its natural history. I recall the excitement of finding the cytopathology of this subtle slow-growing agent in cultured cells inoculated with material from these active and healthy kids. It was my initial introduction to a fascinating world where infection did not mean disease and the factors triggering overt disease

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FIGURE 8.2 Cytopathic effect of CMV growing in a culture of fetal fibroblasts. The cytological changes conform to those observed in infected cells of human tissue. Note the cellular enlargement and the reniform intranuclear inclusion with an amorphous amphophilic nucleoplasm. A clear halo surrounds the inclusion. The indistinct intracytoplasmic inclusion is evident. FIGURE 8.1 The late Dr. Margaret Smith, Professor of Pathology at Washington University, St. Louis. Dr. Smith's pioneering work provided our initial insights into disseminated neonatal disease due to CMV.

tories soon provided insights into this unique virus and its common occurrence as a latent or subclinical infection in members of the general population. As the biology of CMV was elucidated, events in another unrelated area of modern clinical medicine — organ transplantation and therapeutic immunosuppression — crystallized an intense clinical interest in this important virus. With implantation of the first allografted kidneys in 1973, accompanied by the clinical use of the immunosuppressive agents Imuran and prednisone, devastating multisystem CMV disease suddenly appeared (Kanich and Craighead, 1966). To date, these infections continue to occur in organ transplant recipients despite refinements in immunosuppression and transfusion therapy. Those of us acquainted with this story were not surprised when CMV appeared in the profoundly immunosuppressed patient with AIDS.

CYTOMEGALIC INCLUSION BODY CELLS The distinctive morphologic features of the infected cell were vividly described many years ago by Margaret Smith (Figure 8.2). Her observations can be paraphrased as follows (see Smith, 1964):

Its nucleus is enlarged and the cytoplasm increased in amount. The great size which may be attained (25-40 jLim) and the intranuclear inclusion (8-10 |Lim) is a strikingly unique characteristic. The large intranuclear inclusion is surrounded by a clear halo which separates it from a distinct nuclear membrane containing one or more dense basophilic masses. At times, the nuclear membrane appears wrinkled or partially collapsed. The shape of the inclusion usually corresponds to that of the nucleus in which it lies. When stained with hematoxylin and eosin, the intranuclear inclusion may either be acidophilic or basophilic. However, the degree of basophilia of the inclusion is seldom as great as that of the nuclear membrane. The inclusion may appear granular or nodular or uneven in density; at times, the peripheral zone stains less intensely than the center. Frequently, however, the inclusion appears homogeneous. Its outline may be either sharply defined or hazy. The cytoplasm of the cell is acidophilic or amphophilic and frequently contains small basophilic bodies which vary in size and number. The basophilic bodies measure 2-4 jim in diameter, are concentrated usually in one part of the cytoplasm and may be arranged in a concentric manner near the periphery of the cell. The cytoplasm of the largest cells is frequently vacuolated and their contour irregular. The intranuclear inclusion may occur in a cell which does not contain cytoplasmic inclusions; but on the other hand, the cytoplasmic inclusions never occur in cells which contain a nucleus devoid of an inclusion. At times, a nucleus cannot be seen

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in cell-containing cytoplasmic inclusions, either because the cell is so large that the nucleus is not in the plane of the section or because the cell has degenerated. The inclusions are usually found in epithelial cells but may occur in cells of the interstitial connective tissue and in vascular endothelium. Both the intranuclear and cytoplasmic inclusions are Feulgen-positive and the cytoplasmic inclusions stain less intensely than the intranuclear (Figure 8.3). The cytoplasmic, but not the intranuclear, inclusions react positively with periodic acid-leucofuchsin (PAS stain). The complexities of the cellular changes involved in these morphological observations have now been elucidated by electron microscopic studies (Kanich and Craighead, 1972a,b).

EPIDEMIOLOGY A N D NATURAL HISTORY The prevalence of CMV infection in various healthy population groups at different ages is influenced largely by sociological conditions. Those in the lower economic strata of our society and residents of developing countries tend to be infected very early in life. In contrast, fewer than 50% of the population of developed countries have been infected by young adulthood. The majority of healthy men and women who have serological evidence of past infection are carriers of the virus in a latent and inapparent fashion. Transmission between infants and young children and to adults from children usually occurs through saliva or by the respiratory droplet route. In adults, venereal spread by seminal fluid and vaginal secretions occurs. The tissues of infected children and adults often support replication of the virus for extended, if not indefinite, periods of time. CMV can be recovered from the oral cavity and the urine of newly infected children and adults for periods of months or years (Table 8.1). Autopsy studies of children dying of a variety of causes have documented the presence of cells with inclusions in roughly 8-32% of parotid glands and, to

FIGURE 8.3 Electron micrographic illustration of the intranuclear features (N) and the intracytoplasmic inclusions that develop early in the course of the infection in cultured cells (C-1). The inclusions are comprised of mitochondria, Golgi apparatus, free ribosomes, and endoplasmic reticulum. Note the diffusely altered homogenous nucleoplasm in which encapsulated virions can be found. Two nucleoli are seen. The halo, seen so clearly in cells fixed with formalin, is not evident when the tissue is fixed for electron microscopy with glutaraldehyde and in solutions containing heavy metals such as mercury or picric acid. With the passage of time, additional complex changes occur in the cytoplasmic inclusions. In particular, lysosomes in large numbers and viral coat constituents accumulate in the cytoplasm (x9800). Reprinted with permission from Kanich and Craighead (1972a).

a lesser extent, in other organs (Farber and Wolbach, 1932). The virus can be recovered from these same tissues, even though high titers of antibodies and sensitized T cells are present in the circulation. Inapparent infection in the absence of inclusion-bearing cells now has been demonstrated in multiple human organs us-

TABLE 8.1 Duration of CMV Excretion in Congenitally Infected Infants Time from birth to follow-up* Virus isolation

0-3 months

4-12 months

1-2 years

2-3 years

3-4 years

4-5 years

5-6 years

Urine Throat Swab

44/48 44/48

14/18 11/18

6/7 1/4

14/17 5/13

4/8 0/7

9/11 2/11

1/1 0/1

*No. infected/no. tested. Adapted with permission from MacDonald and Tobin (1978).

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ing the refined tools of molecular biology, that is, PCR and in situ hybridization. The prevalence of latent CMV infection in members of the general adult population is doubtless much higher than previously indicated by the findings in serological surveys (Hansen et al, 1994; Powell, 1989; Sachdev, 1990; Kotsimbos et al, 1997).

CONGENITAL C M V INFECTION A N D DISEASE In developed countries, CMV is the most commonly transmitted virus in utero. It can be recovered from the urine of approximately 0.5 to 2.5% of infants shortly after birth. A small proportion of infected concepti abort or are stillborn (Schwartz et al., 1989), whereas the majority of the infants with these congenital infections are asymptomatic at birth and experience no recognizable long-term effects. There is no quantitative information on the prevalence of abortions and stillbirths attributable to CMV. The virus has been recovered from the macerated products of conception, and, on rare occasions, inclusion-bearing cells are found in the tissue (Kriel et al, 1970; Schwartz et al, 1990). Systemic fatal disease occurs in roughly 3% of infected conceptuses, and long-term residual effects are found in approximately 15% of these patients. The outcome depends in part on the immune status of the mother and the gestational age of the fetus when infection occurs. In a recent study, 21% of infants with HIV-1 infections were also actively infected with CMV, whereas only 4% of healthy newborns had detectable CMV in the urine or oropharynx (Doyle et al, 1996). Most congenital infections result from viremia occurring in a nonimmune pregnant woman experiencing a primary infection late in pregnancy. It is uncertain how often the placenta is infected secondary to maternal infection. Jahn et al (1991) found CMV antigens in the placentas of five infants with congenital disease. Inclusion-bearing cells are occasionally located in the placenta, as discussed in more detail below. A small number of newborns contract the virus in the birth canal as a result of cervical infections (Stagno et al, 1986), and an additional few (roughly 8%) by breastfeeding (Hayes et al, 1972; Stagno et al, 1980). Sixty-five percent of young women classified as members of a "high-income" economic group proved to be seronegative, whereas only 23% of those in a 'Tow-income" category lacked detectable serum antibodies, indicating prior and presumed latent infection (Stagno et al, 1986). However, fewer than 2% of the

seronegative "high-income" women experienced primary infections during pregnancy, whereas almost 4% of the "low-income" seronegative mothers were similarly infected. These data reflect the findings of a national survey that demonstrated that mothers of infants with congenital CMV infection tend to be young, primiparous, and black as well as members of a lower socioeconomic class (Istas et al, 1995). With the advent of organ transplantation and improved survival of graft recipients, pregnancies are beginning to occur among women with active systemic CMV infections consequent to immunosuppression. While the numbers are too small to document a pattern, disseminated congenital infections are reported in the offspring of some of these women (Laifer et al, 1995; Evans et al, 1975). Overt CMV disease of the neonate is a rare syndrome with variable clinical and pathological manifestations (Wyatt et al, 1950; Eichenwald and Shinefield, 1962; Stagno et al, 1975, 1977a,b; Stern and Tucker, 1973) (Table 8.2). Superficially, the acute disease resembles congenital rubella, toxoplasmosis, and syphilis. Although its occurrence differs from one socioeconomic group to another, the extensive experience of McCracken et al (1969) in the New York City area indicated an incidence of 1 in 3300 births. While many infants die within weeks or months of birth, a substantial number survive beyond the first year of life, often with profound physical and neurological residue (Figure 8.4). Infection in these infants and children is documented by demonstration of high concentrations of virus in the urine and blood by culture or PCR (Nelson et al, 1995) and cytomegalic inclusion-bearing cells in major organs postmortem. The features of these lesions are described in detail in the sections that follow. Hepatosplenomegaly occurs in 70% of cases. It is usually associated with jaundice and variable degrees of ascites. Biopsies of the liver often reveal hepatitis with focal giant cell transformation and cholangitis accompanied by accumulation of the typical infected cells in the intra- and extrahepatic bile ducts (Alagille et al, 1983). ExtrameduUary hematopoiesis is strikingly evident in both the liver and spleen, and no doubt accounts in part for enlargement of these organs. With prolonged survival, these organs gradually decrease in size, but residual hepatic portal fibrosis is often seen. Over half of the infants manifest disease of the central nervous system during the first 6 months of life (Istas et al, 1995). In the study by McCracken, 40% of infected children exhibited microcephaly at or shortly after birth, and the majority of these infants developed periventricular calcification in the brain within the first

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Cytomegalovirus TABLE 8.2 T h e National Congenital C M V D i s e a s e Registry Case D e f i n i t i o n A. Confirmed symptomatic congenital CMV disease 1. Detection of CMV in urine, saliva, secretions, or tissue obtained within the first 3 weeks of life 2. In a newborn one or more of the following signs, symptoms, or laboratory abnormalities present: Small for gestational age Petechiae Purpura Splenomegaly Hepatomegaly Jaundice at birth Microcephaly Chorioretinitis Unexplained neurological abnormality Intracranial calcifications Hearing impairment Direct hyperbilirubinemia (bilirubin level >3 mg/dL) Platelet count <75,000/mm3 Alanine aminotransferase level >100 U / L AND 3. Exclusion of other diseases that produce these abnormalities B. Possible symptomatic congenital CMV disease 1. Detection of CMV in urine, saliva, secretions, or tissue obtained from 3 weeks to 1 year of life 2. One or more of the signs, symptoms, or laboratory abnormalities listed in section A AND 3. Exclusion of other diseases that produce these abnormalities Reprinted with permission from Istas et ah (1995).

PREDICTED OUTCOME OF CONGENITAL CMV

0.5-2.0% Women acquiring primary infection during pregnancy

50% Fetus not infected

60% Cinically "normal" neonates

Normal children 55%

50% Fetus infected

38% Clinically "abnormal" neonates

2% Stillbirths

Handicapped children

Post natal deaths

With mild handicap 25%

With severe handicap 15%(5%deaf)

FIGURE 8.4 Evolution of clinical outcomes in congenitally infected infants. Adapted with permission from MacDonald and Tobin (1978).

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several weeks. In a second more recent study, 53% had microcephaly at birth. In an additional 20%, microcephaly and hydrocephaly appeared later in life (Boppana et al, 1992; Stagno et al, 1977a). Variable degrees of mental deficiency are documented later in life among the less severely affected survivors, but the extent of the defects may be difficult to evaluate because of the common occurrence of deafness and blindness (Wilson and DuBois, 1923). Lesions in a variety of other organs have been described. Myocarditis occurs rarely in the neonatal disease, and congenital structural lesions of the heart are not found with increased frequency. Abnormalities of anatomic structures derived from the first branchial arch (micrognathia, high arched and cleft palate) are described in some reports. Inclusion-bearing cytomegalic cells are observed in the adrenal cortex and pancreas, particularly in the islets of Langerhans. Interestingly enough, the beta cells are often infected, an observation that has provoked questions regarding the possible role of CMV in type II diabetes mellitus later in life (Onodera et al, 1983). Adrenal insufficiency due to diffuse cortical necrosis has been described. Radiologically lesions of the long bones resembling those associated with the congenital rubella syndrome are noted (Graham et al, 1970). Throughout the length of the bone, the pattern of longitudinal striations is interrupted by irregular areas of radiolucency The histopathology of these bone changes remains to be evaluated. Thrombocytopenia and hemolytic anemia are common features of the congenital disease, possibly because of involvement of the vascular endothelium by the virus. Petechiae occur in about 20% of cases.

PLACENTAL INFECTION A N D DISEASES Lymphoplasmocytic villitis with accumulation of increased numbers of macrophages (Hofbauer cells) and villous necrosis are commonly associated with congenital infections (rubella, varicella, variola, syphilis, and toxoplasmosis). CMV has been recovered from placentas exhibiting these pathological changes, and variable numbers of cytomegalic inclusion-bearing macrophages and infected endothelial cells are noted (Hayes and Gibas, 1971; Monif and Dische, 1972; Mostoufizadeh et al, 1982; Sachdev et al, 1990; Schwartz et al, 1992) (Figure 8.5). Immunohistochemistry and in situ studies employing a viral DNA probe demonstrate infection of Hofbauer cells, trophoblasts, and endothelial cells (Jahn et al, 1991; Sachdev et al.

FIGURE 8.5 Placental villus of congenitally infected stillbirth. Note the scattered macrophages with intranuclear inclusions.

1990; Muhlemann et al, 1992). Garcia et al (1989) published a detailed analysis of the pathology of the placenta at various stages of gestation.

INFECTIONS OF IMMUNOLOGICALLY INTACT CHILDREN A N D ADULTS The sporadic but exceedingly rare occurrence of CMV disease in healthy children and adults has long been recognized by pathologists (Wong and Warner, 1962). In a review, Fisher and Davis (1958) found only 19 adult cases reported in the literature. Of these, eight manifested as an exudative interstitial pneumonia, whereas most of the remaining cases were recognized incidentally by the finding of inclusion-bearing cells in the granulation tissue of ulcerative digestive tract lesions. In a more recent review, Eddleston et al (1997) evaluated 34 clinical cases of severe CMV infection. Central nervous system involvement was suspected in 10, but these patients survived, with only one exhibiting residual neurological problems. Hepatitis as an isolated disease process occurred in many others. Almost

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10% were pregnant women, suggesting that gestation may be a risk factor. Most of these cases occurred before the era of molecular diagnosis, and only serological or virological diagnostic criteria were used. No doubt more sensitive molecular approaches would improve diagnostic sensitivity in cases of subtle infection (Brainard et al, 1994; Kuhn et al, 1995; Toro and Ossa, 1996; Hansen et al, 1997; Mahieu et al, 1997; Rimsza et al, 1996; Eckart et al, 1996).

M O N O N U C L E O S I S A N D THE POSTTRANSFUSION SYNDROME Roughly 10% of clinical cases of mononucleosis are attributable to CMV infection (Klemola, 1967; Lajo et al, 1994). Heterophil antibodies commonly develop during the course of classical EBV-associated mononucleosis, but this type of antibody rarely is elaborated in those infected with CMV, even though cold agglutinins, rheumatoid factor, and/or antinuclear antigen commonly appear in the blood. Kaariainen et al (1966) described a mononucleosis-like syndrome in patients receiving multiple transfusions of blood during open cardiac surgery. It is now appreciated that CMV infection is acquired from virus-infected leukocytes in the transfused blood. With changing clinical practices, the incidence of blood transfusion-related CMV-associated mononucleosis is becoming less common. Refrigerated stored blood rarely is a source of infection. The pathological features of CMV mononucleosis have not been defined, and there is presently no way to know whether comparable tissue changes occur in EBV- and CMV-associated disease. However, atypical lymphocytes, often termed virocytes, are found in both conditions. CMV mononucleosis usually is relatively mild, and information from autopsied cases has not accumulated.

To a large extent, disease is limited to infants with congenitally acquired infections, or patients with underlying immunodeficiency disorders. In the congenitally infected newborn with systemic involvement of multiple organs, CMV encephalitis exhibits a strikingly consistent pattern of lesions. The predominant finding in the central nervous system is a profound periventriculitis with subependymal inflammation and tissue destruction (Figure 8.6). Haymaker et al (1954) characterized the lesion as hyperplastic to reflect the accumulation of reactive proliferating ependymal cells associated with gliosis in the subependymal tissue (Figure S.7). Lesions of this type are described by other authors (Wyatt and Tribby 1952; Worth and Howard, 1950; Smith and Vellios, 1950). This process ultimately results in periventricular calcification, and ventricular dilatation often occurs. It is currently uncertain whether or not spontaneously developing hydrocephalus in young children has CMV as one of its etiologies, but more subtle infections might be manifest in this manner. Ependymal cell involvement of lesions with lesser degrees of severity most probably account for the aqueductal stenosis and the internal hydrocephalus that occasionally develops in congenitally infected children during the first year of life. Microcephaly is either present at birth or becomes evident during the first year of life in many congeni-

NERVOUS SYSTEM INFECTION A N D DISEASE The manifestations of CMV involvement of the central and peripheral nervous systems are protean and incompletely defined pathologically and virologically (McArthur, 1987). Many clinical reports have claimed an etiological role for CMV in various neurologic syndromes based on demonstration of increases in the concentrations of serum antibody spanning the period of illness or isolation of virus from the oropharynx and urine of infected patients. Clearly, these findings are insufficient to establish a cause-and-effect relationship.

^ i ^ ' FIGURE 8.6 Severe ventriculitis and periventriculitis in the brain of an infant with congenital CMV infection. Note the roughened ventricular walls and the necrosis and calcification in tissue subjacent to the ventricular walls. Reprinted with permission from Haymaker et al (1954).

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FIGURE 8.7 (A) Periventricular inflammatory changes associated with gliosis and proliferation of ependymal cells. (B) Depiction of subependymal accumulation of unidentified infected cells.

tally infected children. In one study, microcephaly was present in 53% of children (Boppana et al, 1992), whereas in a second series 60% were affected (Hanshaw and Weller, 1961). Generally, these infants exhibit the typical periventricular calcium deposits described above. It can serve as a marker of CMV disease, although congenital toxoplasmosis is an alternate explanation. Case reports attest to the occurrence of microgyria and cerebellar disorganization as well as other abnormalities of the developing brain in stillbirths with systemic CMV infection (Figure 8.8). A causative association of CMV with congenitally acquired develop-

FIGURE 8.8 Extensive defect involving several adjacent gyri in the parietal lobe of the brain of a congenitally infected infant. Reprinted with permission from Crome and France (1959).

ment abnormalities has not been established definitively because evidence of infection in the involved central nervous system tissue is often lacking (Crome and France, 1959; Ceballos et al, 1976; McCracken et al, 1969). Several studies document a decrease in IQ among children with a background of congenital, but silent, CMV infection (Reynolds et al, 1974; Hanshaw et al, 1976). Lesions of the central nervous system have not been correlated pathologically with the relatively minor reductions in mental capacity found in these children. A diversity of syndromes has been described in both immunologically intact adults and in patients with varying types and degrees of immunosuppression. Aseptic meningitis occasionally occurs in heterophilnegative mononucleosis syndrome (Klemola et al, 1967; Perham et al, 1971; Chin et al, 1973). One-third of the patients with Guillain-Barre syndrome surveyed by Dowling et al (1977) had serological evidence of CMV infection, that is, a significant increase in serum antibody titers. Cases of polyneuritis associated with CMV infection in immunologically intact persons are described (Kabins et al, 1976; Leonard and Tobin, 1971). The pathogenic role of CMV in these conditions is uncertain (Bishopric et al, 1985). Chapter 16 discusses in detail the association of CMV with polyneuritis in HIV-1-infected patients. Encephalitis in normal adults attributable to CMV has been described (Phillips et al, 1977; Back et al, 1977). In one of the cases reported by Phillips et al (1977), seizures occurred and a temporal lobe biopsy revealed gliosis and focal neuronal degeneration. Culture of the brain tissue yielded CMV. Recently, Power et al (1990) demonstrated CMV by in situ hybridization in a diversity of cells in brain biopsies from 7 of 10

Cytomegalovirus

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FIGURE 8.9 (A) Periventricular calcification as demonstrated by magnetic resonance imaging with gastolineum enhancement. Note the increased signaling in the periventricular white matter. (B) Extensive granulations are found over the ventricular surfaces with associated scarring. Reprinted with permission from Kalayjian et al. (1993) and through the courtesy of R. Kalayjian, MD.

patients with Rasmussen's encephalitis. Patients with this syndrome have intractable epilepsy, and the brain tissue exhibits gliosis as well as neuronal loss and perivascular lymphocytic cuffing. This claim remains to be substantiated. To the extent neurological disease attributable to CMV develops in immunologically intact adults, it would appear to be an uncommon event (Chin et al, 1973). CMV involvement of the nervous system in the immunosuppressed patient occurs commonly, with its most striking manifestation being found in AIDS (Morgello et al, 1987). Approximately 30% of patients with advanced AIDS are infected with CMV, but virological documentation is often inadequate unless molecular

approaches are employed. For example, Arribas et al (1995) found greater than 10^/ml copy numbers of CMV genome in the cerebrospinal fluid of patients with severe brain infections. This is important because inclusion-bearing cells are only sporadically apparent in nervous system lesions histologically (Achim et al, 1994). Encephalitis is the most common pathologic finding; it ranges in severity from the presence of scattered microglial nodules in the cortex (Grafe and Wiley, 1989) to a necrotizing encephalopathy most commonly having a periventricular localization (Kalayjian et al, 1993; Morgello et al, 1987) (Figure 8.9A,B). Vasculitis associated with evidence of CMV involvement of the endothelium (Koeppen et al, 1981) (Figure 8.10),

FIGURE 8.10 Severe destructive and proliferative vasculitis in the brain of an adult with AIDS. Note the inclusion-bearing cells at the vascular periphery.

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Pathology and Pathogenesis of Human Viral Disease

FIGURE 8.11 Glial nodule in the brain of immunosuppressed renal allograft recipient. Note the inclusion-bearing neuron surrounded by an assortment of reactive glial cells.

myelitis (Moskowitz ei al., 1984), and radiculitis (Said ei al., 1991) also occur. Morgello ei al. (1987) reported that 6.5% of the glial nodules observed in their cases exhibited an inclusion-bearing cell (Figure 8.11). Necrotizing lesions were found in only 10% of cases with CMV infection. Occasionally, these localized lesions simulate malignancies, that is, pseudotumors (Moulignier ei al., 1996). In these conditions, concomitant pathological changes in the nervous system attributable to HIV-1 infection make a critical assessment of the role of CMV difficult. Because the pathological features of the cognitive/motor syndrome of AIDS overlap those of CMV encephalitis, immunological or molecular in siiu localization studies are required if the pathologist is to define the contribution of the two viruses to the disease. Similarly, both HIV-1 and CMV can contribute to the polyradiculopathies that so commonly occur in AIDS (Kim and Hollander, 1993; Tokumoto and Hollander, 1993; Leonard and Tobin, 1971). This subject is considered in more detail in Chapter 16. Pathological central nervous system changes are less frequently observed in patients undergoing immunosuppressive drug treatment for organ transplantation and corticosteroid treatment for chronic inflammatory diseases. Schneck (1965) evaluated the brains of a series of 34 renal allograft transplant recipients and observed glial nodules in the grey matter of 11 patients. Although CMV was the implied etiology, the specificity of these lesions was not established since inclusionbearing cells were not present.

PULMONARY INFECTION A N D DISEASE In a review in The American journal ofPaihology more than 40 years ago, Hamperl (1956) noted the presence of cytomegalic cells with inclusions in the lungs of infants with plasma cell interstitial pneumonitis, a unique and often fatal disease reported frequently in Europe in the middle of this century. He concluded that the inclusions were related to the basic disease process, an infection with protozoa termed pneumocystitis. Time has clarified this issue and made evident the common concomitant infections with CMV and Pneumocysiis carina in debilitated and immunosuppressed patients. Although recognized before the era of organ transplantation, CMV pneumonitis came of age as a significant disease in the early days of therapeutic immunosuppression when treatment regimens were under intense evaluation and drug toxicity with severe leukopenia was common (Hedley-Whyte and Craighead, 1965; Hill ei al, 1964). Since that time, it has become clear that CMV pulmonary infections are the most common life-threatening complication of immunosuppression in all forms of organ transplantation (Craighead, 1971; Kanich and Craighead, 1966; Baughman, 1997). The outcome of immunosuppression is complicated by the clinical observation that therapeutic agents differ with regard to their effects on the infection per se, and the host response (Fishman and Rubin, 1998).

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The prevalence and severity of CMV pulmonary disease is determined by patients' prior exposure to CMV and the existence of a latent viral state. The type and intensity of immunosuppressive therapy and the occurrence of a graft-vs.-host response in the organ recipient are also important considerations. For example, in an experimental murine model, allogenic stimulation was found to promote differentiation of latently infected peripheral blood monocytes and the release of virus (Soderberg-Naucler et al, 1997). Immunologically naive patients are particularly susceptible, assuming CMV is acquired from the newly engrafted organ or by means of blood transfusion. The mortality of untreated CMV pneumonia in this group is 80% (Baughman,

1997); thus the importance of contemporary efforts to screen donors for serological evidence of prior infection before transplantation of an organ graft into a susceptible recipient. In its most overt form, CMV pneumonia manifests as a diffuse bilaterally symmetrical pneumonia exhibiting interstitial accumulations of mononuclear cells and fluid, accompanied by intra-airspace proteinaceous exudates with hyaline membranes (Figure 8.12). Variable numbers of inclusion-bearing macrophages (Figures 8.13 and 8.14), epithelial alveolar lining cells, and capillary endothelial cells are found (Figures 8.15 and 8.16) (Craighead, 1975a; Craighead and Brody 1975). Under these circumstances, the lungs

F I G U R E 8.12 Interstitial pneumonitis with intra-airspace proteinaceous exudates in the lung of a renal allograft recipient with CMV pneumonia. Prominent inclusion-bearing pneumocytes are seen in the airspace walls. A hyaline membrane lines the wall of the respiratory bronchiole at the base of the photomicrograph.

F I G U R E 8.14 Infected macrophage in the interstitium of the lung associated with interstitial edema and intra-airspace exudation of protein in a lung with CMV pneumonia.

F I G U R E 8.13 Alveolar macrophage with prominent intranuclear inclusion and abundant dust particulates in the cytoplasm of a lung with CMV pneumonia.

F I G U R E 8.15 Luxuriant collection of CMV-infected pneumocytes largely obliterated an airspace in a lung with CMV pneumonia.

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Pathology and Pathogenesis of Human Viral D i s e a s e

FIGURE 8.16 Infected endothelial cells in the lumen of a small blood vessel in the interstitium of a lung with CMV pneumonia.

yield large amounts of infectious virus and the disease is almost invariably fatal (Figure 8.17). At the other end of the spectrum are patients whose lungs yield substantially smaller amounts of virus, and inclusion-bearing macrophages are found only rarely, or not at all, upon microscopical examination of the tissue (Table

8.1). CMV is detected in about half of the bronchoalveolar lavage specimens obtained from patients who lack clinical evidence of pneumonia. Between these two extremes, the pathologist observes disease in the form of localized relatively circumscribed lesions involving closely associated epithelial cells in a cluster

FIGURE 8.17 Lung of a mouse (right) experimentally infected by subcutaneous inoculation of a murine strain of CMV. The photomicrographs on the left show normal lung tissue from a healthy animal. B = bronchus; Br = bronchiole; * = vessels. Note the interstitial inflammatory infiltrate and edema fluid in the airspaces (arrowhead) of the infected lung. The lungs of this mouse were substantially heavier than normal controls and the protein content was significantly increased. Reprinted with permission from Brody and Craighead (1974).

Cytomegalovirus

of airspaces. Alternatively, the pneumonia exhibits a lobular distribution. The infection in these milder cases is not fatal. Although CMV is commonly found in the lungs of AIDS patients, overt interstitial pneumonia is uncommon. Paradoxically, the clinical manifestations of infections in these profoundly immunosuppressed patients contrast strikingly with the picture in allotransplant recipients who develop life-threatening pneumonias when immunosuppressive therapy is excessive. While both groups of patients are immunoincompetent, the nature of the immune deficiency, and their previous experience of the host with CMV, differ. For example, almost all male homosexuals with AIDS are infected with CMV long before the immunodeficiency attributable to HIV-1 develops; thus, they have acquired a degree of immunity to CMV. In contrast, overt fatal CMV pneumonia occurs predominantly in allotransplant recipients undergoing a primary infection, after the initiation of immunosuppressive drug treatment. Experimental models of CMV pneumonia have been developed using various strains of mice and virus. A variety of immunosuppressive regimens have been employed. Differences in the outcome of these experiments may reflect subtle genetic influences that might

99

alter the immune response to CMV. In the lungs of experimentally infected immunologically modified animals, macrophages transport virus to the lungs where endothelial cells of interstitial small blood vessels and pneumocytes lining the air spaces are infected. No doubt this accounts for the interstitial and intra-airspace exudates that develop. In these models, two factors appear to determine the outcome of the infection with regard to the development of life-threatening pneumonia. These are: (1) the concentration of virus in the lung, which may be an expression of the unique pathogenicity of the viral strain, or susceptibility of the animal; (2) the host immune response as reflected in the localization of sensitized CD8+ cytolytic T cells in the lung interstitium. However, development of the exudate pneumonia with interstitial and intra-airspace fluid accumulation might well prove to be due to cytokine mediators elaborated by infected and immunologically active cells (Brody and Craighead, 1974; Murphy et al, 1975; Shanley and Ballas, 1985; Reddehase ei al, 1985; Jordan, 1978; Shanley ei al, 1982; Shanley and Pesanti, 1985; Kaur ei al, 1996) (Figure 8.18). Necrotizing and ulcerative laryngitis, tracheitis, and bronchiolitis accompanied by pathological evidence of CMV infection are reported (Siegel ei al, 1992; Imoto ei

Type I Cell

Type

Early Antigen -^^^ Lat« Antigen -^^^s..^^ Fc Receptor^

F I G U R E 8.18 Hypothetical schema depicting the pathogenesis of CMV pneumonia. Circulating virus-infected macrophages enter lung and migrate into airspaces. The viral-encoded "early"' and "'late'' antigens provoke a CD4+ T lymphocyte response that initiates cytokine-mediated trafficking of CD8H- and B cells. The cytolytic CD8+ cells damage the capillary endothelium and the pneumocytes lining airspaces. Fluid accumulation in the interstitium and airspaces follows. In immunosuppressed patients, the endothelial cells and pneumocytes are often secondarily infected. S. Huber was consulted during the preparation of this cartoon.

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a/., 1990; Vasudevan et al, 1990). It is unclear whether the lesions were caused by the virus, or represent the secondary involvement of endothelial cells and fibroblasts in the granulation tissue of a lesion caused by some other insult. To me, infections of this type could account for the CMV found in respiratory tract secretions of patients having no evidence of pneumonia. The latter possibility seems most probable.

DIGESTIVE TRACT INFECTION A N D DISEASE In 1950, "protozoa-like" cells in gastric ulcers were noted by Hartz (1950). Smith and Vellios (1950) described involvement of the gut in five cases of neonatal disseminated CMV. Since that time, a plethora of clinical and pathological reports have documented the presence of cytomegalic inclusion-bearing cells in lesions of digestive tract from the mouth to the anus (Wong and Warner, 1962; Levine et ah, 1964; Keren et al.,

1975; Sidi et al, 1979). Affected patients include those with preexisting gastrointestinal erosive lesions such as ulcerative colitis who are receiving corticosteroid treatment, organ allotransplant recipients being administered a variety of immunosuppressive regimens (Strayer et al, 1981; Foucar et al, 1981; Owens et al, 1976; Campbell et al, 1977; Kaplan et al, 1989; Merigan et al, 1992; Bombi et al, 1987), and patients with AIDS. In organ transplant cases, the incidence of digestive tract lesions is said to range from 2 to 10%, often occurring in the early months after transplantation in association with disseminated CMV (Buckner and Pomeroy, 1993) (Figure 8.19). Hemorrhagic cecal lesions are a particularly notable problem in these patients, but it is unclear as to whether or not patients are uniquely predisposed to lesions of the proximal large intestine. Surgical resections of the bowel have been required in many of these patients due to uncontrolled bleeding, perforation, toxic megacolon, or pseudotumors representing infected inflammatory granulation tissue (Rich et al, 1992). The common occurrence of a

B

FIGURE 8.19 (A) Perforating lesion in the distal duodenum with an abscess in the serosal retroperitoneal adipose tissue. (B) Inclusion-bearing cells typical of CMV are found at localized sites in the mucosa, and in both endothelial cells and fibroblasts in the granulation tissue in the wall of the abscess. Note the associated hemorrhage. Photographs kindly provided by Washington Winn, MD. (C) CMV inclusions in epithelial cells of the large intestine with an associated lymphocytic infiltrate in the mucosa. Reprinted with permission from Henson (1972) and through the courtesy of D. Henson, MD.

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variety of gastrointestinal lesions in children and adults with AIDS suggests a possible predisposition of the digestive tract to CMV infections (Smith et al, 1988; Schwartz et al, 1989; Dolgin et al, 1990; Kyriazis and Mitra, 1992; Buckner and Pomeroy, 1993). For reasons that are unclear, CMV esophagitis seems to develop commonly in these patients. A case of CMV mononucleosis with gastric ulcers has also been described. Microscopical descriptions and immunological labeling have documented infection of mucosal epithelial cells (Henson, 1972; Freeman ef al, 1977) (Figure 8.19A-C), endothelial cells and fibroblasts in the granulation tissues at the base of ulcers, intravascular mononuclear cells and cells of the autonomic plexus, that is, Auerbach and Meissner ganglion cells (Press et al, 1980; Sonsino et al, 1984;, Anderson et al, 1990) and smooth muscle of the digestive tract wall (Sinzger et al, 1995). Small blood vessels exhibit a variety of changes. Inflammatory, necrotizing, and proliferative changes of the vessel walls, and thrombosis are commonly noted (Goodman and Porter, 1973). PCR has established infection in digestive tract biopsies too small for adequate microscopical evaluation (Yoshida et al, 1996). While the presence of viral cytopathology in ulcerative lesions is clear, the role of the virus in causation of the initial lesion often is less certain. In the oropharynx and esophagus, typical herpes simplex intranuclear inclusions and syncytia are often observed in the proliferating squamous cells of the mucosa adjacent to the CMV lesions in the granulation tissue ulcer crater. CMV appears to preferentially infect newly proliferating endothelial cells and fibroblasts, and it is reasonable to hypothesize that the granulation tissue forming in ulcerative lesions might be secondarily infected by CMV. On the other hand, CMV infec-

Flepatic involvement by CMV was noted in the early comprehensive pathologic studies of neonates with congenitally acquired disease (Smith and Vellios, 1950; Wyatt et al, 1950; Henson et al, 1974) (Figure 8.20). In the enlarged livers of these cases, evidence of chronic necrotizing hepatitis characterized by varying degrees of portal inflammation and fibrosis are found (Zuppen et al, 1986) (Figure 8.21). The bile ducts occasionally exhibit cytomegalic cells with inclusion, and bile pooling is seen in the canaliculi and Kupffer cells. On occasion, giant cell transformation of hepatocytes is present. The hepatitis observed in CMV-infected adult organ transplant recipients (Ware et al, 1979; Demetris et al, 1985) and in children with chemotherapy-treated neoplasms exhibits a similar pattern of disease (Henson, 1972). CMV is the most common pathogen infecting liver allograft recipients, and hepatitis is its most common manifestation (Winston et al, 1995). In these cases, inclusion-bearing cytomegalic cells are also seen in hepatocytes and endothelial cells of the portal venous system. Doubtless the extent of the disease is a reflection of the severity of infection in these patients, but it is not clear why the transplanted liver is a focus of disease. Could it reflect targeting of the host's immune response on this foreign organ?

FIGURE 8.20 Extensive necrosis of the liver parenchyma in an infant with a congenitally acquired CMV infection. Both the hepatocytes and bile duct epithelial cells are infected.

FIGURE 8.21 Localized microabscess in the liver of adult allotransplant recipient. Note the single CMV inclusion-bearing hepatocyte.

tions most probably impede healing of a lesion that would otherwise resolve spontaneously. This possibility is supported by the apparent response of some patients with ulcerative lesions to gancyclovir therapy.

LIVER INFECTION A N D DISEASE

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In analyzing clinical and virological information on children with nonspecific illnesses and hepatic enlargement accompanied by liver function abnormalities, Hanshaw et al. (1965) concluded that CMV was a likely etiologic agent in many cases. This claim has not been substantiated. However, CMV hepatitis clearly is evident in children and adults with CMVassociated heterophil-negative mononucleosis (Snover and Horowitz, 1984; Clarke et al, 1979; Bonkowsky ei al, 1975). Typically, infiltrates of small and large lymphocytes are seen in sinusoids and portal areas of the liver accompanied by variable, but generally modest, degrees of focal necrosis in a pattern suggestive of granulomatous inflammation. Kupffer cell hyperplasia is often evident, and immunohistochemical studies indicate that these cells may be early sites of viral replication (Theise et al, 1993). Histological examination of the liver usually fails to reveal hepatic parenchymal cells with CMV inclusions, and immunocytochemistry has not demonstrated viral antigen in the liver cells (Snover and Horowitz, 1984). Thus, the clinical/pathological picture in CMV-associated liver disease cannot be differentiated from that seen in EBV hepatitis associated with the mononucleosis syndrome (Wills, 1972). Extrahepatic biliary atresia is a rare condition with an incidence in 1 in 10,000 births. Its etiology and pathogenesis are obscure (Gautier and Eliot, 1981). Considerable debate focuses on the possible role of viral hepatitis and CMV in the causation of extrahepatic biliary atresia or congenitally absent/reduced numbers of biliary intrahepatic ductules (Witzelben et al, 1978; Bangaru et al, 1980; Hart et al, 1991; Kage et al, 1993). Inclusions can readily be found in the epithelium of the biliary system in some cases of congenitally acquired hepatic infection (Oppenheimer and Eastley 1973; Balazs, 1987). Landing (1974) postulated that extrahepatic biliary atresia represented a continuum with neonatal hepatitis and intrahepatic biliary tract disease. The pathological observations suggest an association of extrahepatic biliary atresia with CMV are circumstantial and inconclusive. The question will be difficult to satisfactorily resolve (Finegold and Carpenter, 1982). Recently, the so-called vanishing bile duct syndrome occurring in liver allograft recipients has been associated with CMV infection (Fishman and Rubin, 1998).

Wyatt et al, 1950; Monif and Donnelly 1977). It is seen to a lesser extent in the allotransplant recipient and patients with AIDS and Hodgkin's disease. Ductal cells occasionally exhibit inclusions, but the acinar and endocrine elements are more frequently affected. A similar observation has been made in mice experimentally infected with murine CMV. In many human cases, the lesions in beta cells of the pancreatic islets are particularly striking, but it is unfortunate that the identity of the infected islet cells in many reported cases was not established (Hultquist et al, 1973). Interstitial inflammatory cell infiltrates are a variable feature. Although hyperglycemia does not occur in congenital cytomegalic inclusion disease, the consistency of the morphological observation has raised questions as to the possible role of CMV in the pathogenesis of diabetes mellitus (Craighead, 1975b). Evidence to support such a contention is, to a large extent, lacking. Ward et al (1979) documented the onset of type I diabetes in a congenitally infected child at 13 months of age and a similar association in a renal allograft recipient has been claimed. Pak et al (1988) demonstrated CMV by in situ hybridization in the lymphocytes of 22% of patients with type I diabetes, but in only 2.6% of a group of health controls. On the other hand, pancreatic tissue from 44% of patients with type II diabetes yielded CMV genomic material, but control pancreatic tissue was uniformly negative (Lohr, 1990). In situ hybridization showed the CMV signal in the insular tissue of the diabetic patients. Unfortunately, substantiation of these intriguing, but most astonishing, findings currently is lacking. In experimental studies using mice, CMV of murine origin regularly infects the beta cells (Craighead et al, 1991) (Figure 8.22). Onodera and colleagues (1983) found that CMV infection was associated with development of hyperglycemia in mice treated with subdiabetogenic dosages of streptozotocin. This chemical damages beta cells and is diabetogenic in some strains of mice when administered at appropriate dosages. One might hypothesize a synergistic interaction of the chemical and virus (or an additive effect), leading to destruction of beta cells, resulting in diabetes.

GENITOURINARY TRACT INFECTION A N D DISEASE PANCREAS INFECTION A N D DISEASE Infection of the pancreas is common in congenitally acquired CMV disease (Cappell and McFarlane, 1947; Smith and Vellios, 1950; Worth and Howard, 1950;

Viruria occurs commonly in neonates with congenitally acquired infections. It can persist for years, with the highest concentrations of virus in the urine being found shortly after birth (Embil et al, 1972; Betts et al, 1972; MacDonald and Tobin, 1978) (see Table 8.1). Some

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F I G U R E 8.22 Islet of Langerhans in a mouse experimentally infected with a murine strain of CMV. In this study, immunohistochemistry established the infected cells to be beta cells. The islets of infants with congenital CMV disease display similar evidence of beta cell infection.

F I G U R E 8.23 Inclusions in the lining cells of the renal tubules of an infant with congenital CMV infection. These lesions are often sufficiently extensive to permit the demonstration of inclusion-bearing cells in cytological preparations of urinary sediments. In years past, urine cytopathological analyses were used in an attempt to establish a diagnosis.

healthy adults also excrete virus for prolonged periods of time (Davies et ah, 1979; Starr, 1970), and chronic viruria is common during therapeutic immunosuppression and chemotherapy (Hanshaw and Weller, 1961), as well as in AIDS patients (Mueller et al, 1995). Adults with heterophil-negative mononucleosis often are viruric. The prevalence of infection determined in various studies is, in part, a reflection of the sensitivity of the technique used to demonstrate virus. Tissue culture can be a useful tool for routine patient screening. Inclusion-bearing cytomegalic cells are commonly found in the renal tubules at autopsy of congenitally infected infants (Figure 8.23). In the days before congenital infection could be documented using tissue culture isolation of virus from urine, clinicians commonly sought evidence of cytomegalic inclusion disease by microscopical examination of the urinary sediments in an effort to find pathognomonic cytomegalic inclusionbearing cells. This is a difficult and often fruitless exercise. The site of virus replication in the urinary tract of adults and children with less severe nonfatal infections has not been satisfactorily addressed by systematic studies using immunohistochemistry and in situ techniques. As pathologists are well aware, inclusion-bearing cells are not seen in the kidney tissue of children and adults at autopsy. Yet, compelling clinical evidence now indicates that the kidneys of normal healthy graft donors can serve as a source of infection for susceptible transplant recipients (Ho et al, 1975; Betts et al, 1975). Thus, the virus n\ust lie latent in renal tissue for indefinite periods of time. The notion is supported by studies in mice using murine strains of CMV. In this work, kidney tissue was found to regularly yield virus for

periods of over a year after experimental inoculation of a murine strain of CMV. Evidence of an active infection was not otherwise apparent in these animals. Renal glomerular involvement is seen in children with congenital infections (Beneck et al., 1986) and adult recipients of organ allotransplants (Ozawa and Stuart, 1979). The ultrastructural evidence strongly suggests that endothelial cells in the mesangium are the sites of virus replication, but experimental studies in a mouse model have shown that infected macrophages migrate into the glomerulus from other sites (Craighead and Brody 1975; Murphy et al, 1975) (Figure 8.24A,B). This process is associated with a diffuse or lobular deposition of newly deposited stroma in the mesangium. In the model, obliteration of the glomerulus by scar tissue ultimately resulted from the infection. There is currently no definitive evidence to indicate that similar lesions occur in humans. Some observers have argued that an immune complex glomerulonephritis develops in organ allograft recipients, but the evidence is incomplete and the matter unresolved. The problem of defining the pathogenesis of glomerular lesions in transplant recipients is compounded by the overlap of CMV lesions with the nonviral glomerulitis that occurs as an apparent complication of immunosuppressive drug treatment. In male patients with HIV-1 infections, CMV is commonly recovered from semen. It is likely that this is the major means for sexual transmission. Lang and Kummer (1975) recovered CMV from the semen of 2 of 185 healthy men seeking a fertility examination. In the studies of Leach et al (1994), 22% of HIV-1-infected patients excreted CMV in the semen on at least one

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F I G U R E 8.24 (A) Glomerulus of a mouse experimentally infected with a murine strain of CMV Large numbers of infected macrophages accumulate in the mesangium. This influx of cells is associated with focal necrosis of glomerular lobules. The process ultimately results in fibrous obliteration of the glomerulus. (B) Macrophages in the mesangium migrating from the vascular lumina of a mouse experimentally infected with a murine strain of CMV. Note the massive accumulations of encapsulated virions in the cell nucleus. As noted above, when tissues are glutaraldehyde-fixed for electron microscopy a halo does not develop around the inclusion.

occasion and 40% shed it more than once. In a molecular analysis, Rasmussen ei al. (1995) demonstrated fewer than 100 copy numbers of the CMV genome in the semen of 30% of HIV-1 positive patients. Semen

was found to be infected for longer than 8 months in some. Spermatozoa did not appear to be the carrier of the virus, and most studies indicated that the mononuclear cell complement of the semen carries the virus.

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The tissue source of infection in the male is uncertain, and we know of no study in which the male genital tract has been evaluated systematically for evidence of infection. In the female, endocervical infections in the form of cytomegalic cells with inclusions can be documented on rare occasions during routine histological examination of biopsy material (Figure 8.25) (Deppisch, 1981; Goldman et al, 1969) and in cytological studies (Sickel et al, 1991; Brown ei al, 1988; Byard et al, 1991; Henry-Stanley et al, 1993). In various reports, from 3 to 14% of pregnant women have demonstrable CMV infections of the endocervix when biopsies are examined microscopically (Wenckebach and Curry, 1976). Pathologists have also described inclusion-bearing cytomegalic cells in the endometrium and the ovaries (Subietas et al, 1977; Dehner and Askin, 1975; McCracken et al, 1974). There have been no reported comprehensive studies to document infection using immunohistochemistry or in situ molecular techniques for locating virus-infected cells in tissues of the female genital tract. Over 20% of women attending sexually transmitted disease clinics yield CMV when cervical secretions are cultured on only a single occasion. Interestingly enough, the highest prevalence of infection was found in females under the age of 20 years (Collier et al, 1995). As might be expected, cervical infections are found more commonly in prostitutes than in other sexually active women (Shen et al, 1994; Montecalvo et al.

1997). Often, papillomavirus infections occur concomitantly. In some cases, pelvic inflammatory disease has also been attributed to CMV.

MYOCARDIAL INFECTION A N D DISEASE Myocarditis and pericarditis occasionally develop in infants with congenital disease, and adults with heterophil-negative mononucleosis (Koizumi et al, 1974; Wink and Schmitz, 1980; Cohen and Corey 1985; Gonwa et al, 1989; Powell et al, 1989; Ball and Archer, 1976). About 6% of patients with CMV mononucleosis are believed to have myocarditis. Clinically, electrocardiological changes in the ST segment and in T waves are associated with serological evidence of infection. In a few cases, cardiac enlargement and failure were documented. Pathological findings of both lymphocytic interstitial infiltration and inclusion bodybearing myocytes and endocardial cells have been reported in the few cases documented by pathological study. In a fatal case, cardiogenic shock developed 7 weeks after interstitial infiltrates were found in the myocardium (Cohen and Corey, 1985). Using molecular probes, Schonian et al (1995) screened cases of active and healed myocarditis and

FIGURE 8.25 Endocervical biopsy specimen from a 20-year-old woman with a clinical diagnosis of pelvic inflammatory disease. Note the scattered glandular lining cells with CMV inclusions. A luxuriant inflammatory reaction comprised of a mixed mononuclear cell influx with the formation of germinal centers is present, but its cause is uncertain. Reprinted with permission from Wenckebach and Curry (1976) and kindly provided by G. Wenckebach, MD.

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dilated cardiomyopathy. CMV infection was documented in about 10% of the cases of myocarditis, and in approximately 25% of cases of dilated cardiomyopathy. The pathogenic significance of these findings is unknown. Experimental studies in mice have established a key role for CD4+ lymphocytes in the pathogenesis of myocarditis. CMV localizes in the endothelial and endocardial cells, triggering a delayed hypersensitivity reaction characterized by accumulation of lymphocytes around the penetrating small blood vessels and in the interstitium of the heart (Craighead et al, 1991). In contrast to the findings in the study of pneumonia, CD8+ lymphocytes are not involved in this process (Figure 8.26).

EYE DISEASE Chorioretinitis is commonly found in infants with congenitally acquired CMV infections (Lonn, 1972). The prevalence appears to roughly approximate the severity of the systemic infection. In reported studies, 25 to 29% of infected children had clinical evidence of retinal disease (Stagno et al., 1977b; Eichenwald and Shinefield, 1962). Chorioretinitis develops in about 5% of organ allotransplant recipients (Murray et al, 1977; Porter et al, 1972; Egbert et al, 1980).

In AIDS, lesions of the retina are an important clinical problem, with an estimated prevalence of significant disease in 6 to 15% of patients (Faber et al, 1992; Hansen et al, 1994). Eye involvement appears to be a complication of viremia (Hansen et al, 1994; Fiala et al, 1977), and virus (or its DNA) can often be found in tears (Cox et al, 1975) and aqueous humor (Chumbley et al, 1975; Mitchell and Fox, 1995). Clinically the early lesions have a characteristic clinical picture, with punctate opaque areas of necrosis being evident in the retina by ophthalmoscopic evaluation. The retinal changes progress to resemble a "brushfire" over a period of months with a sheathing of vessels by the exudate and hemorrhage being the common feature. Aneurysms of the small retinal vessels occasionally develop and retinal detachment is a common complication of advanced cases (Augsburger and Henry, 1978; Meredith et al, 1979; Broughton et al, 1978). CMV retinitis is a lesion of advanced immunosuppression, for it develops when CD4+ T cell counts are <50/mm^ (Friedberg, 1997). The clinical features of the lesions are often indistinguishable from toxoplasma chorioretinitis, and varicella panophthalmitis (Papanicolaou et al, 1997) (Figure 8.27A-C). In advanced lesions evaluated pathologically postmortem, the retina exhibits extensive necrosis with disorganization of the retinal cellular layers by edema and inflammatory exudate. Inclusion-bearing cytomegalic

FIGURE 8.26 Focal inflammatory lesion in the heart of a thymectomized bone marrow-repleted CMV-infected mouse that was administered 1 x 10^ CD4+ T cells intravenously Note the destructive nature of the lesion, attributable to CD4+ lymphocytes. Focal calcifications frequently were noted in the damaged myocardium of these animals. Lesions of this type fail to develop in animals lacking CD4+ cells. Reprinted with permission from Craighead et al (1991).

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EAR DISEASE

FIGURE 8.27 CMV chorioretinitis in patients with AIDS. (A) Fulminating hemorrhage/necrotic CMV retinitis. (B) The retina several months later. (C) Smoldering retinitis exhibiting punctate lesions surrounding the central retinal scar. Reprinted with permission from Friedberg (1997) through the courtesy of D. Friedberg, MD.

cells are readily identified. To a variable extent, the choroidal layer is infiltrated by lymphocytes and plasma cells. Iritis can develop, but the extent of the inflammatory response is most probably attenuated by the basic underlying disease process, that is, AIDS. Vessels demonstrate variable features; vasculitis and thrombosis are described in occasional cases with endothelial cell involvement by CMV being evident. Optic nerve necrosis is found in some cases. The irreversibility of these lesions is apparent (Smith, 1964).

Infection with CMV has been documented in the middle ear of about 3% of children with acute otitis media. Presumably, these infections result from environmental acquisition of the virus. In one child, a persistent CMV middle ear infection of at least 3 weeks was documented (Chonmaitree et al., 1992). Commonly, respiratory bacterial pathogens are isolated concomitantly from middle ear fluid. Hearing loss occurs in about 50% of infants born with clinically apparent congenitally acquired CMV disease. Approximately 7 to 13% of children with more subtle infections at birth develop a degree of hearing impairment (Davis et al, 1987; Hanshaw eial., 1976). In one study, 10% of congenitally infected children were found to be completely deaf when tested later in life (Harris et al, 1984). Histologic and virological studies of temporal bones from infants with congenitally acquired disease establish the consistent presence of an endolabyrinthitis. The histopathology of the cochlea is variable, with cytomegalic inclusion-bearing cells being found in the scala media, Reissner's membrane, and the stria vascularis, but not the organ of Corti (Strauss, 1990) (Figure 8.28). Davis et al (1977) hypothesize that hematogenous spread of the virus into the endolymph occurs by means of the capillaries of the stria vascularis. Inner ear infections are not an extension of viral disease in the central nervous system, or in the middle ear. Examination of the vestibular apparatus has demonstrated infected cells in the membranous labyrinth, with involvement of the utricle, saccule, and semicircular canal. Evidence of infection in the innervation of the inner ear has not been found (Davis et al, 1977). Examination of the inner ear of congenitally infected persons dying later in life documents extensive destruction and scarring of the involved structures but without evidence of persistent infection (Rarey and Davis, 1993).

POSSIBLE ROLE OF CMV IN ATHEROSCLEROSIS Pathologists have played an important role in bringing to our attention the possible role of herpesviruses in atherosclerosis and the accelerated coronary vascular sclerosis of the transplanted heart allograft. In 1958, insightful studies by Vogel demonstrated the inclusion of CMV in the newly proliferated endothelial cells of a young boy with generalized cytomegalic inclusion disease and established the unique affinity of this virus for capillary endothelial cells of granulation tissue in a

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B

FIGURE 8.28 Middle ear of a 22-day-old infant congenitally infected with CMV. The duration of the gestation was estimated to be 31-32 weeks. (A) CMV inclusion-bearing cells lining the cochlear duct (C). Arrows define Reissner membrane. It impinges on the organ of Corti (arrowheads) and stria vascularis ductus cochlearis (STR). The spiral ligament (SL) is normal. (B) CMV-infected epithelial cells at the base of the crista ampularis of the semicircular canal. The sensory epithelium (between the arrows) and the vestibular branch of the 8th cranial nerve (V) are not infected. (C) CMV-infected cells of Reissner membrane (arrow). An eosinophilic precipitate adheres to the membrane, and is found within the cochlear duct (C). The spiral ligament (SL) is not involved. Reprinted with permission from Davis et al (1977) and through the courtesy of G. Davis, MD.

murine model. Fabricant et al (1983) showed that Marck's disease due to an avian herpesvirus produces atherosclerosis in infected chickens. Shortly thereafter, Benditt et al (1983) detected herpesvirus messenger RNA in human atherosclerotic lesions, an observation confirmed by later investigators exploring the possible role of CMV in this disease. As already noted, CMV inclusion-bearing endothelial and endocardial cells are commonly observed in naturally infected human tissues (Hendrix et al, 1990; Persaud, 1970) and experimental studies have documented the infectivity of human CMV for cultured endothelium (Lathey et al, 1990; Smiley et al, 1988). Infected endothelial cells have been demonstrated in the circulating blood (Grefte et al, 1993).

Soon after initiation of cardiac transplantation, a previously unrecognized and rapidly progressive proliferative disease process of the coronary vessels of the grafted heart was discovered (Pahl et al, 1990). This accelerated atherosclerotic lesion occurs in roughly half of the transplant recipients after 5 years. It substantially compromises the outcome and is a major cause of death. Clinical and epidemiological investigations soon showed that the lesion occurred commonly in patients who develop a CMV infection after transplantation, and studies of the affected coronary vessels demonstrated both CMV antigens and DNA in endothelial cells (Melnick et al, 1990; Grattan et al, 1989; McDonald et al, 1989; Loebe et al, 1990; Koskinen et al, 1994; Forbes et al, 1990; Millett et al, 1991; Wu et al.

Cytomegalovirus

1992; Lemstrom et ah, 1995). Endothelial and smooth muscle cell proliferation are found in experimentally infected rat aortic allografts (Lemstrom et al., 1993). These interesting observations suggest, but do not establish, a role for CMV in the pathogenesis of these newly described vascular lesions (Dong et ah, 1996). Gulizia et al (1995) detected CMV genomes in 20% of allografted coronary vessels, but in only 5% of the vessels of control subjects. This was not considered to be a statistically significant difference. In a recent report, Zhou et al. (1996) documented a significant decrease in the luminal diameter of the coronary arteries of CMV-seropositive patients after coronary atherectomy, but this conclusion has not been substantiated by the work of others (Kol et al, 1995). Recent studies indicate that CMV-infected cells elaborate the growth factor IL-1, and CMV proteins are believed to bind the suppressor gene, p53 (Marx, 1994). This potentially enhances proliferation of smooth muscle cells in vessel walls (Speir et al, 1994; Kovac et al, 1996). Induction of adhesion factors on endothelial cell surfaces during the course of CMV antigenemia has been documented in heart allotransplant recipients (Koskinen et al, 1994), and similar effects on VCAM-1 and ICAM-1 expression are found when CMV-sensitized T cells interact with infected endothelial cells in vitro. Presumably, this is a cytokine-mediated effect (Waldman and Knight, 1996; Waldman et al, 1997). Demetris et al (1997) proposed that altered immunity may account for smooth muscle proliferation in the vessel walls, and Zhu et al (1995) believe that protein products of CMV replication (lEl and IE2) inhibit apoptosis by infected cells. On the other hand, in vitro experiments document an inhibitor effect of CMV on cell cyclic kinetics (Jault et al, 1995; Lu and Shenk, 1996; Bresnahan et al, 1996; Dittmer and Mocarski, 1997). At present, the role of CMV in atherogenesis after cardiac transplantation and arterioplasty remains to be resolved, and it is quite possible the lesion has a multifactorial basis. Carefully designed epidemiological studies that attempt to determine whether or not virus-specific therapy prevents lesion development may provide an answer to this important clinical problem. CMV infection appears not to be a risk factor for coronary artery atherosclerosis in the im^munologically intact adult (Adler et al, 1998).

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Adler, S., Hur, J., Wang, J., and Vetrovec, G. (1998). Prior infection with cytomegalovirus is not a major risk factor for angiographically demonstrated coronary artery atherosclerosis. /. Infect. Dis. 177, 209-212. Alagille, D., Odievre, M., and Hadchouel, M. (1983). Paucity of interlobular bile ducts. In ''Recent Concepts in Biliary Atresia and Its Related Disorders (M. Kasai, ed.), pp. 59-65. Excerpta Medica, Princeton. Anderson, V., Greco, M., Recalde, A., Chandwani, S., Church, J., and Krasinski, K. (1990). Intestinal cytomegalovirus ganglioneuronitis in children with human immunodeficiency virus infection. Ped. Pathol. 10, 167-174. Arribas, J., Clifford, D., Fichtenbaum, C , Commins, D., Powderly, W., and Storch, G. (1995). Level of cytomegalovirus (CMV) DNA in cerebrospinal fluid of subjects with AIDS and CMV infection of the central nervous system. /. Infect. Dis. 172, 527-531. Augsburger, J., and Henry, R. (1978). Retinal aneurysms in adult cytomegalovirus retinitis. Am. J. Ophthalmol. 86, 794-797. Back, E., Hoglund, C , and Malmlund, H. (1977). Cytomegalovirus infection associated with severe encephalitis. Scand. J. Infect. Dis. 9, 141-143. Balazs, M. (1987). Cytoplasmic cytomegalovirus inclusions in human bile duct epithelia. Hum. Pathol. 18, 970-971. Ball, S., and Archer, G. (1976). Myocarditis complicating cytomegalovirus mononucleosis. Postgrad. Med. J. 52,102-105. Bangaru, B., Morecki, R., Glaser, J., Gartner, L., and Horwitz, M. (1980). Comparative studies of biliary atresia in the human newborn and reovirus-induced cholangitis in weanling mice. Lab. Invest. 43, 456-462. Baughman, R. (1997). Cytomegalovirus: The monster in the closet? Am. J. Respir. Crit. Care Med. 156,1-2. Benditt, E., Barrett, T., and McDougal, J. (1983). Viruses in the etiology of atherosclerosis. Proc. Natl. Acad. Sci U.S.A. 80, 6386-6389. Beneck, D., Greco, M., and Feiner, H. (1986). Glomerulonephritis in congenital cytomegalic inclusion disease. Hum. Pathol. 17, 10541059. Betts, R., Douglas, R., Pabico, R., Talley, T., Yakub, Y, and Freeman, R. (1972). Shedding patterns of herpes simplex virus and cytomegalovirus among chronic dialysis patients. Trans. Am. Soc. Artif Intern. Organs 18, 262-265. Betts, R., Freeman, R., Douglas Jr, R., Talley T., and Rundell, B. (1975). Transmission of cytomegalovirus infection with renal allograft. Kidney Int. 8, 385-392. Bishopric, G., Bruner, J., and Butler, J. (1985). Guillain-Barre syndrome with cytomegalovirus infection of peripheral nerves. Arch. Pathol. Lab. Med. 109,1106-1108. Bombi, J., Cardesa, A., Llebaria, C , Rives, A., Carreras, E., Granena, A., and Jimenez de Anta, M. (1987). Main autopsy findings in bone marrow transplant patients. Arch. Pathol. Lab. Med. I l l , 125-129. Bonkowsky, H., Lee, R., and Klatskin, G. (1975). Acute granulomatous hepatitis: Occurrence in cytomegalovirus mononucleosis. JAMA 233,1284-1288. Boppana, S., Pass, R., Britt, W., Stagno, S., and Alford, C. (1992). Symptomatic congenital cytomegalovirus infection: Neonatal morbidity and mortality. Pediatr. Infect. Dis. J. 11, 93-99. Brainard, J., Greenson, J., Vesy, C , Tesi, R., Papp, A., Snyder, P., Western, L., and Prior, T. (1994). Detection of cytomegalovirus in liver transplant biopsies. Transplantation 57,1753-1757. Bresnahan, W., Boldogh, I., Thompson, E., and Albrecht, T. (1996). Human cytomegalovirus inhibits cellular DNA synthesis and arrests productively infected cells in late Gl. Virology 224,150-160.

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C H A P T E R

9 Epstein-Barr Virus (EBV) correct when the association of holoendemic malaria with the lymphoma was discovered. I, like so many young impressionable physicians, was struck by the mystery surrounding the recognition of this extraordinary tumor upon hearing Burkitt speak during one of his numerous trips to North America in the early 1960s. Dr. Anthony Epstein, a young pathologist and experimental virologist at the Bland Sutton Institute in London, was similarly intrigued. He established a collaboration with Burkitt in an effort to recover the then-hypothesized transmissible agent from tumor tissue. After considerable work, Epstein and his associates Barr and Achong (1964) detected a herpesvirus by electron microscopy in proliferating lymphoid cells that had grown from a tumor biopsy in the transporting medium during a prolonged trip from Uganda to London. The delayed arrival of the shipment turned out to be the serendipitous basis for the discovery, since the latent virus replicated in the dividing tumor cells and thus became evident to the electron microscopist. The growth of this new virus in multiplying lymphoblastic cells provided a means for working with it in the laboratory, allowing the studies that now serve as the foundation for our current understanding (Epstein et al, 1964). In the mid-1960s, Gertrude and Werner Henle undertook studies that ultimately demonstrated the association of EB virus with infectious mononucleosis (IM). The chance occurrence of "mono" in a technician at Henle's laboratory provided the opportunity to test the serum of this young woman for development of EBV-specific antibodies. Subsequently, lymphocytes from this patient proved to be a carrier of the virus. This serendipitous finding was complemented by seroepidemiological studies of undergraduate college students with IM whose "acute" and "chronic" serum had been painstakingly stored for future systematic studies of this type. Within a short period of time, an association of EBV infection with IM was found. Work with EBV has progressed with extraordinary rapidity during more recent years, particularly as a result of its demonstrated causative relationship with the lymphoproliferative disorders occurring

INTRODUCTION AND HISTORICAL OVERVIEW 117 CELLULAR AND MOLECULAR BIOLOGY OF EBV 118 INFECTIOUS MONONUCLEOSIS (IM) 120

Neuromuscular Disease 122 Myocarditis and Pericarditis 123 Kidney Disease 123 Lower Female Genital Tract 123 X-LiNKED LYMPHOFROLIFERATIVE DISEASE (XLP) (DUNCAN'S DISEASE) 123 BuRKiTT's LYMPHOMA (BL) 124 LYMPHOFROLIFERATIVE DISORDERS (LPDS) ASSOCIATED WITH IMMUNOSUPPRESSION 127 NoN-HoDGKiN'S LYMPHOMA 129

HoDGKiN's DISEASE (HD) 130 NASOPHARYNGEAL CARCINOMA ( N P C ) 131 LYMPHOEPITHELIOMATOUS GASTRIC CARCINOMA

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SiNONASAL TUMORS 133 PULMONARY DISEASE 134 LYMPHOMATOID GRANULOMATOSIS 135 INFLAMMATORY PSEUDOTUMORS 135 SJOGREN'S SYNDROME AND SALIVARY GLAND TUMORS 135 HAIRY LEUKOPLAKIA (HCL) 136 VIRUS-ASSOCIATED HEMATOPHAGOCYTIC SYNDROME 138 REFERENCES 139

Ex Africa semper aliquid novi [Africa always brings something new] Pliny 23-79 CE.

INTRODUCTION A N D HISTORICAL OVERVIEW Some would call it serendipity, but others consider it an insightful discovery by a trained mind. The history of Epstein-Barr virus (EBV) goes back several decades to when Dennis Burkitt, a British surgeon working in Uganda, described for the first time the unique lymphoma that now bears his name. Because of its common occurrence in young African residents of a relatively warm high-rainfall belt across East Africa, Burkitt hypothesized that the tumor was caused by a vector-transmitted infectious agent. Subsequently, his geo-ecological considerations proved at least partially PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

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in immunosuppressed transplant recipients. The finding of the virus in hyperplastic and malignant cells of patients with a variety of diseases that traditionally are not thought to be infectious has stimulated much additional research. Despite the enormous effort thus far expended to characterize EBV, we still are uncertain as to its role in the causation of many of the clinical syndromes with which it is now found. EBV is associated with a spectrum of clinical conditions that appear to reflect the capacity of this virus to stimulate lymphoid cell proliferation and immortalize B cells (Sugden, 1994). Immune regulation of the infection is key to ultimate outcome, but the resulting interplay of immunological factors with infected cells is accompanied by illness. In the healthy infant and young child, infection is clinically inapparent or accompanied by a minor respiratory illness, whereas in the immunologically naive teenage and adult infection is often, but not invariably, reflected in the infectious mononucleosis syndrome. In various forms of acquired and heritable immune disorders, B cell infection is expressed as an infiltrative lymphoproliferative disorder that can ultimately evolve into a lymphoma.

CELLULAR A N D MOLECULAR BIOLOGY OF EBV EBV is a member of the gammaherpesvirus family, being classified in the lymphocryptovirus genus. The latter term aptly refers to the subtle latency of the virus in B lymphocytes under most biological circumstances. Strikingly similar viruses are found in some Old World primates; however, their role in disease among these animals is not known. Naturally occurring human EBV strains are divided into two closely related types depending upon the unique characteristics of certain of their nucleoproteins. Type 1 strains are found most frequently in members of developed societies; whereas type 2 strains are recovered from roughly half of the infected African population. The medical significance of the type specificity of EBV, if any, is obscure. The EBV virion has structural and biochemical properties similar to other human herpes viruses. Nonetheless, its biological characteristics are unique features attributable to expression of the various proteins formed during the replicative cycle. EBV customarily is acquired by means of oral exposure. A persistent cytolytic infection of the pharyngeal epithelial cells then develops. Virions with linear DNA are recoverable from oropharyngeal secretions and can be found in the blood, free or in association with B cells. As with other respiratory viruses, secretory IgAmost probably serves

to protect the already immune. Trafficking B lymphocytes in and near the mucosa are secondarily infected, and these cells undergo a replicative sequence within 48 to 72 hours (Figure 9.1). Specific receptors on the epithelial cells bind to the glycoproteins of the virion, and a similar, if not identical, receptor on the B lymphocyte serves as the basis for infection of these cells. This receptor is analogous to the type 2 complement receptor. This, then, is another example of virus perversely utilizing preexisting cellular entities for its specific purpose (McClure, 1992). Roughly 10% of the infected B cells acquire the capacity to support physiological replication of the virus indefinitely, both in vivo and in vitro. The immortalization of the lymphocytes is demonstrated in approximately 1 of every 10^ to 10^ circulating bloodstream cells. Shortly after infection, the viral DNA begins to form co-linear circularized viral DNA episomes that characterize the inactive state. Latently, immortalized lymphocytes express a panoply of biochemical constituents that characterize this state. Eleven or possibly more genes are expressed during the latency period. Six nucleoproteins (EBNA-1, -2, -3A-C, and LP), two RNAs (EBER-1 and -2), and three membrane proteins (LMP-1, -2A and -B) are encoded by these genes. These entities are activated in a complex sequence of events, with their expression differing in various forms of EB latency While the details of this complex and incompletely understood series of biochemical events is beyond the scope of this discussion, a voluminous body of experimental evidence leads to the conclusion that at least some of these gene products play a role in immortalization of the cells and their subsequent oncogenicity. The expression products of the various genes have been capitalized upon by pathologists using PCR and in situ hybridization to identify EBV latently infected cells in tissue samples. Immortalization of B lymphocytes results in striking cytological changes. These include enlargement of cells in their subsequent division associated with DNA synthesis and acquisition of several surface activation markers and adhesion factors. These events are similar to those occurring in antigen-stimulated lymphocytes, and some experimental evidence suggests that they are a result of cell stimulation by autocrine growth factors. As noted above, some EBV-infected B lymphocytes sustain a permissive viral growth cycle with a productive synthesis and assembly of complete virions within the cell. This results in cytolysis. The factors influencing development of a permissive cytolytic state are unclear, but various drugs or metabolic conditions that inhibit or alter macromolecular synthesis by the infected cell may be responsible (Harris, 1992).

Epstein-Barr Virus

•?^l-*^-*5^

^V •!> i 8* F I G U R E 9.1 Distribution and immunophenotype of EBV-infected cells in the epithelium of a tonsil affected by IM. (A) Demonstration of numerous EBV-infected (EBER-positive) cells (red-brown nuclei) in all layers of the tonsillar epithelium (EP), (B) Double labeling for EBER (blue-black nuclei) and cytokeratin (brown cytoplasm). The EBER-positive cells appear to be cytokeratin-negative. Original magnification x600. (C) Double labeling for EBER and CD20. Most EBER-labeled cells present in the tonsillar EP do not express the CD20 molecule. (D) Double labeling for EBER and the B-cell marker Ki-B3 (CD45RB). Most, if not all, EBER-positive cells present within the tonsillar EP proved to express the Ki-B3 antigen, indicating their B-cell nature. Reprinted with permission from Anagnostopoulos et at. (1995).

119

120

Pathology and Pathogenesis of Human Viral Disease

INFECTIOUS M O N O N U C L E O S I S (IM) (Custer and Smith, 1948; Cowing, 1975; Tindle, 1983; Childs et al, 1987)

Whereas natural infection of the infant and young child is either asymptomatic or results in mild transient systemic symptoms and fever, it is expressed as the clinical syndrome of infectious mononucleosis (IM) in a small proportion of children under the age of 10 (Baehner and Shuler, 1967), teenagers, and young adults who lack immunity. The acute pharyngitis is undoubtedly consequent to cytolytic EBV infection of the pharyngeal mucosal cells (Niedobitek et al., 1989, 1994). The subsequent infection of trafficking B lymphocytes and those of the lymphoid plexus of the pharynx (Waldeyer's ring) result in immortalization of B lymphocytes. Lymphoid hyperplasia or cytolytic infection of the pharyngeal epithelium no doubt contributes to the acute systemic symptomatology characteristic of IM. These events are compounded by the NK cell and T cell responses to the virus-infected cells that contribute to symptoms through elaboration of interferon and cytokines. The incubation period ranges from 5 to 7 weeks. After an acute and sometimes protracted course, ranging from several days to a few weeks, symptoms gradually abate, to be followed (to a variable extent) by continued malaise and ease of fatigability (Figures 9.2 and 9.3). In addition to these systemic symptoms, the acute illness is characterized by fever and pharyngitis with tonsillar hyperplasia accompanied by generalized lymphadenopathy and hepatosplenomegaly As discussed in more detail below, a wide variety of organ systems can be involved, resulting in a diversity of reversible clinical syndromes. Hete r o p h i l antibodies resulting from a nonspecific immune response of the infected B cells can be detected in

roughly 85% of cases in which EBV is implicated (Paul and Bunnell, 1982). The characteristic presence of circulating so-called atypical lymphocytes in the blood is a well-recognized feature of the disease and reflects cytopathic changes in the immunologically stimulated NK and T cells resulting from the infection (Figure 9.4) (Shiftan and Mendelsohn, 1978; Munoz and Sharon, 1990). Contemporary evidence suggests that these cells are clonally derived. The splenomegaly, most probably, is a reflection of the luxuriant immune response of the host. Virus is not found by in situ hybridization in the spleen. In the review of Custer and Smith (1948), spleen weights as high as 760 g (i.e., roughly 5-7 greater than normal) are reported. Because generalized lymphadenopathy and the clinical features of the disease are so characteristic of IM, lymph node biopsies are only rarely obtained from patients with this disease. Biopsy is occasionally justified in patients who fail to develop heterophile antibodies and the typical IM syndrome or in relatively asymptomatic persons with localized lymphadenopathy. When death occurs as a result of IM, the Xlinked lymphoproliferative syndrome is a likely consideration. This uncommon genetically mediated disease is discussed below. In the lymph nodes of patients with IM, the pathologist observes hyperplasia of lymphoid elements with expansion of the interfoUicular space and infiltration of the capsule. The sinusoids of the node are either compressed by the paracortex or expanded by an infiltrate of pleomorphic B lymphocytes and sinus histiocytes. Occasional plasma cells are found. Small blood vessels proliferate in the interfoUicular areas. Follicular hyperplasia is invariably present with variably shaped germinal centers and abundant phagocytic histiocytes (lochim, 1991). The immunoblast is a large cell that usually exhibits a smooth nuclear configuration and mul-

Abnormal liv«r function tests

^.^^^W^rophil antibody.

14 Day of illness

F I G U R E 9.2 Frequency and duration of major symptoms of uncomplicated IM. Reprinted with permission from Carter and Penman (1969).

14 Day of illness

F I G U R E 9.3 Typical laboratory findings in patients with uncomplicated infectious mononucleosis. Reprinted with permission from Carter and Penman (1969).

Epstein-Barr Virus

B

,;i ^^^^

F I G U R E 9.4 Examples of the atypical lymphocytes (virocytes) seen in peripheral blood smears of patients with IM. The features are nonspecific; similar circulating cells are seen in other acute viral infections. Reprinted with permission and through the courtesy of J. Lunde, MD.

tiple nucleoli associated with a fine chromatin pattern (Figure 9.5A). The presence of immunoblastic cellular elements exhibiting numerous mitoses and variable degrees of atypia in nodules and sheets, or the occasional presence of Reed-Sternberg-like cells, are features of the process that invariably raise for consideration the possibility of a large-cell lymphoma of the immunoblastic type and Hodgkin's disease (McMahon et al, 1970; Tindle et al, 1972; Agliozzo and Reingold, 1971) (Figure 9.5D). Necrosis is usually, but not invari-

121

ably, seen (Figure 9.5B,C). Retention of the lymph node architecture, as well as the polymorphic features of the cellular population, are arguments against a malignant diagnosis. Extreme caution is warranted when considering a malignant diagnosis in a young patient who might in fact have IM (Salvador et al, 1971). B cells in these lymph nodes are commonly infected. The virus is in a replicative phase, and multiple EBV genotypes are found, an indication that at this stage the infection is polyclonal (Strickler et al, 1993). Theoretically, molecular characterization of the infecting viral population would permit differentiation between IM and lymphoma. As the disease resolves, associated with elaboration of specific EBV antibody, lymphoid hyperplasia subsides and the lymph nodes can even appear hypocellular. Germinal centers become quiescent, but a heterogenous population of macrophages and lymphoblasts persist in sinusoids. The profound involvement of major organs sporadically results in infiltrative lesions of the liver and the meninges, and occasionally the perivascular spaces of the brain and the interstitium of the lung. Splenic rupture and partial obstruction of airways of the lungs can occur. Acute pancreatitis has been described (Craighead, 1981). Destruction of the bone marrow has been reported to result in aplastic anemia, autoimmune hemolytic anemia, and thrombocytopenia, but this is rare (Clancy et al., 1971; Lazarus and Baehner, 1981). The typical lymph node morphology of IM may be difficult to differentiate on pathological grounds from the profound reactive lymphoproliferative responses occasionally observed in infections with rubellavirus and vacciniavirus (Hartsock, 1968); the lesions of toxoplasmosis may be similar. Appropriately selected serological assays should resolve this diagnostic quandary, but modern molecular approaches could provide specificity to the diagnoses. Systemic specific organ disorders, as a complication of acute IM, are infrequently reported in the clinical literature. On the other hand, the comprehensive review of Custer and Smith (1948) indicate that lymphocytic infiltrative lesions are commonly found at autopsy in many major organs. This review is of particular importance inasmuch as death in the cases described was due to acute fatal complications of the illness such as splenic rupture or trauma (not the fatal infectious mononucleosis syndrome described before). Orchitis, hepatitis (Chang and Campbell, 1975; Horwitz et al, 1980; Markin et al, 1987), pancreatitis (Everett et al, 1969; Burgess and Menser, 1974), myocarditis (Frishman et al, 1977; Pejme, 1964; Wechsler et al, 1946), interstitial pneumonitis and alveolitis (Schooley et al, 1986; Barbera et al, 1992), interstitial nephritis

122

Pathology and Pathogenesis of Human Viral Disease

FIGURE 9.5 Lymph node pathology of IM. (A) Sinusoid region of the node is replete with large lymphoid cells having immunoblastic features. Mature lymphocytes are sparse. Variable numbers of mitosis are present. (B) Extensive capsular and perinodal infiltration of lymphoid cells. The arrow indicates an area of necrosis. (C) Massive necrosis associated with one focus of viable cells. (D) Reed-Sternberg-like cell. The prominent nucleolus should be differentiated from a viral intranuclear inclusion. B-D Reprinted with permission from Strickler et al. (1993) and through the courtesy of J. Strickler, MD, F. Fedeli, MD, C. Horwitz, MD, C. Copenhaver, MS, and G. Frizzera, MD.

(Mayer et ah, 1996), meningoencephalitis (Lange et ah, 1976; Cotton et ah, 1994), retrobulbar neuritis (Anderson et ah, 1994), Bell's palsy (Grose et ah, 1973), cerebellitis (Dowling and van Slyck, 1966; Bennett and Peters, 1961; Bergen and Grossman, 1975), and Guillain-Barre syndrome (Grose et ah, 1975) have been described. Unfortunately, at this time, information on involvement of the various organs by the virus is limited. Several of the more important organ-related syndromes are discussed in greater detail below. Neuromuscular Disease Involvement of the central and peripheral nervous systems occurs sporadically in acute IM, occasionally in the absence of the typical features of the illness (Grose et ah, 1975; Seltzer, 1953; Schnell et ah, 1966).

Silverstein and colleagues (1972) documented objective neurological involvement in 6% of patients with IM. These disorders are generally transient, and pathological observations are not recorded in the literature. The conditions commonly reported are Guillain-Barre syndrome (Hafstrom, 1963; Smith, 1956), meningoencephalitis (Lange et ah, 1976; Silverstein et ah, 1972; Cotton et ah, 1994; Linnemann et ah, 1973), cerebellitis, at times simulating a space-occupying lesion (Gilbert and Culebras, 1972; Dowling and Van Slyck, 1966; Bennett and Peters, 1961), myelitis, localized neuritis, including Bell's palsy (Grose et ah, 1973), polyneuritis, and retrobulbar neuritis (Anderson et ah, 1994). In a survey conducted by Grose et ah (1975), 7 of 24 cases of Guillain-Barre and 3 of 16 patients with Bell's palsy showed evidence of a recent primary EBV infection. In a literature survey by Singh and Scheld (1996), reports

Epstein-Barr Virus

of five cases of acute rhabdomyolysis with elevated serum concentrations of muscle enzymes were found. Myoglobin deposition in the kidney tubules with renal failure has been described (Mayer et ah, 1996). Pathological studies are not recorded. Myocarditis and Pericarditis EKG changes are observed in a variable number of patients with IM (Pejme, 1964; Wechsler ei al, 1946; Hoagland, 1964). Occasional case reports of heart block are documented (Reitman and Zirin, 1978), and congestive myocardiopathy has been reported (Dec et ah, 1985). A single pathological description of a case of IM with acute and unexpected death is recorded in the literature (Frishman et al, 1977). Microscopical studies of the heart of this teenager revealed extensive areas of myocardial fiber replacement and a cellular infiltrate comprised predominantly of histocytes, and, to a lesser extent, lymphocytes. Fibroblastic activity and regenerating myofibers were noted. In this case, the spleen was 500 grams and exhibited cell populations typical of acute IM. A report of interest documented a nonLangerhans histiocytic interstitial infiltrate in the heart of a young man with fatal IM believed to be consequent to the X-linked immunodeficiency syndrome (Seemayer et ah, 1994). The histocytes in the heart were not infected as shown by in situ hybridization studies, and there was no necrosis of myocardial cells.

Kidney Disease Subclinical renal disease is believed to be relatively common in patients with acute IM. A number of case reports of renal insufficiency and oliguric renal failure are documented in the literature (reviewed in Mayer et ah, 1996). Interstitial lymphocytic infiltrates have been reported in most case studies pathologically, and a few cases of a glomerulonephropathy believed to be due to inimune complexes are documented. The interstitial lymphocytes in the case of nephritis reported by Mayer et ah (1996) proved to be suppressor T cells. The finding suggests that the widespread organ lesions sometimes observed in acute IM may be a T cell response to infection, as is seen in the spleen.

Lower Female Genital Tract Cervicitis accompanied by demonstrable EBV-infected epithelial cells in cytological preparations was documented in some 5 of 28 women studied (Sixbey et

123

ah, 1986). In another case, labial ulcers were found to be EBV infected in a young woman with acute IM (Portnoy et ah, 1984). In this latter case, and several of the former group, cunnilingus apparently occurred, although its causative contribution to the infection is not known. Recently, a number of EBV-associated lymphoepitheliomas of the uterine cervix in Asian women were reported (Tseng et ah, 1997); this subject is discussed in more detail below. Pancreatitis and abrupt-onset insulin-requiring diabetes mellitus have been reported in individual cases of IM, and a causative association has been suggested (Craighead, 1981). Reports of cases of this type are exceedingly rare. Horn et ah (1988) demonstrated similarities between certain EBV polypeptides and the marker (position 57) on the DQP subunit of MHC that predisposes to type 1 diabetes mellitus. This observation supports the concept of viral mimicry of key human proteins in the pathogenesis of this disease.

X-LINKED LYMPHOPROLIFERATIVE DISEASE (XLP) (DUNCAN^S DISEASE) The insightful recognition of a familial predisposition to fatal IM by Dr. David Purtilo brought to our attention this rare but unique condition (Purtilo, 1975; Bar ei ah, 1974). Described for the first time in 1969, XLP is inherited as a maternally derived genetic defect closely linked to one or more loci in a short 2.2 Mb segment of DNA on the Xq25 band of the X chromosome (Figure 9.6). There may be different chromosomal but functionally similar defects among the various kinships (Seemayer et ah, 1993). It is hypothesized that the normal gene is required to effect an appropriate T helper type 2 response after EBV infection. An estimated 1 X 10^ male children are at risk worldwide. There is circumstantial and anecdotal information to suggest that these boys deal with other common viral infections in an adequate fashion (Seemayer et ah, 1993). XLP is characterized by three phenotypic expressions, the most common of which is the occurrence of IM relatively early in life among boys (mean age of onset = 2.5 years). At the outset, this fatal form of IM exhibits the typical clinical features of more benign disease with fever, malaise, generalized lymphadenopathy, and hepatosplenomegaly being characteristic (Penman, 1970; Lukes and Cox, 1958; Purtilo et ah, 1991). However, fulminating necrotizing hepatitis and bone marrow hypoplasia and necrosis develop, resulting in an acute mortality of roughly 40%. Pathologically, there is explosive polyclonal B and T (both CD4+

124

Pathology and Pathogenesis of Human Viral Disease

KEY: Q O • 7 t

Maie.unaffected Female,unoffected Male, affected Abortion Dead

FIGURE 9.6 Pedigree of XLP-affected Duncan family. Reprinted with permission and through the courtesy of D. Purtilo, MD.

and CD8+) cell proliferation in an approximate B:T ratio of 1:1. These cells permeate interstitially into many organs, their presence being accompanied by variable degrees of tissue necrosis and organ failure (Tazawa et ah, 1993). Early on in the infection and for the initial 2-3 weeks, proliferation of lymphoid elements is a predominant feature of the disease, although later hypoplasia often becomes evident. These temporal changes are particularly apparent in the thymus, which initially shows an immunoblastic proliferation. Later destruction of the thymic epithelium by multinucleate giant cells is seen, and calcification of HasseFs corpuscles follows. In the lymph node and the spleen, the infiltrates of polyclonal immunoblastic cells efface the architecture. With the passage of time, depletion of these elements is evident, particularly in the thymusdependent regions of the lymph nodes. Plasma cell accumulations are seen in the late stages of the disease. In situ hybridization demonstrates EBNA-1 and -2 in numerous B cell nuclei, and viral proteins are commonly found in the affected organs. Thus, in contrast to IM in the immunocompetent patient, the infection is a systemic process. The development of an extranodal non-Hodgkin's B cell lymphoma is the second phenotypic expression of EBV in these genetically predisposed males. It may occur independently without an initial bout of IM, or develop after recovery from the acute illness. Roughly 25% of the carriers of the XLP gene exhibit this form of the disease. The lymphoma involves the central nerv-

ous system in the majority of these patients. EBV in the lymphoma is monoclonal, as are the tumor cells. Patients in the third group exhibit profound hypogammaglobulinemia before or after infection associated with hypergammaglobulinemia A or M (or both) (Provisor et al, 1975; Kuis et al, 1985). An inadequate antibody response to the viral capsid antigen (VCA) and nuclear antigens (EBNA-1 and -2) are characteristic (Masucci et ah, 1981). Simply stated, these young lads lack the ability to switch from an IgM to an IgG antibody response due to a T helper cell defect. The immune deficiencies are occasionally associated with opportunistic infections. Seventy percent of boys with XLP die by the age of 10, and survival of susceptible males beyond 40 years of age has not been documented. The immunological abnormalities of XLP are incompletely understood. Seemayer et al. (1993) hypothesize that regulation of the cytotoxic T cell response during infection is accentuated, resulting in fulminating hepatitis and bone marrow depression, with the virus-associated hemophagocytic syndrome occurring commonly.

BURKITT'S LYMPHOMA (BL) Dennis Burkitt can be accorded credit for initially recognizing the clinical uniqueness of this extraordinary lymphoma (Burkitt, 1961). According to him.

Epstein-Barr Virus The seminal observation made at the outset of the BL investigation was that two or more tumors that had hitherto been considered totally different in nature often occurred together in the same patient. This observation demanded an explanation, and a common cause seemed a reasonable possibility. Previously, orbital manifestations had been viewed as retinoblastoma, those in the kidneys or adrenals as neuroblastoma, those involving the ovaries as granulosa-cell tumors, and so on — all these tumors being composed of small round cells. Subsequently, they were shown to be related in the shared but distinctive age distribution of each tumour, and later still their geographical distribution was shown to be identical.

However, it was pathologists working in Uganda who pointed out the distinct histological features of this tumor and probable origin from lymphoid cells (O'Conor and Davies, 1960; Templeton, 1976). By 1967, a World Health Organization (WHO) committee convening at the National Institutes of Health concluded that the tumor was a distinct pathological entity. In the words of the committee, this undifferentiated lymphoma had the following morphological features (WHO, 1969): Histologically, the tumour is classically characterized by a strikingly monotonous proliferation of ''primitive" cells which are 10 to 25 |im in diameter, with round to oval nuclei and two to five prominent basophilic nucleoli. The nuclei approximate in median diameter those of the benign ''starry-sky'' macrophages that are often, but not invariably, present in the tumour. The nuclear membranes of the neoplastic cells are prominent, and their chromatin is coarsely reticulated in a clear parachromatin. The cells possess amphophilic cytoplasm which contains large amounts of ribonucleic acid and is therefore strongly pyroninophilic. Small round cytoplasmic vacuoles may be seen in paraffin sections; however, they are often better appreciated in air-dried Romanovsky-stained imprints. In the latter type of preparation, the cytoplasm is deeply basophilic and frequently contains numerous clear vacuoles. Special stains, in particular, oil red O, have been used to demonstrate

125

that some of these vacuoles contain neutral lipid. The neoplastic cells are characteristically, although not invariably, devoid of glycogen, and manifest little or no hydrolytic enzyme activity. A consistent feature of this tumor is a very high mitotic index. Mitotic figures are seen in approximately 4% of the neoplastic cells and, in association with this high growth fraction, one often observes numerous pyknotic cells and "starrysky" macrophages containing nuclear debris. [See Figure 9.7.] Ultrastructurally, at low magnification, they are strikingly monomorphic. The dominant cells are round to oval and have a high nucleocytoplasmic ratio. Benign-appearing macrophages are usually interspersed and contain phagocytosed cellular debris. The nuclei of the neoplastic cells are round or oval, with only shallow irregular indentations. Projections of the nuclear envelope may be seen as satellites or as nuclear invaginations. Chromatin is abundant and clumped at the nuclear envelope and around the prominent nucleoli, with relatively clear interchromatinic nucleoplasm. Cytoplasm is moderate in amount, the most characteristic feature being the numerous polyribosomes with only rare ergastoplasmic lamellae. Large lipid-filled vacuoles are usually present but may be simulated under the light-microscope by mitochondria, which tend to be dilated and aggregated at one pole of the cell.

The similarity of the Burkitt's lymphoblasts to follicular center cells soon became apparent, and the availability of lymphocyte immunological markers permitted their definitive identification as B cells. Burkitt's lymphoma is now classified in the National Cancer Institute Working Formulation as a high-grade lymphoma comprised of small noncleaved cells. Endemic Burkitt's lymphoma is the most common pediatric neoplasm in Central Africa, occurring in a tropical belt of relatively high humidity where childhood malnutrition is common and Falciprium malaria is holoendemic. In Africa, tumor prevalence proves to relate inversely to latitude, but the incidence is reduced at high altitudes. Thus, it is truly a tropical disease.

FIGURE 9.7 Histological features of B cell lymphoma with the starry-sky macrophages characteristic of Burkitt's lymphoma. The lesion is from the orbit of a 4-year-old African boy. Reprinted with permission from WHO (1969).

126

Pathology and Pathogenesis of Human Viral Disease

F I G U R E 9.8 Location of reported cases of Burkitt's lymphoma in those areas that have an annual rainfall of over 50 cm and an average temperature in the coolest months of over 15.6°C. The shaded area represents that in which, on climatic grounds, Burkitt's lymphoma might be expected to occur. The black squares show the distribution of the cases in the series compiled by Burkitt. Reprinted with permission from Haddow (1963).

However, in Central and East Africa, it often occurs in the highlands (i.e., >4000 ft. above sea level), where temperatures are more moderate, although malaria is rampant (Figure 9.8). Similar, but more restricted, foci of disease occur in native populations of eastern New Guinea and localized areas of tropical South America, where malaria is also prevalent. An identical lymphoma occurs rarely among somewhat older children in temperate regions of Europe, North and South America, and Japan. Although the tumor's histological features are the basis for its identity, similarities and differences in the organ distribution of the lymphoma in the sporadically and endemically occurring conditions are evident. In Africa, involvement of the orbit of the eye, the jaw, and the ovary are common, whereas these organs are less frequently involved in the disease, occurring sporadically in developed countries. For reasons that are totally unclear, the distribution of lesions in African cases is changing (Table 9.1). The unique lesions of the mandible that so characterize the African disease are age related; they occur much less commonly in older children (Figure 9.9). Worldwide, Burkitt's lymphoma has a striking male predominance and rarely occurs in adults except in association with AIDS. It comprises roughly a third of

F I G U R E 9.9 Typical expansive tumor mass of Burkitt's lymphoma originating in the mandible of an African boy.

the B cell lymphomas developing in untreated patients with human immunodeficiency virus (HIV). The role of EB virus in the pathogenesis of Burkitt's is the central issue in the disease. In Uganda, affected children invariably exhibit high titers of viral-specific capsid antibody (VCA) long before the onset of the disease, and evidence of infection is found by various techniques in almost all of the tumors, that is, 95-98%. In contrast, the tumors in only a small proportion of children with BL in Western countries evidence EBV infection. Similarly, evidence of EBV infection is less commonly found in tumors from patients with BL in North Africa. Although EBV cannot be accorded an etiological role in the Burkitt's form of lymphoma, it most probably plays an important contributory role in tumorigenesis, accounting in part for the relatively high prevalence of the disease in Central Africa and in tropical South America, where infections early in life are the rule. The striking geographic association of Burkitt's lymphoma with holoendemic malaria and endemic malnutrition (particularly kwashiorkor) has served as the basis for hypotheses regarding the pathogenesis of the cancer. In endemic areas, the immunological response to the malaria infections is maximal at the time in life when the incidence of BL is greatest. Moreover, BL is believed to occur less frequently and at an older age when malarial infections are less common. It is speculated that the acquisition of EBV relatively early in life might have a lymphoproliferative effect, possibly in conjunction with the superimposed depressing effect

127

Epstein-Barr Virus TABLE 9.1 Tumor Sites i n E n d e m i c and Sporadic Burkitt's Lymphoma (% of cases) Endemic

Central nervous system Peripheral lymph nodes Mandible Bone (excluding mandible) Abdomen/pelvis Gut Liver Spleen Kidney Adrenals

<1964

>1967

Sporadic

8 58 50 34

38 NR 21 27

48 44 44 85 70

19 22 19 40 21

11 17 7 4 67 NR NR NR NR NR

Adapted with permission from Templeton (1976). NR = not recorded.

of malaria and malnutrition on immunity, particularly suppressor cells of the T lymphocyte network. Most probably, the virus immortalizes the cells, as occurs in lymphoid cells infected in vitro and in EB-induced nonBurkitt's lymphomas in experimentally infected primates. Luxuriant expansion of the transformed B cell population might be expected to contribute to the characteristic chromosomal translocations described in detail below. Under the circumstances, the immune response to EBV is attenuated by the effect of Falciparum malaria and malnutrition on regulatory T cell immunity. While these concepts are appealing, the evidence is largely circumstantial. Moreover, they do not account for the sporadic occurrence of the disease in temperate developed countries, where the lymphoma no doubt has an entirely different pathogenesis. One could hypothesize that the EBV infection of lymphoblasts in the endemic disease is an epiphenomenon related to the high background prevalence of infection occurring early in life among residents of Central Africa and other tropical regions. Characteristic karyotypic alterations in the tumor cells are the hallmark of endemic Burkitt's lymphoma. They consist of the reciprocal translocations t(8;14), t(8;22), or t(2:8). The incidence of these cytogenetic alterations in the sporadically occurring tumors is not roughly comparable. In one series of 22 African cases in which tumor cell lines were used, 13 t(8;14), 6 t(8;22), 2 t(2;8), and 1 del(8)(q24) were found. Although similar karyotype alterations have been described in lymphomas and leukemias of other types, their occurrence is exceedingly rare except in the Burkitt's lymphomas developing in patients with AIDS. EBV infection of lymphoblastoid cells per se does not appear to be responsible since chromosomal changes do not develop in infected lymphoblastoid lines of cells in the laboratory. The type of translocation correlates with the presence of detectible immunoglobulin heavy and light

chains on the plasma membrane of the tumor cell. Kappa or lambda light chain expression correlates with the t(8;2) and t(8;22) translocations. These observations account for the prevailing view that immunoglobulin promotors of the respective transposed chromosomal segments activate the c-myc oncogene in or adjacent to the breakpoint of the 8th chromosome accounting for malignant phenotypic expression. Under these circumstances, the c-myc regulatory effects on the cell cycle are altered, leading to cell proliferation, rather than to apoptosis. The picture is more complex, however, for, as shown by Pelicci et al. (1986), the breakpoint is located outside the c-myc locus in endemic BL, whereas in the sporadic cases it is found within a 5' region that includes the first intron and exon as well as the flanking sequence of the gene. In addition, in endemic cases of BL with the (8;14) translocation, the breakpoint in the heavy chain gene occurs in the joining region, while in sporadic cases, it is found in the switch region preceding the constant region (Table 9.2). LYMPHOPROLIFERATIVE DISORDERS (LPDs) ASSOCIATED WITH IMMUNOSUPPRESSION (Ziegler, 1981; Lones et al, 1995; Preiksaitis et al, 1992; Purtilo et al, 1992; Saemundsen et al, 1981; Randhawa et al, 1990,1992; Knowles et al, 1995; Craig et al, 1993; Lager et al, 1993)

A number of complex acquired and genetically mediated syndromes of immunosuppression predispose to the development of EBV-associated LPDs of varying degrees of severity and prevalence (Klein and Purtilo, 1981) (Table 9.3). The condition occurs in solid organ allotransplant recipients who are undergoing immunosuppressive drug therapy (Hanto et al., 1981; Abu-Farsakh et al, 1992; Berg et al, 1992; BorischChappuis et al, 1990; Ho et al, 1985) and is seen in

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Pathology and Pathogenesis of Human Viral Disease TABLE 9.2 Characteristics of Endemic and Sporadic Burkitt's Lymphoma Endemic — (African type) Rate: 8-10 cases/100,000/year Mean age — 7 years EBV infection — 97%« Association with holoendemic malaria Chromosomal translocation long 8 to 14 — 86% (heavy chain) long 8 to 2 (kappa light chain) long 8 to 22 (lambda light chain) breakpoint outside of long 8 myc gene Sporadic — (American type) Rate: 0.1 case/100,000/year Mean age — 11 years EBV infection —15-30% Chromosomal translocation as with endemic breakpoint inside of long 8 myc gene AIDS-associated EBV infection - - 35-50% "Approximately 30 gene copies per cell.

AIDS (Andiman et ah, 1985). It now commands particular attention in AIDS because of the relatively common occurrence of the syndrome and the propensity for the development of non-Hodgkin's lymphomas (see Chapter 16). LPDs now occur in roughly 2% of allotransplant organ recipients at variable intervals after initiation of immunosuppression. In a study of almost 1000 allotransplant recipients, a lymphoproliferative disorder occurred in 0.8% of patients with kidney grafts, 1.62% with hepatic grafts, and 5.9% of those with cardiac allotransplants (Bhan, 1992). The median interval after transplantation is about 6 months. A higher incidence of disease has been reported from several other transplantation centers. EBV infection immediately before or promptly after transplantation has been associated with a high incidence of LPDs in children. LPDs first became evident in patients who had been administered cyclosporin A. Other therapies such as administration of OKT3 immune serum (which effectively blocks cytolytic T cell action) also predispose to the early onset of the condition. LPDs represent a spectrum of disease conditions, and their clinical stages have been described (Hanto et al, 1982, 1983, 1985; Knowles et al, 1995). In their simplest form, these disorders are reflected as an IM-like syndrome that can be confused with immunologic rejection of the allograft and graft vs. host disease. Pathologically, they are reflected in a polymorphic B cell hyperplasia with limited organ infiltration by B

TABLE 9.3 Clinical Classification of Lymphoproliferative Disorders Associated w i t h I m m u n o s u p p r e s s i o n I. Infectious mononucleosis illness a. Acute febrile illness b. Polymorphic B cell hyperplasia c. Response to acyclovir and withdrawal of immunosuppressive therapy II. Infectious mononucleosis illness involving multiple vital organs a. Acute febrile illness resembling fatal XLP b. Polyclonal or oligoclonal B cell hyperplasia c. Response to acyclovir treatment and to the withdrawal of immunosuppressive drugs is variable III. Lymphomas of predominantly brain and digestive tract a. Monomorphic B lymphoblastoid neoplasm b. No response to acyclovir treatment or withdrawal of immunosuppressive drugs

and T lymphocytes such as may occur in IM (Shearer et al, 1985). Often, the lesions tend to be plasmocytic and are distributed in the oropharynx and lymph nodes (Rizkalla et ah, 1997). Almost invariably, a polyclonal EBV infection can be established in the cells of these infiltrates by in situ hybridization of the viral DNA and immunofluorescent demonstration of EBNA in lymphoid cells of the lesion (Oudejans et al, 1995). At this stage, LPDs are readily reversed by reduction or elimination of immunosuppressive therapy, but this, of course, jeopardizes the transplanted organ. A dramatic response to acyclovir administration often occurs, and this is the therapeutic approach currently employed (Hanto et al, 1982). In a somewhat more complex advanced stage, LPDs are characterized by the infiltrative spread of B lymphocytes and immortalized lymphoblastoid cells into lymphoid organs and into extranodal sites throughout the body. The process usually is polymorphic and polyclonal, although oligoclonal accumulations of cells are often identified in isolated organs. As the disease evolves, a monoclonal population appears, but these cells lack the abnormal protooncogene expression and mutant suppressor genes that characterize a lymphoma (Knowles et al, 1995). Nonetheless, the morphological finding alerts pathologists to the possibility of an incipient lymphoma. Disease at this stage often can be reversed by elimination of immunosuppressive treatment and/or acyclovir administration. In their most devastating form, LPDs are reflected as a large-cell (centroblastic or immunoblastic) nonHodgkin's lymphoma in which is found a monoclonal population of malignant B cells. Lesions of this type can occur at localized sites such as the gastrointestinal tract or brain, and polyclonal infiltrates are found in other more distant organs. Thus, the clinical and patho-

Epstein-Barr Virus

logical evidence strongly suggests that the lymphoma evolves as a clonal population of cells from the more complex polymorphic disease. These lymphomas only rarely respond to elimination of immunosuppressive drug treatment. Southern blot analysis of the EBV DNA in these cells shows that the viral genome is monoclonal and latent in the form of a circular episome. Typically, lymphomas develop in older patients long after initiation of transplantation and commonly involve the central nervous system and abdominal digestive tract.

N O N - H O D G K I N ' S LYMPHOMA EBV is implicated in either the causation or development of B cell lymphomas in the post-transplantation lymphoproliferative disorders, and in non-Hodgkin's lymphomas occurring in AIDS and a variety of other immune deficiency states. It has also been strongly associated with Hodgkin's disease and the Ki-1 positive anaplastic large-cell lymphomas that share the CD30 Ki antigen with Hodgkin's disease and may be of T cell origin (DiGiuseppe et al, 1995) (Figure 9.10A-D). Most

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tumors occurring in patients in an immunosuppressed condition are the so-called high-grade non-Hodgkin's lymphomas. The evidence linking EBV to these tumors is based upon the findings of molecular studies conducted in many different laboratories during the 1990s. However, the infrequent presence of EBV genomic products in spontaneously developing non-Hodgkin's lymphoma in nonimmunocompromised persons, as well as the less than absolute association of the disease with evidence of infection in the immunodeficient patient, raises considerable doubt as to the intrinsic pathogenic role of the virus in tumorigenesis. The common finding of both latent and replicative virus infections in the tumor cells of the immunodeficient patient is compatible with a loss of immune control (Birx et al, 1986). Three different patterns of EBV gene expression are found in various of the different morphologic types of lymphoma. This observation, at present, defies explanation from the perspective of a possible role of EBV gene products in the pathogenesis of these tumors. Does EBV predispose to either the development or clinical progression of non-Hodgkin's lymphoma? In my opinion, the evidence, although incomplete, argues for an affirmative response. Of paramount consideration is the established increase in frequency of these

^t ^ - ^ ^ ^ ^

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*i§L. ^ % # FIGURE 9.10 (A,B) Anaplastic large-cell lymphoma. Immunochemistry demonstrated the CD30Ki antigen in tumor cells. (C,D) Examples of Reed-Sternberg cells in Hodgkin's lymphoma. Reprinted with permission and through the courtesy of J. Lunde, MD.

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lesions in immunocompromised states accompanied by the finding of EBV latency and gene expression in a substantial proportion of tumor cells. These tumors commonly are aggressive and occur in extranodal sites and atypical locations (such as lymphomas in the brain of patients with AIDS) (Katz et al, 1986; Chang et ah, 1993; Morgello, 1992). The EBV gene product, LMP-1, has an oncogenic role in B cell lymphoma of the immunosuppressed patient. It seems to act as a transmembrane signaling protein that interacts with intercellular transduction molecules, thus mediating transformation (Liebowitz, 1998). Recent additional evidence suggests that the cellular survival protein, bcl-2, is upregulated in the EBV-infected cell, thus inhibiting apoptosis. Infection of the tumor precursor cell appears to occur before the onset of cellular proliferation, inasmuch as the latent episomes in the cell are monoclonal. It is likely that enhanced mitotic activity and inhibition of apoptosis predispose to the diverse cytogenetic aberrations that characterize the tumors at the time of pathologic diagnosis. In turn, these major changes in the cell genome most probably contribute to tumor progression and the aggressive malignant clinical features of the lesions. While EBV may not be the spark that kindles the fire, it may be the oxygen that turns a smoldering flame into a raging inferno. Infection of T cell lymphomas by EBV is now well documented, but detailed systematic information is lacking both with regard to the identity of the infected cells and the expression of the viral genes (Su et ah, 1991; Ott et al, 1992). As noted above, the C3 complement receptor serves as the viral receptor on both B and epithelial cells, but current evidence suggests that this receptor is not present on T cells. Are there then other, still uncharacterized, EBV receptors present on T cells, or are the C3 receptors so sparsely distributed on the cells that they are undetectable with current technology? Alternatively, could receptors be present on progenitor cells and not on the dedifferentiated tumor cells studied by the pathologists in the advanced tumor? Answers to these questions are lacking at present. Using PCR, Tokunaga et al (1993) found EBV DNA in the tumors of 21 out of 96 cases of adult T cell leukemia/lymphoma. EBV EBER-1 and LMP-1 were demonstrated in the cells of the majority of these cases by either in situ hybridization or immunohistochemistry Presumably, this disease can be attributed to HTLV1 infection since the patients resided in the geographic central focus of HTLV-1 in the southern Japanese island of Kyushu. Less information exists with regard to the association of the sporadically occurring systemic and cutaneous T cell lymphomas unrelated to HTLV that occur worldwide (Cheng et al, 1993).

H O D G K I N ' S DISEASE (HD) The association of EBV infection with HD is now well established, yet the role of the virus in causation and progression of the disease is obscure. This is a particularly interesting issue, inasmuch as EBV infection of the tumor does not result in a shortened lifespan or a more aggressive clinical course (Fellbaum et al, 1992). Several unresolved questions continue to obfuscate the problem. Is HD in reality not one but several different disease processes having as the common denominator the Reed-Sternberg cell? Is HD an infectious process that terminates in an uncontrolled malignancy? Do the various morphologic forms of the disease represent a continuum of biologic responses to a hypothetical infectious agent, or do they reflect differing patterns of response to a single etiological agent? What is the biologic significance of the Reed-Sternberg (RS) cell, and what is its origin? Is the fact that the RS cells are aneuploid and monoclonal an indication of the cell's malignant potential despite its rarity in some forms of HD? Epidemiological evidence provides support for an infectious etiology for at least one of the morphologic types of HD, that is, the lymphocyte predominant type (LP). This form characteristically develops in males during the late teenage years or the twenties. It tends to occur in those with a past history of IM and in members of relatively small families having a higher level of educational attainment and socioeconomic status. These latter demographic features are consistent with the pattern of infectious disease that occurs with devastating severity in those protected from endemic infections early in life (e.g., paralytic poliomyelitis). Three other morphologic forms of HD commonly occur: nodular sclerosing, mixed cellularity, and lymphocyte depleted types. While their epidemiology is complex, HD, manifested as these morphological types, tends to occur in somewhat older persons exhibiting none of the demographic associations exhibited by the LP form. Indeed, the mixed cellularity and lymphocyte depleted types usually develop in patients older than 50 years of age, and the peak incidence is in the 8th and 9th decades of life. Cytomorphological and in situ hybridization studies in adult patients with HD who have no known risk factors demonstrate conclusively an association of RS cells and RS-variant cells (so-called Hodgkin's or H cells) with latent clonal EBV genomic components, specifically EBER-1 and LMP-1 (Brousset et al, 1993). No evidence of a replicative infection is found, and the viral genome appears to be monoclonal. This observation implies that the initial infection developed in the

Epstein-Barr Virus

primordial cells of the tumor and that RS and H cells are derived from a common progenitor cell. However, the prevalence of EBV infection appears to be highest in the mixed cell type, intermediate in the nodular sclerosing HD, and uncommon in the LP type (Shibata . ei al., 1991b). In these forms of the disease, an occasional lymphocyte may carry the viral genome (Khan et ah, 1992). Recently, it was also found in the rare primary cutaneous HD (Kumar et al, 1996). These findings argue against the relevance of a pathogenic association of the LP form with IM, and thus EBV infection as suggested in the discussion. They further indicate a stronger association of EBV infection with the more malignant types of disease that tend to occur in older persons. On the other hand, in the rare cases of HD in the pediatric age range, the association with EBV is greatest in children under the age of 4 years. In one study, only 21% of those in the 10-15-year age group were positive (Andriko ei al, 1997). Evidence of rampant EBV infections are found in HD patients with advanced HIV-1 infections (Rubio, 1994). In the tumor, RS and H cells invariably exhibit latent non-episomal infection with EBV (Siebert et al, 1995; Herndier et al, 1993; Carbone et al, 1996). There appears to be an increased prevalence of HD in AIDS, and the disease tends to be more malignant. Prior to modern drug therapy, HD patients with AIDS had a relatively short survival time. This combination of diseases is commonly terminated by overwhelming opportunistic infections, but it is not known whether EBV contributes to the fatal outcome. Advanced HD is often accompanied by a profound degree of T cell-mediated immunosuppression. This defect is thought to be secondary either to deranged antigen processing and presentation by the HD macrophage or, alternatively, T cell unresponsiveness to IL-1 (Jandl, 1996). Whatever the mechanism, HD patients exhibit defects in delayed hypersensitivity in recall skin tests, such as dinitrochlorobenzene sensitization, and manifest an increased tolerance to skin grafts. In view of these findings, one might ask whether a deficiency in T cell immunity predisposes to EBV infection in those with HD. As noted above, RS and H cells exhibit EBV EBER-1 and LMP-1 gene activation, although information on gene copy number in these cells is lacking. It is intriguing to speculate that LMP-1 may contribute to the oncogenicity of the RS and H cells. The products of LMP1 have a diversity of transforming effects when transfected into fibroblasts and B cells in vitro. LMP-1 alters cell growth in vitro and causes the loss of contact inhibition and promotes anchorage independence. By activating the bcl-2 gene, apoptosis is inhibited, thus tend-

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ing to increase the lifespan of these cells (Khan et al, 1993).

NASOPHARYNGEAL C A R C I N O M A (NPC) Undifferentiated carcinomas of the nasopharynx occur with unusually high frequency in residents of southeast China and in emigrant Chinese elsewhere. They comprise approximately 20% of the cancers occurring in these people. Similar tumors are found in the native population of Greenland and in foci in both Africa and the Mediterranean Basin. Undifferentiated nasopharyngeal carcinomas are rarely seen in Caucasians in developed countries of the world. In these areas, the pharyngeal carcinomas are almost invariably comprised of well-differentiated keratinized squamous epithelium, accompanied by variable infiltrates of lymphocytes (Ablashi and Levine, 1983). Latent EBV infections are consistently demonstrated by in situ hybridization in the cells of these undifferentiated tumors in endemic regions of China. In contrast, evidence of infection is infrequently found in the squamous epithelial cells of the well-differentiated nasopharyngeal tumors developing in Caucasians. Latent infections by EBV also do not occur in normal squamous epithelium after a naturally occurring pharyngeal infection. Additional evidence etiologically linking EBV with these tumors is the demonstrated clonal nature of both the tumor cells and the virus within them (Raab-Traub, 1992; Pathmanathan et al, 1995). Almost invariably, exceptionally high levels of circulating antibodies reactive with specific EBV proteins are found in the blood serum of healthy persons destined to develop the tumor. Despite the consistent association of EBV with the undifferentiated nasopharyngeal carcinomas, the contribution of the virus to the events of tumorigenesis remains uncertain. Epidemiological studies suggest, but fail to prove, an etiologic role for dietary factors, the most likely of which is the common consumption of heavily salted fish, particularly at an early age in life (Yu, 1991). The importance of this food or other environmental factors in the pathogenesis of the tumor is suggested by the dramatic decrease in prevalence of nasopharyngeal carcinomas among first- and second-degree Chinese immigrants to North America, who consume a more Western diet (Table 9.4) (Hildesheim and Levine, 1993; Flores et al, 1986). These findings argue against, but do not entirely exclude, a genetic contribution to carcinogenesis, and at the least suggest a cultural influence. The WHO classification assigns tumors of the nasopharynx into three categories based on morphologic

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TABLE 9.4 Incidence of Nasopharyngeal Carcinoma Among Chinese by Sex and Residence No. cases/1 x 10^ Hong Kong Chinese males Chinese females Los Angeles (males) Foreign-born Chinese U.S.-born Chinese U.S.-born Caucasians Canada (males) Foreign-born Chinese 2nd- and 3rd-generation Chinese Caucasians

30 13 14 3.5 0.6 20 1.3 0.22

differentiation of the malignant components. The first of these categories is the well-differentiated keratinizing squamous carcinoma, whereas WHO 2 and 3 represent the undifferentiated carcinomas. The epithelial cell component in these latter tumors is characterized by relatively uniform cells with vesicular nuclei and prominent nucleoli. These cells often form syncytia that can simulate Reed-Sternberg cells. Inflammatory cell infiltrates comprised of small numbers of plasma cells and eosinophils, as well as a predominant population of small T lymphocytes, are found in the lesions. The tumors exhibit two general morphologic patterns that appear to have no influence upon prognosis. The first, termed "Regaud," is characterized by accumulation of neoplastic epithelial cells into the nests and cords interspersed with the inflammatory cell infiltrates (Figure 9.11A,B). In the second pattern, termed "Schmincke,'' the malignant epithelial cells are intermixed with the predominantly lymphoid infiltrates of cells (Figure 9.11C). The latter tumor type must be distinguished from a malignant lymphoma, which it occasionally resembles. Although the clinical and pathologic evidence is limited, NPCs appear to develop from foci of noninvasive epithelial hyperplasia that are presumed to evolve from dysplasia and carcinoma in situ. Virological studies using in situ techniques demonstrate the EBV-encoded RNA (EBER) and the transforming protein LMP1 in dysplastic and noninvasive carcinoma cells, but not in the lymphoid elements that accompany the epithelial components (Brousset et al, 1992). As would be expected, the pharyngeal mucosal tissue from normal control individuals reveals no evidence of infection (Pathmanathan et al, 1995). The same constituents of EBV are consistently found in high copy number in cells of the invasive advanced tumors. In both the pre-

FIGURE 9.11 (A) The "Regaud" morphological form of a poorly differentiated N P C The cytokeratin reactive epithelial cells (B) are arranged in nests interspersed with lymphocytes. (C) The ''Schmincke" morphological form of N P C Although epithelial cells are present, the infiltrating lymphocytes predominate in this poorly differentiated lymphoma. Reprinted with permission and through the courtesy of R. Grenko, MD.

invasive and advanced lesions, the EBV DNA is not integrated into host cell DNA and is found as a circular episome in the cell. Molecular studies have shown that the virion population in both the preinvasive and invasive tumors is clonal. Thus, the malignant cells would appear to be infected with the progeny of a single virion that may have unique biological properties. How the virus and the presumptive environmental fac-

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tors interact is currently a matter for speculation, since no experimental information is available. In Caucasians, lymphoepithelial tumors comprised of variable amounts of undifferentiated carcinoma and squamous carcinomatous elements have been described in the floor of the mouth, tongue, soft palate and uvula, nasal cavity, upper airways and larynx, the upper digestive tract, and the genitourinary tract (Tseng et al, 1997). But, in some of these lesions an association with EBV has not been established (GuUey et al, 1995).

LYMPHOEPITHELIOMATOUS GASTRIC C A R C I N O M A The etiology of gastric carcinoma is obscure, although host genetic predisposition and environmental factors (including diet) are important influencing factors in the causation of these tumors. While the epidemiology and pathogenesis of gastric cancer in general is beyond the scope of this discussion, the possible role of EBV in the pathogenesis of the lymphoepitheliomatous type gastric carcinoma is considered here (Figure 9.12A). Burke and his associates (1990) detected EBV DNA sequences in the malignant epithelial cells of the rare lymphoepitheliomatous poorly differentiated gastric carcinomas (Shibata et al, 1991a). These tumors comprise approximately 4% of the gastric cancers occurring in Japan, where cancer of the stomach continues to be endemic and relatively common (Watanabe et al, 1976). In situ hybridization of these lesions using an EBV-encoded probe complementary to the small early RNA of the virus (EBER) demonstrated infection of the malignant epithelial cells in about 85% of the lesions (Matsunou et al, 1996) (Figure 9.12B,C). Little or no evidence of infection of the infiltrating lymphocyte population was found. Further studies have demonstrated the monoclonality of the virus and its presence as a latent episomal agent in very early lesions as well as in advanced and metastatic tumors (Harn et al, 1995). Additional studies have now shown that EBV infection is not limited to carcinoma with lymphoid stroma, but occurs in 5 to 15% of carcinomas of all morphological types in Japan and in the United States (Shibata and Weiss, 1992; de Bruin et al, 1995; GuUey et al, 1996).

SINONASAL TUMORS Non-Hodgkin's disease, lymphoproliferative disorders of natural killer N K / T cell derivation in the nasal cavity and paranasal sinuses, comprise only a small

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FIGURE 9.12 (A) Superficial spreading of lymphoepitheliomatous gastric adenocarcinoma. Note the dense lymphocytic infiltrates with lymphoid follicles. (B) Interface between normal and dysplastic gastric mucosa showing reactivity of the dysplastic elements with EBER antibody of EBV by immunohistochemistry. (C) EBER antibody reactivity of the epithelial elements of a gastric carcinoma. Panels B and C reprinted with permission from GuUey et al. (1996) and through the courtesy of M. Gulley, MD.

proportion of the extranodal lymphomas (fewer than 8%) occurring in the Orient and North America (Petrella et al, 1996). They commonly present clinically as the so-called lethal midline granuloma, a lesion characterized by ulceration and necrosis of the tissues infiltrating in and around the nasal cavities. When the lymphoid cells of the tumor are polymorphic and

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Pathology and Pathogenesis of Human Viral Disease

lymphoreticular in character, the lesion is often termed polymorphic reticulosis, particularly when there is an angiocentric pattern. These tumors are clinically aggressive, and patient survival can usually be measured in months. Systematic studies of these unique but rare neoplasms has demonstrated a strong association with latent EBV infection in the clonally derived N K / T cells of the lesions (Jones et al, 1988; Cohen ei ah, 1980; Richel ei al, 1990; Tomita et al, 1995; Arber et al, 1993; Kanavaros et al., 1993). The virus, like its host cells, is clonally derived, an observation suggesting that infection occurred at an early stage in tumorigenesis. Since the infected cells consistently exhibit EBER and LMP-1 when analyzed by in situ techniques, one is obliged to conclude, at least preliminarily, that these oncogenic proteins play an intrinsic role in neoplastic formation (Harabuchi et al, 1990,1996; Arber et al, 1993; Ho et al, 1990). The observations summarized above must be interpreted in the light of studies of lymphocytes in routinely excised nasal polyps. In work carried out in Hong Kong, 85% of the B and T cells in polyps exhibited EBV protein (Tao et al, 1996), and PCR studies of nasal and oral secretions have shown that over 50% of adults have within these tissues actively replicating EBV.

PULMONARY DISEASE During the acute stages of IM, the bronchial epithelial cells are extensively infected with EBV. Chronic infection of the respiratory mucosa also occurs commonly as shown by various virus detection techniques, including in situ hybridization studies on exfoliated cells (Lung et al, 1985). Pulmonary disease, in the form of interstitial pneumonia and lymphoid hyperplasia in the lung, is found in approximately 5% of patients during the acute stages of IM (Mundy, 1972; Sriskandan et al, 1996). Hilar lymphadenopathy occasionally is an accompanying feature (Fermaglich, 1975; Evans, 1967; Baehner and Shuler, 1967). Severe consolidative pneumonia occurs in fatal IM (Custer and Smith, 1948). A diffuse chronic interstitial pneumonitis attributed to EBV in two febrile patients was described by Schooley and his colleagues (1986). EBV infection of pneumocytes and airway cells is demonstrable in over 10% of cases of diffuse interstitial pneumonia examined at autopsy (Oda et al, 1994). In addition, viral capsid antigen indicative of an acute infection was found in the lung tissue of 6 of 12 cases of diffuse interstitial fibrosis of diverse etiologies (Quddus et al.

1997). The importance of the infection in these cases, if any, is unknown. EBV infections of the lymphocytes in lymphoid interstitial pneumonitis (LIP) has been documented by in situ hybridization both in adult patients with a spontaneously developing form of the disease (Barbera et al, 1992) in and children with AIDS-associated LIP (Rubinstein et al, 1986). LIP in the non-HIV-infected patient is a rare condition developing spontaneously in persons of all ages, but most commonly in middle-aged adult women (male:female ratio = 1:5). It is often, but not invariably, associated with a variety of conditions of immune dysregulation, including Sjogren's syndrome, autoimmune hepatitis, pernicious anemia, and myasthenia gravis. Pathologically, LIP is characterized by the presence of a diffuse heterogenous interstitial and peribronchial infiltration of B lymphocytes, plasma cells, and histiocytes. Among 14 selected cases of LIP, 9 had EBV in B cells, but lung tissue from 2 of 10 controls with interstitial fibrosis were also positive (Barbera et al, 1992). About 75% of children with AIDS develop LIP characterized by interstitial infiltration of lymphocytes, predominantly of the CD8+ lineage (Lin et al, 1988; Morris et al, 1987; Guillon et al, 1988). Often, there is a nodular character to the lymphoid accumulations in the lesions that are commonly associated with the walls of the bronchus. Whether or not this lymphoid hyperplastic lesion is pathogenically consistent with the sporadically occurring LIP in the adult is, at present, unclear, but almost invariably, HIV-infected patients exhibit polyclonal hypergammaglobulinemia (Joshi et al, 1985). High titers of EBV capsid antibody are found frequently in the serum. In situ studies by Rubinstein et al (1986) demonstrated EB genomic material in the lymphoid elements of four biopsies from six patients with AIDS manifesting the typical pathological picture of lymphoid hyperplasia. The features of LIP are considered in additional detail in Chapter 16. Fewer than 50 cases of large-cell immunoblastic B lymphomas developing in the pleural cavity after surgically induced pneumothorax for pleural tuberculosis have been reported (Ohsawa et al, 1995). The majority (but not all) of these cases with EBV infections originated in Japan, where this form of treatment for tuberculosis was commonly used in the past. Fukayama (1994) reported elevated EBV LMP-1 and EBNA-1 antigens in tumor cells from all five of the studied patients. EBV infections have also been found in tumors of this type diagnosed in Europe. Lymphoepitheliomatous carcinoma of bronchial origin occurs uncommonly. About 4% of the lung cancers resected surgically in Hong Kong among both smokers and nonsmokers are so classified, but the prevalence in

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Caucasians is much lower (Pittaluga et ah, 1993; Butler et aU 1989; Higashiyama et al, 1995; Chan et al, 1995). An EBV-associated tumor of the lung in an 8-year-old child was reported by Curcio et at. (1997). As in the pharynx, these tumors are comprised of variable-sized islands of poorly differentiated epithelial tumor cells intermingled with lymphocytes and plasma cells. In situ hybridization studies consistently show EBNA in epithelial cells, but not in the lymphocytes. Similar studies of the routinely resected traditional morphological types of bronchogenic carcinomas have consistently proven negative.

LYMPHOMATOID GRANULOMATOSIS This poorly understood lesion was initially described by Leibow and his associates (1972). It is characterized by (i) polymorphic lymphoid infiltrates populated predominantly by T cells and a lesser number of B cells; (ii) angiitis as reflected in the transmural involvement of medium-sized veins and arteries by a lymphoid infiltrate; and (iii) necrosis, resulting in the so-called (but mischaracterized) granulomatosis. The overlap of this lesion with angiocentric T cell lymphomas is vaguely defined, and the two lesions are no doubt confused on occasion (Peiper, 1993). Other possible sources for confusion are the so-called T cell-rich B cell lymphomas (Ramsay et ah, 1988). EBV infection is consistently found in the minor B cell components of the pulmonary lesions of lymphomatoid granulomatosis, but the biological state of the viral genome and its expression have not yet been characterized (Guinee et al., 1994; Haque et al, 1998). Using in situ hybridization, Myers et al (1995) found the EBV DNA to be restricted to the large atypical B cells. T cells were not infected. Lymphomatoid granulomatosis is a rare infiltrative lesion of the skin, lung, liver, kidney, and nervous system in which atypical T lymphocytes, plasma cells, and macrophages accumulate with a distinct relationship to small blood vessels. They are considered by some to be the sinonasal tumors described above. Although believed to be of T cell origin, recent evidence suggests that they may be of natural killer (NK) origin (Salvio, 1996). Interestingly enough, the lymphoid cells in these obscure disease processes are often infected by EBV, as shown by in situ hybridization studies and PCR (Harabuchi et ah, 1996; Katzenstein and Peiper, 1990). Thus, the susceptibility of NK cells requires further study. On the other hand, the large atypical cells in the pulmonary lesions of lymphomatoid granulomatosis prove to be EBV-infected B cells located in a sea of

reactive T cells (Myers et al, 1995). Similarly, the infection has now been found to occur in B-lineage cells of the equally obscure angioimmunoblastic lymphadenopathy and its related lymphomas (Weiss et al, 1992).

INFLAMMATORY PSEUDOTUMORS Inflammatory pseudotumors in lymph nodes, spleen, and liver are commonly infected by EBV as demonstrated by in situ hybridization approaches. The spindle nondendritic reticulum cells are predominantly involved. The importance of this finding remains to be established (Arber et al, 1995; Selves et al, 1996).

SJOGREN'S SYNDROME A N D SALIVARY G L A N D T U M O R S In 1933, the Swedish ophthalmologist H. S. C. Sjogren described the syndrome that now bears his name. It predominantly affects middle-aged women and is characterized by keratoconjunctivitis, sicca (dry eyes), and xerostomia (dry mouth) and is usually accompanied by rheumatoid arthritis. Pathologically, Sjogren's syndrome is characterized by replacement of the involved salivary and lacrimal glands by a dense infiltrate of B cells, often organized into germinal centers, accompanied by T cells, plasma cells, and histiocytes (Figure 9.13A,B). Variable numbers of the so-called epimyothelial islands further typify the lesion. These latter structures represent the residue of the atrophic or apoptotic acinar and ductal epithelial cells. They exhibit varying degrees of dedifferentiation and mitotic activity, resulting in an appearance that can suggest malignancy. EBV DNA is detected focally in the ductal epithelium of the normal adult lacrimal gland, though it is not present in acinar cells (Pflugfelder et al, 1993). A similar distribution of virus genome most probably occurs in the major and minor salivary glands in Sjogren's disease. However, detailed sensitive molecular localization studies have yet to be carried out on these organs. In Sjogren's syndrome, evidence of active virus replication is found in epithelial cells of the salivary and lacrimal glands, and EBV can be recovered from saliva. The infectivity status of the B lymphocytes that characterize these lesions is unclear (DiGiuseppe et al, 1994; Fox et al, 1986; Chan et al, 1994).

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Pathology and Pathogenesis of Human Viral Disease

'^VZ

FIGURE 9.13 Parotid gland lesions of Sjogren's syndrome. (A) Epithelial islands are infiltrated and surrounded by dense accumulations of B lymphocytes. In (B) the epithelial island is reticulated and hyalinized. Reprinted from Foote and Frazell (1954).

While Sjogren's syndrome clearly is considered to be an autoimmune process, the skill of the pathologist is occasionally challenged by the necessity to differentiate the lesions of this disease from the much rarer B cell lymphomas that infrequently occur in the salivary glands. A second equally difficult challenge is diagnostic assessment of the Sjogren's lesion for the poorly differentiated lymphoepithelial tumors that occur in these same glands. These lesions strikingly resemble the EBV-associated pharyngeal lymphoepithelial tumors. They predominantly but not invariably (Kotsianti et al, 1996), occur in Orientals and Native American residents of Greenland. In situ DNA hybridization of the salivary glands in these cases has consistently demonstrated evidence of episomal EBV infection of the poorly differentiated tumor cells but not the lymphocytes (Hamilton-Dutoit et al, 1991). Other investigators have detected EBER-1 in lymphoid infiltrates (Wen et al, 1996). Additional studies by Tsai et al (1996) have failed to demonstrate similar viral constructs in 49 other morphologic types of salivary gland cancers. The observation establishes quite clearly the specificity of EBV infection in the poorly differentiated carcinomas (Saito et al, 1989).

While a cause-and-effect relationship between EBV and the development of Sjogren's disease and the epithelial tumors of the lacrimal and salivary glands has not been established, the admittedly incomplete evidence available at present suggests that the lymphoid response in these unique lesions may focus on EBV virus proteins or altered cells in which the virus resides. Could it be that the lymphoid infiltrates introduce an element of immune control of tumor growth, a proposition consistent with the relatively prolonged survival time of patients with these tumors (Tsai et al, 1996)? Whatever its pathogenetic role in neoplasia, EBV virus would not appear to be the sole etiological factor.

HAIRY LEUKOPLAKIA (HCL) HCL is an elevated, verrucous, and somewhat "hairy" whitish proliferative, but nonmalignant, epithelial lesion developing on the lateral surfaces of the tongue and occasionally at other sites in the oral pharynx. EBV is believed to be the etiological agent. HCL

Epstein-Barr Virus

was initially described among homosexual males with AIDS (Greenspan et ah, 1984) but is now known to develop occasionally in recipients of organ allotransplants with iatrogenic immunosuppression (Itin et ah, 1988; Epstein et al, 1988) and rarely among seemingly immunocompetent adults. EBV is known to replicate in the epithelium of the tongue in both immunocompetent and immunocompromised patients who lack clinically evident HCL lesions, and it is claimed that EBV receptors are located on the lateral aspects of the tongue, but this observation requires confirmation (Greenspan et ah, 1985; Corso et ah, 1989). Indeed, replication sites for EBV in the oropharynx must be common. Ferbas et ah (1992) recovered ElBV from the oral pharyngeal secretions of almost 50% of HIV-positive homosexual males, and 16% proved to be HIV-1 seronegative. In typical HCL, luxuriant EBV replication occurs, as established by the consistent demonstration of EBV virions in cells of the lesion, accompanied by expression of LMP-1 and EBNA and the presence of

137

liner viral DNA. In addition, the lesions respond to acyclovir treatment. About 25% of HIV-1-infected persons develop the lesion; it seems to be a prognosticator barkening the arrival of AIDS. In one study, 30% of HIV-infected patients with HCL developed overt AIDS within a 3-year period, but the presence of lesions does not correlate with CD4+ cell numbers or other risk factors for AIDS. The tongue lesions are characterized by prominent acanthosis and parakeratosis with loose accumulations of keratin on the surface. This gives the lesion the "hairy" appearance noted clinically (Figure 9.14A,B). A spectrum of structural features result in flat lesions in some cases and corrugation of the surface in others. Squamous epithelial cells with intranuclear inclusions characterize the lesion. The majority of inclusion-bearing cells are located in the stratum spinosum. These cells also typically exhibit a ballooning koniocytosis with an eosinophilic ground-glass appearance to the cytoplasm. Fernandez and colleagues (1990) describe

FIGURE 9.14 Hairy cell leukoplakia showing marked acanthosis and parakeratosis with the resulting superficial ''hairy" projections (A). In (B) the slightly corrugated surface of the lesion results from parakeratosis. Ballooned keratinocytes are seen. (C) Keratinocytes of the superficial spinous layer display intranuclear inclusions (arrow) and (D) ground-glass nucleoplasm with a steel-grey hue after hematoxylin and eosin staining of tissue sections. Reprinted with permission from Fernandez et al. (1990) and through the courtesy of J. Fernandez, MD.

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three characteristic inclusions. The first is the typical eosinophilic Cowdry type A inclusion that is surrounded by a clear halo (Figure 9.14C). In the second form, the nucleus exhibits a ground-glass eosinophilic appearance with clumped chromation adjacent to the periphery of the nuclear membrane (Figure 9.14D). These authors also describe a third type having a steelgrey ground-glass nucleoplasm and a clumped, peripheral chromation. Ultrastructurally, the nuclear inclusions characteristically exhibit nonenveloped virions, as is typical with other herpesviruses (Fowler et ah, 1989). Indeed, the nuclear inclusions described above are morphologically similar to those seen in herpes simplex (herpesvirus type 1) infection. However, studies of numerous HCL lesions appear to have ruled out non-EBV herpesviruses. Immunohistochemistry and in situ hybridization studies suggest that EBV can only be found in the superficial epithelial layers of the HCL lesion, but by using PCR in situ hybridization Brandwein et al. (1996) demonstrated EBV DNA in basal and parabasal cells of the epithelium. The finding of the LMP-1 and EBNA-1 proteins in mucosal cells is consistent with a proliferative effect attributable to the oncogenic products of EBV. It also suggests that EBV may lie latent in the epithelium of the tongue and other epithelial cells of the aerodigestive tract to become activated when immune repression occurs. Recently, Palefsky and associates (1996) detected consistent compositional changes in the amino acid motif of LMP-1 in these infected cells. Since the same alterations are found in the EBV-infected cells of nasopharyngeal carcinoma, these

authors hypothesized that modifications in these proteins may have oncogenic potential. However, HCL lesions do not appear to predispose to the development of lingular cancer.

VIRUS-ASSOCIATED HEMOPHAGOCYTIC SYNDROME Two decades ago, Risdall et al. (1979) described 19 childhood and adolescent patients with fever and diverse constitutional symptoms whose bone marrow exhibited histiocytic hyperplasia and hematophagocytosis. Active infection with members of the herpesvirus family were demonstrated in 14 of the 19 patients. Although cytomegalovirus was the most common infection documented in this series, more recently, the syndrome commonly has been associated with EBV. Significant hematophagocytosis is now recognized to occur in three categories of patients. The first group reflects a rare familial predisposition, in which there is an overlap with the fatal infectious mononucleosis XLP syndrome. Hematophagocytosis almost universally is found at autopsy in patients with this condition (Mroczek et al, 1987; Christensson et al, 1987). T cell lymphomas characterize the second group. In these patients, nonmalignant histocytes engorge erythrocytes, but the malignant T cells prove to be latently infected with EBV in a ringed episomal form (Sullivan et al, 1985; Chen et al, 1991; Wong et al, 1996; Jandl,

FIGURE 9.15 (A) Lymph node with hematophagocytosis in a 39-year-old man. Note the depletion of lymphocytes and the expansion of the sinusoids. (B) Liver of a 65-year-old man with prominent hematophagocytosis by littoral cells. The only other morphological abnormality is mild steatosis of the liver parenchymal cells. Reprinted with permission from Gaffey et al. (1993) and through the courtesy of M. Gaffey MD.

Epstein-Barr Virus

1996). The third group of patients manifest acute viral infections (Wilson et ah, 1981; Look et al, 1981; McKenna et al, 1981; Reisman and Greco, 1984). Of these, EBV again proves to be the most common. In the childhood cases described by Gaffey et al. (1993), in situ hybridization showed the infected cells to be small lymphocytes, but an occasional immunoblast was also reactive. The cell types were not further established. In these cases, hematophagocytosis was consistently found in the bone marrow, lymph nodes, liver, and spleen where infected lymphocytes were also located (Figure 9.15A,B). Ross et al. (1991) reported the clinical and pathological features of a severe febrile systemic illness in which a chronic EBV infection was accompanied by hematophagocytosis demonstrable in the spleen. Recently, Su and colleagues (1994) described a fulminating hematophagocytosis syndrome in 15 previously healthy pediatric-age group patients. Of great interest was the consistent demonstration of EBV RNA in T cells as established by immunolabeling. Because of its sporadic occurrence and the difficulty in recognizing hemophagocytosis pathologically, little fundamental mechanistic information has accumulated to explain the phenomenon. One must conclude that it is the consequence of activation of macrophages by the infection, but the possible role of autoantibodies to erythrocytes elaborated in response to infection remains a pathogenic consideration requiring exploration.

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Varicella-Zoster Virus (VZV) years of general practice. This work represents the only published systematic description of the epidemiology and natural history of the condition as it occurs outside the hospital setting. The clinical observations of von Bokay (1909) first raised the possibility of a relationship of herpes zoster in the adult to systemic varicella in childhood. Physicians subsequently noted the occurrence of chickenpox in children exposed to zoster patients, an observation substantiated by experimental induction of the disease in children inoculated with fluid from zoster vesicles. The association was further substantiated by the histological studies of Lipschiitz (1921), who documented the morphological similarity of the lesions in the two conditions. Definitive virological, molecular, and immunological proof of this association ultimately followed isolation of VZV in cultured cells by Weller et al (1958).

INTRODUCTION AND HISTORICAL OVERVIEW 147 DISSEMINATED CHILDHOOD VZV INFECTION OF THE SKIN: CHICKENPOX 147 HEMORRHAGIC VZV INFECTIONS OF THE SKIN 149 CHRONIC VZV INFECTIONS OF THE SKIN 150 NERVOUS SYSTEM DISEASE 151

Herpes Zoster 151 Encephalopathies 154 EYE DISEASE 156 EAR DISEASE 158 PULMONARY DISEASE 158 DIGESTIVE TRACT DISEASE 159 LIVER DISEASE 159 RENAL DISEASE 160 TESTICULAR DISEASE 162 HEART DISEASE 162 JOINT, SYNOVIAL, AND MUSCLE DISEASES CONGENITAL VZV INFECTION 162 REFERENCES 163

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DISSEMINATED C H I L D H O O D VZV INFECTION OF THE SKIN: CHICKENPOX

Few of us recall our initial encounter vvrith varicellavirus (VZV) in the form of chickenpox, but those of US who have experienced herpes zoster will not forget it. Long considered a benign right-of-passage for the youngster, varicella sporadically is responsible for significant disease if the infection occurs in utero or is acquired perinatally, during adulthood, or under circumstances of either immunosuppression or waning immunity. These latter conditions are of increasing importance during the current era of aggressive cancer therapy, organ transplantation, and AIDS. My initial serious thinking about varicella and herpes zoster related to a fortuitous visit with the late R. Edgar Hope-Simpson in Cirencester, England. He was clearly one of medicine's most accomplished family practitioners. He has effectively devoted his intellectual life to the epidemiology of common diseases in the semirural community of the British Isles that he serves. In an extraordinarily insightful paper, Hope-Simpson (1965) summarizes his observations during sixteen PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

The clinical features of chickenpox are known to most parents and are well described in countless publications. The pathological features of the lesions are less appreciated by pathologists, whose opportunities for examining the skin lesions are limited. In the typical case, the vesicles customarily develop over roughly a 4-day period in multiple waves over the thorax and abdomen some 9 to 21 days after exposure. To a more limited extent, they spread with the passage of time to involve the extremities and head. This central predominance of the skin changes may account for the common later occurrence of herpes zoster lesions in the dermatomes innervated by the thoracic and lumbar sensory neurons. Interestingly enough, sunburned and irritated skin tend to develop particularly prominent crops of vesicular lesions (Muckle, 1978). The characteristic distribution of 147

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Pathology and Pathogenesis of Human Viral D i s e a s e

lesions and their cyclic sequence contrast with the eruption of smallpox, which appears as a uniform vesicular rash with a prominent distribution on the extremities, including the palms of the hands and soles of the feet (Chapter 26). The mechanism of acquisition and dissemination of VZV is not completely understood. Presumably, the infection is contracted by the respiratory route, followed by spread of the virus to systemic sites by means of the blood during the clinical latency period. Viremia has been variously documented 1 to 11 days before the appearance of the skin rash, and a virus-specific deoxythymidine kinase is found in the blood 3 to 5 days before the rash appears. Recurrent episodes of viremia presumably seed the endothelial cells of the skin capillaries where viral replication next occurs. Virus has been detected in the blood serum as well as in a variety of circulating cells (i.e., macrophages, B and T cells), but the biologically important mechanism for dissemination of the virus from one site to another is not known. The critical role of the endothelial cell in initiating subsequent viral involvement of the malpighian stratum (but not the stratum basalis) of the epidermis is becoming increasingly appreciated. Although the mechanisms are obscure, the endothelial lesions of the skin capillaries most probably generate vasoactive cytochemicals, locally resulting in the vascular flush that surrounds the developing lesion (the so-called dew drop on a rose petal). Infection of the individual epithelial cells of the skin results in cytoplasm "ballooning'' and the appearance of the intranuclear eosinophilic inclusions that characterize infected cells. The coalescence of adjacent infected "balloon" cells yields multinucleate cells (having, at times, as many as 30 nuclei) and acantholysis in the mal-

phagian stratum that leads to formation of the uniloculate serous fluid-filled vesicles that characterize the early lesion (Figure 10.1 A). The actual mechanistic basis for cell "ballooning" and cell fusion to form giant cells in varicella is unknown. Within days, polymorphonuclear leukocytes accumulate in the vesicle fluid. The superficial granular and keratinizing layers of the epidermis collapse, and the vesicle leaks and then becomes crusted (Figure lO.lB). Because the stratum basalis remains relatively intact and the dermal connective tissue is uninvolved, resolution of the vesicle evolves by epithelial regeneration without scarring (McCormick et al, 1969; McSorley et al, 1974). The immunologically normal child develops detectable humoral and cellular immune responses during the acute stages of the infection. Lymphocyte-mediated immune mechanisms appear to play a key role in resolution of the disease process, but there is no evidence of this response in the form of a mononuclear cell infiltrate in the resolving cutaneous lesions. However, in a wide variety of heritable and acquired cellular immune deficiency conditions, life-threatening systemic disease develops during the initial encounter with the virus, as will be discussed in more detail in what follows (Bullowa and Wishik, 1935; Hook et al, 1968). The importance and role of the humoral antibody in the resolution of skin lesions are uncertain, but antibody is believed to be the critical factor in protective immunity against reinfection. Among enrollees in an HMO in the northeastern United States, the rate of hospitalization proved to be 0.4% (Choo et al, 1995). In one recent study, one of every 200 children under 13 years of age required hospitalization (Yawn and Lydick, 1997). The overall

FIGURE 10.1 (A) Mature skin vesicle of chickenpox. The basal cell and the cornified layers are preserved. Infected cells in the stratum malphagi enlarge by forming cytoplasmic vacuoles — so-called "balloon cells/' The cells subsequently coalesce through the process of acantholysis to form the vesicle. (B) Inflammatory cells, debris, and serum fill the cavity of the vesicle. Regeneration of the basal epithelium occurs during convalescence without scarring.

Varicella-Zoster Virus

death-to-case ratio due to chickenpox is approximately 6.7 per 10,000 cases, but those over the age of 20 years and under 5 are at greatest risk. Thirty-five percent of children with Hodgkin's disease develop VZV infections, and almost a quarter of these manifest disseminated disease (Reboul et ah, 1978). Feldman et al. (1975) reported on the outcome of VZV infections in 60 children receiving cancer chemotherapy. A third of these patients developed visceral disease and 7% died. Although VZV infection clearly is potentially devastating for patients who are chronically ill or immunosuppressed, the majority of the children hospitalized with the complications of chickenpox are believed to be immunologically normal. In one study, 38% of hospitalized children had evidence of viral dissemination with encephalitis, pneumonitis, bacterial super-infection, and Reye's syndrome (Fleisher et al, 1981). Cheatham et al (1956) published detailed autopsy descriptions of the pathological findings in two young children dying with visceral VZV infection. The disease was disseminated and involved all major organs including the nervous system.

HEMORRHAGIC VZV INFECTIONS OF THE SKIN On rare occasions, the vesicular lesions of chickenpox become hemorrhagic during the acute stages of the

149

illness or later during the crusting stage (McKay and Margaretten, 1967). In these cases, thrombocytopenia and capillary fragility occur. The morbidity is low, and fatal complications are exceedingly rare. Purpura fulminans develops on occasion as a complication of VZV infection (Becker and Buckley, 1966). In some of these cases, evidence of intravascular coagulation is observed at autopsy, and hemorrhages are widespread in internal organs. In one series of eight children with purpura fulminans, three died shortly after onset of bleeding, and two additional patients required amputation of an extremity due to hemorrhagic gangrene (Stoesser and Lockwood, 1938). In a comprehensive frequently cited review, Charkes (1961) describes varicella-associated skin conditions that seem to be complications of superimposed bacterial infections (Gonzalez-Ruiz et al, 1995). Leukocytoclastic vasculitis is believed to account for hemorrhagic skin lesions in some patients (Cohen and Trapuckd, 1984; Singh and Deng, 1998) (Figure 10.2). The pathogenesis of hemorrhage in VZV skin infections is unclear, for many cases have not been critically evaluated by biopsy and other modern laboratory approaches. As noted above, thrombocytopenia is common in these patients. Using electron microscopy, Espinoza and Kuhn (1974) documented infection of megakaryocytes in the bone marrow of a fatal case, but additional studies of this phenomenon have not been reported. Intravascular coagulation undoubtedly is a

FIGURE 10.2 Hemorrhagic papulovesicular eruption on the leg of a liver allotransplant recipient. Examination of the dermis showed leukocytoclastic vasculitis, a lesion characterized by fibrinoid degenerative changes of the small blood vessels and infiltrates of leukocytes accompanied by accumulations of nuclear debris. Reprinted with permission from Singh and Deng (1998) through the courtesy of N. Singh, MD.

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Pathology and Pathogenesis of Human Viral Disease

contributing factor in some cases. VZV infects endothelial cells of small blood vessels, and one can readily envision this to be a trigger for thrombosis and the consumption of coagulation products. Acquired protein C deficiency is a complication of the vasculopathy of VZV infections and appears to be an explanation for hemorrhage in some cases. Protein C is a vitamin K-dependent serine protease with antithrombotic properties that is activated by thrombomodulin/thrombin complexes on endothelial cells (Gerson et ah, 1993).

CHRONIC VZV INFECTIONS OF THE SKIN Chronic papillated and verrucous lesions develop in AIDS patients with herpes zoster (HZ). Histologically, there is a marked hyperkerative epidermal hyperplasia with prominent parakeratosis and variable degrees of

acanthoses (Figure 10.3). The cytolysis that contributes to vesicle formation often does not occur, although some lesions are focally necrotic. Keratinocytes with typical intranuclear inclusions and multinucleate cells are consistently found. These lesions are clinically and pathologically reminiscent of the verrucous hairy leukoplakia of the tongue due to EBV that occurs in patients with AIDS. On rare occasions, herpes simplex can produce similar skin changes. PCR analysis of exfoliated or biopsy tissue proves effective in establishing a specific etiological diagnosis (LeBoit et al, 1992). The pathogenesis of these VZV chronic skin lesions is obscure, but it is likely that attenuated cellular immunity plays an important role. Similar skin changes have been described in organ transplant recipients who are receiving immunosuppressive therapy. It has been suggested that the lesions are due to an altered pattern of viral gene expression. Nikkels and colleagues (1997) have hypothesized that

FIGURE 10.3 Verrucous epidermal hyperplasia characterized by marked hyperkeratosis, and pseudocarcinomatous hyperplasia of adnexal epithelium in this lesion caused by VZV. Note columns of the so-called molluscum bodies (A). Numerous multinucleated keratinocytes with the typical HZV nuclear changes are present within the hyperplastic epithelial nests at the base of the lesion (B). Keratinocytes at the sides of the papillations have coarse keratinohyaline granules similar to those seen in verruca vulgaris. An area at the base of the papulation shows laminar keratinization (C). Molluscum bodies form vertical columns in the cornified layer (D). This lesion should not be confused with molluscum contagiosum due to a poxvirus (see Chapter 26).

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Varicella-Zoster Virus

the lesions develop in patients infected with acyclovirresistant VZV strains (Hoppenjans et al, 1990).

NERVOUS SYSTEM DISEASE A diversity of lesions develop in the central nervous system as a consequence of VZV infection. Our understanding of the pathogenesis of these conditions is far from complete, in part because serious nervous system disease develops infrequently and autopsy materials are rarely available for study. Thus, modern tools to establish infection in cells were not applied to this problem until quite recently, and systematic studies using in situ techniques for the identification of virusinfected cells and tissues are only now being reported. While typically VZV induces the so-called Cowdry type A intranuclear inclusions, traditional morphology proves to be an insensitive tool for assessing the localization and distribution of virus in tissue. These problems are confounded by our limited understanding of the virological factors that influence pathogenicity. Herpes Zoster (HZ) Herpes (derived from the same root as herpetology) refers to the serpentine creeping nature of the vesicular eruption. The Greek ''zoster" and "zone'' translate into the English girdle, indicating the distribution of lesions along dermatomes. Cingulus, a Latin word for girdle, is the presumptive derivation of the medical slang term "shingles."

0 1 2 3 4 5 6 7

Cervical

8||0

1 2 3 4 5 6 7 8 9

Dorsal

10111

In immunologically intact persons of all ages, herpes zoster is usually reflected as a unilateral dermatome-limited vesicular rash characteristically closely associated with the distribution of a sensory nerve (Figure 10.4). This relationship was initially suggested by Bright in 1831 and proven 30 years later by Von Barensprung, who first observed evidence of infection in sensory ganglia and nerves at autopsy of patients with HZ. It occurs in persons of all ages and at variable intervals of time after chickenpox. The predisposition of persons of advanced age to HZ (Figure 10.5) was clearly demonstrated by the epidemiological work of Hope-Simpson (1965). In his studies, the overall prevalence of HZ was 3.4 per 1000 in the general population, but 10 per 1000 in octogenarians who had no recognized predisposing medical conditions. Rogers and Tindall (1971) documented an absolute increase in the frequency of trigeminal nerve involvement in geriatric patients (in comparison to patients of all ages). The disease in these older folks was longer in duration and more severe than in younger patients. Its complications are believed to be more severe, and in the elderly the incidence of postherpetic neuralgia increases with age (Kost and Straus, 1996) (Figure 10.6). HZ frequently develops in persons with leukemia, lymphoma (particularly Hodgkin's disease), and a variety of conditions and therapies that result in altered cell-mediated imn\une responsivity. For example, the studies of Atkinson et al. (1980) showed that nearly half of all bone marrow allograft recipients who survive for 6 months or longer develop HZ. Similarly Reboul et al (1978) noted the occurrence of HZ in 20% of children with Hodgkin's

12 3 4 5 0 12 3 4 5

Lumbar Sacral

40

50

60

Age Groups

Reprinted with permission from LeBoit et al. (1992). FIGURE 10.4 Dermatome involvement by HZ among patients of all ages included in the population-based study of Hope-Simpson (1965). Reprinted with permission.

FIGURE 10.5 Age-specific incidence of HZ in the populationbased study of Hope-Simpson (1965). Reprinted with permission.

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Pathology and Pathogenesis of Human Viral Disease

> 12T ^. \

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FIGURE 10.6 Annual incidence of HZ and the proportion of cases with postherpetic neuralgia. Panel A shows the annual incidence of H Z / I x 10^ persons in a general medical practice. Panel B documents the percentage of patients with pain persisting after onset of the HZ-associated rash. Data are from the placebo group in one large double-blind treatment study. Panel C shows the proportion of patients with postherpetic neuralgia according to age. Reprinted with permission from Kost and Straus (1996).

disease. Fifty percent of patients who received chemotherapy and extensive field irradiation developed HZ. Herpetic lesions in these conditions frequently are not confined to a dermatome distribution and often persist for extended periods. Compelling epidemiological and biological evidence establishes HZ as the reflection of a latent varicella infection acquired earlier in life. Gilden et al. (1992) claim that 90% of adults with serological evidence of past infection are carriers of latent VZV genome in sensory ganglia of the trigeminal or thoracic nerves (or both). The biological nature of the latency is a subject of continued research, but evidence recently

A

Skin

Satellite Cell

summarized by Hay and Ruyechan (1994) indicates that VZV RNA and regulatory proteins are detectable by molecular means in cells of the ganglion in the absence of clinical evidence of disease. They hypothesize that viral replication is downregulated but not inactive during periods of latency. The limited published work suggests that the viral genome is located in satellite cells of the ganglion, but not in the sensory neuron per se during latency (Nagashima et al, 1975; Hay and Ruyechan, 1994) (Figure 10.7). Numerous studies have demonstrated viral particles ultrastructurally in infected ganglion cells, and the virus has been cultured from ganglia at autopsy during and after an attack of

B

Skin

Satellite Cell

FIGURE 10.7 A model for VZV latency based on studies of human ganglia and animal models. Following primary infection with VZV, latency is established in some, but not all, cells of the sensory ganglion (A). The primary site of latency appears to be the satellite cells that surround the neurons, although some neurons may also harbor the latent virus. Upon reactivation that leads to HZ, the virus spreads by means of a lytic infection within the ganglion and via axonal transport. It then infects the skin innervated by the neurons (B). Reprinted with permission from Hay and Ruyechan (1994).

Varicella-Zoster Virus

HZ (Esiri and Tomlinson, 1972; Ghatak and Zimmerman, 1973; Bastian et al, 1974). Unfortunately, there are no satisfactory animal models of HZ. The pathology of HZ has been the subject of numerous reports in the past. The classical description was published by Head and Campbell (1900). I quote liberally from their vivid description of the fundamental lesions of this disease. "Changes in the ganglion of the posterior root: If the patient has died with the eruptions still out upon his skin, the affected ganglion will be found to be in a condition of profound inflammation. The interstitial tissue will be crowded with small round cells.... These inflammatory cells may be closely massed into clumps, and such foci may be scattered around the periphery and in the central tissue of the ganglion. These foci of inflammatory cells ... will occasionally be found to be situated around extravasated blood.... In the center of these hemorrhagic foci, the ganglion cells are absolutely destroyed. But in the surrounding zone of small round cells, the remains of ganglion cells can be generally seen.... Over the portion of the ganglion which is inflamed, the sheath generally shows similar changes. The vessels are engorged and occasionally, blood may be extravasated. An arterial branch entering at the distal pole of the ganglion and proceeding towards the lesions differed from similar vessels in the normal part of the ganglion, in that, an abundance of extruded leukocytes occurred along its course.... Ultimately, the focus of inflammation became converted into fibrous tissue and the density and extent of this scar depends on the severity and extent of the original inflammation. Usually, the final result is a scar occurring from 1/6 to 1/2 of the ganglion.... Over the scar is the ganglionic tissue, the sheath is thickened and altered in appearance.... The sheaths may become three times the thickness of the normal, and present a curious nonlaminated appearance, in marked contrast to the normal wavy appearance in the remainder of the sheath.... Thus, in conclusion, the acute changes found in the ganglion in a case of Herpes Zoster consists of (1) extremely acute inflammation with the exudation of small, round deeply-stained cells; (2) extravasation of blood; (3) destruction of ganglion cells and fibers; (4) inflammation of the sheath of the ganglion. "Changes in the posterior nerve-roots: Thirteen days after the eruption first appeared, profound degeneration was found in the posterior roots, and it is probable that this degeneration begins to make its appearance 10 days after the appearance of eruption.... This acute change seems to have reached its height at about 15 days after the appearance of eruption.... If the destruction has been profound, fibrous tissue takes the place of the destroyed nerve fibers.... Fifty-seven days after

153

the eruption, the secondary sclerosis was already quite evident, but 153 days after the eruption, the last remains of the products of acute degeneration could be seen amongst the secondary fibrous tissue. After 272 days, all signs of acute degeneration had disappeared, but the sclerosis was well-marked, more than 1/2 the posterior root being replaced by fibrous tissue.... Thus, the changes we have found in the posterior root correspond to the results that might have been expected from the lesion in the ganglia. They consisted of acute degeneration followed by a greater or lesser amount of secondary sclerosis according to the severity of the acute destruction. "Changes in the peripheral nerves: In the mixed trunks of the peripheral nerves close to the ganglion, these acutely degenerated fibers stand out clearly ... against the normal fibers of the anterior root. The number of these degenerated fibers varies considerably with the severity of the ganglionic lesion.... Eight days after the eruption, no acute degeneration could be found in the peripheral nerves in connection with the affected ganglion, but 13 days after, degeneration was present to a marked degree.... Both posterior primary and anterior primary divisions are markedly affected, but the number of degenerative fibers in the posterior primary division always appears to be greater, probably owing to the almost entirely afferent nature of the branch in the dorsal region. This degeneration can be traced right back to the fine twigs which pass upward into the skin and supply the area over which the eruption is distributed. With time, the products of degeneration are removed from the peripheral nerve, and if the ganglion lesion is not severe, it may be impossible to be certain of any abnormalities in the peripheral nerves. On the other hand, if the lesion has been a severe one of the nerve fibers that have degenerated ... are replaced by fibrous tissue and whole bundles of the nerve may be sclerosed.... Thus, in the peripheral nerves, degeneration seems to appear, to disappear, and to be replaced by sclerotic changes at the same periods after the initial lesions in the ganglion" (also see MuUer and Winkelmann, 1969). "Degeneration in the spinal cord: It is not surprising that such lesions of the ganglion as we have described in the previous section should be attended by acute degeneration of the root-fibers in the posterior columns of the spinal cord. This degeneration probably appears about the 9th or 10th day after the eruption. When dealing with the similar degeneration and the fibers of the posterior roots, we drew attention to the rapidity with which the products of the acute degeneration were removed and replaced by secondary fibrous tissue. In the spinal cord, on the other hand, the products

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Pathology and Pathogenesis of Human Viral Disease

of acute degeneration are removed much more slowly. Thus, ^1 days after the eruption, the products of degeneration in the posterior root showed signs of absorption and these roots were already considerably sclerosed, but the degeneration in the spinal cord was extremely well marked.... One hundred and fifty-three days after the eruption, the acute degeneration in the spinal cord was still profoundly marked, but in 272 days, it had disappeared.... When the degeneration is cleared away from the spinal cord, it leaves no perceptible sclerosis behind it, probably owing to the relatively small number of fibers destroyed" (also see McCarthy and Amer, 1978). Encephalopathies According to Denny-Brown and his colleagues (Denny-Brown el al., 1944), "Four histologic events ... distinguish herpes zoster from other pathological processes." These are: (1) necrotizing ganglionitis; (2) "a poliomyelitis which closely resembles anterior poliomyelitis, but is readily distinguished by its unilaterality segmental localization, and greater involvement of the posterior horn, posterior root and dorsal spinal ganglion"; (3) a mild localized leptomeningitis, and (4) peripheral mononeuritis. Other authors have documented the development of a transverse myelitis in patients with HZ, but the number of cases are small and the incidence of this complication unclear (Hogan and Krigman, 1973; Devinsky ei al., 1991; Gomez-Tortosa et al., 1994; Meylan ei al., 1995; Lionet ei al, 1996; Thomas and Howard, 1972). VZV disease of the central nervous system almost invariably develops in a milieu of relative immunosuppression, yet little quantitative or qualitative information on the status of the immune system in patients with recognized disease has been reported. Thus, our understanding of the immunologic mechanisms influencing the growth and spread of VZV in brain tissues is primitive. Boughton ei al. (1966) documented a wide variety of nervous system disorders in the 2.6% of patients hospitalized with, or following, an episode of chickenpox. These conditions include aseptic meningitis, encephalitis, cerebellar ataxia, optic neuritis, Guillain-Barre syndrome, and Reye's syndrome. The relationship of infection to the neurological problem in these cases has largely been established on the basis of clinical and epidemiological observations and, to a more limited extent, viral serology. Among children with varicella, acute, but generally transient, ataxia attributed to cerebellar infection is the most common neurological problem occurring concomitantly with the rash (Boudewijn ei al., 1978). Almost invariably, this clinical condition is

of limited severity and leaves no residual neurologic abnormalities in its wake. Thus far, pathologic studies have not been reported. Meningoencephalitis of varying degrees of severity have also been described in otherwise healthy children with chickenpox, but often the encephalopathy is first recognized days to weeks after the onset of the rash (Norris ei al., 1970; Wees and Madhavan, 1980) (see Figure 10.15). On rare occasions, it occurs before the appearance of the skin lesions. Autopsy study of these cases has been uncommon (Takashima and Becker, 1979). Perivascular lymphocytic "cuffing" and focal demyelinization of the white matter is found in the brain, but evidence of infection in the form of inclusion-bearing cells is lacking (Appelbaum ei al., 1953). These observations raise for consideration the possibility that many reported cases of alleged varicella encephalitis, in fact, represent post-infectious encephalomyelopathy rather than a specific VZV related lesion. Serious life-threatening VZV encephalopathy sporadically occurs in children with malignant disease, particularly acute lymphogenous leukemia, and Hodgkin's disease, and in adult recipients of chemotherapy and those with AIDS. Commonly, adults who develop an encephalopathy also manifest lesions of herpes zoster. The zosteriform lesions in these cases often are either generalized or involve several dermatomes. Lymphadenopathy occasionally accompanies the rash (Patterson ei al., 1980). However, skin lesions are not invariably present in the so-called zoster sine herpetic syndrome (Lewis, 1958; Manian ei al., 1995). In the study of Jemsek ei al. (1983), 45% of patients with encephalopathy had disease believed to have emanated from cranial or cranial-cervical ganglions. Lesions in the higher levels of the neuraxis are often seen in AIDS. Three fundamental types of lesions have been reported to develop in adults in HZV encephalopathy: 1. Large vessel vasculopathy. The carotid arteries and their various branches into and over the cerebral cortex demonstrate diverse lesions ranging from a lymphocytic angiitis to a granulomatous angiitis. Affected vessels may exhibit intimal proliferation, fibrinoid necrosis, and thrombosis. In the affected vasculature, evidence of infection is found in endothelial cells in the form of intranuclear inclusions or viral particles, demonstrable by electron microscopy, or by in siiu localization of the VZV genome (Bourdette ei al, 1983; Blue and Rosenblum, 1983; Linnemann and Alvira, 1980; Kleinschmidt-DeMasters ei al, 1996; Amlie-Lefond ei al, 1995). Herpes zoster ophthalmicus is the most commonly associated viral lesion. Patients typically exhibit the classical features of a cerebral vascular accident. Because the lesions tend to develop in

Varicella-Zoster Virus

155

FIGURE 10.8 Small arterial branches from the circle of Willis showing the active inflammatory stages of VZV vasculitis (A), and the quiescent advanced stage with marked intimal proliferation (B). Reprinted with permission from Kleinschmidt-DeMasters et at. (1996) through the courtesy of B. Kleinschmidt-DeMasters, MD.

older persons, the classical stroke due to atherosclerotic vascular disease is a clinical diagnostic consideration. In varicella vasculopathy, angiograms reveal characteristic segmental irregularities in the appearance of the affected vessels. Although it almost always occurs in adults, two cases of varicella-associated vasculopathy with cerebral infarction were recently reported in boys aged 7 and 9 years (Shuper et ah, 1990; Tucciarone et al, 1992). 2. Encephalitis. Often, the small blood vessels in affected areas show a lymphocytic vasculitis, but they manifest no evidence of endothelial cell infection (Figure 10.8). Scattered, localized, but well-delineated areas of demyelinization and necrosis, often in a perivascular location, characterize this second type of lesion (Figure 10.9). At the periphery of the areas of damage, oligodendroglia exhibit typical intranuclear inclusions, clearly indicating a direct association of the lesion with an active viral infection (McCormick et al, 1969). Astrocytosis may be present depending upon the age of the lesion. Encephalitis of this type results from hematogenous spread of the virus into distal branches of the vasculature. Typically, lesions are located at the junction of grey and white matter. Clinically, this form of VZV encephalopathy results in nonspecific neurological manifestations such as obtundation and problems of mentation (Kleinschmidt-DeMasters et ah, 1996; Jemsek et ah, 1983).

3. Ventriculitis and periventriculitis. Ependymal cells lining the major ventricular systems of the brain commonly show morphological evidence of infection. With the passage of time, the deeper periventricular tissue is affected. Microscopical examination of the lesions reveals nodular astrocytic proliferation on the ependymal surfaces (Gray et ah, 1994). This change can

FIGURE 10.9 A hemorrhagic infarct secondary to a vasculopathy in the occipital lobe of the brain. Several small ovoid ischemic/demyelinating lesions are depicted by arrowheads. Reprinted with permission from Kleinschmidt-DeMasters et al. (1996) through the courtesy of B. Kleinschmidt-DeMasters, MD.

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Pathology and Pathogenesis of Human Viral Disease

FIGURE 10.10 Periventricular ischemic/demyelinating lesion (arrowhead) adjacent to the lateral ventricle at the level of the amygdala. Reprinted with permission from Kleinschmidt-DeMasters et al. (1996) through the courtesy of B. Kleinschmidt-DeMasters, MD.

be detected by radiological imaging techniques and changes in the cerebral spinal fluid that may be diagnostic. The pathological picture is similar to the ventriculitis that occurs with cytomegalovirus infections in neonates (Figure 10.10).

EYE DISEASE Involvement of the external eye by VZV occasionally occurs in uncomplicated chickenpox. In these cases, the lids and conjunctivae exhibit inflamma-

tion and vesicular lesions. Uncommonly, the cornea is involved, resulting in a superficial or disciform interstitial keratitis (Pavan-Langston and McCuUey, 1973; Karlin, 1993). The lesions are consequent to vesicle formation in the epithelium followed by rupture, leaving shallow ulcerations in the cornea. Direct viral invasion of the internal eye is rare, but it may be a consequence of either hematogenous spread of the virus or transmission along nerves (Cheatham et al, 1956). Under these circumstances, uveitis, cataracts, oculomotor paralysis, and optic neuritis occur (Appel et al, 1988; Robb, 1972; Osfler and Thygeson, 1976; and Lee et al, 1997).

FIGURE 10.11 Residual scarring of the cornea secondary to VZV keratitis involving the nasal branches of the trigeminal nerve. Reprinted with permission from Pavan-Langston and McCulley (1973) through the courtesy of D. Pavan-Langston, MD.

Varicella-Zoster Virus

157

OPTHALMIC BRANCH OF THE TRIGEMINAL NERVE Nasociliary Infratrochlear Division | Nasal Division

Lacrimal • Frontal I

FIGURE 10.12 Anatomical distribution of the branches of the sensory nerve of the trigeminal cranial nerve. Some individual variability in distribution can be expected. HZ of this cranial nerve usually involves only a single branch of the nerve, as illustrated in Figure 10.13.

VZV involvement of the eye is more common in herpes zoster when the ophthalmic branch of the trigeminal nerve is involved as a consequence of a latent infection in its gasserian ganglion (Ostler and Thygeson, 1976; Karlin, 1993) (Figure 10.11). The postganglionic nerve further divides into four branches that individually supply specific anatomic components of the internal and external eye (Figure 10.12) (Edgerton, 1945; Duke-Elder, 1965). The distribution of the virus into these various nerve branches ultimately determines the location of the lesion that develops and accounts for the enormous variability in clinical expression of the infection one patient to another (Figure 10.13). Interestingly enough, herpes simplex virus commonly involves the mandibular branch of the fifth cervical nerve, accounting for lesions on the lips. Few pathological studies of the eye involved by VZV are reported since enucleation rarely is required in nonimmunosuppressed adults. Naumann et al. (1968) recorded detailed observations on enucleated orbits with disease developing as a consequence of VZV ophthalmicus. While the details are beyond the scope of this discussion, inflammatory lesions of the nerves and intraorbital vasculitis were prominent features in many cases. Various anatomic structures had undergone necrosis; retinal destruction with detachment and lens opacification were common. Necrotizing lesions of the retina due to various genera of the herpesviruses are occurring with increasing frequency in patients with AIDS and among recipients of chemotherapy for cancer and organ transplantation

(Porter ei al, 1972; Egbert et al, 1980; Friedman et al, 1993; Wunderli et al, 1996; Galindez et al, 1996; Batisse et al, 1996; Garweg and Bohnke, 1997). Of the responsible agents, cytomegalovirus is clearly the most common. Ocular infections with this virus occur in 20 to 25% of patients with AIDS, and in roughly 2% of organ transplant recipients (see Chapters 8 and 16). The syndrome of acute necrotizing retinitis in the literature is usually consequent to VZV (this is a condition variously termed "acute retinal necrosis syndrome,'' "rap-

FIGURE 10.13 Hemorrhagic HZ eruption in the distribution of the frontal branch of the trigeminal nerve. Reprinted wi\h permission from Pavan-Langston and McCuUey (1973) through the courtesy of D. Pavan-Langston, MD.

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Pathology and Pathogenesis of Human Viral Disease

idly progressive outer retinal necrosis/' and "atypical necrotizing retinopathy"). On rare occasions, herpes simplex and herpes simiae are responsible for similar lesions. In immunosuppressed patients, VZV acute necrotizing retinitis develops as a complication of zoster ophthalmicus, but can occur when a distant dermatome is involved, or in the absence of herpetic lesions of the skin. It is rapidly progressive and usually results in destruction and detachment of the retina with blindness. While initially unilateral, it becomes bilateral with the passage of time in Vs to % of patients. Since the clinical manifestations of the various herpesvirus retinopathy are variable, molecular identification techniques have proven most useful in establishing an etiological diagnosis. While the acute necrotizing retinitis syndrome usually occurs in circumstances of immunosuppression associated with clinical herpes zoster, it has developed in healthy adults on very rare occasions after chickenpox (Barondes et al, 1992).

EAR DISEASE In the only report published to date, Bordley and Kapur (1972) described the inner ear lesions of fatal varicella. In three cases, the mucus membranes of the middle ear showed focal necrosis and lymphocytic infiltration. An exudate of polymorphonuclear cells was found in the lumen. Hunt (1907) described so-called Ramsay Hunt syndrome (or herpes zoster oticus) and noted chronic inflammation of the Vllth cranial nerve. As one recalls, this nerve is comprised of both sensory and motor roots. He suggested (but did not demonstrate) that the geniculate ganglion was similarly inflamed. Infection of this ganglion has been established by PCR (Furuta et al, 1992). Ramsay Hunt syndrome assumes several different clinical forms: (1) herpes zoster oticus without apparent nerve disease; (2) herpes zoster oticus with facial palsy; (3) the second pattern accompanied by auditory symptoms; and (4) the second pattern with "Meniere's complex." In temporal bone studies, facial perivascular, perineural, and intraneural lymphocytic infiltrates are found. Vesicular eruption of the conchal area of the external ear occurs in some cases (Harner et al, 1970). The perivascular lesions are consistent with vasculitis, a lesion seen in larger vessels in herpes zoster ophthalmicus syndrome, as discussed earlier. Inflammatory changes are customarily not observed in the cranial nerve and associated vessels in idiopathic Bell's palsy. Zajtchuk and her associates (Zajtchuk et al, 1972) described in detail the pathology of the inner ear in herpes oticus.

PULMONARY DISEASE Pneumonia is a complication of chickenpox in persons of all ages, but the majority of life-threatening cases occurs in healthy young adults who are experiencing their initial infection with VZV (Bagdade and Melmon, 1966; Rotter and Collins, 1961). In the United States, 25% of the deaths attributed to varicella each year are a result of chickenpox pneumonia (Figure 10.14A-D). However, pneumonia also develops in neonates as a reflection of a disseminated infection, and in children receiving chemotherapy. The overall prevalence in adults is difficult to estimate because of the marked predominance of cases among young males and in pregnant women (Weber and Pellecchia, 1965; Pickard, 1968; Harris and Rhoades, 1965; Fish, 1960; Castleman and Kibble, 1963). In military populations, where hospitalization rates are high, approximately 15% of young male soldiers with varicella exhibited clinical or radiological evidence of pneumonia (Triebwasser et al, 1967). The incidence is much higher in adults admitted to civilian hospitals with chickenpox, which no doubt represent either more severe cases of chickenpox or those with diseases predisposing to opportunistic infections (Fitz and Mieklejohn, 1956; Mermelstein and Freireich, 1961). The clinical illness in seemingly normal adults is variable in severity (Table 10.1). It usually develops shortly after the appearance of the rash (Appelbaum et al, 1953) (Figure 10.15). Mortality rates differ in various reported series. Untreated varicella pneumonia in the adult has a mortality of approximately 10%. Pregnant females represent a unique population with a relatively high mortality (Enders, 1984; Qureshi and Jacques, 1996). In one study, 17% of women with clinical varicella developed pneumonia, and 45% of those in the second or third trimester succumbed (Fish, 1960). Pathologically, the lungs exhibit a diffuse interstitial and intra-airspace noncellular exudative process that tends to accumulate in nodular and occasionally hemorrhagic foci. Necrosis of parenchyma is often observed at these sites (Claudy, 1947). This most probably accounts for the pattern of diffuse nodular densities in the X-ray picture during acute illness. It is occasionally represented by calcified nodularity in roentgenograms of the chest after recovery (Knyvett, 1965; Meyer et al, 1986). Small subtle intranuclear inclusions are observed in a variable number of pulmonary epithelial cells and in the endothelium of scattered pulmonary vessels (Figure 10.16A-C). Vascular necrosis and thrombosis appear occasionally in the lung parenchyma, accounting for the sporadic occurrence of lung infarcts (Click et al, 1972). Viral inclusions also are found in the mesothelial cells of the pleura accompa-

159

Varicella-Zoster Virus

B

^im^

JH

FIGURE 10.14 Disseminated hemorrhagic VZV infection in a young pregnant school teacher (A). At autopsy, the lungs were deeply congested, heavy (B,C), and consolidated with focal areas of hemorrhagic necrosis. (D) Cross-section of lung after formalin fixation.

nied by pleuritis and effusions. The prevalence of pleural disease is uncertain since many pathology reports fail to mention the pleura. Charles and his associates (1986) established the diagnosis of pleural VZV in one of their cancer patients with a disseminated infection using exfoliative cytology. Multinucleate cells and individual cells with intranuclear inclusions were found (Figure 10.17).

viral infections of infants and children have been associated with this condition (see Chapter 2). Reye's syndrome has been described on a number of occasions in children with varicella (Szalay 1972; Eshchar et ah, 1973; Ey et ah, 1981; Fronstin, 1968; Jenkins et ah 1967;

DIGESTIVE TRACT DISEASE Involvement of the bowel with infarction necrosis is reported in corticosteroid-treated patients with disseminated varicella (Chang et al, 1978; Keene et ah, 1978). More frequently, pseudo-obstruction occurs concomitantly with dermatomal HZ (Tribble et ah, 1993). 8

LIVER DISEASE Reye (1963) described the syndrome of acute encephalopathy accompanied by fatty degeneration of the liver that now bears his name. A number of acute

10

12

14

16

18

Days after Onset of Rash FIGURE 10.15 Time of onset of VZV encephalitis after the appearance of the VZV skin eruption. The development of pneumonia follows a similar time course. Reprinted with permission from Appelbaum et al (1953).

160

Pathology and Pathogenesis of Human Viral Disease TABLE 10.1 Clinical Features of Varicella Pneumonia in 27 Seemingly Normal Adults (>20 years of age, 21 males and 6 females)

Cough Dyspnea Cyanosis Hemoptysis X-ray charges

Severe

Moderate

Mild

None

9 8 5 3 7

6 5 2 4 9

12 3 1 5 10

11 19 15 1

-

Adapted with permission from Tables 32.3 and 32.4 in Krugman et al. (1977).

Norman, 1968; Barr et al, 1968; Click et al, 1970). In some cases the patient had not consumed aspirin, the presumptive critical cofactor in causation of the disease (Click et al, 1970; Belay et al, 1999). Central nervous system signs and evidence of liver dysfunction occur within days after the onset of the rash. The pathology of the liver lesions associated with varicella has been described (Lichtenstein et al, 1983). Hepatitis characterized by mononuclear inflammatory and focal hepatic necrosis of the liver with intrahepatocyte nuclear inclusions is reported infrequently in patients infected with VZV (Figure 10.18) (Eshchar et al, 1973). To a large extent, these liver lesions have been found in immunocompromised patients with disseminated infections and in both children and adults with fatal varicella pneumonia. Ross et al (1980) reported

massive hepatic necrosis in a 64 year-old woman without recognized predisposing conditions and skin lesions. Hepatocytes exhibited intranuclear inclusions and serological studies documented HZV infection.

RENAL DISEASE Henoch et al (1884) described four children with clinical features of the nephrotic syndrome developing 3 to 11 days after the onset of chickenpox. Scattered reports since that time have documented a proliferative nephritis similar to poststreptococcal glomerulonephritis in children and adults with the varicella eruption (Denney and Baker, 1929; Krebs and Burvant,

FIGURE 10.16 HZV pneumonia. (A) Note the two nuclei with subtle intranuclear eosinophilic inclusion in cells of the alveolar inflammatory exudate. (B) Alveolar macrophage and a pneumocyte (arrowhead) with intranuclear inclusions. The pleural surface shows a fibrinous exudate. (C) Deeply congested lung with intra-airspace exudation of fluid. Note the alveolar macrophage with tiny intranuclear inclusion associated with prominent clearing of the surrounding nucleoplasm. In the author's experience, varicella inclusions in cells of the lung are subtler. Their location often demands painstaking microscopy.

Varicella-Zoster Virus

161

FIGURE 10.17 Cytological preparation of pleural fluid sediments showing multinucleate mesothelial cells (A) and cells with intranuclear inclusions (B, arrow). Reprinted with permission from Charles et al. (1986).

1972; Minkowitz et al, 1968; Yuceoglu et al, 1967). Viral nuclear inclusions were not observed in pathological material from these patients. Autopsy reports of fatal cases of varicella have not noted the presence of kidney lesions at autopsy. A survey of hospitalized children with varicella documented nephritis in 0.1%. On the other hand, a much higher prevalence of "nephritis with uremia"

was noted among fatal cases of varicella occurring in an outbreak in Cameroon (West Africa). The renal diseases in these alleged cases were not confirmed by pathological study. Recently, a young adult male developed chickenpox accompanied by nephritis 22 months after receiving a single dose of an investigational varicella vaccine (Pillai et al, 1993). The immunological response to the vaccine was apparently poor. This case

FIGURE ^ 10.18 Necrotizing lesion in the liver of an immunocompromised child with a disseminated HZV infection.

162

Pathology and Pathogenesis of Human Viral Disease

vividly poses questions regarding the possible role of viral-immune complexes in the genesis of the renal disease.

TESTICULAR DISEASE In a review of the literature, Liu et al. (1994) found six cases of acute orchitis occurring concomitantly with acute varicella. Both children and adults were affected. During convalescence, the testes were atrophic in four of the five patients evaluated.

HEART DISEASE Myocarditis in children with fatal varicella was first reported by Hackel (1953). Focal interstitial inflammation was present in seven cases, but specific involvement of muscle cells was not found. Sporadic reports since that time have documented myocarditis and arrhythmias occurring in children during and immediately after chickenpox and as isolated lesions most probably responsible for death (Tatter et ah, 1964; Moore et al, 1969; Lorber et al, 1988; Waagner and Murphy 1990). Noren et al (1982) found interstitial myocarditis histologically in two-thirds of fatal cases of varicella in a retrospective autopsy study. Myocarditis had not been suspected clinically in these cases. Only rarely are viral inclusions identified in myocardial cells. Morales et al (1971) described inflammatory disease restricted to the conduction system and a cardiac nerve ganglion in one case of sudden death. Fatal myocarditis also occurs during early pregnancy. In a recent case report, a 12-year-old child developed myocarditis that rapidly evolved into a dilated cardiomyopathy requiring cardiac transplantation (Tsintsof et al, 1993).

JOINT, SYNOVIAL, A N D MUSCLE DISEASE Involvement of the synovium and joints in children with otherwise uncomplicated chickenpox is rare. It customarily is monoarticular (DiLiberti et al, 1977; Smith and Sanford, 1967; Mulhern et al, 1971; Ward and Bishop, 1970; Brook, 1977), but cases of polyarthritis are described (Friedman and Naveh, 1971; Sekanina and Frana, 1973). Symptoms appear 1 to 7 days after development of the skin eruption (Priest et al, 1978),

but cases are described in which arthritis precedes the appearance of the rash (Fierman, 1990). Evaluation of joint aspirate usually reveals a prominent number of lymphocytes. However, in some cases, a marked neutrophil response has been seen, indicating a possible superimposed bacterial infection. Resolution of the acute inflammatory process occurs without complications. VZV can be recovered from joint fluid in about a third of cases. Arthritis is not described in many reported cases of systemic varicella, and pathological studies of joints at autopsy are not described. On rare occasions, myositis develops with varicella and herpes zoster. Pratt et al (1995) described rhabdomyolysis in an adolescent and a young adult with primary varicella infection. There were striking increases in blood concentrations of creatine kinase during the acute stage of the illness when myalgia was a prominent complaint. The biopsy from one patient showed individual muscle fiber degeneration and necrosis; PCR yielded viral genomic material. Norris et al (1969) described perivascular cuffing in a muscle biopsy from a patient with HZ and painful muscles.

CONGENITAL VZV INFECTION The incidence of varicella during pregnancy is estimated to range between 0.2 and 7 per 10,000 live births (Qureshi and Jacques, 1996). Two syndromes occur in infants born of women contracting varicella during pregnancy The first results from an infection during the initial 20 weeks of pregnancy In two recently reported studies, pregnancy loss was approximately 67.5% when infection of the mother occurred during the first 20 weeks of gestation (Enders et al, 1994; Balducci et al, 1992). Oyer and colleagues (1998) demonstrated nuclear inclusions in autolyzed embryonic tissue and confirmed the presence of VZV by immunohistochemistry and electron microscopy. Similar findings have been reported by others. An embryopathy is the outcome among those concepti that survive. Characteristically, the infection is manifest as skin, skeletal, and muscle abnormalities accompanied by ophthalmic lesions (cataracts, chorioretinitis, and microphthalmia) and developmental abnormalities of the brain, that is, microencephaly and hydroencephaly (Anglin, 1973; Cotlier, 1978; Laforet, 1947; McKendry and Bailey 1973; Paryani and Arvin, 1986; Srabstein et al, 1974; Frey et al, 1977; Rinvick, 1969; Magliocco et al, 1992; Charles et al, 1977; Savage et al, 1973). The second syndrome is seen in the offspring of women who contract varicella during the last three

Varicella-Zoster Virus

weeks of pregnancy. Under this circumstance, disseminated varicella with multiple organ involvement develops (Oppenheimer, 1944; Ehrlich et ah, 1958; Newman, 1965; Da Silva et ah, 1990; Purtilo et al, 1977). Mortality among newborns is roughly 30%, and at autopsy most major organ systems are involved. Until recently, the risk of congenital varicella was uncertain because most information was based on isolated case reports. Several studies of maternal infection during the first trimester have now been conducted. The most meaningful was an investigation controlled for spontaneously occurring nonvaricella-related congenital abnornialities. Two percent of live-born infants exhibited the features of the congenital varicella embryopathy. Similarly, the prevalence of disseminated infections acquired late in gestation from mothers with chickenpox was low (Pastuszak et ah, 1994). In a recent study of over 1300 pregnant women with varicella infections, 9 cases (0.7%) of the congenital varicella syndrome occurred. The highest risk was between the 13th and 20th weeks of pregnancy. The fetuses of women with herpes zoster were not at risk, an indication that immunity acquired by the mother earlier in life was protective (Enders et ah, 1994). A limited number of reports document the placental lesions in maternal VZV infection. Necrotizing and chronic inflammatory lesions of the placental villi characterize the pathological findings, but foci of necrosis and infarction are also described. Inclusion-bearing cells have not been found (Oyer et al, 1998; Garcia, 1963). Qureshi and Jacques (1996) recently summarized the diverse pathology literature on this subject.

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C H A P T E R

Herpesvirus Type 6 (HHV-6) n 1986, a previously unrecognized lymphotropic (gamma) herpesvirus was recovered from circulating peripheral blood lymphocytes of several patients with lymphoproliferative disorders and / AIDS (Salahuddin et ah, 1986). Since that time, considerable effort has focused on characterizing this new agent and determining its relevance as a cause of disease in humans. Although an impressive body of information has accumulated during the past ten years, the etiological role of HHV-6 in human disease is incompletely defined, and its pathogenic properties poorly understood. The difficulties in establishing a pathogenic role for HHV-6 are based upon the virus' almost universal presence as a latent infection in the tissues of older children and adults (Suga et al, 1995; Luppi et al, 1995; McCullers et al, 1995; Di Luca et al, 1995; Cuende et al, 1994; Caserta and Hall, 1993; Leach et al, 1994; Hall et al, 1994), and its reactivation often during infections with other herpesviruses, immunosuppression therapy, graft-vs.-host disease (Wilborn et al, 1994), and transplant rejection (Hoshino et al, 1995). Additional problems relate to the cumbersome techniques currently required to isolate the virus from human tissue as well as the probable insensitivity of the available immunohistochemical tests currently required for detecting serum antibody. PCR has been used to detect viral DNA in various tissues and body secretions, but this technique does not differentiate between latent and active infections. Thus, the contribution of PCR to our understanding of HHV-6 disease has thus far been limited. Two variants (A and B) of HHV-6 have been defined, but it is not known whether they differ biologically or in pathogenicity. HHV-6 shares DNA molecular homology with the organizational structure of cytomegalovirus, but nonetheless, it possesses unique DNA sequences and is antigenically distinct from other members of the herpesvirus family. It replicates in both B and T lymphocytes, although clinically it is most often demonstrated in the latter cells, particularly, but not exclusively, those of the

PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

CD4+ lineage. Maternal antibody appears to protect newborns until approximately 6 months of age, after which serologic evidence of infection gradually becomes apparent. Over 90% of children possess serum antibodies before 6 years of age. Thus, asymptomatic or unrecognized infections occur commonly among preschool children. The mode of transmission of the virus is unknown. HHV-6, the established etiological agent of exanthema subitum (syn. roseola infantum, sixth disease), exhibits unique clinical and epidemiological features that are consistent with our knowledge of the epidemiology of HHV-6. It rarely is seen before 6 months of age, and usually occurs before age 4 as a sporadic case or in outbreaks of limited size. Secondary cases in family members are rare, no doubt because of the almost universal presence of immunologically mediated resistance, indicating prior infection in most older persons. The exanthema is preceded by fever that subsides with the appearance of a macular or maculopapillary rash on the trunk and to a lesser extent on the face and extremities. Leukopenia and a relative lymphocytosis accompany the exanthema that rarely persists for longer than 24 hr. Many HHV-6 infections manifest only as fever without clinical evidence of a rash. Variant B viruses are more commonly recovered from patients with exanthema subitum. HHV-6 has also been etiologically associated with a non-EBV/non-CMV heterophil-negative mononucleosis syndrome in young adults (Pruksananonda et al, 1992; Akashi et al, 1993). Like other members of the herpesvirus group, latent HHV-6 appears to be activated in states of immunosuppression such as after organ transplantation and in those infected with HIV-1 (Fairfax et al, 1994). Diseases of the central nervous system (i.e., meningoencephalitis), lung, and liver (i.e., hepatitis) have been attributed to HHV-6 under such circumstances, but the evidence in many cases is inconclusive (Ishiguro et al, 1990; Asano et al, 1992; Suga et al, 1993; Achim et al, 1994;

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Carrigan and Knox, 1994; Caserta et ah, 1994; Drobyski et al, 1994; Wilborn et al, 1994; Knox and Carrigan, 1995; Moschettini et al, 1996). HHV-6 has also been demonstrated in pulmonary tissue of immunosuppressed patients with idiopathic interstitial pneumonia (Carrigan et al, 1991; Cone et al, 1993). A cause-and-effect relationship in these cases has not been established, and the sites of viral replication are unknown. Hepatitis and the hematophagocytic syndrome (see Chapter 9) also have been documented in patients with HHV-6 infections (Asano et al, 1990; Huang et al, 1990). Death of a 13-year-old presumptively immunocompetent girl with a rash and multisystem HHV-6 infection has been described (Prezioso et al, 1992). Autopsy revealed atypical lymphocytes with intranuclear inclusions of the herpesvirus type infiltrating multiple organs. Electron microscopy demonstrated herpes virions in the inclusions, and in situ hybridization established the presence of HHV-6. Of particular interest to diagnostic pathologists is the demonstration of HHV-6 by in situ hybridization in the prominent sinus histiocytes of lymph nodes in seven of nine patients with Rosai-Dorfman syndrome (syn. sinus lymphocytosis with massive lymphadenopathy) (Levine et al, 1992) and in histiocyte necrotizing lymphadenitis (syn. Kikuchi's disease). A number of other hematological syndromes of obscure etiology are currently being investigated in consideration of the possible role of HHV-6 in their causation. The near universal presence of genomic DNA in tissue makes this a difficult task. Immunohistochemistry using a monoclonal antibody is claimed to identify virus in tissue (Pitalia et al, 1993). Should such an approach prove sensitive and valid, pathological studies might yield insightful observations on the role of HHV-6 in disease. The virus or viral DNA can readily be detected in circulating mononuclear cells and in the saliva of patients with exanthema subitum. The salivary glands and possibly bronchial glands are suspected to be a common site of subsequent latent infection. Studies by Caserta and colleagues (1994) suggest that the central nervous system may serve as the locus for latent virus, but the evidence is only circumstantial.

References Achim, C , Wang, R., Miners, D., and Wiley, C. (1994). Brain viral burden in HIV infection. /. Neuropathol Exp. Neurol. 53, 284-294. Akashi, K., Eizuru, Y, Sumiyoshi, Y, Minematsu, T., Hara, S., Harada, M., Kikuchi, M., Niho, Y, and Minamishima, Y (1993). Brief report: Severe infectious mononucleosis-like syndrome and primary human herpesvirus 6 infection in an adult. New Engl. ]. Med. 329,168-175.

Asano, Y, Yoshikawa, T., Suga, S., Yazaki, T., Kondo, K., and Yamanishi, K. (1990). Fatal fulminant hepatitis in an infant with human herpesvirus-6 infection [letter]. Lancet 1, 862-863. Asano, Y, Yoshikawa, T., Kajita, Y, Ogura, R., Suga, S., Yazaki, T., Nakashima, T., Yamada, A., and Kurata, T. (1992). Fatal encephalitis/encephalopathy in primary human herpesvirus-6 infection. Arch. Dis. Child. 67,1484-1485. Carrigan, D., and Knox, K. (1994). Human herpesvirus 6 (HHV-6) isolation from bone marrow: HHV-6-associated bone marrow suppression in bone marrow transplant patients. Blood 84, 33073310. Carrigan, D., Drobyski, W., Russler, S., Tapper, M., Knox, K., and Ash, R. (1991). Interstitial pneumonitis associated with human herpesvirus-6 infection after marrow transplantation. Lancet 338, 147149. Caserta, M., and Hall, C. (1993). Human herpesvirus-6. Annu. Rev. Med. 44, 377-383. Caserta, M., Hall, C , Schnabel, K., Mclntyre, K., Long, C , Costanzo, M., Dewhurst, S., Insel, R., and Epstein, L. (1994). Neuroinvasion and persistence of human herpesvirus 6 in children. /. Infect. Dis. 170, 1586-1590. Cone, R., Hackman, R., Huang, M., Bowden, R., Meyers, J., Metcalf, M., Zeh, J., Ashley, R., and Corey, L. (1993). Human herpesvirus 6 in lung tissue from patients with pneumonitis after bone marrow transplantation. New Engl. J. Med. 329,156-161. Cuende, J., Ruiz, J., Civeira, M., and Prieto, J. (1994). High prevalence of HHV-6 DNA in peripheral blood mononuclear cells of healthy individuals detected by nested-PCR. /. Med. Virol. 43,115-118. Di Luca, D., Mirandola, P., Ravaioli, T., Dolcetti, R., Frigatti, A., Bovenzi, P., Sighinolfi, L., Monini, P., and Cassai, E. (1995). Human herpesvirus 6 and 7 in salivary glands and shedding in saliva of healthy and human immunodeficiency virus positive individuals. /. Med. Virol. 45, 462^68. Drobyski, W., Knox, K., Majewski, D., and Carrigan, D. (1994). Brief report: Fatal encephalitis due to variant B human herpesvirus-6 infection in a bone marrow-transplant recipient. New Engl. J. Med. 330, 1356-1360. Fairfax, M., Schacker, T., Cone, R., Collier, A., and Corey L. (1994). Human herpesvirus 6 DNA in blood cells of human immunodeficiency virus-infected men: Correlation of high levels with high CD4 cell counts. /. Infect. Dis. 169,1342-1345. Hall, C , Long, C , Schnabel, K., Caserta, M., Mclntyre, K., Costanzo, M., Knott, A., Dewhurst, S., Insel, R., and Epstein, L. (1994). Human herpesvirus-6 (HHV6) infection in children: Prospective evaluation for complications and reactivation. New Engl. ]. Med. 331, 432-438. Hoshino, K., Nishi, T., Adachi, H., Ito, H., Fukuda, Y, Dohi, K., and Kurata, T. (1995). Human herpesvirus-6 infection in renal allografts: Retrospective immunohistochemical study in Japanese recipients. Transpl. Int. 8, 169-173. Huang, L., Lee, C , Lin, K., Chuu, W., Lee, P., Chen, R., Chen, J., and Lin, D. (1990). Human herpesvirus-6 associated with fatal haemophagocytic syndrome [letter]. Lancet 336, 60-61. Ishiguro, N., Yamada, S., Takahashi, T., Takahashi, Y, Togashi, T., Okuno, T., and Yamanishi, K. (1990). Meningo-encephalitis associated with HHV-6 related exanthem subitum. Acta Paediatr Scand. 79, 987-989. Knox, K., and Carrigan, D. (1995). Active human herpesvirus (HHV6) infection of the central nervous system in patients with AIDS. /. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 9, 69-73. Leach, C , Newton, E., McParlin, S., and Jenson, H. (1994). Human herpesvirus 6 infection of the female genital tract. /. Infect. Dis. 169, 1281-1283.

Herpesvirus Type 6 Levine, P., Jahan, N., Murari, P., Manak, M., and Jaffe, E. (1992). Detection of human herpesvirus 6 in tissues involved by sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). J. Infect. Dis. 166, 291-295. Luppi, M., Barozzi, P., Maiorana, A., Marasca, R., Trovato, R., Fano, R., Ceccherini-Nelli, L., and Torelli, G. (1995). Human herpesvirus-6: A survey of presence and distribution of genomic sequences in normal brain and neuroglial tumors. /. Med. Virol. 47,105-111. McCuUers, J., Lakeman, R, and Whitley, R. (1995). Human herpesvirus 6 is associated with focal encephalitis. Clin. Infect. Dis. 21, 571-576. Moschettini, D., Balestri, P., Fois, A., and Valensin, P. (1996). Acute encephalitis due to human herpesvirus 6. Clin. Infect. Dis. 23, 397-398. Pitalia, A., Liu-Yin, J., Freemont, A., Morris, D., and Fitzmaurice, R. (1993). Immunohistochemical detection of human herpes virus 6 in formalin-fixed, paraffin-embedded lung tissues. /. Med. Virol. 41,103-107. Prezioso, P., Cangiarella, J., Lee, M., Nuovo, G., Borkowsky, W., Orlow, S., and Greco, M. (1992). Fatal disseminated infection with human herpesvirus-6. /. Pediatr 120, 921-923.

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Pruksananonda, R, Hall, C., Insel, R., Mclntyre, K., Pellett, P, Long, C., Schnabel, K., Pincus, P., Stamey, R, and Damgaugh, T. (1992). Primary human herpesvirus 6 infection in young children. New Engl. J. Med. 326,1445-1450. Salahuddin, S., Ablashi, D., Markham, P., Josephs, S., Sturzenegger, S., Kaplan, M., Halligan, G., Biberfeld, P, Wong-Staal, K, and Kramarsky, B. (1986). Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders. Science 234, 596-601. Suga, S., Yoshikawa, T., Asano, Y, Kozawa, T., Nakashima, T., Kobayashi, L, Yazaki, T., Yamamoto, H., Kajita, Y, and Ozaki, T. (1993). Clinical and virological analysis of 21 infants with exanthem subitum (roseola infantum) and central nervous system complications. Ann. Neurol. 33, 597-603. Suga, S., Yazaki, T., Kajita, Y, Ozaki, T., and Asano, Y (1995). Detection of human herpesvirus 6 DNAs in samples from several body sites of patients with exanthem subitum and their mothers by polymerase chain reaction assay. /. Med. Virol. 46, 52-55. Wilborn, R, Schmidt, C., Brinkmann, V., Jendroska, K., Oettle, H., and Siegert, W. (1994). A potential role for human herpesvirus type 6 in nervous system disease. /. Neuroimmunol. 49, 213-214.

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12 Kaposi Sarcoma-Associated Herpesvirus (KSHV, HHV-8) INTRODUCTION

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progressive neoplastic process of children and male adult residents of the highlands of equatorial subSaharan Africa (Lothe, 1963; Templeton, 1981; Hutt, 1984). More recently, it garnered attention when KS was found with increasing frequency among organ allotransplant recipients being administered immunosuppressive agents (Harwood et ah, 1979; Klepp et ah, 1978; Akhtar et ah, 1984; Vella et ah, 1997). As it turned out, most of these patients proved to be of Jewish origin. They experienced a progressive life-threatening disease that often became systemic and involved internal organs. As already noted, KS was dramatically reintroduced to the medical and general public when it appeared unexpectedly among male homosexuals with HIV-1 infections prior to development of fullblown AIDS (Friedman-Kien, 1981) (see Chapter 16). The infrequent occurrence of KS among aging males, particularly those of Jewish or Mediterranean heritage, provides little hint as to its causation, but over the past century etiologic speculation has been rampant. The common occurrence of the disease in the highlands of Central Africa suggested a likely role of either environmental influences or an infectious agent (or both) in its causation. The epidemiology of KS in male homosexuals conducting unprotected sexual acts with multiple partners of the same gender seemed to be most compatible with a transmissible venereal-acquired infection. But the infrequent occurrence of KS among hen\ophiliacs inadvertently infected with HIV1 by means of blood transfusions or blood concentrates strongly suggested that KS was not caused by HIV-1. The demonstration of a new gammaherpesvirus, now termed KSHV, or HHV-8, in the lesions (Chang et ah, 1994) and in mononuclear cells of the blood of patients with HIV-1 infections before the appearance of the disease (Whitby et ah, 1995) strongly suggests, but does not prove, an etiologic relationship between this "new" virus and the disease. As of yet, HHV-8 has not been shown to induce lesions similar to KS in experimental animals, and attempts to thwart the progression of KS

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LYMPHOMA (BCBL)

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INTRODUCTION As a pathologist, I first confronted Kaposi's sarcoma (KS) as a diagnostic problem in a mission hospital in the central highlands of northern Tanzania. To my inexperienced eye, it proved to be a challenge w^hen attempting to differentiate the atypical lesion from pyogenic granuloma, infected traumatized hemangiomas, and an assortment of other chronic conditions of the skin that commonly occur in the indigenous African population. The frequency of these diagnostic encounters peaked my interest and stimulated further exploration of the clinical problem. The issue proved particularly intriguing, since at the time KS was blossoming forth in the United States in a quite different form among young male homosexuals destined to die of AIDS (see Chapter 16). The outcome of the inquiries by my colleagues and I w^as a paper that addressed the subtle immunologic deficiencies of patients vv^ith endemic KS in Africa who had not experienced an HIV-1 infection (Craighead et ah, 1988). While the research answered a few questions, it engendered a deep curiosity focused on this enigmatic lesion. Many other medical scientists have been similarly intrigued. In 1872, the Hungarian dermatologist, Moritz Kaposi (Figure 12.1) described the unique disease of the skin that now bears his name. It was initially recognized as a rare affliction, primarily occurring in older men of Mediterranean and Jewish heritage (Ross et al., 1985). It was later found to be a relatively common

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Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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FIGURE 12.2 Typical skin lesion of KS of roughly 2 to 3 months duration on the arm of a 60-year-old truck driver with chemotherapy-treated chronic lymphocytic leukemia.

FIGURE 12.1 Moritz Kaposi, the Austrian dermatologist who described the unique lesion of the skin that now carries his name.

with specific antiviral agents directed against HHV-8 are only in their infancy. Although Kock's postulates have yet to be satisfied, the cumulative evidence supporting an etiologic role for HHV-8 in KS is compelling. KS exhibits a number of fascinating clinical and pathological features. With the possible exception of preadolescent children in Africa, it invariably affects males substantially more often than females, even when male homosexuals are excluded (Templeton, 1981). Its appearance seems to be triggered by a defect(s) in immunosurveillance, but HIV-1-infected patients do not typically exhibit the rampant opportunistic infections that characterize advanced AIDS. Hemangiomatous lesions on the trunk and head are common, and progression of the disease with involvement of the upper aerodigestive tract and internal organs is relatively rapid. It accompanies the premorbid deterioration of cellular immunity. To this extent, KS has been a causative or contributing factor in the death of many patients infected with HIV-1. In these cases, fatal involvement of the respiratory and digestive tract by the neoplasm is common (Gottlieb and Ackerman, 1982; Martin et al, 1993). In contrast, the disease is chronic in the sporadically occurring cases (that have no associa-

tion with HIV-1) in the Mediterranean Basin and subSaharan East Central Africa. Overt immunodeficiency is not evident in these patients (Matondo and Zumla, 1996). It initially appears as an indolent superficial violaceous lesion usually of the extremities (Figure 12.2) and evolves through a series of tumors (Figure 12.3), progressing centripetally in the distribution of the lymphatics to later involve the upper extremities, neck, and head (Reynolds et al, 1965; Lospalluti et al, 1995). Later, a "woody" edema of the feet and hands develops (Figure 12.4). Visceral organs are rarely affected until late in the disease (Lothe and Murray 1962; Reed et al, 1974; Port et al, 1982) (see Table 16.11). Chang and his colleagues (1994) reported the identification of a previously unrecognized herpesvirus in

FIGURE 12.3 Multiple confluent tumorous masses of KS on the dorsum of the hand and wrist of a young African man who was otherwise active and relatively healthy Note the edema of the digits. Reprinted with permission from Craighead et al. (1988).

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sified as a gammaherpesvirus and assigned the designation HHV-8 (Moore et al, 1996a,b). This classification now seems fully appropriate inasmuch as the virus can usually be demonstrated in the circulating CD19-positive B cells of patients with KS. More recently, electron microscopy has shown that the virion of HHV-8 is structurally similar to other herpesviruses. In addition, it has been claimed that HHV-8 can be grown in a continuous line of embryonal human kidney epithelial cells by cocultivating susceptible cells with those derived from KS lesions (Foreman et al, 1997). The validity of this finding has been questioned (Blauvelt et al, 1997a). At present, we know very little about KSHV/HHV-8 and the viral genes responsible for the distinctive character of the lesions for which it appears to be responsible. Information is only now accumulating in sufficient depth to permit some understanding of the means by which HHV-8 infection is contracted and disseminated.

EPIDEMIOLOGY

FIGURE 12.4 "Metastatic'' tumor nodules on the leg of a young African male with a KS lesion on the plantar surface of the foot.

homogenates of KS tumor tissue using a relatively new clinical isolation technique (representational difference analysis) and the polymerase chain reaction (PCR) to amplify a 233-bp herpesvirus-like sequence. This observation was confirmed and has been expanded upon by many others since that time (Moore and Chang, 1995; Chuck et al, 1996; Dictor et al, 1996). The viral gene segments originally identified in these experiments represented components coding for elements of the capsid and tegmentum proteins that share genetic markers with Herpesvirus simiae and, to a lesser extent, Epstein-Barr virus (EBV). On this basis, KSHV is clas-

Gaps of enormous proportion exist with regard to our understanding of the distribution of HHV-8 in members of the general population, both in developed countries and in regions of the Mediterranean Basin and Africa with a high prevalence of KS. Much of the currently available information is based on studies of small groups of incompletely characterized persons, using serological tests of unverifiable accuracy and molecular approaches predisposed to error. Thus, apparent conflicts in published data may reflect technical artefacts. Although it is currently impossible to develop firm conclusions, the evidence indicates that the prevalence of latent or active HHV-8 infections among members of the general population is low in the endemic areas of sub-Saharan Africa and the Mediterranean Basin, as well as in members of the United States and Europe populations (Marchioli et al, 1996; Lennette et al, 1996; Corbellino et al, 1996a; Blauvelt et al, 1997b; Cathomas et al, 1998). In various surveys, roughly 10 to 50% of homosexual males have been found to be infected when the peripheral blood mononuclear cells are analyzed (Lefrere et al, 1996; Moore et al, 1996a; De Milito et al, 1996). Almost all patients with AIDS who exhibit KS lesions carry the virus in peripheral blood mononuclear cells. Similarly, there is a strong association between HHV-8 with KS in cases occurring in the Mediterranean Basin and in subSaharan Africa (Dupin et al, 1995; Huang et al, 1995).

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The mode of transmission of HHV-8 among male homosexuals destined to develop KS is uncertain. Epidemiological analysis suggests the possibility of fecal-oral transmission, and studies by Thomas et al. (1996) indicate that HHV-8 is elaborated in the mucosa of the duodenum and small intestine. In this work, HHV-8 was detected in the digestive tract mucosa of 47% male homosexuals by means of endoscopic biopsy, but the identity of the actual cells supporting virus growth was not established. Oral secretions and tissues have proven positive in 75% of AIDS patients with KS (Koelle et al, 1997; Di Alberti et al, 1997), and semen, as well as both prostatic and testicular tissues, also yield evidence of infection in most, but not all, studies (Lin et al, 1995; Monini et al, 1996; Corbellino et al, 1996b; Gupta et al, 1996; Howard et al, 1997; Diamond et al, 1997). While body secretions may serve as a means for spreading the virus, it is not entirely clear whether the infection is intrinsic to epithelial or germinal cells of the reproductive organs, or reflects HHV-8 in resident and inflammatory mononuclear cells of blood origin. In a detailed study of semen using PCR in situ technology, Huang et al (1997) found evidence in both spermatozoa and mononuclear cells. Recently, viral genomic material was demonstrated in the epithelium of the prostate gland by in situ methodology (Staskus et al, 1997). Vertical transmission of HHV-8 from an HlV-l-infected mother with KS to her offspring is reported (McCarthy et al, 1996). On rare occasions, familial occurrence of KS has been documented (Greco et al, 1938; Zeligman, 1960; Finlay and Marks, 1979; Digiovanna and Safai, 1981), and cases of KS occurring among very young African children are recorded in the literature. This suggests, but certainly does not prove, vertical transmission (Lothe, 1963; Dutz and Stout, 1960).

PATHOGENESIS A N D PATHOLOGY The pathogenesis of the lesions of KS is an enigma that remains largely unexplained despite considerable research. Indeed, it is unclear whether the "tumor" represents a "true" neoplasm resulting from malignant cellular transformation or is a localized proliferation of vascular and stromal elements possibly resulting from the autocrine or paracrine influences of a panoply of growth factors elaborated in response to infection (Cornali et al, 1996; Ensoli et al, 1994; Koster et al, 1996; Nickoloff and Foreman, 1996; Nair et al, 1992; Nakamura et al, 1997; Boshoff et al, 1997). Impressive arguments support both possibilities. Evidence arguing for the concept of malignancy is the demonstration of monoclonality among the cells of the multiple "tu-

mors" in some but not all patients (Rabkin et al, 1997), and the seemingly unrestricted invasive and metastasizing growth characteristics of the disease in immunosuppressed patients. Contrariwise, our inability to transplant the "tumor" into animals and to grow "tumor cells" in culture in the absence of growth factors (Nakamura et al, 1988, 1997) is evidence supporting the view that the lesion may be a nonmalignant cellular proliferation responding to a stimulus yet to be defined. Whatever its nature, HHV-8 is intimately involved, as demonstrated by in situ localization of the virus in endothelial cells and the spindle cell elements of the lesions at progressive stages in the development of KS (Boshoff et al, 1995; Li et al, 1996; Dictor et al, 1996; Staskus et al, 1997; Cathomas et al, 1998). However, the finding of the virus in "tumor" cells must be interpreted with caution, for, as noted earlier, many of these patients have evidence of a systemic HHV-8 infection. This large and complex virus is currently being characterized; its pathogenic mechanisms remain to be elucidated (Ganem, 1996; Chang et al, 1996). Immunological influences clearly are reflected in the development and progression of the lesions of KS, but at what stage in the evolution of the infection do they act? Studies of KS in Africa (Craighead et al, 1988) and our understanding of transplantation immunosuppression (Klepp et al, 1978; Harwood et al, 1979; Hoshaw and Schwartz, 1980; Stahl et al, 1982; Rasmussen et al, 1982) and AIDS argue that cell-mediated mechanisms are involved. There would appear to be a delicate imbalance of the various T cell elements since reduction or elimination of immunosuppressive drugs in the transplant recipient (Vadhan-Raj et al, 1986; Santucci et al, 1988; Erer et al, 1997), or the treatment of AIDS often reverses the course of KS (Real and Krown, 1985; Soler et al, 1996). The histopathogenesis of KS is the subject of an abundant literature. The subject is particularly confusing since classification schemata based on epidemiological/clinical staging and microscopical morphology are often used interchangeably in the literature. Clinically, there are four general categories of disease: 1. Classical. The disease initially described by Kaposi. It occurs predominantly among elderly male Jews and men residing in restricted geographic regions of the Mediterranean Basin. This form is frequently manifest as indolent lesions of the extremities, and rarely is a cause of death, despite its chronicity. 2. Endemic. The disease occurs with a relatively high frequency in the equatorial highlands of East and

Kaposi Sarcoma-Associated Herpesvirus

Central Africa with a maleifemale ratio of roughly 15:1. This chronic disease form is often multifocal and evolves as tumorous nodules predominantly on the lower extremities. 3. Lymphadenopathic. A condition observed in African children of both sexes, and in an occasional patient with AIDS. In this fulminating form of KS, the prototypic lesions are found in isolated lymph nodes and the condition is manifest as generalized disease of the lymphoid system. 4. Invasive. Infiltrating tumor invades internal organs, particularly those of the respiratory and digestive tracts. This form is occasionally seen late in the course of African endemic KS and as a fulminating aggressive disease in patients with AIDS and recipients of immunosuppression. See Templeton (1981), Krigel et al (1983), and Lospalluti et al (1995). An understanding of the histogenesis of KS lesions is intrinsic to an appreciation of the variable pathologic features of the disease (see Figure 12.5). Of critical importance is the question of a cell of origin for the proliferating vascular structures that are an intrinsic hallmark of the lesion. Are they derived from blood vessels or lymphatics or primitive mesenchyma? While a restrictive approach to this question may be naive, the bulk of the histologic ultrastructural and immunochemical evidence indicates that the fundamental vascular structures reflect an aberrant series of interconnections between lymphatics and venules (Figure 12.6A,B). Simply, in the earliest lesions, there appear to be lymphatic-to-venous shunts with pooling of lymph and blood in the slit-like and sinusoidal structures of infinite complexity that are found in the lesions (Dictor, 1986; Leibowitz et al, 1980; Cossu et al, 1997). The immunohistochemistry strongly suggests that many of these abnormal vascular channels have a reaction pattern consistent with lymphatics (Beckstead et al, 1985; Jones et al, 1986). Additionally, the fine structural features of these lesions are consistent with this notion of histogenesis as shown by the elegant studies of McNutt et al (1983). As seen by these investigators, capillary dendritic pericytes are consistently lacking in the typical vascular structures of the lesion, and there is a discontinuity between the endothelial cells. In addition, the basal lamina of the vascular channels prove to be thin and fragmented. These features are consistent with an origin in lymphatic vessels (Dorfman, 1986) (Figure 12.6A~D). The origin of the sarcomatous elements of the evolving KS lesion is the second question, particularly when

175

the tumors become large and nodular and exhibit the features of a fibrosarcoma (Figure 12.6C,D). To date, the immunohistochemistry indicates that these proliferating "sarcomatoid" cellular elements are of endothelial origin, although the cells exhibit none of the fine structural features of endothelial cells (Schulze et al, 1987). Assuming the correctness of these conclusions, one could envision KS to be an evolutionary disease process in which the early lesions represent lymphatic proliferations that are potentially multifocal, with semiautonomous growth. Progression occurs when immunological controls are aborted. Where HHV-8 fits into this scenario is totally unclear. It may provide the stimulus for growth through generation of chemokines yet to be elucidated (Li et al, 1996; Boshoff et al, 1997). Typical skin lesions evolve clinically through stages nominally termed (1) patch, (2) plaque, and (3) nodular. In HIV-1-infected patients, the patch stage clinically presents as a small violaceous-pink irregular discoloration of the skin surface often seen initially on the forehead, conjunctiva, tip of the nose, and the glans penis. To me, this pattern suggests that the causative agent may be sensitive to internal body heat, such as is the case with Treponema pallidum. Histologically, these lesions are comprised of irregular loosely organized vascular slits lined by a thin attenuated endothelium. Usually, there is an associated mononuclear inflammatory infiltrate comprised of lymphocytes and plasma cells (Ackerman, 1979). In the plaque stage that follows, the vascular so-called "glomeruloids" become more prominent, with the vascular structures exhibiting a jagged configuration. Occasionally, vascular structures are dilated into sinusoids. A chronic inflammatory infiltrate is evident, and loosely organized spindle cells are interposed between bundles of collagen adjacent to the abnormal vascular structures. Congestion and extravasation of erythrocytes are often prominent in these lesions, and variable amounts of hemosiderin are seen. In the nodular stage that follows, inflammation is not a prominent feature, but the well-circumscribed nodules are comprised of mildly pleomorphic spindle cells and collagen fibers. Templeton (1981) describes a very aggressive form of the disease that evolves from the lesions of nodular KS on the extremities in African patients. I have seen similar cases in East Africa. These tumors transgress the deep fascia of the lower extremities and infiltrate subcutaneously. There is an associated "woody" consistency to the lower extremities, as described above. Sporadic reports from the United States in the preAIDS era document KS occurring in adults in a single, or in a cluster, of lymph nodes (Lee and Moore, 1965; Ramos et al, 1976; Berman et al, 1986; Dutz and Stout,

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Multiple Pathways Involving HHV-8, Integrins^ CD40 and Bcl-x Leading to Emei^ence and Survival of KS Tumor Cells HIV-1 Infected a T-cells

Macrophages | and Dermal Dendritic Cells

Oncostatin M Tat bFGF Scatter Factor Other Gytokines

Resting Endothelial Cells (Epithelioid)

Proliferation via Secretion of Mitogens and Cytokines Activated Endothelial Cells (Spindle Shaped)

Proliferating KS Tumor [Cells and Endothelial Cells 4Cell Survival 4 BCI-XL 4lniegrins ^ Apopiosis 4CD40 FIGURE 12.5 Multistep pathw^ay leading to formation of KS lesions highlighting roles for HHV-8 and other proteins that regulate apoptosis. In the upper portion of this diagram, involvement of HIV-1, various grov^th factors, and cytokines in the initiation and transdifferentiation-dependent phases are portrayed, leading to emergence of activated endothelial cells. The subsequent steps involving proliferation of KS tumor cells and neovascularization are suggested to involve active participation and infection by HHV-8 and overexpression of integrins, extracellular matrix, and various proteins that can prolong the longevity of KS tumor cells and endothelial cells such as BC1-XL and CD40. It is conceivable that certain viral gene products derived from the novel y-herpesvirus HHV-8 can directly influence the biology of the KS tumor cell and endothelial cell. HHV-8 is known to be present in circulating B lymphocytes, and in situ polymerase chain reaction has demonstrated viral transcripts w^ithin the tumor cells and endothelial cells. A vicious cycle can be envisioned in w^hich the activated KS tumor cells and endothelial cells produce cytokines capable of autocrine grov\^th stimulation and recruitment of additional tumor cells into the neoplastic lesion. The exact relationship betv^een participation of the immune system and the presence of HIV-1 and/or HHV-8 in formation of KS lesions is uncertain at this time. It is possible that KS lesions may not be truly sarcomatous lesions but rather represent an abortive immune response to a heterogenous group of virally infected mesenchymal cell types. Nonetheless, the angioproliferative lesions can produce deadly consequences secondary to extensive and uncontrollable hemorrhage. There is precedent for an infectious agent to produce a KS-mimetic phenotype knovv^n as bacillary angiomatosis, which is relatively easily treated by antibiotics. It may be feasible to treat KS lesions most effectively by targeting the infectious agent rather than using cytotoxic chemotherapeutic agents that may exacerbate the immunosuppression associated with HIV-1 infection. In any event, it should be clear that many new therapeutic opportunities exist based on progress made in clarifying etiological and pathophysiological issues related to KS. Reprinted with permission from Nickoloff and Foreman (1996).

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FIGURE 12.6 (A) Subepidermal plaque lesions of KS exhibiting the slit-like vascular structures and the interposed accumulations of mononuclear cells. The absence of erythrocytes in the spaces suggest interconnections with lymphatics. (B) Mixed subepithelial tumorous lesion of KS showing the superficial infiltrating fibroblastoid elements and the deeper sinusoids and accumulations of complex small vascular structures. (C) Sinusoids of a mixed lesion of KS with nodular fibroblastoid lesion at the base. (D) Sarcomatous lesion of KS in a nodular lesion similar to Figure 12.3. Note the prominent pleomorphism and the tumor giant cells. Reprinted with permission from Craighead et ah (1988).

I960; Ecklund and Valaitis, 1962). As noted above, childhood cases with generalized lymph node involvement are relatively common in Africa (O'Connell, 1977; Lothe, 1963). Templeton (1981) claims that these cases comiprise roughly 4% of the KS occurring in Uganda. In contrast to adults, this form of disease generally devel-

ops with equal frequency in boys and girls. In association with the HIV-1 epidemic, involvement of lymph nodes is occurring with increasing frequency among African children and adults (Marquart et ah, 1987), and in male homosexuals in North America and Europe (Finkbeiner et al, 1982).

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Bhana et at. (1970) described two morphologic forms of KS in lymph nodes, a finding confirmed by other authors (O'Connell, 1977). In the first, the node is extensively involved with the KS lesion, and the process seems to emanate from the medulla of the node. Multiple lymph nodes exhibit disease, and the condition tends to progress rapidly. In the second, only a portion of a node is involved, with the KS lesion usually being found in sinusoids and involving the capsule (Ramos et ah, 1976). This form seems to occur in the drainage pattern of cutaneous KS lesions and resembles a metastasis. However, cases are reported in which primary skin lesions are not present (Lee and Moore, 1965). In reviewing the histologic description in numerous early reports, I note the common observation of nodular lymphoid hyperplasia with plasma cell infiltrates and a small blood vessel proliferation developing in portions of the node not involved by KS. The picture strikingly resembles Castleman's disease. Recent authors have described the lesions of KS in nodes exhibiting classical features of Castleman's disease (Rywlin et ah, 1983; Chen, 1984) (see below).

A N G I O S A R C O M A S A N D OTHER VASCULAR LESIONS Malignant non-KS vascular sarcomas occur rarely (ca. 1% of all sarcomas in North Americans) and exhibit diverse morphological and clinical features. To a large extent, these lesions are idiopathic, although some are causatively related to chronic irradiation secondary to thorotrast administration and long-term lymphatic obstruction (i.e., postmastectomy). A relatively large number of angiosarcomas and related lesions have now been examined by PCR in an effort to detect a possible association with HHV-8 infection. The results, at present, are inconclusive and conflict from one report to another. McDonagh et al (1996) found evidence of HHV-8 infection in almost 30% of the angiosarcomas they studied, but only 1 of 20 hemangiomas proved positive. Hemangiopericytomas consistently proved negative. Only 1 of the 50 patients yielding these specimens was immunocompromised. On the other hand, an evaluation of 11 primary vascular tumors of body cavity origin (Lin et al, 1996), 15 capillary hemangiomas (Smoller et al, 1997), and 138 assorted vascular lesions (Jin, 1996) yielded no evidence of HHV-8 infection. Clearly, the possible role of HHV-8 in the pathogenesis of non-KS vascular lesions will require further study, work that no doubt will be published in future years.

BODY CAVITY-BASED NON-HODGKIN'S LYMPHOMA (BCBL) Lymphomatous effusions into the pleural and peritoneal cavities, in the absence of solid non-Hodgkin's lymphomatous masses, are known as BCBLs (Carbone et al, 1996; Hermine et al, 1996). BCBLs are usually comprised of large neoplastic cells with abundant cytoplasm and an irregular pleomorphic nucleus. Several nucleoli are present. The cell populations appear to be a mixture of anaplastic, multilobulated, and multinucleated large cells, many of which have immunoblastic features (Carbone et al, 1996; Hsi et al, 1998) (Figure 12.7). These cancers are rare and occur predominantly, but not exclusively, in males (Said et al, 1996). Patients occasionally develop KS before or after the appearance of the tumor, but die as a result of the complications of the lymphoma, usually less than 6 months after diagnosis. Thus, KS may not have had an opportunity to develop in many cases (Strauchen et al, 1996). BCBLs develop predominantly in patients with AIDS (DePond et al, 1997). EBV infection of the cells with expression of the oncogenic LMP-1 and EBNA-2 proteins is common in AIDS-associated cases, but evidence of EBV customarily is not found in the tumors of patients uninfected with HIV-1. In contrast to most AIDS-associated lymphomas that are of B cell lineage (see Chapter 7), most BCBLs have an indeterminant immunophenotype, although they exhibit a clonal rearrangement of the immunoglobulin genes (Karcher and Alkan, 1997). Individual cells of BCBLs prove to be latently infected with HHV-8 in the form of a circular episome of the viral DNA at a high copy number, but no information is available at present as to the clonality of the virus in the individual tumor cells of a particular patient. Cells of BCBLs from an EBV serologically negative patient with AIDS were recently reported to exhibit enveloped virus particles and in situ genomic signals consistent with HHV-8 (Hsi et al, 1998) (Figure 12.8). Treatment of cultured tumor cells with a chemical tumor promoter has been shown to induce the formation of morphologically identifiable viral particles within the cell, but not their release from the cell (Miller et al, 1997). Surveys using PCR have shown that benign lymphoid proliferations and solid lymphomas of a wide variety of morphological types in non-AIDS patients exhibit no evidence of HHV-8 infection (Pastore et al, 1995; Cesarman et al, 1996; Gaidano et al, 1996; Chadburn et al, 1997).

Kaposi Sarcoma-Associated Herpesvirus

FIGURE 12.7 Representative morphology of body cavity-based lymphoma from HIV-positive males with Kaposi's sarcoma. (A,B) Cytocentrifuge preparation of pleural fluid and histological preparation of pleural biopsy showing large neoplastic cells of relatively uniform size and shape with immunoblastic features (A, WrightGiemsa stain, original magnification x750; B, hematoxylin-eosin stain, original magnification x300). (C) Cytocentrifuge preparation of pleural fluid showing neoplastic immunoblasts with moderate nuclear pleomorphism, mitotic activity, and prominent cytoplasmic vacuoles (Wright-Giemsa stain, original magnification x750). (D) Cytocentrifuge preparation of pleural fluid showing immunoblastic features, marked variation in cell size, and abundant mitotic activity (Wright-Giemsa stain, original magnification x750). Reprinted with permission from Karcher and Alkan (1997) and through the courtesy of D. Karcher, MD, and S. Alkan, MD.

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FIGURE 12.8 A 38-year-old HIV-positive IV drug user with Kaposi's sarcoma. He presented with a pericardial effusion consequent to the neoplasm described here. He died shortly thereafter. (A) Cytologic smear of premortem pericardial fluid showing noncohesive lymphomatous cells with large pleomorphic nuclei and prominent nucleoli. (B) Papanicolaou stain. Electron micrograph of a pericardial lymphoma cell containing 100 nm of nuclear viral particles (arrows) consistent with the appearance of human herpesvirus-8. Inset: high magnification showing enveloped virus budding from nuclear membrane (arrow) with adjacent nucleocapsids also in the cytoplasm. (C) Section of heart that shows pericardial lymphomatous infiltrate that forms a tumor nodule. Inset: high magnification showing infiltration of myocardium. Hematoxylin and eosin (H&E). (D) Lymphomatous infiltrate in pericardium (H&E). (E) Section of heart with lymphomatous infiltrate. Inset: high magnification. Lymphoma cells express epithelial membrane antigen in a Golgi pattern. Immunohistochemical stain with anti-epithelial membrane antigen. (F) Interstitial pulmonary lymphomatous infiltrate (H&E). (G) Hepatic involvement by lymphoma. Note tumor formation at the right side of the panel (arrows) (H&E). Reprinted with permission from Hsi et ah (1998) and through the courtesy of B. Nickoloff, MD.

ANGIOFOLLICULAR LYMPH N O D E HYPERPLASIA (syn. AFLH, multicentric angiofoUicular lymph node hyperplasia, Castleman's disease) (Shahidi et ah, 1995)

In 1956, Castleman and his associates described a localized lymphadenopathy of the mediastinum in which lymph nodes histologically resembled the thy-

mus. However, the so-called Hassel's bodies proved to be vascularized lymphocyte-depleted germinal follicles exhibiting hyalinized collagen, located in a sea of concentrically layered lymphocytes. The condition proved to be benign, and the masses were effectively treated by surgical excision or radiation therapy. A second clinical variant of the disease was reported by Flendrig (1969) and Keller et al (1972). In this condi-

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tion, the patients were febrile and had both generalized lymphadenopathy and splenomegaly accompanied by anemia and hypergammaglobulinemia. Later reports by Gaba et al (1978) and Weisberger et al (1985) described similar cases having a high mortality and short survival period. The histology of the lymph nodes in these cases resembled the original lesions described by Castleman et al (1956), but exhibited sheets of plasma cells in addition to the uniquely vascularized hyaline structures replacing the lymphoid follicles (Figure 12.9). During the 1980s, additional cases of AFLH with generalized or multicentric lymphadenopathy and systemic symptoms were reported in male homosexuals with AIDS (Dickson et al, 1985; Oksenhendler et al, 1996). Many of these patients had KS. With the discovery of HHV-8, an association with AFLH with infection was established (Soulier et al 1995; Gessain et al, 1996;

Chadburn et al, 1997). Among patients with AIDS, the virus was invariably discovered by PCR in the diseased lymph nodes, whereas fewer than half of the nodes from HIV-1-negative cases with multicentric AFLH proved to be HHV-8 positive. Many of the later patients were men older than 50 years of age who had clinical evidence of immune dysregulation. Further studies documented the presence of HHV-8 genomic material in the circulating blood mononuclear cells of HIV-1-infected patients with AFLH (Grandadam et al, 1997). Although our understanding of this complex disorder is currently quite limited, one might hypothesize that AFLH represents a dysplastic lymphoproliferative disorder, the expression of which is modulated by immunological influences (Ohyashiki et al, 1994). The pathogenic role of HHV-8 in AFLH remains to be clarified, but in a recent report antiviral therapy was associated with clinical resolution of the lesions (Revuelta and Nord, 1998). As noted above, KS lesions have been found in lymph nodes exhibiting the classical features of AFLH (Rywlin et al, 1983; Chen, 1984; Tirelli et al, 1996). Currently, evidence to indicate that EBV contributes to development of the lesions is lacking.

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FIGURE 12.9 Angiofollicular lymph node hyperplasia. Note the hyalinized follicular lesion with associated radiating vascular structure at 8:00 o'clock surrounded by mixed population of mononuclear cells. Reprinted with permission and through the courtesy of J. Lunde, MD.

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Lothe, R, and Murray, J. (1962). Kaposi's sarcoma: Autopsy findings in the African. Acta Union Int. Contra. Cancrum. 18, 429-452. Marchioli, C , Love, J., Abbott, L., Huang, Y, Remick, S., Surtento-Redodica, N., Hutchison, R., Mildvan, D., Friedman-Kien, A., and Poiesz, B. (1996). Prevalence of human herpesvirus 8 DNA sequences in several patient populations. /. Clin. Microbiol. 34,26352638. Marquart, K., Muller, H., Hartter, R, Oku, J., and Ayuko, W. (1987). Lymphadenopathic type of Kaposi's sarcoma in a Ugandan child seropositive for LAV/HTLV-III antibodies. /. Trop. Med. Hyg. 90, 93-94. Martin III, R., Hood, A., and Farmer, E. (1993). Kaposi sarcoma. Medicine 11,1^5-1(A. Matondo, P., and Zumla, A. (1996). The spectrum of African Kaposi's sarcoma: Is it consequential upon diverse immunological responses? Scand. J. Infect. Dis. 28, 225-230. McCarthy, G., Kampmann, B., Novelli, V., Miller, R., Mercey, D., and Gibb, D. (1996). Vertical transmission of Kaposi's sarcoma. Arch. Dis. Child. 74, 455-457. McDonagh, D., Liu, J., Gaffey, M., Layfield, L., Azumi, N., and Traweek, S. (1996). Detection of Kaposi's sarcoma-associated herpes virus-like DNA sequences in angiosarcoma. Am. J. Pathol. 149, 1363-1368. McNutt, N., Fletcher, V, and Conant, M. (1983). Early lesions of Kaposi's sarcoma in homosexual men: An ultrastructural comparison with other vascular proliferations in skin. Am. J. Pathol. I l l , 62-77. Miller, G., Heston, L., Grogan, E., Gradoville, L., Rigsby, M., Sun, R., Shedd, D., Kushnaryov, V, Grossberg, S., and Chang, Y. (1997). Selective switch between latency and lytic replication of Kaposi's sarcoma herpesvirus and Epstein-Barr virus in dually infected body cavity lymphoma cells. /. Virol. 71, 314-324. Monini, P., de Lellis, L., Fabris, M., Rigolin, R, and Cassai, E. (1996). Kaposi's sarcoma-associated herpesvirus DNA sequences in prostate tissue and human semen. New Engl. J. Med. 334,1168-1172. Moore, P., and Chang, Y (1995). Detection of herpesvirus-like DNA sequences in Kaposi's sarcoma in patients with and those without HIV infection. New Engl. ]. Med. 332,1181-1185. Moore, P., Kingsley, L., Holmberg, S., Spira, T., Gupta, P., Hoover, D., Parry, J., Conley, L., Jaffe, H., and Chang, Y (1996a). Kaposi's sarcoma-associated herpesvirus infection prior to onset of Kaposi's sarcoma. AIDS 10, 175-180. Moore, P., Gao, S., Dominguez, G., Cesarman, E., Lungu, O., Knowles, D., Garber, R., Pellett, P, McGeoch, D., and Chang, Y (1996b). Primary characterization of a herpesvirus agent associated with Kaposi's sarcoma. /. Virol. 70, 549-558. Nair, B., DeVico, A., Nakamura, S., Copeland, T., Chen, Y, Patel, A., O'Neil, T., Oroszlan, S., Gallo, R., and Sarngadharan, M. (1992). Identification of a major growth factor for AIDS-Kaposi's sarcoma cells as oncostatin M. Science 255,1430-1432. Nakamura, S., Salahuddin, S., Biberfeld, P., Ensoli, B., Markham, P., Wong-Staal, R, and Gallo, R. (1988). Kaposi's sarcoma cells: Longterm culture with growth factor from retrovirus-infected CD4+ T cells. Science 242, 426-430. Nakamura, S., Murakami-Mori, K., Rao, N., Welch, H., and Rajeev, B. (1997). Vascular endothelial growth factor is a potent angiogenic factor in AIDS-associated Kaposi's sarcoma-derived spindle cells. /. Immunol. 158, 4992-5001. Nickoloff, B., and Foreman, K. (1996). Charting a new course through the chaos of KS (Kaposi's sarcoma). Am. J. Pathol. 148,1323-1329.

O'Connell, K. (1977). Kaposi's sarcoma in lymph nodes: Histological study of lesions from 16 cases in Malawi. /. Clin. Pathol. 30, 696703. Ohyashiki, J., Ohyashiki, K., Kawakubo, K., Serizawa, H., Abe, K., Mikata, A., and Toyama, K. (1994). Molecular genetic, cytogenetic, and immunophenotypic analyses in Castleman's disease of the plasma cell type. Am. J. Clin. Pathol. 101, 290-295. Oksenhendler, E., Duarte, M., Soulier, J., Cacoub, P., Welker, Y, Cadranel, J., Cazals-Hatem, D., Autran, B., Clauvel, J., and Raphael, M. (1996). Multicentric Castleman's disease in HIV infection: A clinical and pathological study of 20 patients. AIDS 10, 61-67. Pastore, C , Gloghini, A., Volpe, G., Nomdedeu, J., Leonardo, E., Mazza, U., Saglio, G., Carbone, A., and Gaidano, G. (1995). Distribution of Kaposi's sarcoma herpesvirus sequences among lymphoid malignancies in Italy and Spain. Br J. Haematol. 91,918-920. Port, J., Traube, J., and Winans, C. (1982). The visceral manifestations of Kaposi's sarcoma. Gastrointest. Endoscopy 28,179-181. Rabkin, C , Janz, S., Lash, A., Coleman, A., Musaba, E., Liotta, L., Biggar, R., and Zhuang, Z. (1997). Monoclonal, origin of multicentric Kaposi's sarcoma lesions. New Engl. J. Med. 336, 988-993. Ramos, C , Taylor, H., Hernandez, B., and Tucker, E. (1976). Primary Kaposi's sarcoma of lymph nodes. Am. J. Clin. Pathol. 66, 9981003. Rasmussen, E., Cooper, K., Kang, K., White Jr., C , Regan, D., and Hanifin, J. (1982). Immunosuppression in a homosexual man with Kaposi's sarcoma. /. Am. Acad. Dermatol. 6, 870-879. Real, R, and Krown, S. (1985). Spontaneous regression of Kaposi's sarcoma in patients with AIDS. New Engl. J. Med. 313,1659. Reed, W, Kamath, H., and Weiss, L. (1974). Kaposi sarcoma, with emphasis on the internal manifestations. Arch. Dermatol. 110,115118. Revuelta, M., and Nord, J. (1998). Successful treatment of multicentric Castleman's disease in a patient with human immunodeficiency virus infection. Clin. Infect. Dis. 26, 527. Reynolds, W, Winkelmann, R., and Soule, E. (1965). Kaposi's sarcoma: A clinicopathologic study with particular reference to its relationship to the reticuloendothelial system. Medicine 44, 419443. Ross, R., Casagrande, J., Dworsky, R., Levine, A., and Mack, T. (1985). Kaposi's sarcoma in Los Angeles, California. /. Natl. Cancer Inst. 75, 1011-1015. Rywlin, A., Rosen, L., and Cabello, B. (1983). Coexistence of Castleman's disease and Kaposi's sarcoma. Am. J. Dermatopathol. 5, 277281. Said, J., Tasaka, T., Takeuchi, S., Asou, H., de Vos, S., Cesarman, E., Knowles, D., and Koeffler, H. (1996). Primary effusion lymphoma in women: Report of two cases of Kaposi's sarcoma herpes virusassociated effusion-based lymphoma in human immunodeficiency virus-negative women. Blood 88, 3124-3128. Santucci, M., Pimpinelli, N., Moretti, S., and Giannotti, B. (1988). Classic and immunodeficiency-associated Kaposi's sarcoma. Arch. Pathol. Lab. Med. Ill, 1214-1220. Schulze, H., Rutten, A., Mahrle, G., and Steigleder, G. (1987). Initial lesions of HIV-related Kaposi's sarcoma — a histological, immunohistochemical, and ultrastructural study. Arch. Dermatol. Res. 279, 499-503. Shahidi, H., Myers, J., and Kvale, P. (1995). Castleman's disease. Mayo Clin. Proc. 70, 969-977. Smoller, B., Chang, P, and Kamel, O. (1997). No role for human herpes virus 8 in the etiology of infantile capillary hemangioma. Mod. Pathol. 10, 675-678.

Kaposi Sarcoma-Associated Herpesvirus Soler, R., Howard, M., Brink, N., Gibb, D., Tedder, R, and Nadal, D. (1996). Regression of AIDS-related Kaposi's sarcoma during therapy with Thalidomide. Clin. Infect Dis. 23, 501-503. Soulier, J., GroUet, L., Oksenhendler, E., Cacoub, R, Cazals-Hatem, D., Babinet, P., d'Agay, M., Clauvel, J., Raphael, M., Degos, L., and al, e. (1995). Kaposi's sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman's disease. Blood 86,1276-1280. Stahl, R., Friedman-Kien, A., Dubin, R., Marmor, M., and ZollaPazner, S. (1982). Immunologic abnormalities in homosexual men: Relationship to Kaposi's sarcoma. Am. J. Med. 73,171-178. Staskus, K., Zhong, W., Gebhard, K., Hemdier, B., Wang, H., Renne, R., Beneke, J., Pudney, J., Anderson, D., Ganem, D., and Haase, A. (1997). Kaposi's sarcoma-associated herpesvirus gene expression in endothelial (spindle) tumor cells. /. Virol. 71, 715-719. Strauchen, J., Hauser, A., Burstein, D., Jimenez, R., Moore, P., and Chang, Y. (1996). Body cavity-based malignant lymphoma containing Kaposi sarcoma-associated herpesvirus in an HlV-negative man with previous Kaposi sarcoma. Ann. Intern. Med. 125, 822-825. Templeton, A. (1981). Kaposi's sarcoma. Pathol. Ann. 16, 315-336. Thomas, J., Brookes, L., McGown, I., Weller, I., and Crawford, D. (1996). HHV8 DNA in normal gastrointestinal mucosa from HIV seropositive people. Lancet 347,1337-1338.

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Tirelli, U., Gaidano, G., Errante, D., and Carbone, A. (1996). Potential heterosexual Kaposi's sarcoma-associated herpesvirus transmission in a couple with HIV-induced immunodepression and with Kaposi's sarcoma and multicentric Castleman's disease. AIDS 10, 1291-1304. Vadhan-Raj, S., Wong, G., Gnecco, C , Cunningham-Rundles, S., Krim, M., Real, P., Oettgen, H., and Krown, S. (1986). Immunological variables as predictors of prognosis in patients with Kaposi's sarcoma and the acquired immunodeficiency syndrome. Cancer Res. 46, 417-425. Vella, J., Mosher, R., and Sayegh, M. (1997). Kaposi's sarcoma after renal transplantation. New Engl. J. Med. 336,1761. Weisberger, D., Nathwani, B., Winberg, C , and Rappaport, H. (1985). Multicentric angiofollicular lymph node hyperplasia: A clinicopathologic study of 16 cases. Hum. Pathol. 16,162-172. Whitby, D., Howard, M., Tenant-Flowers, M., Brink, N., Copas, A., Boshoff, C , Hatzioannou, T., Suggett, F., Aldam, D., Denton, A., Miller, R., Weller, I., Weiss, R., Tedder, R., and Schulz, T. (1995). Detection of Kaposi sarcoma associated herpesvirus in peripheral blood of HIV-infected individuals and progression to Kaposi's sarcoma. Lancet 346, 799-802. Zeligman, I. (1960). Kaposi's sarcoma in a father and son. Bull. Johns Hopkins Hosp. 107, 208-212.

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C H A P T E R

13 Herpesvirus Simiae Virus (Herpes B) appears to be a major means for spread of the virus in breeding colonies (Weigler et al, 1993, 1995). While herpes B infection in its natural host is relatively benign, when the virus is inoculated into subhuman primates of unrelated species devastating central nervous system infections often evolve. The occurrence of fatal disease in humans appears to be comparable. After cutaneous inoculation of a primate, the virus replicates locally, resulting in a crop of vesicles and localized inflammation before spreading by means of the lymphatics to regional lymph nodes. Lymphadenitis often develops, and focal areas of necrosis and hemorrhage are found in the nodes. Similar lesions are occasionally found in HSV-infected humans (see Figures 7.23 and 7.24). Systemic spread of the virus usually follows with the development of circumscribed necrotic lesions in the liver, adrenals, spleen, and kidney. The neural route of centripetal spread of the virus by means of peripheral nerves to the central nervous system results in a myelitis, ultimately followed by development of a diffuse encephalitis. In striking contrast to HSV, herpes B does not favor the limbic system of the brain (Weigler, 1992). Detailed information on the pathological features of herpes B in humans is surprisingly limited. Although considerable variability between individual cases is well documented, in general, the neuropathological picture is one of a diffuse chronic active infection involving the spinal cord, brainstem, and cerebral cortex (Hummeler, et al, 1959; Sabin and Wright, 1934; Sabin, 1949; Nagler and Klotz, 1958). Typical intranuclear eosinophilic inclusions are the hallmark of the virus. The overt hemorrhagic necrosis of the temiporal lobe so characteristic of HSV encephalitis (see Figures 7.9 and 7.14) is not observed. With increasing awareness, the risk for workers in research facilities and in vaccine production has been substantially reduced. Considerable effort now focuses on identification of latent and subclinical infections in subhuman primates and establishment of

^/% oughly 35 different herpesviruses are known ^ ^ X to infect various species of nonhuman priM^L mates. Of these agents, only "herpes B" (syn. r • ^ Herpesvirus simiae, Cercopithecine Herpesvirus 1) is a recognized pathogen for man. It is an enzootic virus of the Old World Macaca mulatta rhesus monkey that has virological similarities to Herpes simplex virus (HSV). Twenty-five cases of disease in humans have been documented, and several additional cases are suspected but not proven. To a large extent, these infections were acquired inadvertently in an occupational setting as the result of monkey bites or exposure to cultured cells of Macaca origin. One instance of person-to-person spread by mechanical means has been documented (Holmes et al, 1990). Twenty-two of the 25 recognized infections in humans progressed to encephalomyelitis, resulting in 16 deaths (Weigler, 1992). Many of the surviving patients had significant residual neurological disease, some requiring institutionalization. In contrast to HSV, herpes B infections result in a diffuse encephalitis and transverse myelitis, anatomically related to the initial primary site of infection. The incubation period before development of local lesions ranges from 2 days to more than 10 years. At the local site of inoculation, vesicles and a promiinent soft tissue reaction are observed in some patients. In one case, an ophthalmic herpes zoster clinical picture developed initially, to be followed by a rapidly evolving encephalomyelitis (Fierer et al, 1973). Based on studies of naturally and experimentally infected subhuman primates, it is reasonable to conclude that the biological properties of herpes B and HSV are similar. For example, the incidence of subclinical infections in primate colonies increases with age. Although these infections usually become latent and are clinically unimportant, virus can be recovered sporadically from the oral cavity, conjunctiva, and genital secretions of individual, seemingly healthy, monkeys (Weigler, 1992). Indeed, infection by the venereal route

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virus-free colonies of animals for research purposes and tissue culture production. Herpes B infections should rarely occur in the future (Wells ei a\., 1989; Holmes ei al, 1995).

References Fierer, J., Bazeley, P., and Braude, A. (1973). Herpes B virus encephalomyelitis presenting as ophthalmic zoster: A possible latent infection reactivated. Ann. Intern. Med. 79, 225-228. Holmes, G., Hilliard, J., Klontz, K., Rupert, A., Schindler, C , Parrish, E., Griffin, D., Ward, G., Bernstein, N., Bean, T., Ball, M., Brady J., Wilder, M., and Kaplan, J. (1990). B virus {Herpesvirus simiae) infection in humans: Epidemiologic investigation of a cluster. Ann. Intern. Med. Ill, 833-839. Holmes, G., Chapman, L., Stewart, J., Straus, S., Hilliard, J., and Davenport, D. (1995). Guidelines for the prevention and treatment of B-virus infections in exposed persons. Clin. Infect. Dis. 20, 421-439. Hummeler, K., Davidson, W, Henle, W, LaBoccetta, A., and Ruch, H. (1959). Encephalomyelitis due to infection with Herpesvirus

simiae (Herpes B virus): A report of two fatal, laboratory-acquired cases. New Engl. ]. Med. 261, 64-68. Nagler, R, and Klotz, M. (1958). Fatal B virus infection in person subject to recurrent herpes labialis. Can. Med. Assoc. J. 79, 743-745. Sabin, A. (1949). Fatal B virus encephalomyelitis in physician working with monkeys. /. Clin. Invest. 28, 808. Sabin, A., and Wright, A. (1934). Acute ascending myelitis following monkey bite, with isolation of virus capable of reproducing disease. /. Exper. Med. 59,115-136. Weigler, B. (1992). Biology of B virus in Macaque and human hosts: A review. Clin. Infect. Dis. 14, 555-567. Weigler, B., Hird, D., Hilliard, J., Lerche, N., Roberts, J., and Scott, L. (1993). Epidemiology of cercopithecine herpesvirus 1 (B virus) infection and shedding in a large breeding cohort of Rhesus macaques. /. Infect. Dis. 167, 257-263. Weigler, B., Scinicariello, R, and Hilliard, J. (1995). Risk of venereal B virus (Cercopithecine Herpesvirus 1) transmission in Rhesus monkeys using molecular epidemiology. /. Infect. Dis. 171,1139-1143. Wells, D., Lipper, S., Hilliard, J., Stewart, J., Holmes, G., Herrmann, K., Kiley, M., and Schonberger, L. (1989). Herpesvirus simiae contamination of primary Rhesus monkey kidney cell cultures: CDC recommendations to minimize risks to laboratory personnel. Diagn. Microbiol. Infect. Dis. 12, 333-336.

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14 Adenoviruses INTRODUCTION

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DISEASE IN IMMUNOSUPPRESSED PATIENTS GENITOURINARY TRACT DISEASE DIGESTIVE TRACT DISEASE MYOCARDIAL DISEASE

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Since the time of this initial work in the early 1950s, an enormous body of information has accumulated on what has proven to be a large family of viruses that has the capacity to infect a diversity of organs. At the time of this writing, 42 antigenically distinct serotypes of adenoviruses have been recovered from humans, and many more types are found in lesser animal species. The human adenoviruses are now nominally classified into seven groups, based largely on their biological features (Table 14.1). While they share similar structural and biochemical features, only about a third of the 42 strains are commonly recovered from clinical specimens, and only a few of these strains are known to cause disease. The adenovirus virions range from 60 to 90 nm in diameter. The viruses are comprised of a linear cord of double-stranded DNA made u p of nine translational units surrounded by a complex capsid of 252 unit capsomeres arranged in icosahedral symmetry. Pentamers, to which are attached fibers of variable length, form at the 12 vertices of the capsid, whereas the remaining capsomeres are hexamers. The fibers of the pentamers functionally serve to attach the virus to receptors of an unidentified composition on cell surfaces. Internalization of the virions into the cell is facilitated by integrins (Goldman and Wilson, 1995). After pinocytosis, the virion is uncoated, and synthesis of the "early" proteins (E1/E3) begins using the molecular machinery of the cell. These proteins turn off cellular DNA and protein synthesis, inducing apoptosis. Elaboration of the structural so-called "late" proteins of the virus follows, after which assembly of the progeny virions occurs in the nucleus (Figure 14.1). These particles are often organized into crystalline arrays (Figure 14.2). Because of the distinctive morphological features of the virus crystals, adenovirus-infected cells are readily detected by electron microscopy in human tissue. A substantial excess of the structural components of the virus are manufactured and accumulate in the cell nucleus during the replication cycle. These deposits appear to account, in part, for the intranuclear inclusions that are

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INTRODUCTION Inflammation and hyperplasia of the tonsils and adenoids invariably develop during the countless episodes of acute respiratory disease experienced by otherwrise healthy infants and young children. Despite its almost universal occurrence, the pathogenesis of common childhood oropharyngeal lymphoid hyperplasia is incompletely understood. To explore this intriguing question, Wallace Rowre and his colleagues (1953) cultured tonsillar and adenoid tissue for viruses and tediously monitored the events that evolved in the cells that grew in vitro from these tissues. Over the ensuing wreeks, the cells gradually developed the cytopathology of a new and previously unrecognized family of viruses. Not one, but several, antigenically different agents were recovered from lymphoid tissues in this manner. One group proved to be the prototype strains of what were initially termed the adenopharyngeal conjunctival (APC) agents, now known as adenoviruses. The second was cytomegalovirus (see Chapter 8). Subsequent studies have shown that adenoviruses of several different serotypes can be recovered from 60 to 75% of tonsil and adenoid specimens evaluated in the manner explored by Rowe (Van der Veen and Lambriex, 1973; Strohl and Schlesinger, 1965). More recent studies have shown that the virus is fully assembled and is present in a latent state in roughly 1 of every 10^ lymphoid cells in the oropharynx. On rare occasions, intranuclear inclusions typical of this family of viruses have been found in the squamous epithelial cells of the tonsils and adenoids (Brown, 1974).

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Pathology and Pathogenesis of Human Viral D i s e a s e TABLE 14.1 Classification of A d e n o v i r u s Serotypes Origin and group Respiratory C Bl Urinary Tract B2 Keratoconjunctivitis Bl D E Enteric Infections A F

Disease associations

Serotype

I, 2, 5, 6 3, 7, 16, 21

Minor acute upper respiratory illnesses; commonly are latent; persist in tonsils/adenoids Pneumonia

II, 14, 34, 35

Cystitis and nephritis; types 34 & 35 commonly disseminate in immunocompromised patients

3,7 Multiple serotypes 4

Sporadic and epidemic keratoconjunctivitis

12,18, 31 40,41

Diarrhea

FIGURE 14.1 The nucleus of a cultured epithelial cell 48 hr after infection with adenovirus type 2. The arrow depicts inclusions of several types (I, II, and III), the most prominent of which is the paracrystalline array. These components contribute to the inclusions observed by light microscopy N defines the nucleolus. Virions are distributed throughout the nucleus in this cell but not in crystalline arrays (12,000x). Reprinted with permission from Weber and Stich (1969).

the typical cytological features of the adenovirus-infected epithelial cell. The presence of viral proteins in the so-called "smudge" cells that typify adenovirus-infected tissues has not been established. However, infected epithelial cells often fail to exhibit diagnostic morphological changes (Ladenheim et ah, 1995). Adenoviruses are ubiquitous and most of us, no doubt, are latent carriers. Infections with the common endemic types (1-2, 5-7,14) customarily occur in childhood, resulting in relatively transient upper respiratory illnesses characterized by rhinitis, pharyngitis, tracheitis, and more generalized systemic symptoms. When patients exhibit unilateral or bilateral follicular conjunctivitis, the illness is termed pharyngeal conjunctival fevers. Overall, adenoviruses are responsible for roughly 5% of the upper respiratory illnesses of children. Perhaps 10% of the uncomplicated cases of childhood pneumonitis are due to these viruses. Thus, adenovirus infections are a "right of passage" for us all, and they cause serious illness on only rare occasions. The diseases considered in this chapter prove to be the more serious and often life-threatening. In the discussion, I have deliberately avoided a listing of specific adenovirus serotypes associated with various syndromes, since the information can be found in any standard virology text, and this detail to a large extent is meaningless when dealing with a clinical problem. Adenoviral lesions are characterized by a distinctive cytopathology and an associated acute inflammatory response. The cellular necrosis that follows is attributed to both the turn-off of cell protein synthesis and cell-mediated immune mechanisms (Ginsberg et al, 1990). However, information on the immunopathology of adenovirus infections is limited, and the relative contribution of immune mechanisms in the clearance of virus and recovery is uncertain. The common occurrence of fulminating adenovirus disease in recipients of

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FIGURE 14.2 Region of the nucleus of a cultured epithelial cell 96 hours after infection with adenovirus type 18. Note the fine structural features of the paracrystalline arrays (A). In (B) a similar array is closely associated with a crystal of virions (v) (42,000x). Reprinted with permission from Weber and Liao (1969).

immunosuppressive agents such as organ allograft recipients, and in patients with AIDS, emphasizes the important role of immunity in the control of infection. Recent experimental evidence indicates that the E1/E3 "early" proteins elaborated by the virus increase the expression of ICAM and class I major histocompatibility antigens by an infected cell (Pilewski et ah, 1995; Ginsberg et al, 1989). Epithelial cell susceptibility to the destructive effects of tumor necrosis factor alpha and other cytokines may also be influenced by these products elaborated "early" in the replicative cycle (Duerksen-Hughes, 1989). The inflammatory response customarily observed in the submucosa of the infected respiratory tract appears to contribute to both virus eradication and destruction of the mucosa.

RESPIRATORY TRACT DISEASE Most children are infected with adenoviruses at an early age, and by the age of 5 years, at least 50% of children have serological evidence of a past experience with as many as four different viruses of the pneumotropic groups Bl and C. The infection may be asymptomatic or manifest as a nonspecific transient respiratory illness, ranging in severity from nasal congestion to croup, but usually accompanied by fever. Thus, adenovirus illnesses cannot be differentiated clinically

from minor respiratory disease due to a variety of other common viruses. In total, however, adenoviruses account for only about 5% of the relatively mild respiratory infections experienced by infants and young children. The virus is usually transmitted by either the fecal-oral route, or by the respiratory droplet route, and customarily does not spread in epidemic form. In children, approximately 5% of adenovirus infections of the oropharynx spread to involve the lower respiratory tract, with bronchitis, bronchiolitis, and pneumonia being the most common clinical manifestations. They represent a substantial component of the pneumonias of young children requiring hospitalization. Adenoviruses types 3, 5, 7, and 21 are the most frequently involved serotypes (Collier et ah, 1966; Chany et al, 1958; Becroft, 1967; Benyesh-Melnick and Rosenberg, 1964). Newborn infants are affected uncommonly, although neonatal infections acquired in the birth canal or transplacentally from an infected mother are reported (Pinto et al, 1992; Benyesh-Melnick and Rosenberg, 1964; Abzug and Levin, 1991; Chiou et al, 1994). Outbreaks of nosocomial disease in hospital settings are particularly devastating (SinghNaz et al, 1993). About 15% of infected children died in the largest outbreak of adenovirus pneumonia thus far studied. The clinical features of this febrile respiratory infection focus on the airways and lungs, with a pertussis-like cough being common (Collier et al, 1966). Heart failure and evidence of central nervous system

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B

''¥M-SA\7 FIGURE 14.3 (A) Adenovirus pneumonia with destruction of the bronchial mucosa and plugging of the lumina with mucus secretions and debris. A proteinaceous exudate and focal interstitial pneumonitis are seen in the parenchyma of the lung. (B) The cytolytic changes in the bronchial mucosal cells are seen at higher magnification.

involvement are common features in severe cases (Connor, 1970; Klenk et al, 1972; Schonland et al, 1976). Some reports suggest that Asians are unusually susceptible to adenovirus pneumonia (Lang et al, 1969). Inclusion-body pneumonia in children was first described by Goodpasture et al. (1939), although its viral etiology was not appreciated at the time. The causative role of adenoviruses in this disease is now established, and numerous case reports document the devastating pathological features. Typically, the respiratory mucosa of the bronchi and bronchioles is destroyed and desquamated, with chronic inflammatory cell accumulations being evident in the lumina and submucosal tissues (Figure 14.3A,B). The submucosal glands in the trachea and bronchi are often involved and exhibit a necrotic epithelium. This appears to be a characteristic of adenovirus infections of the airways. In the distal branches of the bronchial tree, accumulated debris comprised of necrotic epithelium accompanied by serous and cellular exudates form eosinophilic plugs that obliterate the lumina of these channels. In situ hybridization establishes the presence of the virus in epithelial cells lining airways (Hogg et al, 1989). Cytological evidence of infection in the form of smudge and inclusion-bearing cells is variable in large part, because the destructive effects of the virus leave few intact mucosal cells (Figures 14.4A-F and 14.5A-F) (Kawai, 1959; Pinkerton and Carroll, 1971; Becroft, 1971; Herbert et al, 1977). Similar cytological changes can be seen in type II cells of the pulmonary alveolar parenchyma when the lungs are involved. In these cases, interstitial inflammation is often evident. A substantial body of evidence attests to the devastating long-term effects of adenovirus infections on the airways and lung parenchyma (Figure 14.6). To a variable extent, bronchiolitis obliterans, bronchiectasis (Becroft, 1971), and interstitial pulmonary fibrosis and

FIGURE 14.4 Adenovirus type 7 infection of an organ culture of differentiated human trachea. (A-D) illustrate the ''honeycombing'' of the nucleus and the small compartmentalized inclusions characteristic of the early virus-induced nuclear alterations. E demonstrates the nucleus of a typical smudge cell and F illustrates a distinctive intranuclear inclusion. The changes in E and F develop relatively late in the course of the infection.

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Adenoviruses

B

FIGURE 14.5 Diverse cytological alterations observed in an organ culture of human tracheal epithelium infected with adenoviruses. They can be characterized as intranuclear inclusions (A-C) and smudge cells (D-F). In evaluating tissue, the pathologist must be acquainted with the spectruni of changes that customarily develop as a result of an adenovirus infection.

chronic inflammation have been described in these cases (Kawai et ah, 1976; Simila et al, 1971; Warner and Marshall, 1976; Lanning et al, 1980; Zarraga et al., 1992). Although the prevalence of these late complications is difficult to assess, a follow-up report of an outbreak of type 21 adenovirus documented permanent residual lung damage in 60% of infected young children and saccular bronchiectasis in 20% (Lang et al, 1969). Chany et al (1958) detected long-term radiological changes in 27% of children 9 to 12 years after recov-

ery from adenovirus pneumonia. On the other hand, Simila et al (1971) noted radiologic evidence of pulmonary fibrosis in only 2 of 29 (7%) documented cases of adenovirus pneumonia and bronchiectasis in an additional 7%. An approximate 50% reduction in the caliber of the small airways was found in the lungs of young dogs experimentally infected with a canine strain of adenovirus (Castleman, 1985). The anatomic changes were attributed to scarring of the airway walls (Wright et al, 1979,1964).

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FIGURE 14.6 A 38-year-old man with AIDS and adenovirus pneumonia established by lung biopsy. Computer axial tomograph demonstrates nodular and alveolar densities with airspace consolidation in both lungs. Reprinted with permission from Maslo et al. (1997).

Chronic persistent adenovirus infections of the respiratory tract have been documented in humans (Chanock, 1974) and experimentally infected animals. Using PCR, Pacini ei al (1984) and Matsuse et al (1992) claimed to have found the adenovirus ElA gene in the lung tissue of a substantial proportion of patients with chronic obstructive pulmonary disease. The significance of these findings with regard to the pathogenesis of chronic lung disease is uncertain. Studies by Hogg et al (1989) and Bateman et al (1995) fail to document an increased prevalence of chronic adenovirus infections in the bronchial tree of patients with follicular bronchitis. This lesion is characterized by the accumulation of prominent lymphoid follicles within the dilated bronchiectatic walls of the airways and associated lung. While saccular bronchiectasis can be a residual effect of adenovirus infections, there is currently no evidence to suggest that chronic adenovirus infections contribute to development of the lesion. As noted above, chronic bronchiolitis and bronchiolitis obliterans are often found in the lungs of those recovering from adenovirus infections. Adenovirus lower respiratory tract disease occurs on sporadic occasions in members of the adult population, but the infection is not an important cause of pneumonia in older age groups. In contrast, outbreaks of infection with certain virus strains prove to be a common cause of pneumonia among recent inductees

into the military in the United States and Europe. The responsible strains of virus in the two geographic regions thus far studied differ, with serotypes 4 and 7 being prevalent in the United States and types 14 and 21 in Europe. The respiratory droplet mode of transmission appears to account for outbreaks on military bases (Chanock, 1974). Although asymptomatic infections are common among recruits, acute respiratory illness develops in approximately 50%, and pneumonia occurs in 5 to 15% of military inductees. Rare fatalities have occurred as a consequence of a progressive pneumonia (Dudding et al, 1972; Loker et al, 1974; Field et al, 1978). Autopsy reveals an extensive acute bacterial pneumonia, and the typical cytological changes indicative of adenovirus infection are not found, even though virus, in high concentrations, can occasionally be recovered from lung tissue. In some of these cases, disseminated intravascular coagulation and rhabdomyolysis of striated muscular masses has been documented (Wright et al, 1979). Because of its common occurrence in young military recruits, oral vaccines were developed, and, to a large extent, routine prophylaxis has eliminated the problem on military bases. It is surprising and unexplained why similar adenovirus outbreaks do not occur in other semiclosed populations such as among college students. However, a recent report documents an outbreak of pneumonia in a semiclosed chronic psychiatric facility. Fourteen

Adenoviruses

percent of the residents developed pneumonia sufficiently severe that 36% of them required mechanical ventilation and 7% died. An uncommon serotype of adenovirus (type 35) was responsible (Sanchez et ah, 1997; Klinger et al, 1998).

DISEASE IN I M M U N O COMPROMISED PATIENTS Disseminated adenovirus infections occur commonly in immunocompromised patients with genetically acquired defects in cellular immunity such as thymic aplasia (Wigger and Blanc, 1966; Aterman et ah, 1973; Charles et al, 1995), X-linked lymphoproliferative syndrome (Purtilo et al, 1985), AIDS (Gelfand et al, 1994; Maddox et al, 1992; Anders, 1990-91), cancer patients undergoing chemotherapy, and after renal (Myerowitz et al, 1975), lung (Ohori et al, 1995), hepatic (Michaels et al, 1992), and bone marrow (Flomenberg et al, 1994) transplantation (Strickler et al, 1992). Infections in allotransplant recipients seem to develop during episodes of graft-vs.-host disease, as documented either clinically or in kidney biopsies (Umekawa and Kurita, 1996; Yagisawa et al, 1995), and during epi-

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sodes of rejection. Both endogenous reactivation (Shields et al, 1985; Ohori et al, 1995) and exogenous nosocomial acquisition of the virus occur (Pingleton et al, 1978). In addition to the lungs, a wide variety of organs exhibit lesions in individual cases, with the parotid (Gelfand et al, 1994), liver (Wigger and Blanc, 1966; Rodriguez et al, 1984; Koneru et al, 1987; Krilov et al, 1990; Purtilo et al, 1985), lung, gall bladder (Hedderwick et al, 1998), colon (Figure 14.7) (Michaels et al, 1992; Janoff et al, 1991; Maddox et al, 1992), brain (Anders et al, 1990-91), pancreas (Niemann et al, 1993), kidney (Figure 14.8), and the lower urinary tract (Ito et al, 1991) being sites of disease. In a systematic study of patients with AIDS, viremia proved to be common (Ferdman and Ross, 1997). In disseminated disease among immunocompromised adult patients, the pulmonary lesions are similar to those described earlier in this chapter (Maslo et al, 1997; Sencer et al, 1993; Zaltzman et al, 1994; Yagisawa et al 1995; Umekawa and Kurita, 1996). The liver exhibits multiple circumscribed foci of coagulation necrosis, with occasional hepatocytes showing the cytologic features of infection. In the colon, adenovirus-infected mucosal cells are readily identified microscopically They exhibit intranuclear inclusions that can be differentiated from the typical "owl eye"

FIGURE 14.7 (A) Colonic mucosa exhibiting typical smudge cells (arrow) established to be due to adenovirus by immunohistochemistry (arrow) (B). The patient died with AIDS. Reprinted with permission from Hedderwick et al. (1998) and through the courtesy of S. Hedderwick, MD, and J. Greenson, MD.

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F I G U R E 14.8 Kidney of a 17-year-old bone marrow recipient who died with cytomegalovirus pneumonia 3 months after transplantation. Adenovirus was isolated from the kidney tissue postmortem but not from the lungs and other major organs. In (A) the tubules are mildly dilated and show focal changes in the epithelial lining cells. Immunohistochemistry (B) with antibody against the adenovirus hexon protein demonstrated viral infection of tubular lining cells without involvement of the glomerulus. Photographs reprinted with permission and through the courtesy of R. Hackman, MD.

inclusions of cytomegalovirus. Superficial cells of the mucosa, but not those of crypts, are usually involved; frequently, the infected cells appear necrotic. The submucosa usually exhibits a chronic inflammatory cell infiltrate. In contrast, cytomegalovirus commonly infects macrophages, vascular endothelial cells, and stromal cells, that is, fibroblasts and smooth muscle cells of the colon in immunosuppressed patients. Involvement of mucosal epithelial cells by cytomegalovirus is less common. In a prospective study of bone marrow transplant recipients, adenoviruses were the most frequently recovered enteric virus. The majority of the patients had diarrhea and many died, but the cause of death and the possible contribution of the adenovirus were not established (Yolken et al, 1982).

GENITOURINARY TRACT DISEASE Renal and tubulo-interstitial disease and hemorrhagic cystitis are major causes of morbidity in allotransplant recipients. In a study of 977 bone marrow transplant recipients, 135 (14%) developed hematuria and dysuria. Of these patients, 60 had severe cystitis requiring aggressive treatment. Twenty-two percent also developed renal failure, presumably attributable to nephritis (Steigbigel et al, 1978; Ito et al, 1991; Tomoe et al, 1994; Green et al, 1994; Usami et al, 1997; Hackman et al, 1997). In kidney biopsies, there are extensive changes in the tubular lining cells attributable to the adenovirus infection and interstitial chronic inflamma-

tion (Figure 14.8). Some, but not all, of the patients studied yielded virus when the urine was cultured. A canine strain of adenovirus (the etiology of an often fatal systemic disease in immunologically intact dogs) induces lesions in the kidney that are similar to those found in immunosuppressed humans (Morrison et al, 1976). Numazaki et al (1968) described the spontaneous occurrence of acute hemorrhagic cystitis in otherwise healthy children associated with infection by type 11 adenovirus. Subsequent studies by these and other investigators (Numazaki et al, 1973; Mufson et al, 1971, 1973; Mufson and Belshe, 1976; Lee et al, 1996) confirmed these findings and established a causative relationship between viruria and disease of the urinary bladder and kidneys. Adenovirus type 11 is rarely found to infect sites other than the urinary tract; it appears to account for approximately 80% of the cases of hemorrhagic cystitis developing spontaneously in children in Japan, but only 23% of cases in the United States. The mode of infection of the urinary tract is not understood, and it is possible the cystitis results from an activated latent virus. The clinical evidence, although incomplete, suggests that disease of the urinary tract is not acquired as a result of viremia and is not a complication of a generalized infection. In both Japan and the United States, males are 2 to 4 times more likely to be affected than females. Systematic studies have not been conducted among persons of various age groups, and the occurrence of this syndrome other than in Japan and the United States has not been documented. The accumulated information is largely clinical. Pathological studies have not been reported, although im-

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munocytochemical studies of exfoliated urinary tract epithelial cells document infection. A case of orchitis in a 5-year-old child with a concomitant adenovirus infection has been reported (Naveh and Friedman, 1975).

DIGESTIVE TRACT DISEASE Adenoviruses often are recovered using traditional cell culture techniques from the stools of healthy children and those with diarrheal disease. Early attempts to associate these virus isolates with disease proved futile, for they were so frequently recovered from health study subjects (Sterner et al, 1961; Richmond et ah, 1979). During the early 1970s, a new class of adenovirus was demonstrated in the stools of children with diarrhea using the technique of immune electron microscopy. It proved impossible to recover these viruses using traditional cell cultures, and they were therefore considered to be uniquely fastidious (Whitelaw et al, 1977). Accordingly, other sensitive diagnostic approaches were developed (i.e., the ELBA and DNA restriction analyses). Only somewhat later were cell cultures found that proved susceptible to these so-called fastidious adenoviruses (Cukor and Blacklow, 1984). Systematic studies have now established specific serotypes (40 and 41) of adenovirus as a cause of diarrhea in infants and children. Indeed, these agents are believed to be the second most common etiology of viral diarrhea (Kapikian, 1993). Affected children usually experience a protracted illness of as long as 10 days, accompanied by fever and occasional respiratory symptoms (Uhnoo et al., 1986). Pathological studies of the digestive tract tissue of children with adenovirus diarrheal disease have not been reported. Intussusception, an acute and often life-threatening surgical emergency in infants and young children, has many causes. However, in the majority of cases, pathologic study demonstrates hyperplasia of the lymphoid tissue of Peyer's patches at the lead point of the intussusception in the ileum, or at the ileocecal junction. Boys are affected twice as often as girls. A growing body of information associates adenovirus infections of the intestinal mucosa and mesenteric lymph nodes with this condition (Prince, 1979). Children hospitalized with intussusception may be more susceptible to infection, as they appear to have a lower prevalence of serum antibodies to the common adenovirus serotypes and virological work has documented infection of the gut in a substantial proportion of cases. In the studies of Ross et al. (1962), serotypes 1, 2, 5, 6, and 7 were

isolated. In other reports, no specific virus type predominated (Bell and Steyn, 1962; Ross et al, 1962). The so-called enteric adenoviruses that are commonly associated with diarrheal disease are not customarily recovered from patients with intussusception (Bhisitkul et al, 1992). Morphologic evaluation of the mucosa of surgically excised gut and appendices have demonstrated typical adenovirus cytopathology in the intestinal mucosa of roughly one-third of cases (Yunis et al, 1975; Montgomery and Popek, 1994).

MYOCARDIAL DISEASE Several case reports document the sporadic occurrence of lymphocytic interstitial myocarditis in newborns and children infected with adenoviruses (Towbin et al, 1994). To date, virus has not been recovered from heart tissue, and cells exhibiting the typical cytological features of an adenovirus infection have not been found in the myocardium; but in two cases, PCR of formalin-fixed paraffin-embedded heart tissue demonstrated the presence of adenovirus DNA (Lozinski et al, 1994). The evidence supporting an adenovirus causation in many clinical cases is circumstantial, based largely on the demonstration of a concurrent infection in other body tissues (Chany et al, 1958; Sterner, 1962; Van Zaane and Van der Veen, 1962; Berkovich et al, 1968; Henson and Mufson, 1971). In mice, a murine strain of adenovirus replicates in endothelial cells and myocytes, causing a destructive interstitial myocarditis and a valvulitis (Blailock et al, 1968). Towbin and his colleagues (1994) recently accomplished the diagnosis by PCR using blood from an acutely ill mother and her fetus, which had dilated cardiomyopathy and hydrops. In a case of sudden death with myocarditis, adenovirus DNA was demonstrated in myocardial cells (Shimizu et al, 1995). By their nature, the reports referred to above document fatal cases studied pathologically. The prevalence of myocarditis in adenovirus-infected persons with a nonfatal outcome is unknown.

CENTRAL NERVOUS SYSTEM DISEASE Meningoencephalitis of varying degrees of severity has been described in infants and children and, on occasion, in adults with and without systemic manifestations of infection, in particular, pneumonia (Chany et al, 1958; Gabrielson et al, 1966; Huttunen, 1970; Simila et al, 1970; Chou et al, 1973; Kelsey 1978; Kim and Gohd, 1983; Koskiniemi and Vaheri, 1982; Lelong et al.

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1956; West et a/., 1985; Anders et al, 1990-91). Brain biopsies have yielded adenoviruses type 2 (West et al, 1985), type 3 (Faulkner and van Rooyen, 1962), type 4, type 5 (Faulkner and van Rooyen, 1962), type 7 (Lord et al, 1975), type 11 (Osamura et al, 1993), and type 32 (Roos et al, 1972). In the small number of surgical brain biopsies thus far studied, the pathological changes have ranged from perivascular cuffing and gliosis to frank necrosis of parenchyma. Comprehensive autopsy studies have not been reported, and much remains to be learned about the pathological features of the central nervous system disease and its pathogenesis. On rare occasions, Reye syndrome has been reported in children with adenovirus pneumonia (Ladisch et al, 1979).

EYE DISEASE During mobilization for the Second World War, workers at scattered shipyards in the United States developed a severe, unilateral or bilateral, painful, and purulent chronic conjunctivitis. The condition often followed visits to eye clinics for the treatment of minor industrial injuries to the eyes. Commonly, the disease occurred in outbreak form and was ultimately traced to the instrumentation, solutions, and hands of ophthalmologists. The work of Jawetz (1959) established the etiological role of adenoviruses of several different serotypes. Later outbreaks were documented in family clusters, such as among playmates attending swimming pools, and among sexual partners (where venereal transmission by means of the adenovirus-infected genital secretions was a consideration). In a recent study, 5% of cases of conjunctivitis occurring over a 10-year period were attributed to adenovirus with, types 3, 4, and 7 being the predominant serotypes isolated (O'Donnell et al, 1993). During the acute stages of the infection, pseudomembranes often form on the corneal surfaces followed by the appearance of subepithelial corneal infiltrates. Occasionally, hemorrhage occurs in the eyes. Finally, a superficial punctate keratitis develops. These exudative lesions, and the associated visual problems persist for weeks or months, or at times even longer. Immunohistochemistry, in situ hybridization, and negative staining electron microscopy can be used to establish the diagnosis in scrapings from the cornea. Adenoviruses are often readily recoverable from the conjunctival exudates during the acute stages of the illness. In the exudates, polymorphonuclear leukocytes predominate. Cells with the specific cytopathic effects of adenoviruses have not been described.

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Montgomery, E., and Popek, E. (1994). Intussusception, adenovirus, and children: A brief reaffirmation. Hum. Pathol. 25,169-174. Morrison, W., Wright, N., and Cornwell, H. (1976). An immunopathologic study of interstitial nephritis associated with experimental canine adenovirus infection. /. Pathol. 120, 221-228. Mufson, M., and Belshe, R. (1976). A review of adenoviruses in the etiology of acute hemorrhagic cystitis. /. Urol. 115,191-194. Mufson, M., ZoUar, L., Mankad, V, and Manalo, D. (1971). Adenovirus infection in acute hemorrhagic cystitis. Am. J. Dis. Child. Ill, 281-285. Mufson, M., Belshe, R., Horrigan, T., and Zollar, L. (1973). Cause of acute hemorrhagic cystitis in children. Am. J. Dis. Child. 126, 605-609. Myerowitz, R., Stalder, H., Oxman, M., Levin, M., Moore, M., Leith, J., Gantz, N., Pellegrini, J., and Hierholzer, J. (1975). Fatal disseminated adenovirus infection in a renal transplant recipient. Am. J. Med. 59, 591-598. Naveh, Y, and Friedman, A. (1975). Orchitis associated with adenoviral infection. Am. ]. Dis. Child. 129, 257-258. Niemann, T., Trigg, M., Winick, N., and Penick, G. (1993). Disseminated adenoviral infection presenting as acute pancreatitis. Hum. Pathol. 24,1145-1148. Numazaki, Y, Shigeta, S., Kumasaka, T., Miyazawa, T., Yamanaka, M., Yano, N., Takai, S., and Ishida, N. (1968). Acute hemorrhagic cystitis in children: Isolation of adenovirus type 11. New Engl. J. Med. 278, 700-704. Numazaki, Y, Kumasaka, T., Yano, N., Yamanaka, M., Miyazawa, T., Takai, S., and Ishida, N. (1973). Further study on acute hemorrhagic cystitis due to adenovirus type 11. New Engl. ]. Med. 289, 344-347. O'Donnell, B., McCruden, E., and Desselberger, U. (1993). Molecular epidemiology of adenovirus conjunctivitis in Glasgow, 19811991. Eye 7 (Pt. 3, Suppl.), 8-14. Ohori, N., Michaels, M., Jaffe, R., Williams, R, and Yousem, S. (1995). Adenovirus pneumonia in lung transplant recipients. Hum. Pathol. 26,1073-1079. Osamura, T., Mizuta, R., Yoshioka, H., and Fushiki, S. (1993). Isolation of adenovirus type 11 from the brain of a neonate with pneumonia and encephalitis. Eur. J. Pediatr. 152, 496^99. Pacini, D., Dubovi, E., and Clyde Jr, W (1984). A new animal model for human respiratory tract disease due to adenovirus. /. Infect. Dis. 150, 92-97. Pilewski, J., Scott, D., Wilson, J., and Albelda, S. (1995). ICAM-1 expression on bronchial epithelium after recombinant adenovirus infection. Am. J. Respir Cell Mol. Biol. 12,142-148. Pingleton, S., Pingleton, W, Hill, R., Dixon, A., Sobonya, R., and Gertzen, J. (1978). Type 3 adenoviral pneumonia occurring in a respiratory intensive care unit. Chest 73, 554-555. Pinkerton, H., and Carroll, S. (1971). Fatal adenovirus pneumonia in infants: Correlation of histologic and electron microscopic observations. Am. ]. Pathol. 65, 543-548. Pinto, A., Beck, R., and Jadavji, T. (1992). Fatal neonatal pneumonia caused by adenovirus type 35. Arch. Pathol. Lab. Med. 116, 95-99. Prince, R. (1979). Evidence for an aetiological role for adenovirus type 7 in the mesenteric adenitis syndrome. Med. J. Aust. 2, 56-57. Purtilo, D., White, R., Filipovich, A., Kersey, J., and Zelkowitz, L. (1985). Fulminant liver failure induced by adenovirus after bone marrow transplantation. New Engl. J. Med. 312,1707-1708. Richmond, S., Dunn, S., Caul, E., Ashley, C , Clarke, S., and Seymour, N. (1979). An outbreak of gastroenteritis in young children caused by adenoviruses. Lancet 1,1178-1180.

Rodriguez Jr, F., Liuzza, G., and Gohd, R. (1984). Disseminated adenovirus serotype 31 infection in an immunocompromised host. Am. J. Clin. Pathol. 82, 615-618. Roos, R., Chou, S., and Rogers, N. (1972). Isolation of an adenovirus 32 strain from human brain in a case of subacute encephalitis. Proc. Soc. Exp. Biol. Med. 139, 636-640. Ross, J., Potter, C , and Zachary, R. (1962). Adenovirus infection in association with intussusception in infancy. Lancet 2, 221-223. Rowe, W, Huebner, R., Gilmore, L., Parrott, R., and Ward, T. (1953). Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc. Soc. Exp. Biol. Med. 84, 570-573. Sahler, O., and Wilfert, C (1974). Fever and petechiae with adenovirus type 7 infection. Pediatrics 53, 233-235. Sanchez, M., Erdman, D., Torok, T., Freeman, C , and Matyas, B. (1997). Outbreak of adenovirus 35 pneumonia among adult residents and staff of a chronic care psychiatric facility. /. Infect. Dis. 176, 760-763. Schonland, M., Strong, M., and Wesley, A. (1976). Fatal adenovirus pneumonia: Clinical and pathological features. South Afr. Med. ]. 50,1748-1751. Sencer, S., Haake, R., and Weisdorf, D. (1993). Hemorrhagic cystitis after bone marrow transplantation: Risk factors and complications. Transplantation 56, 875-879. Shields, A., Hackman, R., Fife, K., Corey, L., and Meyers, J. (1985). Adenovirus infections in patients undergoing bone-marrow transplantation. New Engl. J. Med. 312, 529-533. Shimizu, C , Rambaud, C , Cheron, G., Rouzioux, C , Lozinski, G., Rao, A., Stanway, G., Krous, H., and Bums, J. (1995). Molecular identification of viruses in sudden infant death associated with myocarditis and pericarditis. Pediatr. Infect. Dis. J. 14, 584-588. Simila, S., Junppila, R., Salmi, A., and Pohjonen, R. (1970). Encephalomeningitis in children associated with an adenovirus type 7 epidemic. Acta Paediatr Scand. 59, 310-316. Simila, S., Ylikorkala, O., and Wasz-Hockert, O. (1971). Type 7 adenovirus pneumonia. /. Pediatr 79, 605-611. Singh-Naz, N., Brown, M., and Ganeshananthan, M. (1993). Nosocomial adenovirus infection: Molecular epidemiology of an outbreak. Pediatr Infect. Dis. ]. 12, 922-925. Steigbigel, R., LaScolea Jr, L., and Marx, G. (1978). Renal hematuria associated with adenovirus 7a infection. Am. J. Dis. Child. 132, 208-210. Sterner, G. (1962). Adenovirus infection in childhood: An epidemiological and clinical survey among Swedish children. Acta Paediatr, Suppl. 142,1-30. Sterner, G., Gerzen, P., Ohlson, M., and Svartz-Malmberg, G. (1961). Acute respiratory illness and gastroenteritis in association with adenovirus type 7 infections. Acta Paediatr 50, 457-468. Strickler, J., Singleton, T, Copenhaver, C , Erice, A., and Snover, D. (1992). Adenovirus in the gastrointestinal tracts of immunosuppressed patients. Am. J. Clin. Pathol. 97, 555-558. Strohl, W, and Schlesinger, R. (1965). Quantitative studies of natural and experimental adenovirus infections of human cells, II: Primary cultures and the possible role of asynchronous viral multiplication in the maintenance of infection. Virology 26, 208-220. Tomoe, H., Onitsuka, S., Nishino, S., Suzuki, M., Yago, R., Goya, N., and Toma, H. (1994). Adenovirus-induced kidney graft pyelonephritis following renal transplantation [Japanese]. Hinyokika Kiyo [Acta Urologica Japonica] 40,1005-1008. Towbin, J., Griffin, L., Martin, A., Nelson, S., Siu, B., Ayres, N., Demmler, G., Moise Jr, K., and Zhang, Y.-H. (1994). Intrauterine adenoviral myocarditis presenting as nonimmune hydrops fetalis: diagnosis by polymerase chain reaction. Ped. Infect. Dis. J. 13,144-150.

Adenoviruses Uhnoo, L, Olding-Stenkvist, E., and Kreuger, A. (1986). Clinical features of acute gastroenteritis associated with rotavirus, enteric adenoviruses, and bacteria. Arch. Dis. Child. 61, 732-738. Umekawa, T., and Kurita, T. (1996). Acute hemorrhagic cystitis by adenovirus type 11 with and without type 37 after kidney transplantation. Urologia Internationalis 56,114-116. Usami, T., Mugiya, S., Ushiyama, T., Suzuki, K., and Fujita, K. (1997). Systemic infection resembling hemorrhagic fever with the renal syndrome caused by adenovirus type 11. /. Urol. 157, 617-618. Van der Veen, J., and Lambriex, M. (1973). Relationship of adenovirus to lymphocytes in naturally infected human tonsils and adenoids. Infect. Immunol. 7, 604-609. Van Zaane, D., and Van der Veen, J. (1962). Quelques symtomes cliniques particuliers chez les enfants attenints d'une infection a adenovirus. Presse Med. 70,1021-1022. Warner, J., and Marshall, W. (1976). Crippling lung disease after measles and adenovirus infection. Br J. Dis. Chest 70, 89-94. Weber, J., and Liao, S. (1969). Light and electron microscopy of virusassociated, intranuclear paracrystals in cultured cells infected with types 2, 4, 6, and 18 human adenoviruses. Can. J. Microbiol. 15, 841-845. Weber, J., and Stich, H. (1969). Electron microscopy of cells infected with adenovirus type 2. /. Virol. 3,198-204. West, T., Papasian, C , Park, B., Parker, S., and Kremzier, J. (1985). Adenovirus type 2 encephalitis and concurrent Epstein-Barr virus infection in an adult man. Arch. Neurol. 42, 815-817.

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Whitelaw, A., Davies, H., and Parry, J. (1977). Electron microscopy of fatal adenovirus gastroenteritis. Lancet 1, 361. Wigger, H., and Blanc, W. (1966). Fatal hepatic and bronchial necrosis in adenovirus infection with thymic alymphoplasia. New Engl. J. Med. 275, 870-874. Wright, J., Couchonnal, C , and Hodges, G. (1979). Adenovirus type 21 infection: Occurrence with pneumonia, rhabdomyolysis and myoglobinuria in an adult. JAMA 241, 2420-2421. Wright Jr, H., Beckwith, J., and Gwinn, J. (1964). A fatal case of inclusion body pneumonia in an infant infected with adenovirus type 3. /. Pediatr 64, 528-533. Yagisawa, T., Nakada, T., Takahashi, K., Toma, H., Ota, K., and Yaguchi, H. (1995). Acute hemorrhagic cystitis caused by adenovirus after kidney transplantation. Urologia Internationalis 54, 142-146. Yolken, R., Bishop, C , Townsend, T., Bolyard, A., Bartlett, J., Santos, C , and Saral, R. (1982). Infectious gastroenteritis in bone-marrow transplant recipients. New Engl. J. Med. 306,1009-1012. Yunis, E., Atchison, R., Michaels, R., and DeCicco, F. (1975). Adenovirus and ileocecal intussusception. Lah. Invest. 33, 347-351. Zaltzman, J., Honey, R., and Struthers, N. (1994). Adenovirus-induced hemorrhagic cystitis in association with cytomegalovirus infection in renal allograft recipients. Transplantation 57,1405-1406. Zarraga, A., Kerns, F., and Kitchen, L. (1992). Adenovirus pneumonia with severe sequelae in an immunocompetent adult. Clin. Infect. Dis. 15, 712-713.

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C H A P T E R

15 Retroviruses: General Principles

r

he family Retroviridae is comprised of three subfamilies. These are the spumaviruses, the oncoviruses, and the lentiviruses. The spumaviruses are curiosities. To the best of our knowledge, they play no role in disease of humans and lower animals. The oncoviruses are endogenous to a variety of animal species, including subhuman primates. They are the traditional tumor viruses that were the subject of considerable cancer research in the past. This large and complex subfamily of viruses infect a wide variety of animal species (at last count, over 20) but are not known to be pathogenic for humans. The oncoviruses are now divided into five genera. The

virions are often carriers of integrated protooncogenes that represent biologically important genes acquired from the animal host in which the virus is replicated. Familiar agents of historical importance are the Rous sarcoma virus of avian species, various murine leukemia viruses, the feline leukemia virus, and the mouse mammary tumor virus. The human T cell leukemia/lymphoma viruses 1 and 2 (HTLV-1 and HTLV-2) are pylogenetically related to agents endogenous to certain subhuman primates. They are genetically distinct and only distantly related to the two lentiviruses of great human importance: human immunodeficiency viruses types 1 and 2 (HIV-1

FIGURE 15.1 Budding of HIV-1 virions from the surface of a cultured cell as demonstrated by scanning electron microscopy (7000x). Reprinted with permission and through the courtesy of P. Roingeard and D. Brand.

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and HIV-2). The genome of the human T cell leukemia/lymphoma viruses and the human immunodeficiency viruses are similar. The RNA of the virion is comprised of three critical structural genes {gag, pol, and env) and two accessory regulatory genes. The pol gene codes the reverse transcriptase enzyme that is characteristic of this family of viruses. This enzyme converts the two identical 9.2-kb single strands of viral RNA to double-stranded DNA and, in turn, integrates this DNA with the DNA of the cell, ultimately resulting in formation of the progeny provirus. The env gene is responsible for formation of the viral envelope, and the gag gene directs synthesis of the virions matrix protein. The regulatory genes of HIV-1 and -2 {rev and tat) and of HTLV-1 and -2 {tax and rex) play important roles in the pathogenicity of the viruses, and contribute to the unique functions of the virus. HIV-1 and -2 have sev-

eral luxury genes, the roles of which are incompletely defined, but they no doubt endow the virion with important biological properties yet to be elucidated. Structurally, the nucleocapsid of the virion encompasses its genetic reservoir. It is surrounded by the envelope, which is formed as the virus buds from the surface of the infected cell (Figures 15.1 and 15.2). The lentiviruses of hun\ans are similar to several virus agents of importance in domestic animals. These include visna/maedi, sheep viruses of historical significance, and the more recently recognized feline and bovine immunodeficiency viruses. There are, in addition, a number of endogenous lentiviruses of subhuman primates. Several of these, the simian immunodeficiency viruses (SIVs), have proven to be valuable models for HIV-1 infection in hunnans.

FIGURE 15.2 Murine leukemia virus, a nonhuman retrovirus. (A) Budding of virions from the plasma membrane of a cultured cell. (B) Electron microscopy of negatively stained virions showing surface features. (C) The concentric arrangement of the core, shell, and nucleoid of the virion. (D) The hexagonal arrangement of the subunits of the shell around the core of the virion is recognizable. Bars = 100 nm. Reprinted with permission from and through the courtesy of H. Frank and W. Schafer.

C H A P T E R

16 Human Immunodeficiency Viruses HUMAN IMMUNODEFICIENCY VIRUSES 1 AND 2 (HIV-1 AND HIV-2) 205 HIV-1 CLINICAL COURSE IN ADULTS 207 HIV-1 CLINICAL COURSE IN INFANTS AND CHILDREN 210 PERSISTENT GENERALIZED LYMPHADENOPATHY (PGL) (SYN. PROGRESSIVE GENERALIZED LYMPHADENOPATHY) 212 DISEASES OF THE HEMATOPOIETIC SYSTEM 215 DISEASES OF THE CENTRAL NERVOUS SYSTEM 216 Acute Meningitis 216 HIV-1 Encephalopathy 216 Cognitive/Motor Complex (Syn. Dementia Complex) 217 Myelopathy and Myelitis 219 Neuropathy 219 Myositis 220 Opportunistic CMV Infections of the Central and Peripheral Nervous Systems 221 DISEASES OF THE RESPIRATORY TRACT 222 Diffuse Alveolar Damage (DAD) 222 Lymphoid Interstitial Pneumonia (LIP), Nonspecific Interstitial Pneumonia (NIP), Follicular Bronchitis/Bronchiolitis (FBB) 222 Pulmonary Hypertension and VascularOcclusive Disease 223 Opportunistic Infections of the Lung 225 DISEASES OF THE HEART 226 DISEASES OF THE VASCULATURE 227 DISEASES OF THE KIDNEY 228 DISEASES OF THE TESTIS 229 DISEASES OF THE DIGESTIVE TRACT 230 TUBULORETICULAR STRUCTURES (TRSs) AND CYLINDRICAL CONFRONTING CISTERNAE (CCC) 231 LYMPHOMAS 231 KAPOSI'S SARCOMA 232 CERVICAL CANCER 234 REFERENCES 234

promiscuity in the early 1980s. The origin of the virus is shrouded in mystery. Very recent evidence suggests that it may have been a naturally infected chimpanzee. The route by which it gained access to the homosexual community is unknown. However, the environment for the dissemination by sexual means of this heretofore unrecognized highly infectious agent was propitious at the time. The clinical appearance of cases of Kaposi's sarcoma (due to sexually transmitted HHV-8; see Chapter 12) and Pneumocystis carinii pneumonia (originating from obscure sources) proved to be harbingers of the cellular immune deficiency caused by the agent originally termed human T cell lymphotropic virus (HTLV-3) and later. Type 1 Human Deficiency Virus (HIV-1). The clinical term Acquired Immunologic Deficiency Syndrome (AIDS) was coined soon thereafter, but the clinical definition of this syndrome underwent several revisions as our knowledge grew. The appearance of the virus in male homosexuals in San Francisco was soon followed by outbreaks in Los Angeles and New York, where concentrations of sexually active male homosexuals clustered. The pandemic had begun, and it was not long before a new population of susceptibles turned up with AIDS, that is, the sufferers of hemophilia who were recipients of blood concentrates derived from pooled human plasma. As events unfolded, the source of the blood products used for treatment of these unfortunate patients all too frequently proved to be a donor with a subclinical HIV-1 infection. In 1985, HIV-1 was recovered in laboratories in Paris and Bethesda, Maryland. Sadly, the unfortunate controversy of priority tarnished the brilliance of the pathfinding virological work conducted on both sides of the Atlantic. The more recent history of the worldwide AIDS pandemic is only too well known. While HIV-1 continues to be a threat for male homosexuals engaging in sexual interactions without prophylaxis, it is a latent hazard for the i.v. drug abuser and his/ her sexual consort. Heterosexual intercourse is increasingly serving as a major route of viral transmission, by means of semen, saliva, and genital secretions, in

H U M A N IMMUNODEFICIENCY VIRUSES 1 A N D 2 (HIV-1 A N D HIV-2) The sexual revolution of the 1970s set the stage for the appearance of HIV-1 in epicenters of homosexual PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

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populations where the background prevalence of infection is high and promiscuity an accepted practice. Increasingly, sex workers (e.g., prostitutes) serve as the vector. In Africa, where the prevalence of subclinical infection among the members of the general population is exceedingly high, transmission appears to occur largely by heterosexual routes. A recent report from New York illustrates the changing patterns of viral dissemination, as reflected in the prevalence of autopsies on patients with AIDS in a major metropolitan hospital. During the 1980s, male homosexuality was a risk factor in 25% of HIV-1 cases coming to autopsy, whereas in the period 1989-95 it comprised only 13% of AIDS autopsies. Twenty-four percent of AIDS autopsies were done on heterosexual patients in the 1980s, while in the 1989-95 time period the incidence was 36%. The number of cases of AIDS occurring in intravenous drug users proved to be stable over the period 1980-96 (Concepcion et al., 1996). As illustrated by Tables 16.1, 16.2, and 16.3, opportunistic infections are the hallmark of the AIDS syndrome. In these patients, the commonest lifetime opportunistic infection is Pneumocystis carinii, with lifethreatening pneumonia occurring in about two-thirds of AIDS cases. Kaposi's sarcoma develops in about 50% of cases, and disseminated infections by bacteria of the Mycobacterium avium complex or Mycobacterium tuberculosis are documented in approximately a third. Cytomegalovirus-related disease in various organ systems are observed in 20% of patients with AIDS, although subclinical infections with this virus exist in the majority of those with clinical AIDS. As discussed in more detail below, the number of cases of Kaposi's sarcoma has decreased since the early 1980s, whereas mycobacterial infections are increasingly common. Undoubtedly, the opportunistic infections of importance to AIDS patients will continue to change as new antimicrobial therapies are introduced and treatment of the primary disease improves. As with other lentiviruses, HIV-1 and its less common sibling, HIV-2, possess two identical strands of RNA of about 9.2 kb that are converted into a proviral double-stranded DNA in the nucleus of the infected cell by a unique enzyme, reverse transcriptase, during the early stages of replication. The viruses exhibit similar morphological features and are approximately 120 nm in diameter. An enormous amount of new information has accumulated on the composition of the human lentivirus genome, and on the genes unique to both HIV-1 and HIV-2. The virus genes code for a functionally complex panoply of enzymes and proteins that are elaborated and produced during the course of replica-

TABLE 16.1 C o m m o n Opportunistic Central N e r v o u s S y s t e m Infections and D i s e a s e of Patients w i t h A I D S (in order of frequency) Clinical/pathological process

Etiologic agents

Encephalitis and polyradiculitis

Cytomegalovirus Herpes simplex, type 1 (Chapters 7, 8)

Pseudotumor

Toxoplasma gondii

Meningitis

Cryptococcus neoformans Mycobacterium avium complex

Progressive leukoencephalopathy

Papovirus (BK, JC) (Chapter 22)

B cell lymphoma

Epstein-Barr virus (Chapter 9)

TABLE 16.2 C o m m o n Opportunistic Lung Infections and D i s e a s e i n Patients w i t h A I D S (in order of frequency) Clinical/pathological process

Etiologic agents

Interstitial pneumonia

Cytomegalovirus (Chapter 8) Pneumocystis carinii

Abscess-forming pneumonia with granulomatous and purulent inflammation

Cryptococcus neoformans Mycobacterium tuberculosis Histoplasma capsulatum Coccidioides immitis

Purulent pneumonia with or without abscesses

Streptococcus pneumoniae Haemophilus influenzae Pseudomonas aeruginosa Klebsiella pneumoniae Staphylococcus sp.

TABLE 16.3 C o m m o n Opportunistic D i g e s t i v e Tract Infections i n pre-AIDS and A I D S (in order of prevalence) Oral/oral pharynx^ Candidiasis

Intestinal^ Candidiasis

Hairy cell leukoplakia (Chapter 9)

Cytomegalovirus (Chapter 8)

Kaposi's sarcoma (Chapter 12)

Microsporidiasis

Melanotic macules

Mycobacterium avium complex

Herpes simplex virus, type 1 (Chapter 7)

Cryptosporidia

Condyloma accuminatum (Chapter 21) Amoeba sp. Molluscum contagiosum (Chapter 25) Bacterial glossitis ''Barone et al (1990). ^Greenson et al (1991).

Salmonella sp.

Human Immunodeficiency Viruses

tion. The enveloping lipid membrane of the virion and its constituent glycoproteins are critical to viral attachment and replication, as well as the tropism of the virus for the specific receptors on the target cell. Of these, the external surface glycoprotein, gpl20, and the transmembrane glycoprotein, gp41, are key. In addition, the membrane incorporates several of the important marker antigens of the virus. Infection of susceptible cells by HIV-1 (and, presumably, HIV-2) requires interaction of the major capsid glycoprotein, gpl20, with the CD44- receptor on its surface. Other receptors of the CCR4/CCR5 chemokine family serve as coreceptors and are required for uptake of the virus by the cell. The coreceptor for virions that are macrophage-tropic are CCR5, whereas the comparable coreceptor for the CD4+ cell is CCR4. These receptors are found on the dendritic cells (the so-called Langerhans cells) of the skin and mucus membranes that are the usual primary site of infection. The gp41 protein also participates by facilitating transport of the virion across the plasma membrane, where it is subsequently uncoated. The gene coding membrane glycoprotein gpl20 has highly mutable segments that serve to alter the antigenic makeup of the virus. Since the antigens on the surface of the virions in an individual patient are in an evolutionary state of flux, traditional host immune mechanisms often prove impotent as control measures. Major antigenic differences in gpl20 also serve as the basis for classification of the viral genotypes currently catalogued by the designations A through I. These represent families of viruses that predominate in various regions of the world. There is currently no evidence to indicate that the members of these various families differ in pathogenicity (Janssens et aU 1997). HIV-2 is a genetically distinct lentivirus closely related to a simian immunodeficiency virus endemic in West African Sooty Mangabey monkeys. While virologically similar to HIV-1, the RNA molecular sequences of the two viruses differ. HIV-2 occurs commonly in the coastal equatorial countries of the West African bulge (Guinea Bissau, Senegal, and Gambia), with a much lower frequency elsewhere in Africa, Europe, and North America. HIV-2 is believed to spread by heterosexual intercourse. The virus appeared before HIV-1 in its endemic areas of West Africa, but it is gradually diminishing in prevalence among the population. On the other hand, HIV-1 is spreading into West Africa, even though the incidence of new infections is still substantially lower than in East and Central Africa. Among women of reproductive age in Abidjan, Cote de Ivoire during 1992, the incidence of HIV-2 infection was 1.7%, whereas 9.4% of the same population had serological evidence of HIV-1 infection

207

(De Cock et al, 1993). In the United States, the prevalence of infection, as determined by screening of blood donors between 1992 and 1995, was 2 cases per 7.4 x 10^ units of blood. HIV-2-associated AIDS has not been reported in North America (O'Brien et a/., 1992). HIV-2 appears to be less infectious, and blood concentrations of the virus tend to be lower than in persons infected with HIV-1. While the development of AIDS from HIV2 is well documented in its endemic region, the clinical illness seems to be less severe (De Cock et al, 1993; Ariyoshi et al, 1996), although the distribution and types of lesions occurring in HIV-2- and HIV-1-infected patients with AIDS are similar (Lucas et al, 1991). Thus, the epidemiological and clinical evidence strongly suggests that HIV-2 originated in West Africa as the result of one or more interactions between humans and the Sooty Mangabey monkey. Because of its relatively low infectivity the virus may have now reached an epidemic peak and is on the decline. There is little evidence to suggest that it will spread pandemically in a fashion similar to HIV-1.

HIV-1 CLINICAL COURSE IN ADULTS HIV-1 infections typically are acquired when the virus transits the genital or oral mucosa and is taken u p by the dendritic Langerhans cells in the lamina propria of the epithelium. The transmitted viruses (the socalled R5 strains) customarily are the macrophagetropic variants that use the gpl20 receptor and the CCR5 coreceptor on the plasma membrane of the Langerhans cell to facilitate uptake. The nasopharyngeal tonsils and adenoids are also richly endowed with dendritic cells, and no doubt can serve as a site of virus entry. Thus, the Langerhans macrophage appears to ''screen" a heterogenous population of viruses, permitting entry of R5 variants, agents that are much less pathogenic than the T cell-tropic syncytialforming R4 variant viruses that evolve relatively late in the course of a naturally acquired infection. Persons whose cells lack CCR5 coreceptors through a homogenous deletion of its genes are relatively resistant to infection with "wild" strains of HIV-1. Clearly, defects in cytokine receptors, of which there may be many, dictate, at least in part, the ultimate course of the infection (McNicholl et al, 1997; Smith et al, 1997). After acquisition of the virus, Langerhans cells fuse with circulating CD4+ T lymphocytes, which, in turn, disseminate the virus. Lymph nodes draining the site of infection yield virus when tested 48 hours after exposure.

208

Pathology and Pathogenesis of Human Viral Disease 1200

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FIGURE 16.1 Clinical course of HIV-1 infection in typical patient demonstrating a dramatic reduction of CD4+ cells during the acute episode shortly after infection and subsequent gradual deterioration in the CD4+ T cell reserves until the AIDS syndrome evolves. The concentrations of virus in the blood increase dramatically during the ''acute'' episode of illness that occurs shortly after infection, and during the terminal AIDS syndrome. The concentrations of HIV-1 RNA in the blood parallel the virus concentrations. Reprinted with permission from and through the courtesy of A. Fauci, MD.

and viremia can be documented shortly thereafter. Persons who inadvertently acquire the virus parentally from patients with advanced HIV-1 infections may receive an inoculum of the more pathogenic R4 variants. Figure 16.1 depicts certain parameters of the typical clinical course of disease progression. In 50 to 70% of patients, the first evidence of HIV-1 infection proves to be a "mononucleosis-like" syndrome that develops several weeks after exposure and persists for about 2 to 4 weeks thereafter (mean duration = 25 days) (Table 16.4) (Clarke et al, 1991; Vanhems et al, 1997; Kahn and Walker, 1998). The syndrome is often not diagnosed because HIV-1 antibodies are not yet detectible in the blood. There is a high level of viremia with widespread dissemination of the virus, but the major sites of virus replication at this stage are undefined. Virological and immunological events may dictate the future course of the infection. CD8+ cytotoxic T cells targeted against the virus appear to play a key role in the host's initial response to the virus. As they appear in the blood, viremia abates. Humoral antibodies appear much later and do not seem to be important actors in the body's initial response to infection. The relative height of the viremia in the early stages of the infection (often greater than 10^ RNA molecules per milliliter) correlates inversely with the duration of patient survival (Wong et al, 1996). Although the

TABLE 16.4 Percentage of Patients w i t h Acute HIV-1 Infections Manifesting S i g n s and S y m p t o m s >50%

Fever (temperature >38°C) Lethargy Cutaneous rash Myalgia Headache

>25%-50%

Pharyngitis or sore throat Cervical adenopathy Arthralgia Oral ulcer Odynophagia

5-25%

Weight loss Nausea Diarrhea Night sweats Cough Anorexia Abdominal pain Vomiting Photophobia Sore eyes Tonsillitis Depression Dizziness Adenopathy Oral candidiasis Genital ulcers

Adapted with permission from Vanhems et al. (1997).

Human Immunodeficiency Viruses

relative pathogenicity of the virus may be a factor accounting for the duration of the latency period, as has been found in a few cases (Cao ei al, 1995; Operskalski et al, 1997), host factors appear to be of primary importance. Of these, the major histocompatibility complex (MHC) type of host cells, and its complement of cytokine receptors, may be critical. At present, evidence supporting the notion that histocompatibility antigens may dictate the intensity of the infection or the luxuriance of the immune response is limited and based largely on the finding of a loose but statistically significant association of AIDS with certain MHC types. Several mechanisms whereby molecules encoded by MHC genes might predispose an individual to infection are worthy of brief consideration. First, certain MHC class I and II alleles could limit progression of the HIV-1 infection by serving as a restriction element for one of several immunodominant HIV-1 T helper or cytolytic T cell epitopes (thus promoting a salutary immune response to HIV-1). Alternatively, a lack of protective MHC alleles could predispose to development of AIDS because of a poor immune response. Certain MHC alleles could also predispose an individual to pathogenic immune responses against viral epitopes in various tissues. Lack of an AIDS-promoting MHC allele type might protect against pathogenic responses of the immune system to HIV-1 (Haynes et al, 1996; Hill, 1996). The duration of the clinical latency period and its outcome appear to be dictated at least in part by the cellular and antibody-mediated immune responses to HIV-1 antigens. CD8+ cytolytic T cells are of paramount importance, for they can destroy virus-producing dendritic cells. The elaboration of neutralizing antibodies also may contribute to control and dissemination of extracellular virus, but the role of humoral immunity is less clearly defined. It may also participate in antibody-dependent cell-mediated immunity. For unknown reasons, individual infected patients differ substantially in terms of the briskness and degree of their immune response to the virus. This ultimately determines virus concentrations in the blood and tissue, considerations that appear to influence the duration of the latency period. Differences in the immunoresponsivity of the host are compounded by evolving rapid changes in the antigenic makeup of the virion due to the hypermutability of certain viral genes. On a continuous basis, this may abrogate the effectiveness of a previously elaborated protective antibody. One can envision an evolving immune response to an ever-changing series of viral epitopes.

209

About 10% of HIV-infected persons develop AIDS within 2 to 3 years after acquisition of the virus. Disease in these patients can be fulminating (Michael et al, 1997) with, for example, a rapidly progressive dementia (Bassiri et al, 1995; Holland et al, 1996). Roughly 10 to 15% of those infected with HIV-1 will remain AIDSfree indefinitely, whereas the remainder (about 75%) develop the disease with a median latency period of 10 years (Haynes et al, 1996). The basis for these differences in disease progression is currently unclear, although much new information relevant to the question is accumulating. Race and gender do not appear to influence susceptibility and progression of HIV-1 infections. The factors that correlate with the duration of the latency period are: (1) the levels of cell-associated virus in the blood, and the plasma RNA concentration (Fang et al, 1997), (2) the relative proportion of circulating syncytium-inducing (SI) variants of the virus (Koot et al, 1996; Spijkerman et al, 1998). Patients infected initially with the syncytium-inducing HIV-1 variant usually develop AIDS within 5 years. These variants have the ability to infect and replicate in both macrophages and T cells (Yu et al, 1998). The tendency of infected CD4+ cells to undergo apoptosis (Liegler et al, 1998) is an additional factor. Certain virus variants are highly pathogenic and have specific tropism for the CD4+ T lymphocytes. The blood virus and RNA concentrations correlate inversely with CD4+ cell counts, and the amounts increase substantially as the CD4+ cell numbers fall to a critical level. At this stage, the CD4+-tropic SI virions predominate in the blood and tissues. Longterm HIV-1 survivors possess CD4+ T cells that are resistant to apoptosis, whereas the disease in patients with T cells that readily undergo apoptosis tends to progress more rapidly (Liegler et al, 1998). AIDS in the adult is a clinical syndrome that customarily appears when CD4+ lymphocyte counts in the blood fall to less than 200/mm^ (see Figure 16.1) and high concentrations of virus and viral RNA are found in the blood and tissues (Bagasra et al, 1996). It is usually manifest as one or a combination of opportunistic infections and, to a variable extent, Kaposi's sarcoma and malignant lymphoma. The U.S. Centers for Disease Control and Prevention (CDC) has established a schema for categorizing individual cases, primarily for use in research and epidemiology (Table 16.5). It distinguishes between category B illnesses and lifethreatening infections of major organ systems of category C (Table 16.6). In this chapter, the more common infections and neoplasms associated with AIDS will be considered below.

210

Pathology and Pathogenesis of Human Viral Disease TABLE 16.5 1993 R e v i s e d Classification S y s t e m for HIV-1 Infection and Expanded A I D S Surveillance Case D e f i n i t i o n for A d o l e s c e n t s and A d u l t s Clinical category (A) asymptomatic acute (primary) HIV or PGL''

CD4+ Tcell category

(B) symptomatic. not (A)or(C) conditions

(C) AIDS-indicator conditions*"

(1)>500/^1

Al

(2)200-199/^1

A2

Bl B2

CI C2

(3)<200/^l

A3

B3

C3

"PGL = persistent generalized lymphadenopathy. Clinical category A includes acute (primary) HIV infection. ^See Table 16.6.

TABLE 16.6 C D C Category C Example Infections (see Table 16.5) Candidiasis a. vulva or vagina b. oropharynx and esophagus c. bronchi, trachea & lungs Cytomegalovirus infections of major organ systems, including the eye (retina) (Chapter 8) M. tuberculosis and M. avium complex infections of major organ systems Herpes simplex virus, oropharynx, esophagus, respiratory tract (Chapter 7) Brain Abscess — Toxoplasma gondii Progressive multifocal leukoencephalitis — JB and BK polyomaviruses Cryptococcus Digestive tract Crytosporidiosis, Microsporidiosis Isophora sp. Herpes simplex, type 1 (Chapter 7) Lung Bacterial pneumonia Pneumocystis carinii Histoplasma capsulatum, Coccidioidomycosis immitis Cancer Cervix — HPV type 16 and 18 (Chapter 21) Kaposi's sarcoma — HHV-8 (Chapter 12) Non-Hodgkin's and Hodgkin's lymphomas — EBV

HIV-1 CLINICAL COURSE IN INFANTS AND CHILDREN According to the World Health Organization, by the year 2000, 1 x 10^ children worldwide will have been infected with HIV-1. Roughly 7 x 10^ infants are born to HIV-1-infected women each year in the United States. About 25% of these mothers transmit the virus to their

offspring either during the intrapartum period or by postpartum breast feeding (Davis et al, 1995; Blanche et al, 1994; Pitt et al, 1997; Peckham and Gibb, 1995; Wilfert et al, 1994). In contrast, the incidence of neonatal infection among infants born to HIV-1-positive women approaches 50% in the developing regions of the world, where many important risk factors exist. In 1992, HIV-1 infection was the 11th leading cause of

Human Immunodeficiency Viruses

infant mortality in the United States, and over 90% of these infections were acquired vertically from the mother. Approximately 50% of HIV-1-infected nonpregnant women shed viral RNAin cervical secretions (Goulston ei al., 1998). However, advanced disease in the mother as reflected by low CD4+ blood lymphocyte counts and high blood concentrations of virus (and/or viral RNA) are the most critical factors associated with infection in the offspring (Mayaux et al, 1996,1997; Steketee et al, 1997; Sperling et al, 1996). And, as noted above, the relative pathogenicity of the virus may be a consideration (Balotta et al, 1997). There is an increased prevalence of infection in the infant if the chorioamniotic membranes are ruptured for a prolonged period preceding birth (Umans-Eckenhausen and Lafeber, 1996; Landesman et al, 1996). And, as of yet, genetic influences on the immune responsivity of the child may be critical (Bryson et al, 1995; Pizzo, 1997). Interestingly enough, class I HLA concordance between mother and infant increases perinatal transmission (MacDonald et al, 1998). Most fetal infections occur late in pregnancy or during parturition; however, infection of embryos as early as the 8th week of gestation has been documented (Langston et al, 1995). In situ hybridization studies of tissues from a 12-week-old pregnancy demonstrated virus in multiple organs as well as in the placenta and decidual endometrium. In one study, two of three midterm HIV-1-infected fetuses showed growth retardation with small placentas and a strikingly abnormal placentakfetal weight ratio. The trophoblasts, endothelial cells, and Hofbauer cells evidenced viral antigens, or RNA, and are believed to be infected. However, the role of the placenta in the pathogenesis of infection in the offspring is unclear. The spontaneous abortion rate in pregnancies between 12 and 32 weeks of gestation is about 9%, whereas the incidence of spontaneous abortions in a control population of uninfected pregnant women proves to be less than 3% (Langston et al, 1995). Substantial loss occurs in the third trimester of pregnancy among congenitally infected fetuses. Roughly half the newborns who are infected acquire the virus in utero. These infants usually have detectable virus or RNA in the blood within 48 hours of parturition. The remaining infants are infected during birth, or from breast milk. As noted above, about 50% of HIV-infected women have virus in cervical and vaginal secretions. Generally, this occurs when the concentration of

211

virus in the blood is also relatively high (Douglas and King, 1992). Caesarean section reduces the risk for babies of infected mothers. In various studies, breast feeding is believed to account for some 7 to 22% of cases. The absence of virus or viral RNA in the blood for the first postpartum week strongly suggests that an infected infant acquired the virus during parturition or subsequently by breast feeding. When present, the most striking pathological finding in aborted relatively mature fetuses is profound atrophy of the thymus (Figure 16.2A-D) where in situ hybridization studies have documented infection. The organ is depleted of lymphocytes and the corticomeduUary junction is indistinct. There is a paucity or absence of Hassall's corpuscles and both acute inflammation and fatty infiltration of the parenchyma are often seen (Joshi and Oleske, 1985). Only about 15% of children born with an HIV-1 infection develop functional thymic deficiency as measured by CD4+ and CD8+ cell counts below the fifth percentile postnatally (of values in healthy children). The pathogenic basis for differences between individual children is unclear, but HIV-1 strains have been found to differ with regard to their ability to infect the thymic epithelium and impede thymogenesis (Nahmias et al, 1998). The outcome of a neonatal HIV-1 infection is largely dictated by the route of infection and the immune status of the infant, which most probably reflects the degree of thymic damage. As noted above, the height of the viral and HIV RNA blood concentrations (and viral antigens) (Papaevangelou et al, 1996) inversely correlates with survival time (Shearer et al, 1997; Dickover et al, 1996). Childhood HIV-1 infection is often a more precipitous disease than its counterpart in adults, in whom "latency periods" of 10 or more years are common. Seventy percent of infants with evidence of thymic deficiency are dead within 2 years of birth. They manifest the childhood AIDS syndrome accompanied by opportunistic infections. In contrast, only 37% of a control group with seemingly normal or near normal immune function die (Nahmias et al, 1998). They develop AIDS more slowly. Currently, about 50% of these young patients survive for as long as 10 years. In general, blood concentrations in children are higher than in adults. In the following section, I consider the major tissue changes that characterize HIV-1 infection in persons of all age groups. To provide perspective, the relative concentrations of viral RNA in the tissues at autopsy are summarized in Table 16.7.

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Pathology and Pathogenesis of Human Viral D i s e a s e

FIGURE 16.2 Comparison of thymic histology in HIV-positive and -negative fetuses. (A) Thymus from a 12-week-gestation HIV-1-negative fetus shows early architectural development with beginning condensation of cortical lymphocytes and aggregate of epithelial cells (center and inset) forming early Hassall's corpuscle (bar = 40 |Lim). Inset: bar = 10 jim. (B) Thymus from a 22-week-gestation HIV-1-negative fetus shows well-developed thymic architecture with well-defined cortex and medullary region containing abundant Hassall's corpuscles (multiple between arrows), bar = 40|Lim. (C) Thymus from a 15-week-gestation HIV-1-positive fetus shows marked depletion of lymphocytes with no evident corticomeduUary demarcation and no Hassall's corpuscles, bar = 40)im. (D) Thymus from a 27-week-gestation HIV-1-positive fetus shows somewhat shrunken cortex with blurring of corticomeduUary demarcation and relatively few Hassall's corpuscles (arrows) showing microcystic change. Reprinted with permission from Langston et al. (1995) through the courtesy of C. Langston MD.

PERSISTENT GENERALIZED LYMPHADENOPATHY (PGL) (Syn. PROGRESSIVE GENERALIZED LYMPHADENOPATHY)

During primary HIV-1 infection, the follicles of lymph nodes and other lymphoid organs are major sites of virus replication (Wenig et al, 1996). The follicular dendritic cell is the lead actor in the ensuing drama. It is "a cell that looks like an octopus, with tentacles or dendritic processes that are capable of surrounding lymphocytes" (Tingley, 1996). By mechanisms yet to be defined, these dendritic cells are believed to both present antigens to B cells and transfer the infectious virus to the CD4+ T cells. Tacchetti and colleagues (1997) claim that HIV-1-specific IgM generated locally by plasma cells from complement-fixing

immune complexes facilitate uptake of the virus by the dendritic cells. As noted above, when the dendritic cells sustain a productive infection, they are potential subjects for lysis by neighboring cytolytic CD8+ cells. The infected CD4+ T cells also may be targets for immune lysis, but they can die or undergo apoptosis by other mechanisms. Chemokines such as MlPla, MIP1.3, RANTES, and the interferon-inducing protein IP-16, which are generated by infected resident macrophages, most probably contribute to the pathologic response by attracting a flow of inflammatory cells to sites of virus replication. Thus, luxuriant accumulations of lymphocytes are found at loci of active virus replication during the early stages of the disease. But, as the infection progresses, the dendritic cells and macrophages are destroyed, and the lymphoid cells are reduced substantially in number.

213

Human Immunodeficiency Viruses

TABLE 16.7 Distribution of HIV-1 DNA in Representative Organs of Children and Adults at Autopsy after Death Due to AIDS and Opportunistic Conditions Geometric means HIV-1 DNA copies/ ng of tissue ( # p o s / # tested)

Organ Cerebrum Cerebellum Spinal cord Lung Heart Liver Kidney Spleen Lymph nodes Colon Skin Muscle Testis/ovary

Adults''

Children^

0 (0/3) 0(0/3) 0.3(1/3) 11 2.5(5/7) 4.4(4/5) 5.6(5/6) 99(7/7) 453(7/7) 10.6(6/7) 21.4(6/7) 1.7(3/7) 3.1(6/7)

38(6/7) 3 (5/8) 14.5(4/6) 42.5 (8/9) 8.2 (6/9) 10.3 (6/8) 13.4(8/9) 496 (9/9) 1631 (9/9) 140(8/9) 27.7(6/9) 7.5(5/9) 7.8(5/9)

Adapted with permission from Sei et al. (1994). Tatients died with opportunistic infections and Kaposi's sarcoma. ^Patients had HIV-1-associated encephalopathy.

Systemic lymphadenopathy occurs commonly during the prodromal stages of AIDS. As many as 40% of adult patients with AIDS experience lymphadenopathy at some time prior to the onset of the terminal stages of the disease. The PGL syndrome is defined as lymphadenopathy (with nodes greater than or equal to 1 cm in diameter) in extrainguinal noncontiguous sites for a period of 3 or more months in the absence of a contemporary infection or neoplasm, repeated immunization, persistent dermatitis, i.v. drug abuse, or treatment with certain therapies. The nodes often fluctuate in size and consistency, and can range to as large as 4 cm in diameter. Three morphologic patterns define the spectrum of lymph node changes observed by the pathologist, but none of the histologic features are pathognomonic of HIV-1 infection (loachim, 1990; Baroni and Uccini, 1993; Ewing et al, 1985; Stanley and Frizzera, 1986). These stages, variously termed either 1, 2, 3 or A, B, C, roughly parallel the evolution of the disease, with stage 3 (C) being a grave prognostic indicator. Changes similar to those in lymph nodes are found in the spleen (Burke, 1993) and nasopharyngeal/palatine tonsils (Wenig et ah, 1996). As discussed in the section concerned with AIDS in children, thymic atrophy is a prominent feature of the disease in younger age groups, but pathological information on the thymus in adults with HIV-1 infection is limited. Although macrophages and dendritic cells in the lymph nodes have

been consistently shown to be infected with HIV-1, simultaneous superinfection of nodal tissue with HHV1, HHV-6, HHV-8, CMV, and EBV, as well as a variety of bacteria and fungi, can contribute to, and complicate, the pathological picture. Summarized below are the cardinal morphological features of the lymphoid tissue: Stage 1 (A). Lymph nodes at this stage exhibit the nonspecific pattern of an acute viral infection (Figure 16.3A). Several features stand out. First, there is a florid seemingly explosive follicular hyperplasia with a prominent geographic configuration resulting in serpiginous and "hourglass" shapes to the follicles. These follicles are located in both the cortex and the medulla. The germinal centers are hyperplastic and show abundant numbers of mitoses, but they also exhibit extensive cytolysis with nuclear debris and occasional focal hemorrhage. Macrophages with "tingible bodies" are often prominent. Aggregates of B lymphocytes and plasma cells as well as rare accumulations of neutrophils are present. Hematophagocytosis and lymphophagocytosis are found in about 40% of patients with enlarged lymph nodes (Ewing et a/., 1985). The second fundamental feature of the follicle is attenuation of the mantle zones (Figure 16.3B), with occasional foci of lymphocyte penetration into the follicle. Finally, multinucleate cells typical of HIV-1 infection are commonly found (see Figure 16.4). In two studies, these virus-infected giant cells were seen in approxi-

214

Pathology and Pathogenesis of Human Viral Disease

B

FIGURE 16.3 (A) Acute lymphadenopathy that customarily occurs early in the course of an HIV-1 infection. There is marked follicular hyperplasia with a prominent irregular geographical configuration to the hyperplastic follicles. The mantle zone is disappearing. Reprinted with permission and through the courtesy of J. Said, MD. (B) An irregular follicle with loss of the mantle zone. Note the immunoblastic characteristics of the follicular cells. Reprinted with permission and through the courtesy of J. Said, MD. (C) Marked lymphoid depletion of a lymph node during the advanced stage of HIV-1 infection. (D) Spleen of the patient with advanced HIV-1 infection. Note the marked depletion of the lymphoid elements.

mately 60% of cases during this stage of the disease (Burke et al, 1994; Ewing et al, 1985). In morphometric studies of the parafollicular lymphoid population, Raphael and colleagues (1985) found a consistent inversion of the CD4+:CD8+ cell ratio. This feature is most probably unique to the hyperplastic lymph node of patients with AIDS. The European Lymphoma Study Group has further dissected staging by describing substages with and without fragmentation of the germinal centers (Ost et al, 1989). Stage 2 (B). This is a transitional stage reflecting residual features of stage 1, and the lymphoid depletion pattern of stage 3 described below. The follicles typically are small with decreased numbers of follicular center cells. The mantle zone cannot be defined, and fibrosis of the node is often evident. Stage 3 (C). This stage is customarily seen during clinical AIDS and is characterized by disappearance of

the lymphoid follicles and depletion of lymphocytes (Figure 16.3C). The entire lymph node exhibits prominent sinusoids and cords. Similar changes are found in the spleen (Figure 16.3D). Sinus histiocytosis and erythrophagocytosis are almost invariably present. Plasma cells, Russell bodies, neutrophils, and an occasional eosinophil are observed in the stroma of the node. As in stages 1 and 2, multinucleate cells typical of HIV-1 infection are seen in the majority of cases (Figure 16.4). In addition, the effects of systemic opportunistic infections and Kaposi's sarcoma may be evident. The observations recorded above describe the lymph node pathology in persons with seemingly typical pre-AIDS and AIDS. In recent years, it has become apparent that a small proportion of those who contract HIV-1 experience a nonprogressive infection of as long as 20 years or more. Pantaleo and colleagues (1995) characterized the lymph node pathology in a series of

Human Immunodeficiency Viruses

215

F I G U R E 16.4 Medullary region of a hyperplastic lymph node with prominent multinucleate polykaryocytes. The multinucleate cells have no distinguishing characteristics and vary in configuration. They represent fused macrophages. Reprinted with permission and through the courtesy of J. Said, MD.

long-term survivors who had exceedingly low concentrations of HIV-1 RNA in the blood plasma. Morphologically, the lymph nodes showed quite a different pattern. The germinal centers were relatively small and tended to assume a round or oval configuration. Evidence of infection of dendritic cells in these germinal centers could be demonstrated by in situ hybridization, but often with great difficulty. Thus, it is clear that virus replication is reduced in comparison to adults with typical AIDS. These findings are consistent with the notion that the dendritic cells in lymph nodes and other lymphoid organs (and, most probably, the skin), are major sites of virus replication in the HIV-1-infected subject (Sei et al, 1994).

DISEASES OF THE HEMATOPOIETIC SYSTEM Anemia, neutropenia, and thrombocytopenia are common in AIDS, but they occur infrequently during the latent stages of HIV-1 infection. As might be expected, pathogenesis is usually multifactorial, for the changes may be a nonspecific manifestation of chronic illness and opportunistic infections. The anemia is

rarely significant clinically, but thrombocytopenia occasionally results in bleeding problems (Glatt and Anand, 1995). The bone marrow in patients with AIDS usually exhibits both granulocytic and erythrocytic hyperplasia. Lymphoid accumulations, eosinophils, and plasma cells are commonly found in increased numbers (Geller et al, 1985; Khalil et al, 1996). The fat in the bone marrow often shows serous atrophy, most probably a reflection of the generalized inanition of these patients (Mehta et al, 1992). Megakaryocytes are infected by HIV-1 and may be targets for cytolytic T cells, but this possibility has yet to be evaluated. Examination of the bone marrow of HIV-1-infected patients with AIDS consistently reveals the so-called "naked nuclei" of megakaryocytes. These cells appear to be depleted of cytoplasmic platelets and exhibit a hyperlobulated but condensed nucleus. Bauer and colleagues (1992) believe that the ratio of "naked" megakaryocytic nuclei to normal cells is diagnostic of the AIDS syndrome. I know of no direct evidence that correlates this change with infection of the megakaryocytes, and it may only be a feature of apoptosis attributed to the cytochemical environment of the bone marrow. Platelets also appear to be carriers of HIV-1 and are occasionally subject to immune sequestration in the spleen resulting in shortened life span.

216

Pathology and Pathogenesis of Human Viral Disease

HIV-1 Encephalopathy

DISEASES OF THE CENTRAL NERVOUS SYSTEM

The pathogenic mechanisms resulting in central nervous system involven\ent by HIV-1 are incompletely defined. Shortly after HIV-1 seroconversion, the cerebrospinal fluid often yields virus and detectable viral RNA, but there is no evidence to indicate that the parenchyma of the nervous system is infected at this time. The CSF HIV-1 RNA concentrations correlate with the amounts of viral RNA in the plasma and do not change during the evolution of neurological disease (Bossi et ah, 1998). As noted above, the lymphocytes in the meninges that appear early in the infection, and in the chronic meningitis that may follow, are not infected. Prior to the development of full-blown AIDS, the central nervous system tissue exhibits little morphological evidence of infection, save for focal perivascular cuffing at scattered sites in the brain of an occasional individual. With the onset of the AIDS dementia syndrome, virus appears for the first time in the microglia that appear on the scene (Bell et ah, 1993). Recent studies also document its presence in dendritic cells of the choroid plexus. These latter cells may serve as a reservoir in the central nervous system for subsequent dissemination of the virus into the brain parenchyma (Petito, 1996). Current evidence indicates, however.

(see Figure 16.5)

Acute Meningitis Clinical evidence of meningeal inflammation occasionally is observed during the acute febrile "mononucleosis-like" illness that characterizes the initial systemic response to HIV-1 infection. This transient episode develops in about 5% of those who are infected and appears at about the time of serological conversion, roughly 3 to 6 weeks after the virus is acquired. Typical neurological symptoms of meningismus are headache and photophobia, but encephalitic features are sometimes also seen. A mononuclear lymphocytic pleocytosis and increases in the concentrations of protein are found in the cerebrospinal fluid of about 30% of patients. Virus can be isolated and viral RNA demonstrated in the cerebrospinal fluid, but the lymphocytes apparently are not carriers of virus at this time (Pratt et ah, 1996). Overall, about a third of patients manifest clinical or laboratory evidence of meningeal involvement (Carne et al, 1985; Hawley et al, 1983). A chronic meningeal reaction may follow the acute episode, but this is less well characterized.

-H500

_

H400

o

Brain Atrophy

6

7

8

Years after Infection FIGURE 16.5 Evolution of neurological changes that typically develop during HIV-1 infections. During the acute episode that occurs in early infection, the meninges exhibit a mononuclear cell infiltrate to a variable extent. However, cells in the meninges and cells in the brain parenchyma are not infected. This response is part of a generalized syndrome. The changes in HIV-1 encephalopathy evolve subsequently to a variable extent, as illustrated here.

Human Immunodeficiency Viruses

217

that the macrophage-tropic variant of the virus is carried to the brain in mononuclear cells, where it localizes to initiate the infectious process (the so-called "Trojan horse theory"). These macrophages and the microglia they form serve as the primary site of viral replication in the brain. Neurons are not infected (Sharer et al, 1996). Cognitive/Motor Complex (Syn. Dementia Complex) The brain shows pathological features of infection in more than 75% of persons contracting HIV-1. Neurological disease is variable, both temporally and with regard to its severity, and is influenced by therapy. In the majority of patients, the onset of clinical AIDS precedes the appearance of the signs and symptoms of encephalitis, but occasionally evidence of encephalitis anticipates the appearance of the overt AIDS syndrome, or occurs concomitantly. To a variable extent, patients exhibit cognitive impairment, usually accompanied by sensory, motor, and behavioral signs and symptoms (McArthur, 1987) (Table 16.8). About a third of terminal patients with AIDS exhibit profound dementia. A consensus classification of clinical syndromes associated with HIV-1 infection has been published by a working group of the American Academy of Neurology (Anonymous, 1991). Two distinct pathological pictures are observed. These are: (1) HIV-1 encephalitis (Figure 16.6), and (2) HIV-1 /ewfcoencephalitis, as defined in a recently published consensus report of neuropathologists (Budka et ah, 1991) (Table 16.9). In encephalitis, the pathologist

TABLE 16.8 Neurological S i g n s and S y m p t o m s Associated w i t h A I D S D e m e n t i a , Myelopathy, and Peripheral N e u r o p a t h y Behavior Apathy Agitation Depression Blunting of affect Cognition1 Slowing of mentation Loss of memory Inability to concentrate Motor Ataxia Poor coordination Unsteady gate Muscle weakness and atrophy Sensory Loss of fine touch, heat/cold perception and proprioception

FIGURE 16.6 A microglial nodule in the brain of an AIDS patient with HIV-1 encephalitis. Reprinted with permission and through the courtesy of C. Petito, MD.

TABLE 16.9 R e c o m m e n d e d N e u r o p a t h o l o g y - B a s e d Terminology of HIV-Associated D i s e a s e of the N e r v o u s S y s t e m A. Central nervous system 1. HIV-1 encephalitis 2. HIV leukoencephalopathy 3. Vacuolar myelopathy and vacuolar leukoencephalopathy 4. Lymphocytic meningitis 5. Diffuse poliodystrophy 6. Cerebral vasculitis including granulomatous angiitis B. Peripheral nervous system 1. (HIV-1 associated) acute inflammatory demyelinating polyradiculoneuropathy 2. Chronic inflammatory demyelinating polyradiculoneuropathy 3. HIV-1 associated (predominantly sensory)/ axonal neuropathy 4. Ganglionitis, ganglioradiculitis, polyradiculoneuropathy 5. Necrotizing vasculitis, vasculitic neuropathy Adapted with permission from Budka et al. (1991).

finds focal accumulations of microglia and macrophages in the cortex of the brain, resulting in the formation of variable numbers of macrophage-derived multinucleate cells and mononuclear stellate cells with altered morphology. These cells tend to have a perivascular localization, but the typical perivascular inflammatory cuffs of lymphoid cells seen in many viral encephalopathies usually are not a prominent feature. The lesions are particularly evident in the cortical regions of the grey matter, often in the telencephalon, but changes can also occur diffusely (Grassi et al, 1997). The number of microglia in the brain substances is believed by some pathologists to correlate with the degree of dementia, but this claim has not been established quantitatively. These cells contain HIV-1 by in situ hybridization and electron microscopy. In contrast.

218

Pathology and Pathogenesis of Human Viral Disease

FIGURE 16.7 HIV-1 multinucleate polykaryocytes in the brain of a patient with HIV-1 encephalitis. Reprinted with permission and through the courtesy of C. Petito, MD.

the HIV-1 leukoencephalitis picture is one of diffuse noninflammatory degeneration of the white matter and is manifest ultimately as focal myelin rarefaction and degeneration. So-called myelin pallor is observed, a change attributable to the seepage of serum proteins and fluid into the interstitial spaces. Spongiform or vacuolar changes in the white matter also occur. Demyelinization parallels these changes, but oligodendroglia are not infected by the virus. The lesions in the white matter can be sufficiently severe so as to grossly resemble those of multiple sclerosis. The histologic changes in the central nervous system overlap those

caused by CMV and other opportunistic viral infections, as discussed in more detail in a later section. The presence of the typical multinucleate cells is the most definitive morphological indicator of HIV-1 involvement of the brain, aside from specific immunohistochemical or molecular evidence (Figure 16.7). Atrophy of both the grey and white matter accompanied by astrocytosis and microgliosis are found in the brain in AIDS encephalopathy, but the extent of the changes are variable, one patient to another (Gray et al, 1991). Decreases in cortical neurons have been demonstrated quantitatively, particularly in the frontal lobes. In a study by Ketzler et al. (1990), neuronal density was reduced by 18% and the volume of the perikaryon diminished by over 30%. Neuronal losses of 20% have been documented in the temporal and parietal cortex (Wiley et al, 1991) and 50% to 90% in the hippocampus (Masliah et al., 1992). In the remaining neurons, the dendrites can be tortuous and both vacuolated and dilated. In addition, the length of the neurons is reduced and branching attenuated. The mechanisras of neuronal damage in HIV-1 encephalopathy have been the subject of considerable research. As noted above, neurons rarely, if ever, are infected by HIV-1, yet are profoundly damaged in AIDS dementia. Studies by Petito and Roberts (1995) and Shi and colleagues (1996) strongly suggest that apoptosis of the neurons is the primary mechanism

FIGURE 16.8 Selected photomicrographs from a case of HIV encephalitis. Panel A illlustrates the numerous infiltrating microglial cells that are infected with HIV-1. A collection of small round hyperdense nuclear fragments surrounded by a thin rim of eosinophilic cytoplasm are consistent with apoptotic bodies (B). The center of the field in C shows a vacuole with a prominent apoptotic body containing hyperdense chromatin material. Reprinted with permission from Petito and Roberts (1995).

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(Figure 16.8), but the means by which this occurs is uncertain. The infected macrophages appear to be the key actor in this tragic drama. The products of these profoundly activated cells are believed to be major mediators of injury, but their identity is uncertain. HIV-1infected macrophages have been shown to generate TNFa, platelet activating factor, arachidonic acid metabolites, nitric oxide, free oxygen radicals, and excitatory amino acids (Shi et ah, 1996), TGPP (Johnson and Gold, 1996), prostaglandin E2, and Beta2 microglobulin (Griffin, 1997). In laboratory experiments, these diverse substances have direct or indirect cytotoxic effects. On the other hand, cytokines produced by HIV1-infected macrophages and microglia might also damage neurons (Licinio and Wong, 1997). Using brain tissue from subhuman primates of several species that had been infected experimentally with Simian immunodeficiency virus (SIDS), Sasseville and associates (1996) demonstrated elevated histochemical expression of C-C chemokines, macrophage inflammatory protein 1 alpha and beta, RANTES, monocyte chemotactic protein-3, and the C-X-C chemokine interferon-inducible protein-10. The receptors for several of these cytochemicals were then demonstrated on cells of the mononuclear infiltrates and both resident neurons and glia (Westmoreland et ah, 1998). Soluble products of the HIV-1 virion also have cytotoxic properties, specifically the gpl20 glycoprotein of the viral envelope and the tat regulatory protein. One might envision the release of these substances from the infiltrating infected macrophages and microglia (Magnuson et ah, 1995). Thus, the stage is set for a drama potentially of complex proportions whereby the products of infected macrophages subtly mediate on a chronic basis injury to key populations of neurons. Clearly, much additional research will be required before the end of this story is written. The contemporary clinical and experimental evidence has been successfully summarized by Lipton and Gendelman (1995). Myelopathy and Myelitis Two patterns of injury are observed in the spinal cords of patients with AIDS. These are (1) vacuolar myelopathy and (2) myelitis. In vacuolar myelopathy (Figure 16.9), the pathologists find discrete or coalescent vacuoles, 10 to 50 microns in diameter, containing cellular debris and macrophages. In particular, it involves the posterior and/or lateral columns of the cervical, thoracic, and lumbar segments of the spinal cord. In advanced stages, changes in the axons and myelin

can also be seen. In HIV-1 myelitis, the following features are observed: (1) multiple intraparenchymal or perivascular foci of inflammatory cells, that is, microglia, histiocytes, multinucleate cells, and occasional perivascular lymphocytes; and (2) HIV-1-positive perivascular macrophages/multinucleate cells a n d / o r intraparenchymal microglia. The prevalence of spinal cord lesions differs for unknown reasons in various patient series. In one report from the United States, only a third of patients had cord lesions, whereas almost 80% of patients studied in France were thus affected (Henin et al, 1992). The proportion of patients with one or the other of the two types of lesions also varied. Only 2 of 24 of the spinal cords from patients with AIDS studied by Rosenblum et al (1989) had evidence of cord infection using both immunochemistry and in situ hybridization. On the other hand, in a study by Burns et al (1991a), 35% of HIV-1-infected patients with encephalitis had vacuolar myelopathy. Several authors have noted the striking similarity between the cord lesions of HIV-1 infection and those of subacute combined degeneration as seen in profound vitamin Bi2 deficiency. The pathogenesis of vacuolar change is obscure and its ultrastructural features remain to be defined. Rosenblum et al (1989) analyzed the lesions for multinucleate cells, an indication of active infection, and attempted to locate virus in the tissue. There was evidence of infection at the sites of vacuolar myelopathy in less than 10% of patients. Other studies have shown that the concentrations of virus are extremely low and may represent background. Neuropathy The clinical course of HIV-1 infection almost invariably is confounded by neuropathies of uncertain causation and pathogenesis. In roughly a third of patients, the resulting neuromuscular disease can be debilitating. On rare occasions, an acute inflammatory demyelinization occurs during and following the acute "mononucleosis-like" syndrome. It resembles GullianBarre syndrome, but differs because a pleocytosis is usually found in the CSF. A sensory or mixed sensory/motor mono- and polyneuropathy that typically involves the distal extremities commonly develops as a complication of the AIDS syndrome. Nerve biopsies reveal both axonal degeneration and demyelinization, occasionally accompanied by a vasculitis. This clinical syndrome, and the associated pathological lesions, cannot be differentiated from the neuropathy of CMV (see below). Drug toxicity is an alternate pathogenic consideration.

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1.^ ^ \

FIGURE 16.9 Panel A is a cross-section of the spinal cord showing the diffuse vacuolar changes typical of this form of myelopathy. The cystic spaces are illustrated in panel B. Note the absence of inflammation. The pathogenesis of this lesion is not understood. Reprinted with permission from Rosenblum ei a\. (1989) through the courtesy of M. Rosenblum, MD.

Myositis Lesions of the peripheral skeletal muscle occur both acutely and during the late stages of HIV-1 infection. Myalgias during the early "mononucleosis-like" syndrome have, on occasion, been accompanied by rhabdomyolysis (Guillaume ei ah, 1995). Dalakas et ah (1986) described two patients who lacked evidence of

complicating EBV and CMV infections. In one young adult with muscle weakness, blood concentrations of muscle enzymes (creatine kinase and lactase dehydrogenase and both aspartate and alanine aminotransferase) were increased three- to fourfold. A myopathic picture was found by electromyography, and biopsy showed polymyositis with muscle fiber necrosis and an associated interstitial inflammatory infiltrate com-

Human Immunodeficiency Viruses

prised of HIV-1-positive mononuclear cells. The second patient was a young man with proximal muscle weakness and myalgias accompanied by progressive wasting of the proximal muscles of all four extremities. Biopsies showed variability in muscle fiber size with interstitial infiltrates of HIV-1-positive CD4+ lymphocytes. Active phagocytosis of muscle by macrophages was seen. As might be expected, the muscle enzyme levels in this patient were elevated five- to sixfold. In a single case described by Bailey et al. (1987), electrophysiologic testing established a combination of peripheral nerve and functional abnormalities of skeletal muscles in a middle-aged man with AIDS whose distal lower extremities were atrophic. Biopsy showed focal perimysial mononuclear and multinucleate giant cell infiltration as well as mild variation in fiber size and prominent central nuclei. Demyelinization and axonal loss associated with mononuclear cell infiltrates tended to be located perivascularly These case reports provide some insight into the features of myositis attributable to HIV-1. At present, systematically accumulated information on the prevalence and distribution of muscle lesions in HlV-l-infected patients is lacking. The evidence, albeit limited, indicates that striated muscle cells are not infected, the virus being restricted to infiltrating inflammatory cells. Clearly, the mechanism of the disease process is obscure. Opportunistic CMV Infections of the Central and Peripheral Nervous Systems A diverse variety of infections involve the nervous system in AIDS. The prevalence roughly parallels the severity of the immune suppression (see Table 16.1). In some autopsy series, infections of the central and peripheral nervous systems are recognized in 50% of patients with AIDS. Since the lesion of CMV can simulate those of the AIDS dementia complex and polyneuropathy, the pathologist is confronted with an imposing diagnostic challenge, particularly since effective treatment for systemic CMV infections is in the armamentarium of the clinician (Domingo et ah, 1994). Moreover, it is becoming increasingly evident that CMV may enhance the growth and spread of HIV-1 in tissue. The risk of developing AIDS is increased 2.5-fold in patients with active CMV infections (Webster, 1991). CMV involvement of the brain, retina, spinal cord, and peripheral nerves is a reflection of a systemic infection in half the patients with advanced HIV-1 disease. In the central nervous system, the infection is reflected by three lesions: (1) vasculitis, most probably resulting from infection of the endothe-

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lial cells (Koeppin et al, 1981); (2) a diffuse encephalitis in which glial nodules are scattered in the brain with viral inclusions, antigen, and DNA being identifiable in neurons or glial cells, or both; and (3) an ependymitis, resulting in periventricular wall damage and reactive gliosis. A more detailed discussion of CMV is found in Chapter 8. Lesions of the ependyma due to direct involvement of the individual lining cells of the ventricle and aqueducts are the hallmark of the CMV nervous system syndrome in AIDS. The pathological findings in the patient with AIDS strikingly resembles the lesions seen in neonatal CMV disease. The infectious process seems to evolve in a centrifugal fashion from the ventricular lining cells to involve the subjacent tissue and destroy adjacent brain structures. A fibrinous exudate envelopes the ventricular surfaces, potentially compromising aqueduct function. Almost invariably, the cranial nerves and retina are also involved (Kalayjian et al, 1993; Bylsma et al, 1995). In both the cranial and peripheral nervous systems, the infection presents as a polyradiculopathy either due to direct involvement of the dorsal and ventral neurons and Schwann cells, or as a result of infection of the small endoneurial and epineurial blood vessels. To a variable extent, these vessels exhibit a vasculitis characterized by a mononuclear cell infiltrate that includes plasma cells, and focal thrombosis. As a result, there are sites of localized necrosis or a loss of myelinated fibers accompanied by axonal degeneration with denervation and atrophy of the associated striated muscles. The endothelium of the vasculature of muscles also can be infected by CMV While the clinical manifestations of these neuromuscular lesions are protean, the Cauda equina syndrome often occurs. In this condition, lower extremity flaccid paralysis is typically accompanied by urinary and fecal incontinence (Moskowitz et al, 1984; Eidelberg et al, 1986; Behar et al, 1987; Cornford et al, 1992). In a recent study, the DNA of CMV was demonstrated by PCR in 70% of CSF samples from patients with AIDS (Achim et al, 1994). Similarly ELISA assays of CSF for CMV antibody showed a high degree of reactivity The source of virus in these cases is unclear, but at least meningeal infection in some cases is likely, since increased protein and a pleocytosis is a common finding in the CSF. While these laboratory procedures are specific and highly sensitive indicators of CMV infection, they fail to establish the role of the virus in central and peripheral nervous system disease in AIDS patients. Marmaduke et al (1991) describe large atypical cells that reacted with CMV antibody immunohistochemically in the CSF of a patient who at autopsy had typical CMV ependymitis. Cytological study of the

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CSF supplemented by immunohistochemical assays for virus may provide diagnostic information useful as a basis for treatment. It is noteworthy that MRI studies of both children and adults with AIDS have shown central brain atrophy in persons with presumptive CMV lesions of the ventricular system. This pattern differs from the typical pattern of diffuse atrophy so commonly observed in a variety of CNS diseases, and in the aging (Scarmato et al, 1996).

DISEASES OF THE RESPIRATORY TRACT Diseases of the lungs are a major contributor to morbidity and mortality among those infected with HIV-1. These various conditions are considered here. Diffuse Alveolar Damage (DAD) Diffuse alveolar damage is commonly observed at autopsy among those dying with AIDS. In most patients, the etiology is obscure, although in many it may be secondary to an agonal pneumonia and septicemia or prolonged positive pressure oxygen therapy Some observers have proposed that cytochemicals generated during the course of HIV-1 infection are cytotoxic for the cells lining airspaces, while others have suggested that the toxic properties of various HIV-1 proteins could be responsible. At present, the pathogenesis of diffuse alveolar damage in terminal AIDS is obscure. There may, in fact, be many etiologies. Lymphoid Interstitial Pneumonia (LIP)/ Nonspecific Interstitial Pneumonia (NIP)/ Follicular Bronchitis/Bronchiolitis (FBB) Not all pathologists would agree on the terminology and the descriptive features of the various lesions listed immediately above. Indeed, the conditions most probably overlap morphologically and represent a spectrum of tissue reactions. Alternatively, they could either be temporal stages in a pathogenic continuum, or represent somewhat different histologic reflections of the same disease process. Problems of tissue sampling compound the problem. Rarely is it possible to adequately examine the lungs in the early stages of a disease such as AIDS since tissue is usually sampled only by transbronchial biopsy. Follicular bronchitis/bronchiolitis (FBB) is characterized by the presence of lymphoid follicles centered around the bronchioles accompanied by a minor inter-

stitial inflammatory reaction (Nicholson et al, 1995). Nonspecific interstitial pneumonia (NIP) is seen as peribronchial, perivascular, septal, and pleural lymphoid infiltrates, whereas in lymphoid interstitial pneumonitis (LIP) prominent interstitial infiltrates are distributed throughout the lungs, including the alveolar septae, with spotty accumulations of lymphoid follicle (Griffiths et al, 1995; Kornstein et al, 1986; Travis et al, 1992). NIP and FBB are commonly found in HIV-1-infected adults. They are rarely symptomatic and are customarily not evident radiologically Little more can be said about the clinical aspects of these morphologically defined conditions, since correlative clinical/pathological studies have not been carried out. LIP occurs in as many as half the children with AIDS. Adults with AIDS develop LIP on rare occasions (Andiman et al, 1985). LIP typically occurs during the first 2 years of life in HIV-1-infected infants and children (Moran et al, 1994) (Figure 16.10). Respiratory symptoms can be severe and life threatening, and the radiologically defined infiltrates have a reticular or nodular appearance. On occasion, LIP is a reflection of a systemic lymphoproliferative disorder in these children, and evidence of a monoclonal EBV infection of the lymphoid elements is found. Before the AIDS epidemic, over two decades ago, Carrington and Leibow (1966) described LIP as a new entity. In this initial report, 13 patients were described, only three of whom were children. Sporadic case reports of the disease in adults have been published since that time (Halprin et al, 1972; Montes et al, 1968). Adult LIP often occurs in patients with Sjogren's syndrome and dysglobulinemia, but the numbers of recorded cases are so few that other possible disease associations have not been established. The interstitial infiltrates of LIP are polymorphic and comprised of macrophages, as well as both B and T cells. The latter cells tend to predominate, and the majority are CD8+ cytolytic cells. In contrast, in NIP lesions, B cells and plasma cells are often found in lymphoid aggregates, and the infiltrating T cells are equally divided into populations of CD4+ and CD8+ cells (Griffiths et al, 1995). Insights into the pathogenesis of LIP can be derived from studies of experimental sheep infected with ovine pneumonitis virus, a nonhuman lentivirus infection of considerable economic importance. In mature sheep with a naturally occurring infection, the severity of the lymphocytic pulmonary infiltrative disease is variable but correlates with the amount of virus detected in the alveolar macrophages, the apparent cellular site of replication of this virus in the lung (Brodie et al, 1992). In contrast, diffuse pulmonary lesions similar to these seen in children with LIP can be induced in newborn lambs by intratracheal instillation of ovine pneumonitis virus. Inoculation of adult animals by this same

Human Immunodeficiency Viruses

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F I G U R E 16.10 Lymphoid interstitial pneumonitis in the lung of an infant with perinatally acquired HIV-1 infection. Note the diffuse interstitial infiltrate of a m.ixed population of macrophages and lymphoid elements. At scattered sites, lymphoid follicles are found, but they are not a prominent feature of the disease.

route often fails to produce disease, or results in pulmonary infiltrative lesions only after prolonged incubation periods (Lairmore ei al., 1986). Cohen ei al. (1997) reported on a series of studies with a lentivirus that produces an AIDS-like illness in mice. In this model, a mixed population of macrophages and B and T cells accumulate in the lungs within weeks after intraperitoneal inoculation of the virus. Striking increases in interferon-y, TNpp, and IL10 are found in the lung tissue. While the biologic role of these cytokines remains to be defined, their presence emphasizes the dynamic nature of the disease and indicate that, as with ovine pneumonia virus, the interstitial disease can best be attributed to lentivirus infection. There is currently no evidence to suggest that LIP in AIDS is a direct effect of an HIV-1 infection in the lungs. Pulmonary Hypertension and Vascular-Occlusive Disease Classically, primary pulmonary hypertension is a clinical syndrome of unknown etiology that affects

young women more often than men. It occurs in the absence of underlying cardiovascular and pulmonary parenchymal disease. The typical pathological findings are well-described (Edwards and Edwards, 1977; Legoux ei al., 1990; Coplan ei al., 1990; Jacques ei al., 1992; Pietra ei al., 1989; Aarons and Nye, 1991; de Chadarevian ei al, 1994; Mette ei al., 1992). While the lesions are customarily believed to be restricted to muscular arteries and arterioles in the lungs, Wagenvoort and Mooi (1989) described cases of vascular-occlusive disease in which proliferative lesions similar to those seen in arteries were found in pulmonary veins. Similar lesions have been described in patients with AIDS (Ruchelli ei al., 1994). Two types of arteriolar lesions are observed in the lung histologically In the first, small blood vessels are concentrically obliterated by intimal proliferation, whereas in the second plexiform lesions of the arteriolar lumina are found at scattered sites (Figure 16.11A,B). Cool and her associates (1997) hypothesize that there is a chronological continuum in primary and secondary pulmonary hypertension proceeding from early proliferative concentric vascular obliterative lesions. These workers postulate that the plexiform le-

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FIGURE 16.11 Pulmonary vascular changes associated with AIDS. Panel A illustrates concentric proliferative changes in a pulmonary arteriole. Factor Vlll-associated antigen demonstrated by immunohistochemistry provides the contrast. Panel B shows the plexiform lesions that develop concomitantly. Again, the lesion is stained to demonstrate Factor Vlll-associated antigen indicating the endothelial origin of these complexes. The pathogenesis of the lesion is not understood. Reprinted with permission and through the courtesy of C. Cook, MD.

Human Immunodeficiency Viruses

sions represent a form of adaptive angiogenesis due to influences of chemokines and growth factors. Thus, endothelial cell proliferation is the basic pathogenic process involved in the genesis of the lesions. Kim and Factor (1987) documented pulmonary hypertension in homosexual males with HIV-1 infections. Since that time, numerous additional cases have been described in the literature, and several patients with associated pulmonary vascular-occlusive disease have also been reported. The pathogenesis of these lesions is obscure, but the occurrence of pulmonary hypertension in an HIV-1-infected hemophilic patient suggests, but does not prove, that the lesions are HIV-1 related, and not due to other extraneous opportunistic agents (e.g., HHV-8) (Schulman et al, 1988). However, attempts to demonstrate HIV-1 infection of endothelial or smooth muscle cells in the vascular walls in these cases have thus far proven negative. While the pathogenesis of these idiopathic lesions of both the pulmonary arterial and venous systems is obscure, it has been suggested that they may have a viral etiology. One might speculate that the lesions of the vessels are caused by chronic exposure to circulating products of HIV-1 infection, possibly cytochemicals. Clearly, much additional research is needed. Recently, cases of abrupt-onset pulmonary hypertension accompanied by plexiform vascular lesions were reported among consumers of the pharmacological diet suppressors Fenfluramine and Phentermine (Mark et ah, 1997). The occurrence of this unique vascular disease in drug-treated persons and its sporadic development in female members of the general population (unassociated with drugs) are consistent with an idiosyncratic response to an unknown agent, possibly influenced by individual host susceptibility. The effect of a serotonin-like agent has been postulated based on the morphological features of the cardiac valvular lesions that occur in these same patients. Opportunistic Infections of the Lung In the early 1980s, Pneumocystis carinii pneumonia (PCP) among adult homosexuals proved to be the harbinger of the AIDS epidemic. Over a decade later, a diverse variety of opportunistic infections complicate the clinical course of AIDS (Table 16.2). Although our understanding of the mechanistic basis for susceptibility to the various agents is incomplete, insights are beginning to accumulate. Briefly reviewed here are relevant studies that provide a clearer understanding of the altered function of the defense mechanisms of the lungs in persons infected with HIV-1. Cells recovered by bronchopulmonary lavage of the lungs of healthy nonsmoking adults are sparse, but

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over 90% are macrophages. In patients infected with HIV-1, an increase in the number of T cells is usually found in fluids obtained by bronchoalveolar lavage, even in those who are asymptomatic. In the more advanced stages of the infection, macrophage populations are also increased. These cells appear to be activated; they release a broad spectrum of cytokines that include IL-1, IL-6, IL-8, IL-10, IL-12, and GM-CSF, and macrophage inflammatory protein (Agostini et al., 1995). While the basis for macrophage activation is unclear, one possible explanation is the presence of either proviral DNA or active HIV-1 replication in these cells (Schuitemaker and Miedema, 1996). Cytokines generated by infected alveolar macrophages appear to stimulate antigen presentation by increasing the expression of class II MHC and adhesion molecules by these cells. On the other hand, the cytokines generated by the infected macrophages also might enhance polyclonal B cell activation. Alternatively, they could promote recruitment of T cells, or encourage T cell proliferation. Finally, IL-1 and TNPP generated by macrophages could damage the cells lining airspaces either directly or by recruiting neutrophils to the scene. This might account for the diffuse alveolar damage often seen at autopsy in patients with AIDS as described above. At present, we know very little regarding the possible role of cytokines and the cellular elements of the lung in the biology of the opportunistic pulmonary infections that occur so commonly in AIDS (Agostini et al, 1996; Denis and Ghadirian, 1994). Interactions of macrophages with several common pathogens is altered by HIV-1 infection (Lipman et al, 1997). In one study, phagocytosis of Cryptococcus neoformans by monocytes from patients with reduced CD4+ cell counts was normal, but generation of hydrogen peroxide and beta glucuronidase by the cells was reduced (Harrison and Levitz, 1997). In a second study, the pulmonary alveolar macrophages of HIV-1-positive persons exhibited defects in the binding of Histoplasma capsulatum yeast, but phagocytosis otherwise proved to be normal (Chaturvedi et al, 1995). Macrophages from almost half of the patients included in this latter study supported replication of the organism in contrast to cells from healthy control subjects. A decrease in phagocytosis of opsonized Staphylococcus aureus by monocytes from HIV-1-infected patients was found in a third study (Szkaradkiewicz, 1992), whereas in another investigation monocytes effectively phagocytized several different types of yeast forms (Washburn et al, 1985). Macrophages recovered from HIV-1 patients permit the luxuriant intracellular growth of organisms of the Mycobacterium avium complex (Harrison and Levitz, 1997). While these diverse investigations address only one aspect of the complex

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pathogenesis of opportunistic infections, the evidence suggests that alveolar macrophages of the lung from HIV-1-infected patients may fail to effectively eliminate common pathogens from the lungs. Indeed, at times, the cells may support their replication. A common defect in these studies is the lack of documentation of the status of the HIV-1 infection in the alveolar macrophages. Blood and tissue concentrations of HIV-1 RNA and virus dramatically increase concomitant with the appearance of AIDS. This seems to be somewhat paradoxical, for it occurs when the number of circulating CD4+ lymphocytes are reduced, and lymphoid tissues are undergoing involution. At this time, circulating monocytes/phagocytes customarily exhibit little or no evidence of active infection. However, HIV-1 DNA provirus or fully replicating virions are found in pulmonary alveolar macrophages. Infections by a diverse variety of opportunistic agents seem to upregulate HIV-1 expression, presumably accelerating the onset of the terminal stages of AIDS. The mechanistic basis for this effect is obscure and most probably differs substantially in various types of infections. Recent studies by Orenstein et al (1997) document upregulation of HIV-1 replication in macrophages and lymph nodes infected with Mycobacterium tuberculosis and members of the Mycobacterium avium complex. A similar phenomena occurs with Pneumocystis carinii infection. Not only is the number of cells supporting HIV-1 increased, but virion production is enhanced sufficiently to permit detection of cellular and extracellular virions in the tissues by electron microscopy. Within the lungs, HIV-1 grows more luxuriantly in alveolar macrophages obtained from Mycobacterium tuberculosis-iniected patients (Nakata et al, 1997). Experimental evidence suggests that TNFa elaborated by macrophages from these mycobacteria-infected patients plays a critical, but undefined, role in this process. Nakata et al. (1997) claimed that the events accompanying Mycobacterium tuberculosis infection were associated with a change in the Gpl20-N3 glycoprotein membrane of the virion. Interestingly enough, treatment of the tuberculosis infection seems to retard progression of HIV-1-associated disease (Pape et al, 1993; Manoff et al, 1996). HIV-1 expression by a macrophage cell line infected with a single HIV-1 provirus sequence was triggered by infection with Cryptococcus neoformans and Candida albicans (Harrison et al, 1997). As with M. tuberculosis, the effects seemed to be mediated by TNFa. Similar effects of CMV on the course of an HIV-1 infection have been found (Lathey et al, 1994). The role of CMV is particularly intriguing because of the almost

universal existence of active infections in HlV-l-infected individuals and the demonstrated occurrence of concomitant dual infections with CMV and HIV in macrophages and in the peripheral blood (Mole et al, 1997). However, the phenomena is particularly dramatized when cells that are semi-permissive for the two viruses are employed. Under experimental circumstances, HIV-1 virions were only elaborated when the cells were also infected with CMV. As shown by Lathey et al (1994), primary isolates of CMV enhanced production of the HIV-1 p24 antigen by 5- to 15-fold in peripheral monocytes. The growth of the so-called macrophage-tropic primary isolates of HIV-1 was influenced by CMV infection, whereas T cell-tropic strains of HIV1 were not affected. In contrast to the studies with M. tuberculosis and pathogenic yeast summarized above, TNFa plays no apparent contributory role in CMV infections. Members of the herpesvirus group elaborate chemokines that can influence uptake of HIV-1 by the cell. Thus, generation of "new" viral receptors may be important (Pleskoff et al, 1997). Clearly, the manner by which CMV influences the course of an HIV-1 infection remains to be defined. Activation of macrophages may contribute to HIV-1 expression in some clinical settings. Cultured alveolar macrophages from cigarette smokers (presumably activated cells) express greater amounts of HIV-1 p24 antigen than do infected cells from nonsmokers. Whether this phenomenon occurs in vivo is unclear, for the results of clinical studies conflict with regard to the effects of cigarette smoking on the progression of AIDS (Burns et al, 1991b; Coates et al, 1990; Galai et al, 1997; Halsey et al, 1992; Abbud et al, 1995).

DISEASES OF THE HEART Abnormalities of the heart are commonly found in AIDS at autopsy, but they rarely become evident before the terminal stages of HIV-1 infection (Table 16.10) (Lewis and Grody 1992; Levy et al, 1989; Calabrese et al, 1987; Grody et al, 1990). Aside from the diverse lesions of the heart attributed to opportunistic infections and tumors, a lymphocytic interstitial myocarditis accompanied by varying degrees of focal myocardial necrosis has been reported in approximately 50% of AIDS patients in three studies (Anderson et al, 1988; Beschorner et al, 1990), and in a greater number of cases in the series of Klatt and Meyer (1988). Pericarditis is known to occur in additional patients, the majority of whom have pericardial effusions (Table 16.10). Opportunistic infections and Kaposi's sarcoma

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TABLE 16.10 Autopsy Findings in Hearts of Patients with AIDS Pathological finding Pericardial effusion Cardiomegaly Lymphoma Marantic valvular vegetation Focal infarction Cytomegalovirus Candida sp. Kaposi's sarcoma Mycobacterium avium-intracellulare Coccidioides immitis Cryptococcus neoformans Histoplasma capsulatum Mycobacterium tuberculosis Toxoplasma

No. of cases 33 17 8 7 4 3 3 3 2 2 1 1 1 1

Reprinted with permission from Klatt and Meyer (1988).

involving the pericardium result in effusions and, on occasion, cardiac tamponade. Mycobacterial infections are the commonest infectious cause of pericardial effusions in AIDS. It is currently unclear whether AIDS myocarditis is a specific lesion attributable to HIV-1 infection, or is consequent to an increased susceptibility to incidental infections, presumably in large part of viral origin (Beschorner et al., 1990). The demonstration of HIV-1 in arteriolar endothelial cells and occasional cardiac myocytes argues for, but does not establish, the specificity of the lesion (Bagasra et al, 1996). On the other hand, insufficient numbers of studies have been done using modern diagnostic tools to reasonably exclude alternate explanations for the lesions. And, there is the everpresent concern that the lesions are attributable to antiretrovirus drug toxicity (Lamperth et al, 1991; Walter et al, 1991; Dalakas and Ilia, 1990; Lipshultz et al, 1992). Pentamidine (Stein et al, 1991), ganciclovir (Cohen et al, 1990), and interferon-a2a (Deyton et al, 1989) may be cardiotoxic. Appropriately or not, this possibility has led some pediatricians to withhold zidovudine (AZT, Retrovir) therapy in children. Dilated cardiomyopathy commonly develops in children and adults with AIDS. In one study, progressive left-ventricular dilatation with thinning of the chamber wall was documented by echocardiography. Over the period of observation during which zidovudine was administered, the ventricular mass increased, although it was insufficient to support adequate cardiac output. In a second study, heart weights among AIDS children proved to be increased in weight

by 184% of normal. About a third of children with AIDS exhibit features of dilated cardiomyopathy. In adults, cardiomyopathy appears to occur less commonly than in children. It is consistently accompanied by myocarditis (Anderson et al, 1988). The biologic basis for the myocardial hypertrophy in these cases of dilated cardiomyopathy is unknown. Herskowitz et al (1989) suggest that the cardiac changes may have an autoimmune pathogenesis, but the evidence supporting this conjecture is limited. In an analysis of the lesions of AIDS-associated myocarditis, Beschorner et al (1990) found the cardiac infiltrates to be comprised primarily of non-CD4+ T cells. Natural killer cells and histocytes were notably absent, but CD8+ T cells were found in the infiltrates in 40% of cases. The endothelium in these hearts showed activation of class I and IIMHC antigens, a finding consistent with the effects of cytokines generated by cells of the cardiac infiltrates. A syndrome consistent with an autonomic neuropathy of the heart has been described in patients with AIDS (Becker et al, 1997).

DISEASES OF THE VASCULATURE A microvasculopathy occurs in 70 to 80% of HIV-1positive patients and in more than half the patients with AIDS (Cunningham and Margolis, 1998). Various specific vascular lesions have been noted in individual cases. These are leukocytoclastic and nonleukocytoclastic vasculitis, granulomatosis angiitis, eosinophilic arteritis, and periarteritis nodosa (Winchester et al, 1987; Gisselbrecht et al, 1997). Inflammatory and necrotizing lesions are found in small arterioles, in capillaries, and in postcapillary venules in the skin, in peripheral and cranial nerves, and in the central nervous system. Vasculitis has been identified in two-thirds of the muscle biopsies from cases of peripheral neuropathy/myopathy. Although it is often only discovered at autopsy, patients with clinically overt symptoms and signs of vasculitis (acrocyanosis, digital gangrene) are reported (Gisselbrecht et al, 1997; O'Grady and Sears, 1996). A vascular change characterized by hyalinization of small vessel walls has also been described, but it occurs sporadically and at scattered sites throughout the body. A noninflammatory arteriopathy of small and medium-sized arteries has been found in the tissues of children with AIDS. The lesions are characterized by intimal fibrosis and fragmentation of the elastica of the media, resulting in both luminal narrowing and

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aneurysm formation. Calcification of these vascular lesions is also seen. In considering the pathogenesis of the vascular lesions associated with HIV-1 infection, attempts have been made to exclude alternate etiologic explanations such as immunologic events, responses to infection with hepatitis B and C viruses (conditions that result in circulating immune complexes), and drug reactions (such as those secondary to zidovudine). HIV-1 has been found by in situ hybridization in inflammatory infiltrating cells in a necrotizing vasculitis of peripheral nerve. HIV-1 is known to infect endothelial cells. Moreover, during the course of infection, one can envision that the endothelial lining of vessels is subject to the diverse influences of inflammatory mediators and immune phenomena such as circulating immune complexes. Plasma concentrations of markers of endothelial cells such as von Willebrand factor, angiotensinconverting factor, and endothelial and tissue pharminogen activator as well as its inhibitory fibronectin have been found to be increased during HIV-1 infections (reviewed by Zietz et ah, 1996). In one recent study (see Zietz et at., 1996), the expression of vascular cell adhesion molecules and E selectin as well as both HLA-DR and IL-IB by the aortic endothelial cells increased in HIV-1-infected patients. These perturbations of endothelial cell regulation are accompanied by the appearance of cytological changes, including giant cell formation and an accentuation of leukocyte adhesion. One can only imagine that profound alterations in the vascular endothelium occur as reflected by these findings. Unfortunately, the effects of viruses such as CMV and HHV-8 have not been rigorously ruled out in many clinical studies. Thus, the role of HIV-1, to the exclusion of other infectious agents, is uncertain. Much further characterization of the widespread vascular lesions occurring in AIDS is needed.

DISEASES OF THE KIDNEY A rapidly progressing nephropathy occasionally accompanies HIV-1 infection. It is now well established as a discrete clinical and pathological entity (Pardo et ah, 1987) (see Figure 16.12). For unknown reasons, the nephropathy occurs predominantly among infected African-Americans, and as a result there is a marked geographic distribution of cases in the United States with clustering in major urban areas, where large numbers of blacks reside. The nephropathy may be the initial presenting clinical feature of HIV-1 infection, and its appearance as the nephrotic syndrome often

precedes clinical onset of the AIDS syndrome. This HIV-1-associated nephropathy exhibits a progressive course leading to renal failure quite independent of AIDS. At autopsy, the kidneys of patients with the nephrotic syndrome are enlarged and the cortices pale. A mean renal weight of 487 g was documented in one series (Pardo et ah, 1986). Distinctive lesions are found in the glomeruli, and additional somewhat variable changes occur in the tubules. The glomeruli exhibit focal and segmental sclerosis with vacuolated hyperplastic epithelial cells. The underlying capillary walls are collapsed to a variable extent. As the process progresses, lipid-laden macrophages, plasma proteins, and mesangial matrix-basement membrane material accumulate in the glomeruli. Often, the tubules are dilated, and their epithelial lining cells are flattened and show degenerative changes. Ultimately, tubular cysts develop at the corticomedullary junction. These dilated tubules fill with proteinaceous casts (Figure 16.12). Segmental deposits of IgM and C3 correspond to the morphological changes in the glomeruli, but the pattern is not typical of an immune complex lesion. Electron microscopy shows the foot processes of the epithelial cells to be effaced and the cells to be vacuolated. To a variable extent, endothelial cells of the glomeruli, peritubular capillaries, arteries, and veins exhibit the complex tubulo-reticular structures that prove to be nonspecific but are commonly found in both lupus erythematosus and HIV-1infected tissues. This microscopical description is based on the writings of Cohen and Nash (1992). These workers are major contributors to our understanding of the pathology of HIV-1-associated nephropathy. A variety of immune-complex glomerulopathies have been described in HIV-1-infected persons, but these lesions do not appear to be related specifically to HIV-1. Circulating immune complexes are found in roughly 50% of patients with AIDS, presumably in whole or in part due to the opportunistic systemic infections these patients so often suffer. Recently, a "new," but still relatively uncommon, HIV-1-associated IgA immune complex-mediated nephropathy was described in which HIV-1-specific IgG and/or IgM antibodies were identified in circulating immune complexes and in elutes of renal tissue. In addition, HIV-1 genomic material is found in the kidney (Kimmel et ah, 1992). This condition is biologically and morphologically distinct from HIV-1 nephropathy. As noted above, HIV-1 nephropathy does not appear to be an immune complex-related disease. Be-

Human Immunodeficiency Viruses

229

FIGURE 16.12 This kidney biopsy illustrates characteristic features of an HIV-1-associated nephropathy. The patient was a 29-year-old HIV-1-infected man with nephrotic syndrome, renal insufficiency, and a history of intravenous drug abuse. He had no clinical stigmata of AIDS. Panel A shows extensive interstitial fibrosis, chronic inflammation, tubular dilatation, and tubular atrophy. Panel B shows that the majority of glomeruli are obsolescent. However, some glomeruli demonstrate a mild to moderate increase in mesangial matrix, material, and mesangial hypercellularity. A few glomeruli are well preserved. In some patients, the tubules are dilated and compacted with protein, changes that can be detected grossly. The biopsy findings fit into the spectrum of focal segmental glomerulosclerosis. However, the rapid progression clinically and the marked tubulo-interstitial involvement differentiates HIV-1-associated nephropathy from most cases of focal segmental glomerulosclerosis. Tubulo-reticular inclusions observed in glomerular and endothelial cells ultrastructurally provide another differentiating feature. The nephrotic syndrome accompanied by focal segmental glomerulosis in a patient at risk for HIV-1 infection should prompt an investigation for this agent. Reprinted with permission and through the courtesy of B. MacPherson, MD.

cause many African-Americans with this form of renal disease are i.v. drug abusers, the possibility of heroin addiction nephritis is a diagnostic consideration. In this lesion, mesangial hypercellularity and focal glomerulosclerosis are found, and there is both interstitial inflammation and fibrosis. Abusers of opiates also develop glomerulosclerosis and a necrotizing angiitis with interstitial nephritis. These drug-related renal disorders tend to have a more protracted clinical course.

DISEASES OF THE TESTIS In a comprehensive controlled autopsy review of 100 AIDS cases, Rogers and Klatt (1988) noted the testicular changes summarized in Table 16.11 (see Figure 16.13). Since the series represented relatively young men, alterations are not attributable to aging. Patients with liver cirrhosis were excluded. The pathogenic mechanisms involved are obscure.

230

Pathology and Pathogenesis of Human Viral Disease

TABLE 16.11 Testicular Lesions in Men with AIDS Microscopical lesion"

Age-matched controls (%)

Perivasculitis Loss of germ layer Interstitial fibrosis Lymphocytic infiltrate

2 23 29 10

AIDS patients (%) 12 11 52 29

Adapted with permission from Rogers and Klatt (1988). ''Differences between controls and AIDS Patients = ip < 0.001.

DISEASES OF THE DIGESTIVE TRACT A diverse variety of acute and chronic infections involve the oropharynx in AIDS patients (see Table 16.3) (Barone ei al., 1990). No doubt, these conditions contribute to the poor nutritional status of many of these patients. Diarrheal disease is an imposing clinical problem for many patients with AIDS. While the diarrhea that occurs so commonly in homosexuals usually has an identifiable infectious etiology, that is, the socalled "gay gut syndrome," the histological changes seen in the gut of about 15% of patients with HIV-1 infection have no recognized etiology (Greenson ei al., 1991). Small bowel biopsies in the late stages of AIDS often show villous atrophy and crypt hypertrophy but a relatively low mitotic count even in patients without diarrhea. Bartlett ei al. (1992) suggested that this non-

specific lesion is, in fact, etiologically associated with HIV-1 infection. If so, its pathogenesis is obscure. Batman ei al. (1991) found a reduction in the axonal density of the autonomic nerves in the villi and pericryptal lamina propria of the jejunum. The denervation tended to be greatest in patients with severe diarrhea. This lesion may be a reflection of the autonomic neuropathy that is attributable to HIV-1 infection (Blanshard ei al., 1993). Obviously, further investigation of this interesting problem is needed. The anus is often traumatized as a result of receptive homosexual intercourse. Ulceration and inflammation is common, and it is likely that trauma predisposes to the papillomavirus infections that are so common at this site (see Chapter 21). HIV-1 is released from the anal mucosa and may infect the insertive sexual partner. In one study, 60% of some 374 HIV-1-positive male homosexuals were found to shed HIV-1 RNA when the anal mucosa was swabbed on only a single occasion. Many of these men had normal numbers of CD44- T lymphocytes in their blood. Shedding of virus correlated with inflammation in the anal mucosa, but not with the level of circulating HIV-1 RNA (Kiviat ei al., 1998). Clinical acute pancreatitis has been reported in 17% of infants and 22% of adults with AIDS (Kahn ei al., 1995; Dowell ei al., 1990). It also occurs during the acute systematic illness that follows shortly after acquisition of an HIV-1 infection. Autopsies have failed to identify specific lesions attributable to HIV-1, although opportunistic infections frequently involve the pancreas. In a

FIGURE 16.13 Testis of a man with AIDS exhibiting interstitial fibrosis and thickening of the walls of tubules associated with the loss of germ cells. Reprinted with permission from Rogers and Klatt (1988).

231

Human Immunodeficiency Viruses

controlled autopsy study of adults with AIDS, Dowell and his colleagues (1990) did not identify lesions of the pancreatic parenchyma in excess of the number observed in matched controls. In a series of 71 HlV-l-infected children, Kahn et al (1995) found pathological changes frequently but concluded that they were mild and only reflected systemic disease.

TUBULORETICULAR STRUCTURES (TRSs) A N D CYLINDRICAL CONFRONTING CISTERNAE (CCC) These intracellular structures have perplexed electron microscopists since the time of their first description (Schaff et al, 1985) (see Figure 16.14). Located in association with the rough endoplasmic reticulum, they initially were believed to represent the nucleocapsids of myxoviruses (see Figure 3.1), but cytochemical studies showed them to be constructed of membrane phospholipids and glycoproteins, not nucleoproteins. In addition, the dimensions are inconsistent with a viral origin. According to Luu et al. (1989), "TRSs appear to consist of reticular aggregates of branching membranous tubules ... [which] range from a typical compact honeycomb-like pattern to a pattern characterized by long, loose tubules laying in an amorphous material of low electron density." On the other hand, CCC "appear as relatively long (up to 3.5 |im) cylinders of fused membranous lamellae that are composed of two or more cisternae of endoplasmic reticulum, one inside

the other, with a 24-nm layer of electron dense material present in the cytosol between the cisternae." Both TRSs and CCC are found by ultrastructural analysis in the cells of 83% of patients with AIDS. CCC have been similarly identified in 41% of AIDS patients. The structures are less commonly detected in HlV-l-infected patients without AIDS. Because of their common presence, it is probable they would be found to be present in persons with advanced HIV-1 infections if sufficient effort were to be expended to search for them in tissues. TRSs and CCC are not retrovirus constituents, and their biologic significance in the AIDS syndrome is uncertain. A detailed and amply illustrated review of this interesting topic has been published (Luu et al, 1989).

LYMPHOMAS These neoplasms appear to be consequent to immune suppression, superimposed upon genetic alterations in the tumor precursor lymphoid cells. EBV most probably is a contributing pathogenic factor in some lesions, but its role is often uncertain. Cytokine dysregulation may also contribute. AIDS-related nonHodgkin's lymphomas are believed to evolve from a polyclonal hyperplasia of lymphoid elements into a monoclonal proliferation of B cells which have acquired structural chromosomal alterations. The incidence of non-Hodgkin's B cell lymphomas is increased by roughly 70-fold in patients with AIDS.

FIGURE 16.14 Tubuloreticular structures (thick arrows) in continuity with cylindrical confronting cisternae (thin arrows) in cultured lymphoblastoid cells (4.5 x 10*). Reprinted with permission from Luu et al. (1989).

232

Pathology and Pathogenesis of Human Viral Disease

Before the introduction of protease inhibitor therapy, it was estimated that roughly 1.2 x 10^ new cases of HIVrelated lymphomas would arise annually in the United States (Knowles ei al, 1988; Rabkin and Yellin, 1994). Improvements in HIV-1 treatment no doubt will make this estimate moot. Many non-Hodgkin's lymphomas in patients with AIDS are only discovered by autopsy. In one reported series from Italy, 50% of cases were diagnosed for the first time after death (Ridolfo ei al., 1996). These tumors almost invariably are either highgrade immunoblastic lymphomas or large-cell lymphomas with an immunoblastic component. A variable, but substantially fewer number, of small noncleaved Burkitt's lymphomas also occur, particularly in teenagers and young adults with AIDS, but the incidence of these tumors appears to be decreasing. Burkitt's lymphomas tend to occur early in the course of AIDS, and are often the first indication that an HIV-1 infection is progressing into AIDS. Typically, the HIV-1associated lymphomas are extranodal, and about 30% involve the central nervous system, where they prove to be clinically aggressive. The lymphoraas in the brain tend to display histologic features consistent with an immunoblastic/plasmacytoid neoplasm and are much less heterogeneous than tumors observed in systemic sites. Roughly 60% of the extranodal tumors are associated with the abdominal digestive tract, but a few lesions are located in the oropharynx, anus, and salivary glands (loachim ei al., 1991). Occasionally, the lungs are involved (Eisner ei al., 1996). In AIDS-associated Burkitt's lymphoma, the cytogeneticist consistently finds the chromosomal translocations described in Chapter 9. As a result, the c-myc protooncogene is transcriptionally deregulated and often exhibits mutations. Translocated c-myc alleles can transform B cells in culture and cause lymphomas in transgenic mice (Gaidano ei al, 1998). In contrast to endemic Burkitt's lymphoma, only about a third of these tumors are infected with EBV, and when infection does occur the transforming proteins of EBV are not expressed by the tumor cells. Thus, EBV in this setting appears only to be a nonpathogenic ''passenger" virus. Large-cell lymphomas of B cell origin are now the commonest type of non-Hodgkin's lymphoma developing in patients with AIDS. These tumors customarily appear late in the course of the AIDS syndrome, when immunosuppression is profound. Almost invariably, these B cell lymphomas associated with AIDS carry the EBV genome and the infected cells express the presumptive oncogenic LMP-1 gene of the virus. Largecell lymphomas developing in non-HIV-1-infected members of the general population are not so endowed. Thus, EBV may play a critical role in the patho-

genesis of non-Burkitt's non-Hodgkin's lymphomas in patients with AIDS (Camilleri-Broet ei al, 1997). AIDSassociated, body cavity-based, non-Hodgkin's lymphomas are considered in Chapter 12. A modest increase in the prevalence of Hodgkin's disease has been reported in some but not all AIDS case series (Biggar ei al, 1987; Rabkin and Yellin, 1994; Bellas ei al, 1996; Serraino ei al, 1997). Interestingly enough, Hodgkin's disease appears to be substantially more common among patients with AIDS in Europe, than in the United States. Geographic variability in the prevalence of the tumor among HIV-positive males may be influenced by diagnostic criteria and the methods employed for case ascertainment. The mixed cell and lymphocyte-depleted types predominate among relatively young men with AIDS, whereas in the general population lesions of these morphological types tend to occur in older persons. The disease is often advanced at the time of diagnosis. In these cases, the prognosis proves to be grim and the response to standard therapy poor. Clearly, Hodgkin's disease in patients with AIDS is more aggressive than the disease in members of the general population. As with non-Hodgkin's lymphoma, LMP-1 expression resulting from EBV infection is consistently found in the tumor cells of persons with AIDS, whereas its prevalence in the Hodgkin's tumors occurring in the general population is substantially lower (Bellas ei al, 1996). The LMP-1 gene in the majority of these cases exhibits a 30-base pair deletion near the 3' end. The basis for this molecular defect is unknown, and its pathogenic importance is obscure (Sandvej ei al, 1994). However, it would not appear to reflect the spread of a particular strain of EBV among patients with HIV-1 infections. A similar defective gene is found in about a third of the non-HIV-infected persons with Hodgkin's disease whose tumors express LMP-1.

KAPOSrS SARCOMA (see Chapter 12)

Kaposi's sarcoma (KS) occurred with extraordinary frequency among young homosexual males in the early stages of the AIDS epidemic during the 1980s. The absolute number of cases increased thereafter, with an apparent epidemic peak in 1987. Subsequently, the number of patients with AIDS who present with KS has dropped precipitously. In San Francisco, during 1981, 58% of cases of AIDS manifested KS as the first sign of the disease, whereas in 1989 only 19% had KS as the initial manifestation (Rutherford ei al, 1990). Not only was there a decrease in the absolute number of cases of

233

Human Immunodeficiency Viruses

KS in AIDS patients, but, in addition, the disease developed later in the course of the HIV-1 infection. A study from Denmark showed a similar change in the time of onset of KS, but the proportion of AIDS patients with KS remained the same (Lundgren et ah, 1995). These changing patterns over the years have been accompanied by modifications in sexual practices among male homosexuals, as well as by the appearance of AIDS in other high-risk groups, specifically, i.v. drug abusers. KS occurs rarely in these latter patients. While the incidence of KS is increasing among women with AIDS, transmission would appear to be consequent to a sexual relationship with a bisexual male. Women of Caribbean and African origin seem to be at greatest risk (Beral, 1991). Some studies suggest that women experience a more fulminating course of KS. Among males, epidemiological studies suggest an association of KS with exposure to fecal material and semen of the homosexual partner. A recent report documents a significant risk with insertive oroanal intercourse, that is, so-called rimming or rose-leafing (Grulich et ah, 1997). These patients also have a higher prevalence of sexually transmitted diseases in comparison to AIDS patients who fail to develop KS. Molecular virological work has identified a specific subtype of HHV-8 associated with cases of KS accompanying HIV-1 (Zong et al, 1997), and serological studies document the acquisition of HHV-8 serum antibodies before the development of KS in the same patient group (Gao et al, 1996). The epidemiological evidence clearly establishes KS as a sexually transmitted disease among male homosexuals and temporally relates its epidemic appearance to HHV-8 transmission among the male homosexual/bisexual population. A possible exception may be found among male Haitians, who experience a high prevalence of KS but apparently do not practice homosexual intercourse commonly. KS in AIDS is a multicentric, often monoclonal neoplasm with a highly aggressive clinical course that is frequently manifest as internal disease. About 70% of patients present initially with lesions of the skin, arms, trunk, head, and neck. Lesions of the penis, nose, eyelids, conjunctiva, and forehead are particularly common. They can be single, but more frequently are multiple and appear as pink, red, or violaceous oval or elongated macules, papules, and nodules. In roughly 30% of cases, the skin is not involved and KS is only found in internal organs (Table 16.12). The oropharynx is commonly involved (Patow et ah, 1984), and as the disease progresses, the upper airways develop lifethreatening obstructive KS lesions (Greenberg et al, 1985). The lungs often exhibit lesions that are either

infiltrative or nodular (Garay et al, 1987; Kornfeld and Axelrod, 1983). Both the upper and lower gastrointestinal tracts are affected. About 30% of patients have esophageal lesions, but the most prominent changes are found in the small intestines, where the lesions are submucosal. Initially, they appear as pink/red to violaceous nodules, but the tumors later protrude into the lumen, where they ulcerate and bleed. In some cases, KS lesions are found in virtually every internal organ. Disease was found in the lymph nodes of 47% of cases in one series (Safai et al, 1985). KS in AIDS patients is restricted to lymph nodes on rare occasions. Although the clinical course in AIDS patients is variable, KS often contributes to morbidity and mortality (Reichert et al, 1983; Safai et al, 1985; Moskowitz et al, 1985). The histopathology of KS is considered in Chapter 12. The aggressive character of KS in AIDS has been compared to the picture seen in rare cases of KS occurring among children in Africa. My review of the literature does not support this conclusion, although similarities exist. Typically, the childhood African case presents with generalized lymphadenopathy, but skin lesions are usually not present. Biopsy demonstrates complete replacement of lymph nodes by tumor. Lesions of internal organs in these youngsters rarely are seen, although the number of cases that have been examined by autopsy would appear to be few (Bhana et al, 1970; Davies and Lothe, 1962; O'Connell, 1977; Slavin et al, 1970). The disease in African children is rapidly fatal. TABLE 16.12 Skin and Viscerallnvolvement with Kaposi's Sarcoma^

Organ Skin GI tract Lymph nodes Lung Liver Heart Spleen Bladder Prostate Gallbladder Pancreas Thyroid Eyes

Patients w/ skin involvement (n = 17)

Patients w/o skin involvement (n = 7)

Total (n = 24)

17 9 9 6 2 2 2 2 2 1 1 1 1

0 3 3 3 0 0 0 0 0 0 0 0 0

17 12 12 9 2 2 2 2 2 1 1 1 1

Adapted with permission from Lemlich et al. (1987).

234

Pathology and Pathogenesis of Human Viral Disease

The clinical course and distribution of lesions in African adults with the endemic forms of KS differs substantially from the disease in patients in developed countries with AIDS. In African males who are not infected with HIV-1, lesions usually appear as an indolent process in the distal lower extremities and slowly progress centrally over a period of years. However, in the rare case that has been examined by autopsy, the internal organs are involved (Table 16.13) (Lothe and Murray 1962). TABLE 16.13 Distribution of Kaposi's Sarcoma Lesions in 19 Adult African Patients Established by Autopsy Site of lesion

No. of cases

Skin

14

Lymph nodes

14

Small intestine

10

Large intestine

5

Adrenal glands

6

Liver

6

Salivary glands

2

Lungs

4

Pericardium and heart

5

Larynx

2

Pharynx

2

Tonsil

2

Voluntary muscle

2

Pancreas

2

Spleen

3

Adapted with permission from Lothe and Murray (1962).

CERVICAL CANCER (see Chapter 21)

In 1993, the CDC categorized cervical cancer as an AIDS-defining illness. Although the incidence of HPV infections and cervical neoplasia are strikingly increased in HIV-positive-women, a cause-and-effect relationship between infections with the two involved viruses has been difficult to establish because of the obvious social factors confounding the analysis. The compelling evidence summarized here now supports the notion that patients with HIV-1 are more susceptible to infection with HPV, most probably on the basis of their attenuated immunological capabilities. While it

is possible to envision an interaction between HIV-1 and HPV that enhances the pathogenicity of the latter (as proves to be the cases with the herpesviruses), no such virological phenomenon has been demonstrated thus far. Indeed, in the cervix, the two viruses fail to interface. The papillomaviruses appear to be restricted to the epithelium, and HIV-1 is located in submucosal macrophages (Nuovo ei ah, 1991). In systematic studies comparing HIV-1-positive and -negative sexually active women (Sun et ah, 1997), 95% of those infected with HIV-1 and having fewer than 500 CD4+ cells/ml of blood acquired one or more types of HPV in the genital tract when evaluated on four occasions over an approximate 2-year period (Figure 16.15). Slightly more than 20% of these women acquired a ''high-risk'' HPV during this time period. Persistent HPV infections were documented in 24% of the females with HIV-1, but in only 4% of control subjects (Vernon et al, 1994). Increased amounts of virus have been found in the genital tracts of HIV-1-positive women (in comparison to healthy women), and the amounts tend to correlate inversely with degree of immunosuppression. Since persistence of "high-risk" virus types in the genital tract is a critical risk factor for development of intraepithelial neoplasms and invasive cancer, the evidence argues compellingly that infectious HIV-1 and its immunosuppressive effects critically influence the development of cervical cancer.

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235

Human Immunodeficiency Viruses

HIV-Seronegative Women

o—©—^—_^^o -^^-

<m~

HIV-Seropositive Women

1#3

-tllp-

^p"—-mm^- - —mM^^— ^^^

-O -^maGy

<2y~ 300

-46>' ••

-I'll-

16,31 900

600


Foliow-up (days) FIGURE 16.15 Pattern of HPV type 16 shedding in the genital tract of HIV-1 seronegative and HIV-1 seropositive women. Studies were done routinely on four occasions as illustrated. Each line represents the results from a single subject. All of the patients had HPV type 16 infections, but the illustration demonstrates the chronicity of these infections and the concomitant infection by other HPV types in patients with HIV-1 infections. The chronicity of the infection increases with virus concentration and may account for the common occurrence of cervical neoplasms in HIV-1-positive women. Reprinted with permission from Sun et at. (1997).

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17 Human T Cell Leukemia/Lymphoma Viruses (HTLV-1 and -2) INTRODUCTION 243 T CELL LEUKEMIA/LYMPHOMA (TLL) SYNDROME TROPICAL SPASTIC PARAPARESIS (TSP) 247 INFLAMMATORY CONDITIONS ASSOCIATED WITH

bution in certain population groups. These various strains of HTLV-1 do not appear to differ in pathogenicity. It is estimated that 10 to 20 million persons have contracted HTLV-1 worldwide, with there being an approximate 5% life risk of serious disease developing as a consequence of this infection. Although the virus is now widely distributed among residents of developed countries, the initially endemic foci were first identified in the southern islands of the Japanese archipelago (including Okinawa) (Figure 17.1), the Caribbean Islands and adjacent regions of northeast South America, and West Africa, particularly the Congo Basin. It also occurs sporadically in residents of the southeastern United States and in restricted clusters of Iranian Jewish immigrants in Israel, Native Americans in British Columbia, and in certain tribes in New Guinea. Members of these latter clusters are infected with strains carried during tribal migrations in the distant past (Yamashita et ah, 1996). Three modes of transmission of the virus are documented. Maternal infection of infants by breastfeeding is the primary route in most endemic societies. Transplacental infection is believed to be an unlikely route. In Japan, viral transmission in endemic regions is substantially reduced when seropositive mothers abstain from breastfeeding, or when breast milk is stored at 4°C for 24 hr. Acquisition of infection in the postnatal period appears to result in the greatest risk of acquiring T cell leukemia/lymphoma. Infection also occurs commonly by means of blood transfusion and to a lesser extent by intravenous drug usage. Fresh frozen plasma is not infectious, an indication that the cellular components of the blood serve as the vector of the virus. As might be expected, storage of blood at 4°C for 24 hr reduces infectivity substantially. The rate of acquisition of infection from transfused blood increases dramatically when the cell con-

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HTLV-2 249 REFERENCES

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INTRODUCTION HTLV-1 and -2, 90 run in diameter, are C-type retroviruses having specific tropism for human helper/inducer T cells. HTLV-1 was initially isolated from the T cells of a patient with a cutaneous lymphoma mistakenly thought to be mycosis fungoides/Sezary syndrome (Poiesz ei ah, 1980). Cell cultures of hairy cell leukemia yielded the first strains of HTLV-2. The molecular make-up of these two viruses is similar to other retroviruses, but they possess, in addition, a unique X region in the genome that endows the viruses with exceptional biological properties. The two genes in this X region are now termed tax and rex. HTLV-1 and -2 are closely related to a leukemia/lymphoma virus of bovine origin that has a cosmopolitan distribution and sporadically causes malignant disease in infected cattle. Detailed molecular analyses establish a close link between HTLV-1 and similar agents in subhuman Old and New World primates. Transmission of HTLV-like simian virus to humans may have happened on at least three occasions, but these rare hypothetical events most probably occurred in man's ancient past. As a result, three different subtypes of HTLV-1 are established in various populations worldwide. The most widely distributed type was most probably disseminated by means of the slave trade in centuries past. The other strains of HTLV-1 exhibit a more endemic distri-

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•

Bi \m \m •

110SB-iie M~ 88 20~5B ~20

Kyushu F I G U R E 17.1 Adult T cell leukemia/lymphoma incidence rates (per 1 x 10^ persons) in the Japanese archipelago. Reprinted with permission from Anonymous (1996).

centration of virus is greater than 10^. The risk of infection from a unit of contaminated blood is estimated to range from 15 to 60%. Persons acquiring the virus by means of transfusion seem to be at particularly high risk of developing the HTLV-1 neurological disease described below. Serological screening for HTLVl/HTLV-2 blood for transfusion was initiated in the United States in 1988. Sexual transmission also serves as a route of infection; women are almost invariably at risk. In one Japanese study, the risk for transmission from husband to wife over a 10-year period was estimated to be 60%, whereas wife-to-husband transmission almost never occurred. In another study from Japan, 50% of wives of HTLV-1 seropositive men were infected after 4 years of marriage. Horizontal infection of the wife by the husband and vertical infection of children from the mother most probably account for the clustering of cases in individual families and in communities. The incidence of HTLV-1 among female sex workers in a South American city was found to increase with duration of prostitution. After 6 years the prevalence of seropositivity was 16%. There is currently circumstantial evidence to indicate that an infection acquired sexually can result in disease, although it occurs later in life (Araujo and Andrada-Serpa, 1996), but the possibility exists that sexually transmitted infections of other types may enhance replication of HTLV-1 in infected persons (Anonymous, 1996).

T-CELL LEUKEMIA/LYMPHOMA (TLL) SYNDROME In 1977, Japanese hematologists described a unique T cell lymphoproliferative disease affecting middleaged adults in Kyushu, the southernmost island of the Japanese archipelago (Uchiyama et ah, 1977). Five years later, Catovsky ei al. (1982) documented a cluster of black adults in the Caribbean with a clinical and pathological entity identical to the Japanese syndrome. Later studies in the United States further characterized the leukemia and ultimately resulted in recovery of the etiologically responsible retrovirus (Poiesz et al, 1980; Bunn et al, 1983). HTLV-1 TLL is characterized by an indolent progressive course that may or may not terminate in a precipitous acute phase. Initially, an HTLV-1 carrier is asymptomatic, although a few abnormal lymphocytes may be found in the blood. Occasionally, there is fever and ill health. The outcome of this phase is variable in duration, but most HTLV-1 carriers manifest no further progression of the disease. A smoldering form of TLL is also recognized. In this condition, erythematous, papillary, and nodular skin lesions develop in association with the presence of atypical lymphocytes in the blood as described above. The clinically overt stage of TLL due to HTLV-1 is rapidly progressive and of short duration, presumably

Human T Cell Leukemla/Lymphoma Viruses

occurring after a prolonged prodromal period. It is characterized by lymphocytosis comprised of morphologically distinctive malignant CD4+ cells. While the numbers of lymphocytes in the peripheral blood are variable over a wide range, the numbers are on average in the neighborhood of 25,000 mm^. Anemia is seen in fewer than half of patients, and thrombocytopenia does not occur. Lymphadenopathy, hepatosplenomegaly and hypercalcemia with or without accompanying lytic bone lesions occur concomitantly. About 60% of patients with adult TLL exhibit infiltrative skin lesions that pathologically show both dermal and epidermal involvement with Pautrier's microabscesses. Adult TLL leukemia is characterized by the presence of medium-sized, often pleomorphic, lymphocytes in the blood (Figure 17.2A-D). These cells have a convoluted multilobulate nucleus. They are sometimes termed "flower cells." The cells vary in size in individual patients, as well as among patients of the same ethnic background. They exhibit the T3+T4+T8- phenotype of mature peripheral helper T cells. The cells typically demonstrate chromosomal abnormalities during the acute stages of disease, but the patterns of the karyotype abnormalities do not measurably differ from those found in T cell leukemias not associated with HTLV-1 infection. TLL peripheral blood cells can, at times, be confused with the circulating cells of Sezary syndrome, which have a typical hyperchromatic cerebriform nucleus. Bone marrow examination reveals variable numbers of malignant lymphoid cells and, at times, osteoclastic activity with evidence of bone reabsorption. In one series, 40% of patients had radiologically demonstrable lytic bone lesions (Jaffe et ah, 1984). Hypercalcemia was consistently present in those with bone disease, but it can be seen in the absence of a leukemic infiltrate. The enlarged lymph nodes accompanying the disease exhibit a diffuse loosely scattered population of cells of mixed size, including a large pleomorphic variant cell. Follicular nodularity is not evident (Lukes and Collins, 1992) (Figure 17.2E-G). The liver shows infiltrates in the portal region that extends into the adjacent parenchyma. Other sites of infiltrative involvement are the lung, digestive tract, and kidney, as well as the meninges of the central nervous system. The predominant form of adult lymphatic leukemia is of B cell origin. It exhibits a relatively indolent prolonged course, extending over years or decades. In contrast, T cell leukemias in adults are much less common but have a more aggressive clinical course and are often rapidly fatal. Mycosis fungoides/Sezary syndrome is a chronic largely cutaneous form of T cell

245

lymphoma. The skin lesions of this syndrome must be differentiated from those occurring in TLL. Systematic molecular studies of skin lesions from cases of cutaneous T cell lymphoma, large cell lymphoma, lymphomatoid papulosis, and Hodgkin's disease have failed to reveal any evidence to suggest that these conditions are HTLV-1 related (Kikuchi ei al, 1997; Arai et al, 1994; Wood et al., 1997). Finally, some hematologists include lymphoproliferative disorders comprised of T-gamma cells and characterized by the presence of large granular lymphocytes. The lesions have the features of malignant natural killer cells (Knowles, 1986; Pandolfi et al, 1992). While the classical clinical features of these various skin conditions are distinctive, exclusion of an HTLV-1 etiology for a skin problem in individual cases of leukemia/lymphoma may require virological evaluation. What mechanisms are involved in the transformation of CD4+ T lymphocytes by HTLV-1? At present, we have no specific answers to this question, but intriguing observations support feasible hypotheses. As noted above, TLL occurs sporadically and is rare, even in populations with a high frequency of endemic infection. The disease requires many decades to develop, during which time evidence of a reversible preleukemic state slowly evolves. When leukemia develops, only a small proportion of malignant cells contain identifiable viral protein or virions, whereas most cells exhibit no evidence of viral transcription. Unlike many of the retroviruses of lesser species, that is, the so-called oncornaviruses, the HTLV-1 genome does not incorporate cellular oncogenes acquired during growth in animal tissue. In addition to the constitutive retrovirus genes (gag, pol, and env), the HTLV-1 genome contains a sequence known as pX. The pX gene codes at least three proteins — p21. Tax and Rex. The latter two proteins are intrinsic to viral replication. Tax activates transcription of the viral genome, whereas Rex is a negative regulatory factor. Thus, the interaction of these two gene products plays an intrinsic role in continued virus expression and, accordingly, replication. However, Tax has additional functions, for it activates a plethora of cellular genes including those of the lymphokines such as IL-2 and IL-6, and their receptors, as well as various nuclear protooncogenes and cell surface molecules. It is not surprising that in experimental settings the Tax protein can extend the lifespan of lymphocytes, increase the growth of fibroblasts in agar, and act synergistically with the Ras oncogene to transform rodent cells. Incorporation of the Tax gene in the germline of mice results in a transgenic animal having a marked predisposition for the development of neurofibromas. Findings of this

246

Pathology and Pathogenesis of Human Viral Disease

F I G U R E 1 7 . 2 Adult T cell leukemia associated with HTLV-1 infection. The illustrations are from from a single case and are reprinted with permission from Lukes and Collins (1992). (A) Peripheral blood. A peripheral blood smear shows small lymphocytes with extraordinary variation in nuclear configuration with deep indentation and cloverleaf patterns. The cytoplasm is slightly azurophilic and vacuolated. Small nucleoli are noted. Wright's stain. xlOOO. (B) Peripheral blood. Occasional large blasts are present in this peripheral blood smear, although their percentage is usually low. They have finely acidophilic nuclear chromatin and small nucleoli. The cytoplasm is azurophilic and contains multiple small vacuoles. Wright's stain. xlOOO. (C) Peripheral blood. A cytospin preparation of peripheral blood reveals marked accentuation of the nuclear configurational changes with indentation and lobulation. Wright's stain. xlOOO. (D) Peripheral blood. A plastic-embedded section of buffy coat shows only limited nuclear invagination, illustrating that the degree of indentation and lobulation varies considerably with the conditions of the preparation. xlOOO. (E) Lymph node. In this illustration, there is diffuse involvement without any follicular nodulation. Sinuses are dilated and often contain loosely scattered neoplastic cells. The capsule is not involved. H&E. x32. (F) Lymph node. This section shows remarkable variation in size and nuclear configuration of neoplastic cells. This variation is more apparent than in the peripheral blood illustrations. H&E. x600. (G) Lymph node. An MGP stain emphasizes the abundance of the lightly pyroninophilic character of the cytoplasm. Prominent nucleoli are seen. MGP. x600.

Human T Cell Leukemia/Lymphoma Viruses

type show that Tax is, in fact, a classical oncogene. Conceivably, then. Tax and its cell products could play both an autocrine and a paracrine role in stimulating the growth of infected populations of cells. The extraordinary Tax protein has additional capabilities, for it binds cell cycle inhibitors in a manner similar to protein products of classical DNA tumor viruses. These proteins appear to suppress the naturally occurring cell negative regulators, Rb and p53, thus allowing for runaway cell growth. The various Tax functions described above have, to a large extent, been documented by in vitro studies. What occurs in humans is unclear. The evidence thus far presented does not necessarily serve as a basis for concluding that infection can result in malignant transformation. Rather, one might hypothesize that, as of yet, unidentified cofactors are critical determinants of malignancy. Chromosomal alterations in the HTLV-1infected cell, not dissimilar from those found in nonviral T cell leukemia, may result in a malignant genotype. At present, we just don't know (Hjelle, 1991; Pandolfi et al, 1992; Hollsberg and Hafler, 1993; Yoshida, 1996).

TROPICAL SPASTIC PARAPARESIS (TSP) (syn. HTLV-1-Associated Myelopathy [HAM])

TSP was initially associated with HTLV-1 infection by serological means in studies of patients residing in Jamaica (Cruickshank, 1956; Gessain et ah, 1985). A similar syndrome was found to occur throughout the Caribbean basin as well as in the Cote dTvoire and in the Seychelle Islands. Shortly thereafter, the syndrome was described in Japan, but the designation of HAM was applied there. TSP and HAM are now believed to be identical clinical and pathological entities associated worldwide with HTLV-1 infection (Touze et ah, 1996). Although the etiological relationship of HTLV-1 with TSP/HAM is now well established, the incidence in endemic clusters is low, that is, 0.25% in Jamaica. However, the prevalence of the disease among seropositive persons differs greatly in various geographic regions. For example, the incidence in Martinique is roughly 1.5 to 3%, while in southern Japan it is less than 0.1%, a 15to 30-fold difference (Kaplan et al., 1990). In the same geographic region of the Democratic Republic of the Congo, a threefold difference in prevalence of the disease among various isolated tribes is found. Environmental factors and host genetic influences may be important considerations in the pathogenesis of the

247

disease, but the relative virulence of different virus strains and the mode of acquisition of the virus could play an important role. TSP/HAM is a chronic, but progressively evolving, demyelinating myelopathy primarily, but not exclusively, involving the lateral corticospinal, spinocerebellar, spinothalamic, and posterior corticocerebral tracts (Figure 17.3). Posterior sensory tracts are occasionally affected, but to a relatively minor degree. The lesions are most prominent in the cervical and thoracic cord. At autopsy, axonal loss is often extensive in the cord areas of demyelinization (Levin et al, 1997) (Figure 17.4). Foci of gliosis and chronic perivascular lymphocytic infiltration are sometimes seen (Rosenblum et al., 1992). Typically, the leptomeninges are thickened by fibrosis and hyalinized perivascular fibrosis is seen. Variable numbers of lymphocytes are found in association with small blood vessels in the meninges. In the brain, lymphocytic perivascular cuffing is occasionally found in the medulla and pons, as well as in the white matter of the cerebrum and cerebellum (Akizuki et al, 1987). Patients exhibit a spastic paresis of the lower extremities accompanied by urinary bladder sphincter problems, male impotency, and intestinal obstipation (Domingues et al, 1995). Sensory complaints occur, but they are not a prominent feature of the syndrome. In Japan, the mean age of onset is 44 years, but children in the first decade of life are occasionally affected. The latency period proved to be less than 3.4 years in half of those who developed the infection consequent to blood transfusion (Ijichi et al, 1996). The pathogenic role of HTLV-1 in TSP/HAM is unclear, but there is no evidence to indicate that the disease results from direct involvement of the spinal cord by the virus. Indeed, the clinical data suggest that a chronic inflammatory process (in which HTLV-1-infected CD4+ and CD8+ lymphocytes are found) precedes development of the destructive lesions of the cord. This observation has led some investigators to conclude that TSP/HAM is an idiosyncratic hypersensitivity process mediated immunologically. If so, the target antigen(s) have yet to be identified (Jacobson, 1996). Alternatively, other observations indicate that the infiltrating activated virus-infected lymphocytes may release cytochemicals and cytokines directly, damaging nervous system tissue (Ijichi et al, 1996). Patients tend to have a hyperactive immune system with high concentrations of CD8+ HTLV-1-sensitized lymphocytes and HTLV-1 DNA in the blood and spinal fluid (Levin et al, 1997). Additional pathological characterization of the early stages of this fascinating but

248

Pathology and Pathogenesis of Human Viral Disease

10mr^-k

''-'\Wr\ _^:]^J^] -

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FIGURE 17.3 HTLV-1 spinal cord demyelinating lesion. Cervical cord (top) showing the loss of myelinated axons, largely from lateral corticospinal and posterior spinocellular tracts, but also to an extent in fasciculi gracii as reflected in pallor and shrinkage of these regions. Thoracic segment of cord (bottom) exhibiting diffuse pallor of the lateral and anterior columns. Both HTLV-1-infected patients had HIV-1 associated AIDS. Vacuolar changes are not conspicuous in these specimens, but circumferential leptomeningeal fibrosis and thickening of meningeal blood vessels is evident (Luxol fast blue/hematoxylin-eosin stains). Reprinted with permission from Rosenblum et al. (1992).

poorly understood myelopathy are needed in order for us to assess more critically assess the pathogenic mechanisms involved in this disease.

INFLAMMATORY C O N D I T I O N S ASSOCIATED WITH HTLV-1 INFECTION Several inflammatory disorders of presumptive autoimmune pathogenesis have been described in HTLV-1-infected persons, some of whom are also afflicted with TSP/HAM. Of these, polymyositis, arthritis, and uveitis are best documented. At present, a mechanistic basis for these various lesions is lacking, but the evidence suggests that activated infiltrating HTLV-1-infected CD4+ cells initiate the release of a

plethora of cytokines and cell surface receptors, thus triggering localized inflammation. Interestingly enough, transgenic mice carrying the Tax gene of HTLV-1 develop an inflammatory arthritis and a Sjogren-like parotitis (Green et al, 1989; Iwakura et al, 1991). The polymyositis is characterized by the gradual onset of proximal muscle weakness and an electromyographic picture consistent with inflammation. Histologically, the striated muscles exhibit atrophy, necrosis, and fibrosis associated with prominent infiltrates of macrophages and lymphocytes. The predominant infiltrating lymphocytes are CD8+, cytolytic T cells. These cells have been shown to carry the HTLV1 genome (Osame et al, 1986; Sherman et al, 1995); however, there is no evidence to suggest that muscle cells are infected.

249

Human T Cell Leukemla/Lymphoma Viruses

B

FIGURE 17.4 Immunohistochemical analysis of inflammatory cell infiltrates in spinal cord of HTLV-1-infected women with paraplegia accompanied by dysphagia and dysarthria. Reactive cells stain brown. (A) Perivascular T cell infiltrates. The infiltrate is largely comprised of CD8+ cells (B) that are activated, as shown by their reaction with CD45RO antibody (C). Panel D documents a robust macrophage response. Reprinted with permission from Levin et al. (1997).

The arthropathy is chronic and customarily monarticular. It involves predominantly, but not exclusively, the large joints. Older women who are long-term carriers of HTLV-1 comprise the majority of those so affected. Rheumatoid factor is not elaborated in response to the disease. Arthroscopy shows a villous synovial hyperplasia, an observation confirmed by biopsy. There is, in addition, radiological documentation of destructive changes in the bone and cartilage of the joint. Pathological study reveals synovial cell atypicalities and infiltrates of CD8+ lymphocytes. Some studies have suggested that the synovial cells are infected, but the matter remains to be resolved. Both the infiltrating lymphocytes and synovial fluid are infected with HTLV-1, as demonstrated by PCR. An increased prevalence of uveitis in young adults with HTLV-1 antibodies has been reported from Japan. Bronchitis and alveolitis are also believed to occur in some HTLV-1-infected patients. The inflammatory cell infiltrates in the eye carry the HTLV-1 genome (Mochizuki et ah, 1996). In endemic areas, both thyroiditis of the Hashimoto stroma type and Sjogren's syndrome are found, with

increased prevalence in persons who possess HTLV-1 serum antibodies (Nishioka, 1996).

HTLV-2 Although HTLV-1 and HTLV-2 share 65% of their nucleotide sequences and are morphologically similar, the two viruses differ with regard to their disease-causing potential and population distribution. HTLV-2 was first recovered from a patient with atypical hairy cell leukemia in 1982 (Kalyanaraman et al., 1982); and a second isolate from a similar case was reported 4 years later (Rosenblatt et ah, 1986). This uncommon leukemia tends to occur in middle-aged men (Chang et ah, 1992) and is probably of B cell origin. The possible association of HTLV-2 with hairy cell leukemia would, in retrospect, seem to be fortuitous, for no evidence of an etiologic relationship has accumulated in more recent years. Similarly, infection with HTLV-2 has been demonstrated in a few patients with TSP/HAM, but a

250

Pathology and Pathogenesis of Human Viral Disease

causative link has not been established. Thus, at present, HTLV-2 reniains an orphan with no recognized disease-causing potential for humans. HTLV-2 has a unique geographic distribution, with endogenous clusters having been identified in many, but certainly not all American Indian tribes in North, Central, and South America (Figure 17.5). These include the Pueblos of New Mexico/Arizona, the Seminoles of Florida, and the Yakima of the Pacific Northwest. In Panama, a focus exists in the Guaymi tribes, but not in the nearby closely related San Bias tribal groups. In South America, the virus is distributed widely among scattered tribes from the Caribbean coast to southern Argentina, but no apparent ethnic link serves as a common denominator (Figure 17.5).

YAKIMA NAVAJO

PUEBLO

(nb) GUAYMI

(nb) TOBA

(nb) MATACX)

(nb) FIGURE 17.5 Native American populations with endemic HTLV-2 infections. Reprinted with permission from Anonymous (1996).

In a carefully conducted study of members of the Gran Chaco tribe in western Paraguay and northern Argentina, the overall prevalence of serological reactivity proved to be 22%, with the incidence increasing with advancing age. As with HTLV-1, infection appeared to be transmitted sexually by the male, and postnatally by breastfeeding (Ferrer et al., 1996). As a result, clustering of cases within families proved to be common. For reasons that are totally unclear, serologic evidence of infection is found commonly in i.v. drug users in North America and Europe. In the United States, HTLV-1 seropositive donors of blood are more often infected with HTLV-2 than with HTLV-1. There is also epidemiologic evidence to suggest that HTLV-2 is

spread sexually by non-Native American populations (Weiss, 1994; Khabbaz et al, 1992). Because HTLV-1 and -2 share antigenic determinants, some concern exists regarding the accuracy of the customary ELISA screening methods. More refined techniques (i.e.. Western blot analysis and radioimmunoprecipitation assays) are required to differentiate the two viruses. The sensitivity of the serological assays also proves it to be a potential problem. As a result, serological survey results and populations studies may not detect all who have been infected (Hall et al, 1994; Rios et al, 1994). References Akizuki, S., Nakazato, O., Higuchi, Y, Tanabe, K., Setoguchi, M., Yoshida, S., Miyazaki, Y, Yamamoto, S., Sudou, S., Sannomiya, K., and Okajima, T. (1987). Necropsy findings in HTLV-I associated myelopathy. Lancet 1,156-157. Anonymous (1996). '"Human Immunodeficiency Viruses and Human T-Cell Lymphotropic Viruses.'' "lARC Monographs on the Evaluation of Carcinogenic Risks to Humans/' Vol. 67. WHO, Lyons. Aral, E., Chow, K., Li, C , Tokunaga, M., and Katayama, I. (1994). Differentiation between cutaneous form of adult T cell leukemia/lymphoma and cutaneous T cell lymphoma by in situ hybridization using a human T cell leukemia virus-1 DNA probe. Am. J. Pathol. 144,15-20. Araujo, A.-C, and Andrada-Serpa, M. (1996). Tropical spastic paraparesis/HTLV-I-associated myelopathy in Brazil. /. Acquit. Immune Defic. Syndr. Hum. Retrovirol. 13 (Suppl. 1), S33-S37. Bunn Jr., R, Schechter, C , Jaffe, E., Blayney, D., Young, R., Matthews, M., Blattner, W., Broder, S., Robert-Guroff, M., and Gallo, R. (1983). Clinical course of retrovirus-associated adult T-cell lymphoma in the United States. New Engl. J. Med. 309, 257-264. Catovsky, D., Greaves, M., Rose, M., Galton, D., Goolden, A., McCluskey, D., White, J., Lampert, L, Bourikas, G., Ireland, R., Brownell, A., Bridges, J., Blattner, W, and Gallo, R. (1982). Adult T-cell lymphoma-leukaemia in blacks from the West Indies. Lancet 1, 639-643. Chang, K., Stroup, R., and Weiss, L. (1992). Hairy cell leukemia: Current status. Am. J. Clin. Pathol 97, 719-738. Cruickshank, E. (1956). A neuropathic syndrome of uncertain origin: Review of 100 cases. West Indian Med. J. 5,147-158. Domingues, R., Muniz, M., Pinho, J., Bassit, L., Jorge, M., Alquezar, A., Marchiori, R, Chamone, D., and Scaff, M. (1995). Human T lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis in Sao Paulo, Brazil. Clin. Infect. Dis. 20,1540-1542. Ferrer, J., Esteban, E., Dube, S., Basombrio, M., Segovia, A., PeraltaRamos, M., Dube, D., Sayre, K., Aguayo, N., Hengst, J., and Poiesz, B. (1996). Endemic infection with human T cell leukemia/lymphoma virus type IIB in Argentinean and Paraguayan Indians: Epidemiology and molecular characterization. /. Infect. Dis. 174, 944-953. Gessain, A., Barin, R, Vernant, J., Gout, O., Maurs, L., Calender, A., and De The, G. (1985). Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis. Lancet 2, 407410. Green, J., Hinrichs, S., Vogel, J., and Jay, G. (1989). Exocrinopathy resembling Sjogren's syndrome in HTLV-1 tax transgenic mice. Nature 341, 72-74.

Human T Cell Leukemia/Lymphoma Viruses Hall, W., Kubo, T., Ijichi, S., Takahashi, H, and Zhu, S. (1994). Human T cell leukemia/lymphoma virus, type II (HTLV-II): Emergence of an important newly recognized pathogen. Sem. Virol. 5,165-178. Hjelle, B. (1991). Human T-cell leukemia/lymphoma viruses: Life cycle, pathogenicity, epidemiology, and diagnosis. Arch. Pathol. Lab. Med. 115, 440^50. HoUsberg, P., and Hafler, D. (1993). Pathogenesis of diseases induced by human lymphotropic virus type I infection. New Engl. J. Med. 328,1173-1182. Ijichi, S., Nakagawa, M., Umehara, R, Higuchi, I., Arimura, K., Izumo, S., and Osame, M. (1996). HAM/TSP: Recent perspectives in Japan. /. Acquir Immune Defic. Syndr. Hum. Retrovirol. 13 (Suppl. 1), S26-S32. Iwakura, Y, Tosu, M., Yoshida, E., Takiguchi, M., Sato, K., Kitajima, I., Nishioka, K., Yamamoto, K., Takeda, T., and Hatanaka, M. (1991). Induction of inflammatory arthropathy resembling rheumatoid arthritis in mice transgenic for HTLV-I. Science 253,1026-1028. Jacobson, S. (1996). Cellular immune responses to HTLV-I: Immunopathologic role in HTLV-I-associated neurologic disease. /. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 13 (Suppl. 1), SIOOS106. Jaffe, E., Blattner, W., Blayney, D., Bunn Jr, P., Cossman, J., RobertGuroff, M., and Gallo, R. (1984). The pathologic spectrum of adult T-cell leukemia/lymphoma in the United States. Am. J. Surg. Pathol. 8, 263-275. Kalyanaraman, V., Sarngadharan, M., Robert-Guroff, M., Miyoshi, I., Blayney, D., Gould, D., and Gallo, R. (1982). A new subtype of human T-cell leukemia virus (HTLV-II) associated with a T-cell variant of hairy cell leukemia. Science 218, 571-573. Kaplan, J., Osame, M., Kubota, H., Igata, A., Nishitani, H., Maeda, Y, Khabbaz, R., and Janssen, R. (1990). The risk of development of HTLV-I-associated myelopathy/tropical spastic paraparesis among persons infected with HTLV-I. /. Acquir. Immune Defic. Syndr. 3,1096-1101. Khabbaz, R., Onorato, I., Canon, R., Hartley, T., roberts, B., Hosein, B., and Kaplan, J. (1992). Seroprevalence of HTLV-I and HTLV-II among intravenous drug users and persons in clinics for sexually transmitted diseases. New Engl. J. Med. 326, 375-380. Kikuchi, A., Ohata, Y, Matsumoto, H., Sugiura, M., and Nishikawa, T. (1997). Anti-HTLV-1 antibody positive cutaneous T-cell lymphoma. Cancer 79, 269-274. Knowles III, D. (1986). The human T-cell leukemias: Clinical, cytomorphologic, immunophenotypic, and genotypic characteristics. Hum. Pathol. 17,14-33. Levin, M., Lehky, T., Flerlage, A., Katz, D., Kingma, D., Jaffe, E., Heiss, J., Patronas, N., McFarland, H., and Jacobson, S. (1997). Immunologic analysis of a spinal cord-biopsy specimen from a patient with human T-cell lymphotropic virus type I-associated neurologic disease. New Engl. J. Med. 336, 839-845. Lukes, R., and Collins, R. (1992). ''Tumors of the Hematopoietic System,'' 2nd ed., 28, p. 409. Armed Forces Institute of Pathology, Washington, DC.

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Mochizuki, M., Ono, A., Ikeda, E., Hikita, N., Watanabe, T., Yamaguchi, K., Sagawa, K., and Ito, K. (1996). HTLV-I uveitis. /. Acquir Immune Defic. Syndr Hum. Retrovirol. 13 (Suppl. 1), S50-S56. Nishioka, K. (1996). HTLV-I arthropathy and Sjogren syndrome. /. Acquir. Immune Defic. Syndr Hum. Retrovirol. 13 (Suppl. 1), S57S62. Osame, M., Usuku, K., Izumo, S., Ijichi, N., Amitani, H., Igata, A., Matsumoto, M., and Tara, M. (1986). HTLV-I associated myelopathy, a new clinical entity. Lancet i, 1031-1032. Pandolfi, F., Zambello, R., Cafaro, A., and Semenzato, G. (1992). Biologic and clinical heterogeneity of lymphoproliferative diseases of peripheral mature T lymphocytes. Lab. Invest. 67, 274302. Poiesz, B., Ruscetti, R, Gazdar, A., Bunn, P., Minna, J., and Gallo, R. (1980). Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. U.S.A. 77, 7415-7419. Rios, M., Khabbaz, R., Kaplan, J., Hall, W., Kessler, D., and Bianco, C. (1994). Transmission of human T cell lymphotropic virus (HTLV) type II by transfusion of HTLV-I-screened blood products. /. Infect. Dis. 170, 206-210. Rosenblatt, J., Golde, D., Wachsman, W., Giorgi, J., Jacobs, A., Schmidt, G., Quan, S., Gasson, J., and Chen, I. (1986). A second isolate of HTLV-II associated with atypical hairy-cell leukemia. New Engl. J. Med. 315, 372-377. Rosenblum, M., Brew, B., Hahn, B., Shaw, G., Haase, A., Maroushek, S., and Price, R. (1992). Human T-lymphotropic virus type I-associated myelopathy in patients with the acquired immunodeficiency syndrome. Hum. Pathol. 23, 513-519. Sherman, M., Amin, R., Rodgers-Johnson, P., Morgan, O., Char, G., Mora, C , lannone, R., Collins, G., Papsidero, L., Gibbs Jr., C , and Poiesz, B. (1995). Identification of human T cell leukemia/lymphoma virus type I antibodies, DNA, and protein in patients with polymyositis. Arthritis Rheum. 38, 690-698. Touze, E., Gessain, A., Lyon-Caen, O., and Gout, O. (1996). Tropical spastic paraparesis/HTLV-I-associated myelopathy in Europe and in Africa: clinical and epidemiologic aspects. /. Acquir Immune Defic. Syndr. Hum. Retrovirol. 13 (Suppl. 1), S38-S45. Uchiyama, T., Yodoi, J., Sagawa, K., Takatsuki, K., and Uchino, H. (1977). Adult T-cell leukemia: Clinical and hematologic features of 16 cases. B/oorf 50, 481-492. Weiss, S. (1994). The evolving epidemiology of human T lymphotropic virus type II. /. Infect. Dis. 169,1080-1083. Wood, G., Schaffer, J., Boni, R., Dummeer, R., Burg, G., Takeshita, M., and Kikuchi, M. (1997). No evidence of HTLV-I proviral integration in lymphoproliferative disorders associated with cutaneous T-cell lymphoma. Am. J. Pathol. 150, 667-673. Yamashita, M., Ido, E., Miura, T., and Hayami, M. (1996). Molecular epidemiology of HTLV-I in the world. /. Acquir Immune Defic. Syndr. Hum. Retrovirol. 13 (Suppl. 1), S124-S131. Yoshida, M. (1996). Multiple targets of HTLV-1 for dysregulation of host cells. Sem. Virol. 7, 349-360.

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18 Hepatitis Viruses Although hepatitis has been recognized clinically since the time of the earliest recorded medical history, knowledge of its epidemiology was exclusively observational. Perhaps the most insightful information on the illness developed in the 1960s, when the late Saul Krugman and his associates conducted studies on the residents of Willowbrook State Hospital in Staten Island, New York. This work provided for the first time definitive clinical evidence that two different transmissible agents with clearly defined incubation periods were involved. Their insightful work, which later proved to be the target of much public criticism, was based on inoculation of experimental samples of blood containing virus into institutionalized mentally handicapped children, who would have otherwise naturally contracted the infections early in the course of their residence in this long-term custodial setting. My next experience with hepatitis occurred in the early 1970s, when I worked with June Almeida, whose unique skill was with negative staining of viruses, and whose interest at the time was the newly discovered Australian antigen, a morphologic enigma in the blood of patients with the long-incubation form of hepatitis (Almeida et al., 1971). At the time, I was astonished when we regularly discovered this antigen in the blood of healthy Africans while conducting studies on serum samples brought to us from his homeland by the dynamic young Nigerian pathologist A. O. Williams. This finding provided a preliminary insight into the medical importance of chronic subclinical hepatitis B virus infections in Africa, and the potential importance of the viral carrier state so common in Africans and Asians. During the ensuing years, as work with hepatitis B provided interest for legions of investigators, others turned to explore the etiology of the short-incubation hepatitis described as MS-1 by Krugman and his colleagues at Willowbrook. Using the electron-microscopic technique of negative staining, Feinstone and his colleagues (1973) demonstrated the presumptive causative virus in stool samples from patients with acute hepatitis; six years later. Provost and Hilleman

INTRODUCTION 253 ORALLY ACQUIRED SHORT-INCUBATION-PERIOD ACUTE HEPATITIS 254

Hepatitis A Virus (HAV) 254 Hepatitis E Vims (HEV) 255 PARENTALLY ACQUIRED LONG-INCUBATION-PERIOD ACUTE AND CHRONIC HEPATITIS 257

Hepatitis B Virus (HBV) 257 Hepatitis D Virus (HDV) (Delta Agent) 260 Hepatitis C Virus (HCV) 260 CHRONIC HEPATITIS ( C H ) 262 HEPATOCELLULAR CARCINOMA ( H C C ) 264 AUTOIMMUNE HEPATITIS (AH) 270 PAPILLARY ACRODERMATITIS (GIANOTTI-CROSTI SYNDROME; GCS) 271 GLOMERULONEPHRITIS 272 REFERENCES 273

INTRODUCTION It was one of those inadvertent needle pricks self-inflicted while culturing the blood of my newly admitted patient with a fever of unknown origin. I thought little about it at the time, but in retrospect, I was pleased to learn a few days later that the bacterial blood cultures were sterile. The patient was soon discharged afebrile, but without a diagnosis. I can only assume now that it was subclinical hepatitis, for just 30 days later the incident immediately came to mind when my urine exhibited the deep mahogany tint that can only be attributed to bilirubin. I suddenly felt ill, and indeed I soon was, with fever, overwhelming malaise, anorexia, and the obvious jaundice that serves as the basis for the clinical diagnosis of acute hepatitis. At the time, I was aware of the so-called hepatitis A, that is, the short-incubation form, and hepatitis B, the so-called long-incubation form of hepatitis, but little else. As an intern in 1957,1 also possessed some knowledge of postnecrotic cirrhosis, but how and under what circumstances it developed was obscure. There were no diagnostic tests for the hepatitis viruses, and treatment was limited to bed rest and a good diet.

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TABLE 18.1 Features of Hepatitis Viruses: Their Associated Liver Disease Typical transmission mechanism Virus Virus family Nucleic acid type Virion diameter Genotypes Incubation period (days) Viremia Virus in stool Acute mortality Chronic hepatitis Cirrhosis Hepatocellular carcinoma

Perinatal/ parenteral/ sexual

Fecal-oral/ ,waterborne HAV Picornavirus RNA 28 7 15-50 Brief + <1% No No No

HEV Calcivirus RNA 32 3 15^5 More prolonged +


HBV Hepadnavirus DNA 42 5 28-160 Indefinite 0 1% Yes Yes Yes

HDV Deltavirus DNA 43 3 NA Indefinite 0 >20% Yes Yes Yes

HCV Flavivirus RNA 38-50 9 14-160 Indefinite 0 1% Yes Yes Yes

NA = Not applicable ''20-40% in pregnancy.

(1979) cultured it in vitro. Thus, the stage was set for a remarkable era of discovery during which the epidemiology and pathogenesis of both hepatitis A and B were elucidated, and several new actors (hepatitis C, D, E, and G) were recognized. This chapter provides an overview of the clinical and epidemiological features of these various infections and specifically addresses the pathogenesis and pathology of the resulting liver disease (Table 18.1).

ORALLY ACQUIRED SHORT-INCUBATION-PERIOD ACUTE HEPATITIS The viruses of hepatitis A (HAV) and hepatitis E (HEV) cause a morphologically identical hepatic inflammatory disease of relatively short duration that almost invariably resolves without persistent liver damage. Both viruses are transmitted by the fecal-oral route and have an incubation period ranging from 15 to 60 days. Hepatitis A Virus (HAV) Hepatitis A is caused by a small (27 nm in diameter) RNA-containing virus that is structurally and biochemically similar to the enteroviruses and rhinoviruses (i.e., picornaviruses) (see Chapters 1 and 2) (Figure 18.1). Because of its unique characteristics, HAV has been assigned to a new genus termed hepatavirus. Although HAV causes hepatitis in various spe-

cies of subhuman primates, one of the features that distinguishes it from other picornaviruses is the reluctance of the virus to grow in and destroy (i.e., cause the cytolysis of) cultured cells of a diversity of types in the laboratory. It is perhaps incorrect to refer to HAV in the singular form, since at least seven antigenically identical but biologically dissimilar genotypes of the virus have been recovered from humans around the globe. HAV has a worldwide distribution. The overall prevalence of infection inversely correlates with the socioeconomic status of the community, and thus the potential for pollution of water sources by raw sewage. In the developed countries of North America and Europe, HAV is a sporadic cause of illness in persons of all ages, and the majority of the population lack serological evidence of infection. On the other hand, infection invariably occurs at a relatively early age in developing countries, and most adults possess serum antibodies as an indication of a prior infection earlier in life. Thus, the epidemiological features of HAV resemble the common human enteroviruses to which it is closely related. Hepatitis due to HAV is an acute necro-inflammatory disease that usually resolves without clinical complications after an illness in adults of a few weeks duration. Infections in young children commonly are asymptomatic and anicteric. The incubation period in adolescents and adults ranges from 2 to 6 weeks. Typically acquired by the oral route, the virus makes its way to the liver by an as-of-yet undefined route where it replicates in hepatocytes without obvious cytolytic effects. A brief period of viremia and excretion by

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FIGURE 18.1 HAV particles demonstrated by immune electron microscopy. These particles can be demonstrated in suspensions of stool during the acute stages of hepatitis. Bar = 100 nm. Reprinted with permission and through the courtesy of J. Marshal, PhD, and I. Gust, PhD.

means of the biliary tract begins before the onset of clinical illness. To the best of our knowledge, cells of other major organs do not support growth of this virus. Although definitive clinical and experimental evidence is lacking, hepatitis A may be the outcome of immune-mediated disease attributable to cytotoxic CD8+ T cells or the cytochemicals generated by the inflammatory response (Vallbracht et ah, 1989). The disease is characterized by the accumulation of T and B cells and, to an extent, plasma cells and neutrophils in the portal triads, where they are intimately associated with hepatocytes (Polotsky et ah, 1996). The cells of the liver microscopically are somewhat in organizational disarray, and, to a variable extent, ballooning of the cytoplasm or evidence of apoptosis is seen. The "ballooned" hepatocyte exhibits cytoplasmic rarefaction, and the cells may ultimately undergo necrosis. The apoptotic cell shrinks and assumes a more geometric configuration with an eosinophilic dense cytoplasm and a pyknotic nucleus. These so-called acidophilic bodies are then extruded in the sinusoids, where they are often phagocytized. Kupffer cells lining the sinusoids become prominent and exhibit accumulations of lipofuscin, which represents the residua of phagocytized necrotic or apoptotic cells. Cholestasis is a variable feature. With the passage of time, evidence of liver parenchymal damage is accompanied by the features of hepatocyte regeneration in the form of mitotic activity, and binucleate hepatic parenchymal cells are commonly observed. Recovery generally is complete 3 months after the onset of illness.

As noted above, HAV is not directly cytolytic for cultured primate cells, in contrast to its close relatives, the enteroviruses. In HAV-infected humans, maximal excretion of virus in the stools occurs 14 days before peak serum concentrations of glutamic pyruvic transaminase (SGPT) are found in the blood. Evidence of virus excretion via the gut continues during convalescence (Krugman et a/., 1959; Thornton et ah, 1975; Mao et al, 1980). Based on these observations, one might conclude that injury to the liver cells is not caused by the virus. The presence of HAV-sensitized CD8+ cells in the liver during acute episodes of hepatitis strongly suggests that hepatocyte injury is due to the cytolytic T cell response or the products of these cells (Vallbracht et al., 1989). Humoral immunity does not appear to be involved. The mechanisms whereby the cytoplasm of cells balloon, or apoptosis occurs, are poorly understood. These are two different mechanistic disease processes. Fulminating necrosis of the liver parenchyma develops on only the rarest of occasions in persons infected with HAV (Lee, 1993).

Hepatitis E Virus (HEV) Hepatitis E is believed to be caused by an as-yet unclassified small RNA virus having morphologic features of the calicivirus, and certain molecular characteristics of rubella virus (Krawczynski, 1993; Reyes, 1993). Like other hepatitis viruses, HEV replicates and

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causes liver disease in a variety of subhuman primates. It also grows with great reluctance in cultured cells, and does not directly cause cytolytic damage to the liver in vivo. The epidemiology of HEV differs from HAV. Although detailed information worldwide is still lacking, it appears to be a major cause of both waterborne epidemics of sizable proportions and sporadic cases of hepatitis in developing countries, particularly in subSaharan Africa, the Indian Subcontinent, and Southeast Asia. HEV is a rare cause of hepatitis in the United States and Europe, where it primarily occurs among i.v. users of drugs. Although clinical illness customarily develops in adolescents and young adults in endemic areas of the world, subclinical or anicteric hepatitis seems to occur commonly in children. Serological evidence of infection in endemic regions is found in fewer than a third of the older members of the adult population. This may be due to waning immunity with the passage of time among persons infected in childhood. In areas of the world where clinical hepatitis due to HEV rarely, if ever, occurs, serological evidence of subclinical infection is found in a small proportion of the population. The meaning of this finding remains to be

resolved. It may only indicate that residents of developed countries are infected commonly with an antigenically similar, but as yet unidentified virus of no known health importance. The incubation period of HEV is 40 days on average, but there is great variability. In contrast to HAV, the viremia is more protracted. The virus is excreted from the liver by means of the biliary tract into the gut. HEV hepatitis is customarily more severe than the liver disease due to HAV, and the clinical course is more protracted. While the fatality rate is low, pregnant females are at particular risk, with a high mortality in the third trimester (20-40%) and a high rate of spontaneous abortion. The basis for this tragic outcome associated with gestation is unknown (Asher et ah, 1990). The hepatitis of HEV is similar to HAV microscopically, but chronic cholestasis is a common feature. The liver parenchymal cells also tend to be organized in pseudoglandular arrays. This particular morphologic feature served as the basis for the conclusion in the early 1960s that an epidemic of hepatitis in Ghana was due to a new and unique virus. This suspicion, based on the morphology of the liver disease, was proven to be correct many years later.

FIGURE 18.2 A large HBsAg immune complex in the liver homogenate digested with Pronase and reacted with HbsAb. Some of the long filaments are thick, but the others appear to show headings. There are many more small HBsAg positive particles. Five Dane particles (arrows) show rupture of the envelope, thus partially exposing the inner core particles. There are two uncoated virus-like particles (V) having identical morphology with the internal core of Dane particles. The inset shows a tadpole form composed of a Dane particle and a tail filament in an immune complex. The electron opaque spherical bodies are artifacts. Reprinted with permission from Huang and Groh (1973).

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PARENTALLY ACQUIRED LONG-INCUBATION-PERIOD ACUTE A N D CHRONIC HEPATITIS Hepatitis B Virus (HBV) Few experiments in medicine illustrate better the role of serendipity in science than the discovery of HBV. In 1963, Baruch Blumberg, a medical anthropologist, was exploring the geographic distribution of inherited polymorphic traits when he detected a novel antigen in the blood of a native Australian that reacted with antibodies found in the serum of an adolescent American with hemophilia who had been the recipient of multiple blood transfusions. The antigen proved to be common in the blood of Africans and Asians, but was infrequently found in North Americans and residents of Europe. Over the ensuing years, the common presence of the antigen in the blood of patients with acute and chronic clinical hepatitis B was demonstrated. Much additional research showed that Australian antigen was, in fact, the surface antigen of a new virus family, the hepadnaviridae, now etiologically linked with clinical hepatitis of the type having a prolonged incubation period (Figure 18.2). HBV turned out to be the only human pathogen of this new family, but viruses strikingly similar to it are found in wild North American woodchucks, ground and tree squirrels, herons, and domestic ducks. Infections in these animals now serve as models of HBV in humans. Since useful cell culture methodologies for growing this virus in vitro have not yet been developed, large amounts of purified virus have not been available for scientific study.

The HBV virion, 42 nm in diameter, has as its genetic material a partially double-stranded circular DNA that encodes four genes (Figure 18.3A,B). One of these genes is responsible for the protein coat of the virion (i.e., Australian antigen or HBsAg), and the second codes the template for the viral DNA core (HBcAg). There is also the X gene that encodes regulatory proteins responsible for increasing by many fold the expression of a variety of viral and cellular genes, and a gene encoding the polymerase that catalyzes DNA replication. This latter enzyme is unique to the hepadnaviridae, for it makes possible the reverse transcription of an RNA pregene to DNA. In humans and experimentally infected chimpanzees, the virus is found predominantly in hepatocytes, but it can also be detected in low concentrations in blood mononuclear cells and several internal solid organs, but little is known about the biology of the infection outside of the liver. Multiplication of HBV in the hepatocyte follows attachment of the virus to the cell plasma membrane and uptake into the cytoplasm by mechanisms yet to be defined. The viral receptor on the cell surface has not been identified. Intracellular virion replication is a complex event because it depends on reverse transcription of the genomic DNA to an intermediary pregenomic RNA, which in turn is responsible for DNA synthesis. These events occur in the nucleus of the infected hepatocyte. The virion is further assembled in the cytoplasm by budding through intracellular membranes. The glycoprotein membrane is acquired during this step. Intracellular recycling of these events also occurs, thus amplifying multiplication of viral progeny. Although the cells' synthetic mechanisms are profoundly committed to virus replication, the hepato-

"™-HBsAg (Envelope) —HBcAg/HBeAg {Nucleocapsid) [—DNA polymerase --Circular DNA (HBV genome)

-~-™-? X protein FIGURE 18.3 (A) HBV particles in the blood as demonstrated by negative staining immune electron microscopy (122,000x). Reprinted with permission from Huang and Groh (1973). (B) Schematic representation of HBV illustrating key antigenic components. Reprinted with permission from Gerber and Thung (1985).

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FIGURE 18.4 (a) ''Ground glass" hepatocytes in a liver biopsy from a patient with asymptomatic chronic hepatitis, (b) Hepatocytes stained by immunochemistry to identify HBsAg. Reprinted with permission from Afroudakis et ah (1976).

cytes of the infected liver do not lyse as a result. In general, HBsAg is synthesized in excess; it accumulates in the cytoplasm, resulting in the typical ground-glass appearance of the infected cell cytoplasm (Figure 18.4). It also spills over into the blood, where it is found as the pleomorphic spherical and tubular noninfectious antigen particles known as Australian antigen (see Figure 18.2). Customarily, these particles are intermixed with variable numbers of the so-called Dane particles, which are the true virions of HBV in the blood. In the developed countries of North America and Europe, HBV is transmitted in blood products and by the needles and syringes used by consumers of illicit addictive drugs. Sexual interactions are an additional means of transmission, but the mechanism involved is not understood. With rigorous but highly effective screening of blood products for transfusion, new infections are now almost exclusively limited to the subculture of drug abusers and those who engage in promiscuous sexual activity. Many of these individuals are also HIV-1 positive. Subclinical, anicteric hepatitis proves to be the rule in infants and children. Asymptomatic anicteric hepatitis occurs in roughly 60 to 80% of those acquiring the virus during adulthood. The remainder exhibit chemical or overt clinical acute hepatitis after latency periods of 1.5 to 4 months. While the majority of patients recover after variable periods of illness without significant liver damage, about 1% develop fatal fulminating hepatitis with massive destruction of the liver. The disease in a small number (2-10%) evolves into chronic

hepatitis. The risk of a chronic infection inversely relates to age. Ninety percent of infants infected in the perinatal period fail to clear the virus, whereas only 10% of newly infected adults develop chronic disease. These clinical conditions and their pathogenesis will be considered below. In subSaharan Africa, Southeast Asia, the People's Republic of China, and the Mediterranean Basin, transmission of HBV commonly occurs during the perinatal period. The likelihood that the infant will be infected at the time of parturition relates to the replicative activity of the virus in the mother at the time and, consequently, her virus load. The means by which the virus is transmitted from mother to infant are not well defined, but transplacental infection is likely, and bleeding at the time of birth must result in infection of some newborns. Presumably, the immature immune system of the very young child permits the virus to replicate and spread in the liver without a significant protective response. Infection of newborns is common, and 90% of these infants develop chronic hepatitis. In contrast, only 30% of children exposed after the perinatal period, but before the age of 6 years, develop chronic liver disease. Worldwide, over 2 x 10^ people are chronically infected with HBV. The pathogenesis of HBV hepatitis has been the subject of considerable research. The availability of animal models, including transgenically infected mice, and an abundance of clinical information from naturally infected humans, has made major advances possible.

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However, despite the elegance and comprehensiveness of the investigative work, gaps of considerable magnitude preclude a full appreciation of the pathogenesis of clinical hepatitis and the variable outcomes in humans of different ages and societies. Our current understanding of the mechanisms involved in HBV hepatitis are summarized in great detail in a recent review by Chisari and Ferrari (1997). The evidence now indicates that cellular immune mechanisms are key factors in the development of the hepatic lesions. CD8+ cytolytic T cells appear to be the major actors. These cells directly interact with HLA class I-expressing hepatocytes that are endogenously generating the virus. As a consequence, two independent mechanisms of cell injury may be invoked, resulting in either apoptosis or cytolysis of the liver cells (Figures 18.5 and 18.6). The first is a direct consequence of the actions of at least two types of molecules released after contact of the effector lymphocyte with its target, the infected hepatocyte. The pore-forming perforins and a lymphocyte-specific granular serine esterase are the products released by the T cells that are believed to cause cytolysis of the

hepatocytes. Apoptosis, on the other hand, may be due to a Fas-based mechanism, whereby receptors on infected target cells interact with the Fas ligands of the effector T cell (Figure 18.7). The complex mechanisms involved in these events have been recently reviewed (Schulte-Hermann et ah, 1995; Moretta, 1997). Presumably, through cytolysis or apoptosis of the hepatocyte, the infection is terminated. Regenerating liver cells are protected from reinfection either by means of cellular or humoral immune mechanisms. Contemporary thinking suggests that certain cellularly immune mechanisms downregulate the infections in the liver cells. However, direct evidence indicating that this occurs in human HBV hepatitis is currently lacking.

FIGURE 18.6 Liver of a patient infected with HCV. Note the apoptotic hepatocytes (a) and cells exhibiting ballooning of the cytoplasm (b). These are the two major nonspecific changes observed in liver parenchymal cells during hepatitis.

cytotoxic lymphocyte macrophages lymphocytes

EXECUTION

FIGURE 18.5 Electron micrograph of an apoptotic body derived from liver cells. Note the infiltrating lymphocyte (L). The hepatocyte (H) at the top of the figure appears to be unaffected. The electrondense granules in the hepatocyte are glycogen. Reprinted with permission from Ishak (1994).

HEPATOCYTE

FIGURE 18.7 Three major factors are believed to contribute to apoptosis and death of liver cells. TGFp and TNF may interact with plasma membrane receptors, resulting in cell injury. The FAS ligand also can interact APO-FAS to promote apoptosis. DAG = diacylglycerol; TCR = T cell receptor; CTL = cytotoxic lymphocytes. Reprinted with permission from Schulte-Hermann ei al. (1995).

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While the above mechanisins most probably account in whole, or in part, for cell injury and recovery as a consequence of HBV infection in the liver of adults, the pathogenic basis for chronic hepatitis remains poorly understood. In neonatally infected infants, immunologic tolerance would appear to be the likely explanation, but in the adult with chronic hepatitis our understanding is less clear.

Hepatitis D Virus (HDV) (Delta Agent) About 1% of those infected with HBV develop acute liver failure associated with massive hepatic necrosis. Roughly 10% of newly infected adults and a much larger percentage of infants progress to chronic hepatitis, as noted above. When patients with unresolved HBV infections manifest fulminating necro-inflammatory disease or chronic hepatitis that evolves into cirrhosis, the possibility of a superinfection with the delta agent (HDV) is a consideration (Rizzetto el al, 1983; Lee, 1993; Smedile ei al, 1982). HDV is a defective virus that relies on HBV to provide its envelope protein. Thus, it parasitizes the intracellular synthetic systems of the HBV-infected cell to complete its own replicative cycle. This allows the virus to spread from cell to cell. HDV is a small (36 nm in diameter) virion that has a limited store of genetic material in a single strand of RNA that is circularized when located in liver cells (Wang ei al., 1986). While HDV is cosmopolitan in its distribution, it is usually found when the HBV carrier status of a population is relatively high, or when i.v. drug use is prevalent. However, it has also been associated with outbreaks of severe hepatitis among native populations in South America, where these risk factors are not found (Adler ei al., 1984; Ljunggren ei al., 1985; Fonseca and Simonetti, 1987). The means whereby the virus was introduced into these isolated native populations is obscure, and it is not known how the virus spreads from person to person in this setting. In developed areas such as the United States and Western Europe, fewer than 20% of the blood donors who have evidence of chronic HBV infections are also infected with HDV The acute hepatitis due to HBV cannot be dififerentiated clinically and pathologically from HBV associated with HDV (Verme ei al., 1986). Evidence of infection is detected by immunohistochemistry, or by means of in siiu hybridization. Generally, only a small proportion of the liver cells of patients with acute HBV hepatitis are positive for HDV The virus is found more commonly in the livers of patients with chronic hepatitis and cirrhosis. Not surprisingly, chronic hepatitis is the

outcome in about 20 to 25% of HDV-infected patients, and the disease frequently progresses to cirrhosis in these individuals (Rizzetto ei al., 1983). Like other hepatitis viruses, HDV is not cytolytic in vitro, and the mechanism whereby it causes liver cell injury is not understood. Hepatitis C V i m s (HCV) Before the discovery of HCV in 1989, approximately 20 to 25% of recipients of blood transfusions in urban North America and Europe developed the so-called non-A and non-B hepatitis, that is, acute hepatitis of unknown but presumptive viral etiology. More than 80% of these infections progressed to chronic hepatitis, and in a few the disease evolved into cirrhosis and hepatocellular carcinoma. By screening cDNA "expression" vectors derived from RNA in the plasma of chimpanzees inoculated with serum from patients with non-A/non-B hepatitis, clones of a new virus, termed HCV, were found. This finding, the outgrowth of a truly unique laboratory approach to viral diagnosis, was followed by an extraordinary effort to elucidate the biology of HCV and establish its clinical and epidemiological features. The development of several efficient blood screening methodologies has now largely eliminated this virus as a threat for the recipient of blood transfusions. At present, HCV is unclassified virologically, but it possesses many of the molecular and structural features of members of the flaviviridae family (see Chapters 19 and 24) (Cuthbert, 1994). It is a 30- to 34-nm-indiameter enveloped virus having a single-stranded RNA genome surrounded by an envelope derived from the cell in which it grows. The virus has not been successfully grown in cultured cells; thus, much of the information we possess today is derived from investigations conducted in experimentally infected chimpanzees. HCV has a long open reading frame that contains genomic material for the synthesis of several component proteins of the virion, and one of these genes is highly mutable. Accordingly, as many as 12 genotypes of HCV have already been identified, and because of the high mutation rate, it is quite likely that new mutations appear in the patients as a virus adapts to its host. Thus, changes in the antigenicity of the virus may account for the chronicity of the infection as the virus eludes the infected person's immune response. Although immunologic studies at present are limited, both humoral and cellular immune mechanisms are involved in the host's response to infection. However, there is presently no evidence to suggest that the lesion in the liver of the HCV-infected patient has an immunopathologic basis, as is the case in HBV infection. As

Hepatitis Viruses

would be expected, the humoral immune response does not have the capacity to resolve the infection when the virus is sequestered in hepatocytes. Indeed, humoral immunity may promote selection of new pathogenic mutants in vivo on an ongoing basis. Worldwide, the incidence of HCV infection as assessed by immunologic surveys of the population varies. In Scandinavia, fewer than 1% of the general population are infected, whereas in Egypt a prevalence of 12% has been reported. Four million An\ericans are currently believed to be chronically infected with HCV and approximately 8 x 10^ to 1 x 10^ deaths occur annually as a result of this infection. The great majority of infections with HCV have, in the past, resulted from blood transfusions, but transplacental infection of the fetus and perinatal infections are documented (Lin et ah, 1994; Ohto et ah, 1994; Tovo et al, 1997), particularly in the offspring of women with high blood concentrations of virus. Sexual transmission has not been established as a mode of spread, but evidence of familial clustering of seroreactivity exists and female sex workers who have not been the recipients of blood transfusions exhibit a higher incidence of seroreactivity to the

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virus than do female members of the general population. After exposure, HCV RNA is detected in the blood within 1 to 3 weeks. Studies in chimpanzees have shown that concentrations of virus in the liver are exceedingly high at this time. Evidence of liver cell injury in the form of increased blood levels of serum alanine aminotransferase is demonstrated, but most patients are asymptomatic and jaundice develops infrequently. An occasional patient complains of malaise, weakness, and anorexia, and a few become anoretic. The morphological features of the acute disease are illustrated in Figure 18.8. Only about 15 to 25% of patients appear to recover, whereas the remainder enter the chronic hepatitis stage during which an insidious progression of liver disease evolves over a period of decades. Nonspecific symptoms of hepatitis are noted by the occasional patient with chronic HCV hepatitis, but the disease is rarely symptomatic, and almost never disabling. Over a period of two or more decades, about 20% of patients with chronic hepatitis due to HCV develop cirrhosis, and roughly 5% of these patients subsequently develop hepatocellular carcinoma. It is likely that differences in

FIGURE 18.8 (A) Chronic hepatitis. Note the subtle disorganization of the liver parenchyma and the accumulation of lymphocytes adjacent to a necrotic hepatocyte. (B) Prominent "balloon" degeneration of hepatocytes. Note the inflammatory cells and the "dropout" of liver parenchymal cells. (C) Note the aggregates of lymphoid cells in the portal areas. (D) At higher resolution, the lymphoid cell accumulations in the liver illustrated in A encompass bile ducts. Note the normal appearance of the glycogenated cytoplasm of the hepatocyte. Reprinted with permission from Ishak (1994).

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the pathogenicity of virus strains may account for differing degrees of disease progression. Cofactors influencing host susceptibility to the infection most probably are also an important consideration. Patients infected with both HBV and HCV seem to develop progressive disease frequently (Hytiroglou ei al., 1995). Alcoholic beverage consumption in excess is believed to be a risk factor. Although intravenous drug users with HIV-1 also develop HCV infections, HIV-1 does not appear to significantly influence the course of the disease. While fulminating hepatitis is a rare complication of HCV infection, an occasional liver transplant recipient will develop rapidly progressive hepatic disease due to the virus. Currently, a substantial proportion of the liver transplantations done in the United States are due to HCV liver damage. Recurrence of HCV infections in the grafted livers invariably occurs after transplantation. Interestingly enough, in one study the infection did not affect the life expectancy of the graft recipient over a 4-year period, but there was a higher incidence of graft rejection among HCV-infected patients (Lumbreras ei al., 1998). The majority of the recipients of these liver transplants exhibit a relatively benign course. This contrasts with the substantial mortality observed in liver transplant patients infected with HBV (Lake and Wright, 1991). The majority of the recipients of these liver transplants exhibit a relatively benign course. In one study, moderately severe chronic hepatitis developed after transplantation in 27% of patients over the 3-year period, and cirrhosis was found after a latency period of 4 years in 8% (Cane ei al., 1996). An etiological association of mixed cryoglobulinemia with HCV chronic hepatitis is now established (Ferri ei al, 1991; Agnello ei al, 1992). Most Italian patients with mixed cryoglobulinemia appear to possess HCV antibodies, but in the United States the prevalence is somewhat lower (Levey ei al, 1994). These patients exhibit petechial hemorrhages and ecchymoses in the skin of the lower extremities, and often have arthralgias, hepatosplenomegaly and glomerulonephritis. In the serum, viral RNA and virions are complexed with the cryoproteins. Three clinical categories of cryoglobulinemia have been identified (Brouet ei al, 1974). In type I, the immunoglobulins are homogenous and monoclonal. They are generally the result of a lymphoproliferative disorder such as multiple myeloma or Waldenstrom's macroglobulinemia. In the type II form, mixtures of a monoclonal immunoglobulin with anti-IgG activity (rheumatoid factor) and polyclonal IgG are found. Most cases of HCV-associated cryoglobulinemia are of this type (Agnello ei al, 1992). In the type III form, a mixture of heterogenous, and

polyclonal IgM and IgG molecules are present in the blood. Type II is associated with a diversity of infectious processes and autoimmune diseases (Bloch, 1992). The pathogenesis of cryoglobulinemia in HCV is obscure. Other forms of hepatic disease do not produce these profound abnormalities of serum proteins. About 15% of transfusion-associated hepatitis is not attributable to HBV and HCV, and it is not due to HAV or HEV. In 1967, a candidate virus was isolated from the blood of a surgeon (whose initials were G. B.) with acute hepatitis. The virus, which proved to be a flavivirus, was similar to HCV, for it was found to induce hepatitis in marmosets, a small New World primate. With further study, the agent proved to be not one but two viruses, termed GBV-A and GBV-B. These agents now appear to be indigenous to Tamarin monkeys and may be members of an entirely new, previously unrecognized genus of flaviviruses (Bukh and Apgar, 1997). Later, a third, similar virus, GBV-C, was isolated from patients (Fiordalisi ei al, 1996) with hepatitis, and a fourth, designated HGV, was similarly recovered (although it may be a genotype of GBV-C). The discovery of this confusing plethora of incompletely characterized viruses created a stir in the hepatitis research community, but it may be that the interest was unwarranted. Despite the persistence of these viruses in humans, and their association with HCV infections, the GB viruses do not appear to cause hepatitis in humans and do not play a copathogenic role with HCV in enhancing the severity of the liver disease (Alter ei al, 1997; Colombatto ei al, 1997; Hadziyannis, 1998; Loya, 1996; Brown ei al, 1997). We now know that GBVC / HGV is carried in a chronic viremic state by roughly 1 to 2% of Americans and can be transmitted by transfusion and from mother to offspring (Linnen^f al, 1996; Lin ei al, 1998; Masuko ei al, 1996; Stark ei al, 1996; Thomas ei al, 1997).

C H R O N I C HEPATITIS Chronic hepatitis is a clinical and pathologic syndrome, not a single disease. Patients may be asymptomatic, but more often they experience variable degrees of malaise and fatigue intermittently. Serum concentrations of the liver enzymes alanine and aspartate aminotransferase are usually increased, but alkaline phosphatase and gamma-glutamyl transpeptidase, bilirubin, albumin, and the various coagulation factors are customarily found in normal concentrations. Thus, the synthetic capacity of the liver parenchyma is intact.

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•

W

0 FIGURE 18.9 Chronic hepatitis, low-power assessment of morphologic patterns. (A) Portal hepatitis involves an increase in mononuclear cells (dots), almost entirely confined to portal areas. At scanning magnification, this results in the portal areas being sharply delimited. (B) In periportal hepatitis, an increase in mononuclear cells (dots) in the periportal parenchyma (zone 1) occurs, commonly associated with piecemeal necrosis and lobular inflammation of variable degree. The result is a low-power impression of portal-dominant inflammation; however, the portal areas are less sharply defined than in portal hepatitis. (C) Lobular hepatitis is characterized by lobular inflammation, with or without disarray and necrosis. Pure lobular hepatitis is a feature of acute hepatitis; however, lobular hepatitis in conjunction with considerable portal and periportal inflammation is typical of '"flares" of chronic viral or autoimmune hepatitis. Reprinted with permission from Batts and Ludwig (1995).

Pathologically, chronic hepatitis is defined as a progressive necro-inflammatory disease of variable severity not associated with the features of chronic cholestasis, steatosis, and Mallory body formation (Ishak, 1994) (Figure 18.9). Portal fibrosis, cirrhosis, and, in some cases, hepatocellular carcinoma, are the final outcome. The term "chronic hepatitis'' commits to obsolescence a confusing nomenclature that has accumulated in the field of hepatology since the 1960s (Table 18.2) (Desmet et al, 1994; Party 1995). HBV, with or without the delta agent, and HCV are the common etiologies of chronic hepatitis in most clinical situations, but autoimmune hepatitis is responsible in sporadic cases. Superimposed infections and nutritional or toxic insults to the liver may accentuate the severity of the process. Piecemeal necrosis is the hallmark of chronic hepatitis. It is reflected as the expansive degeneration and destruction of the periportal limiting plate of liver cells, ultimately resulting in the confluence of adjacent portal areas and leading to bridging fibrosis between portal triads. The fibrosis that follows in the wake of the liver parenchymal injury (Figures 18.10-18.12) is progressive and can ultimately terminate in cirrhosis (Figure

18.13). Degenerative changes in individual liver cells consist of either cytoplasmic swelling and rarefaction or the clumping of cytoplasmic organelles (or both). Apoptotic bodies (syn. acidophilic bodies) are also seen (see Figure 18.5). To a variable extent, the portal areas

TABLE 18.2 Chronic Hepatitis and Cirrhosis: Obsolete Terms Chronic hepatitis and related conditions Chronic active hepatitis, chronic aggressive hepatitis, chronic active liver disease, plasma cell hepatitis, lupoid hepatitis, and other synonyms for autoimmune hepatitis Chronic persistent hepatitis Chronic lobular hepatitis Chronic nonsupportive destructive cholangitis Pericholangitis Cirrhosis Portal cirrhosis Postnecrotic cirrhosis Posthepatitic cirrhosis Reprinted with permission from Batts and Ludwig (1995).

264

Pathology and Pathogenesis of Human Viral Disease TABLE 18.3 Scoring S y s t e m for Grading and Staging of Liver S p e c i m e n s w i t h Chronic Hepatitis Grade of necro-inflammatory activity

Stage of fibrosis/ cirrhosis

0 = no necro-inflammatory activity

0 = no fibrosis

1 = mild piecemeal necrosis and lobular activity

1 = mild fibrosis (= portal fibrosis without fibrous septum formation)

2 = moderate piecemeal necrosis and lobular activity

2 = moderate fibrosis (= fibrous septa extending into lobules, but not reaching terminal hepatic venules and other portal tracts)

3 = Severe piecemeal necrosis and lobular activity with or without bridging necrosis

3 = severe fibrosis (= fibrous septa extending to adjacent portal tracts and terminal hepatic venules, indicating transition to cirrhosis) 4 = cirrhosis

Reprinted with permission from Batts and Ludwig (1995).

are infiltrated by B lymphocytes, plasma cells, and macrophages, which, on occasion, accumulate into follicles. Intraacinar infiltrates of inflammatory cells and activated Kupffer cells line sinusoids in the usual histological picture (Figure 18.14). The pathological changes in chronic hepatitis have been categorized semiquantitatively by Batts and Ludwig (1995) into a grading schema for clinical application (Table 18.3; Figure 18.15).

HEPATOCELLULAR CARCINOMA (HCC) (see Figures 18.16-18.19)

Numerous risk factors have been identified that are believed to cause, or contribute to, the development of HCC (Table 18.4). HCC most probably is an example of multistage carcinogenesis in which endogenous and exogenous influences act in concert to transform the hepatocyte, possibly in persons who are genetically

FIGURE 18.10 Post-transfusion HBV with submassive necrosis in a leukemic patient. A tongue of necrotic tissue bridges between lobules of liver. Reprinted with permission from Ishak (1976).

265

Hepatitis Viruses

FIGURE 18.11 Chronic hepatitis. Adjacent portal areas expanded by fibrosis are linked as demonstrated by a reticulin stain that denotes collagen. Reprinted with permission from Ishak (1994).

^"*4SS»%%'i'^V--?vt'*>/'r •'••• •:••

FIGURE 18.12 Marked periportal fibrosis with extension into the lobular structure of liver.

FIGURE 18.13 Nodular cirrhosis secondary to HCV chronic hepatitis. Note the regenerating nodules of liver parenchymal cells separated one from another by bands of connective tissue. Chronic inflammatory cell infiltrates in the fibrous tissue constitute a nonspecific change.

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Pathology and Pathogenesis of Human Viral Disease

FIGURE 18.14 Mixed inflammatory cell infiltrate comprised of lymphocytes, plasma cells, and macrophages in the parenchyma of the liver from a case of chronic HCV infection.

FIGURE 18.15 Staging of chronic hepatitis, schematic diagram. (A) Portal fibrosis (stage 1) characterized by mild fibrous expansion of portal tracts. (B) Periportal fibrosis (stage 2) showing fine strands of connective tissue in zone 1 with only rare portal-portal septa. (C) Septal fibrosis (stage 3) manifested by connective tissue bridges that link portal tracts with other portal tracts and central veins, minimally distorted architecture, but no regenerative nodules. (D) Cirrhosis (stage 4) showing bridging fibrosis and nodular regeneration. Reprinted with permission from Batts and Ludwig (1995).

Hepatitis Viruses TABLE 18.4 Nonviral Risk Factors for Hepatocellular Carcinoma (HCC)

267 TABLE 18.5 Geographic Distribution of H C C 5-20«

20-15(F Aflatoxin Bj contamination of food Alpha 1 antitrypsin deficiency Anabolic and estrogenic steroid consumption Ethanol consumption Hemochromatosis Nutritional deficiencies Thorotrast diagnostic procedures Tobacco smoking

FIGURE 18.16 Extensive involvement of the liver by hepatocellular carcinoma. Note the hemorrhage and necrosis in the large central nodular mass. Elsewhere, the parenchyma is interdigitated by bands of connective tissue of varying thickness. At the resolution of the naked eye, it is often impossible to differentiate neoplastic tumor from cirrhotic liver. Reprinted from Craig et ah (1989).

predisposed. To date, no specific molecular markers have been associated with the transformational event. The pathogenic role of HBV and HCV in the neoplastic process should be considered in this context. Worldwide, some 7 X 10^ HCC deaths occur annually. The incidence of HCC exhibits great geographic variability (Table 18.5), being highest in subSaharan Africa and Southeast Asia, and lowest in the developed countries of Europe and North America. However, pockets of increased prevalence are found where immigrants from endemic areas have settled and retained their traditions as well as the diets of their former homes. HCC in North America is typically a disease of the sixth or seventh decades of life, but in areas of endemicity, it tends to appear clinically at an earlier age. For unknown reasons, HCC occurs predominantly in males in endemic areas of high disease prevalence.

<5"

subSaharan Africa

Mediterranean countries

Europe

Southern China

Japan

North America

Southeast Asia

South America India

Adapted with permission from Anthony (1984). ''Incidence per 100,000 population per year.

FIGURE 18.17 Hepatocellular carcinoma separated into nodules so as to mimic cirrhosis. The cytology of tumor cells is the distinguishing characteristic. Reprinted from Craig et al. (1989).

In regions of the world where HCC is common, HBV infection is acquired by newborns in the perinatal period and persist in the form of chronic antigenemia with smoldering chronic hepatitis for the lifetime of the individual. To the extent that the infection plays a direct role in the causation of HCC, the latency period of this tumor is extraordinarily long. However, in the Orient, occasional cases of HCC are described in young children chronically infected with HBV (Shimoda et al, 1980; Tanaka et ah, 1986), an indication that the latency period need not be long. In the People's Republic of China (Yeh et al, 1989), Taiwan (Beasley et al, 1988), Japan (Ijima et al, 1984; Obata et al, 1980), and in the Alaskan native populations (McMahon et al, 1990), the incidence of HCC is increased 30- to 100-fold in persons chronically infected with HBV. In addition, the HBV genetic material is demonstrated in the cytoplasm of neoplastic cells in over 85% of tumors (Edamoto et al, 1996; Robinson, 1994). Although there is a clear epidemiological association of HBV with HCC, the mechanism of carcinogenesis remains to be fully defined. It has been sug-

268

Pathology and Pathogenesis of Human Viral D i s e a s e

FIGURE 18.18 Hepatocellular carcinoma. Note the nuclear pleomorphism and the loss of orientation of the malignant cells. Atypical mitoses are variable in number. The clear circumscribed bodies contain glycogen. Reprinted from Craig et al. (1989).

FIGURE 18.19 Ground-glass cells in hepatocellular carcinoma. The cytoplasmic changes in the tumor cells mimic "ground-glass" changes in the HBV infected hepatocyte, but immunohistochemistry and electron microscopy show them to be comprised of non-membrane-bound amorphous fibrillary material. HBsAg can occasionally be identified in the cytoplasm of hepatocarcinoma cells, but ground-glass cytoplasmic changes due to HBsAg are a rare finding. Reprinted with permission from Stromeyer et al (1980).

Hepatitis Viruses

gested that HBV is oncogenic, but, as briefly summarized below, the evidence is unconvincing. In those who are chronically infected with HBV, the genome of the virus is integrated in a seemingly random fashion into the DNA of the transformed hepatocyte. These integration sites are usually associated with microdeletions of the cell's DNA, but major changes in the genome are not found. In the tumors, the integrated DNA is monoclonal and detectable in all tumor cells. While this, in fact, proves to be the case, it does not necessarily link the infection causitively with the neoplasm. Indeed, the weakness of the argument that HBV is oncogenic focuses on the lack of specificity of the integration site in the cell chromosomes, and our failure to demonstrate an association of the HBV genome with a critical cellular gene(s) recognized to be intrinsic to the process of carcinogenesis. Regardless, skepticism is sobered by the fact that woodchucks and ducks infected with their specific hepadnavirus almost routinely develop HCC when maintained in captivity in the absence of other possible liver carcinogens. One might speculate regarding alternate mechanisms whereby HBV contributes to the development of HCC. First, a component of the integrated genome of HBV might serve as an independent protooncogene initiating the neoplastic process by a mechanism yet to be defined. Although an attractive hypothesis, there is, at present, no evidence to support it. Second, the HBV genome could provide transcriptional transactivating genes that elaborate products which stimulate hepatocyte proliferation. Two such genes that code for the so-called "X'' protein and the pre-S2 activators have been identified (Hildt ei al, 1996). The HBV "X" protein might then inactivate regulatory suppressors such as the p53 gene or stimulate elaboration of positive growth regulators such as the insulin-like growth factors 1 and 2 (Feitelson and Duan, 1997; Kasai et ah, 1996; Kobayashi et ah, 1997). And, finally, chronic inflammation of the liver parenchyma accompanied by ongoing destruction and regeneration (that is, chronic hepatitis) might lead to neoplastic transformation due to the effects of inflammatory cell-generated oxidants on the hepatocyte DNA or possibly the effects of either endogenous or exogenous promoters or cocarcinogens (Grisham, 1995; Brechot et al, 1982; Popper et a/., 1988). Evidence supporting this attractive concept is based, in part, on the demonstrated common occurrence of cirrhosis in HBV-infected persons who develop HCC. Presumably, the tumor originates in one of the regenerating liver nodules found in this lesion. The mycotoxin food contaminant aflatoxin B^ (and related compounds) is an established carcinogen that could play a contributory role in the pathogenesis of

269

HCC. This oncogenic toxin contaminates food supplies almost universally in areas of high HCC endemicity. In one study from a geographic area where HCC is prevalent, urinary excretion of aflatoxin Bi guanine adducts was increased almost eightfold in patients with HCC (Bradbear et al, 1985). Interestingly enough, aflatoxin Bi induces specific mutations of the p53 suppressor gene that are identical to those found in the malignant hepatocyte of HCC (Bressac et al, 1991; Hsu et al, 1991). The etiological role of HCV in the pathogenesis of HCC is now established, based on epidemiological studies worldwide (Resnick and Koff, 1993; Tsukuma et al, 1993) and the consistent demonstration of replicating virus in the cells of the tumor (Sansonno et al, 1997; Tang et al, 1995; Saito et al, 1997; Edamoto et al, 1996). Considerable geographic variability in the prevalence of HCV-associated HCC is found that no doubt relates to differences in the prevalence of the virus infection in different populations. Since only a third of infections can be traced to blood transfusions, social influences determine the proportion of the population that are infected. The number of liver cancers that are related to HCV in the general population ranges from 72% in Spain to 5% in South Africa, with intermediate percentages being documented in Japan, Europe, and North America (Caselmann and Alt, 1996; Kew et al, 1997; Colombo and Covini, 1995). Because of their similar mode of transmission, many patients infected with HCV are also carriers of HBV One might ask whether or not the two viruses can act synergistically to promote viral transformation of the liver. At present, there is no epidemiological or experimental evidence to support such a possibility. We know very little as to how HCV might act to initiate or promote carcinogenesis. There is no evidence to indicate that viral genes are integrated into the hepatocyte DNA, or act either as transactivating substances or oncogenes (el-Refaie et al, 1996). Since the tumor commonly develops after a prolonged latency period (Castells et al, 1995) in persons with chronic hepatitis and cirrhosis (Reigler, 1996; Shiratori et al, 1995; Silini et al, 1996), malignant transformation of cells in regenerating foci in the cirrhotic liver seems to be the most likely explanation. In one study, 43% of HCCs were found to develop in regions of the liver demonstrating nodular regenerative hyperplasia (Nzeako et al, 1996). Alcoholic beverage consumption may be a cofactor in some cases (Kubo et al, 1997; Bruno et al, 1997), and accumulating epidemiological evidence suggests that specific types of HCV are exceptionally pathogenic (Bruno et al, 1997; Tanaka et al, 1996; Hatzakis et al, 1996; Zein et al, 1996).

270

Pathology and Pathogenesis of Human Viral Disease

A U T O I M M U N E HEPATITIS (AH) Approximately 20% of patients with chronic hepatitis in Europe and North America have an autoimmune form of disease unrelated to virus infection (Holdstock ei ah, 1983). AH is a chronic necro-inflammatory idiopathic liver disease associated with autoantibodies against tissue constituents in the blood and high serum IgG blood concentrations (Johnson and MacFarlane, 1993; Krawitt, 1996). Although the etiology(s) is unknown, it appears to be a disease of disordered immune regulation possibly due to defects in suppressor T cell function (Mondelli et al, 1988; Meyer zum Buschenfelde and Lohse, 1995). Since AH is effectively treated with antiinflammatory and immunosuppressive drugs, differentiation from viral hepatitis and other forms of autoimmune liver disease (primary biliary cirrhosis and sclerosing cholangitis) is a critical clinical challenge. However, to the pathologist's eye, there are no discriminating morphologic features that allow one to differentiate AH from severe chronic viral hepatitis (Figures 18.20 and 18.21). However, portal infiltrates of B cells and helper/suppressor T cells are often prominent in this disease. These features may implicate humoral immune mechanisms in the patho-

genesis of AH. AH tends to progress to cirrhosis rapidly in comparison to chronic viral hepatitis. Since the disease is often subtle at the outset, patients often present with advanced hepatic fibrosis or cirrhosis. Hypergammaglobulinemia and autoantibodies are, on occasion, elaborated by patients with chronic hepatitis having a viral etiology, and approximately 10% of patients with viral hepatitis have circulating autoantibodies (Pawlotsky et al, 1993). However, the titers are usually higher in the autoimmune form of the disease. In addition, patients with AH will, on occasion, possess serum antibody evidence of a prior measles or either an HBV or HCV infection (Pawlotsky et a/., 1993). In these patients, low titers of antiviral antibody do not necessarily imply previous infection. AH typically presents subtly as jaundice in a young woman (male:female ratio in some patient series has been as high as 1:8), possessing certain histocompatibility antigen markers (HLA class I B8 class II DR3 or DR4) and a variety of serum autoantibodies (Table 18.6). Commonly, without treatment, these patients evolve to a chronic stage, resulting in progressive liver parenchymal fibrosis and, ultimately, cirrhosis. A variety of other autoimmune disorders may occur concomitantly in the occasional patient.

FIGURE 18.20 Chronic nonviral autoimmune hepatitis with portal inflammation. The arrow points to a cell exhibiting balloon degeneration. Reprinted with permission from Ishak (1994).

271

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FIGURE 18.21 Chronic nonviral autoimmune hepatitis showing extensive bridging fibrosis. Reprinted with permission from Ishak (1994).

TABLE 18.6 Autoantibodies in Autoimmune Hepatitis

Type 1 (classic)

2 (anti-LKM-1)

Characteristically present autoantibodies Antinuclear Anti-smooth muscle Antiactin Anti-asialoglycoprotein receptor Anti-LKM-1 Anti-liver cytosol 1

Autoantibodies occasionally present Antimitochondrial Anti-soluble liver antigen Anti-liver-pancreas protein Antineutrophil cytoplasmic Anti-liver cytosol 1" Antinuclear''

Reprinted with permission from Krawitt (1996). 'Rare.

PAPILLARY ACRODERMATITIS (GIANOTTI-CROSTI SYNDROME; GCS) GCS is a childhood exanthematous papillary-vesicular symmetrical dermatitis of the face and extremities accompanied by inguinal and axillary adenopathy (Figure 18.22). It customarily occurs during the first 5 years of life, resolving after 2 to 3 weeks without residue. The pathogenesis is obscure. Originally described in youngsters with acute hepatitis B (Ishimaru et ah, 1976; Gianotti, 1973; Toda ei al, 1978; San Joaquin et al, 1981; Draelos et al., 1986), it now appears to also be a

complication of a wide variety of other acute viral infections, including Epstein-Barr virus (Hofmann et ah, 1997; Lacour and Harms, 1995; Mempel et al, 1996), cytomegalovirus (Caputo et al, 1992; Tzeng et al, 1995; Haki et al, 1997), parainfluenza and mumps (Hergueta-Lendinez et al, 1996), respiratory syncytial virus, and vaccinia (Hofmann et al, 1997). Pathologically, the cutaneous lesions exhibit nonspecific perivascular histiocytic and lymphocytic infiltrates in the papillary dermis. The enlarged lymph nodes show pleomorphic histiocytic proliferation with a prominence of endothelial cells in the small blood vessels, lymphatics, and

272

Pathology and Pathogenesis of Human Viral Disease

FIGURE 18.22 Papular lesions of Gianotti-Crosi syndrome on the face and extensor surface of the arm. Reprinted with permission from San Joaquin ef fl/. (1981).

sinuses. The enlarged lymph nodes can persist for several months. Apparently, attempts to detect viral antigens and virions in the skin lesions have failed.

GLOMERULONEPHRITIS Glomerulonephritis (GN) now is a well-recognized outcome of HBV antigenemia (Combes et al, 1971). HBV-associated GN (HBGN) develops frequently in children, most often males, and, to a lesser extent, adults in endemic areas where the prevalence of HBV infections in early life is high (Knieser et al, 1974; Morzycka and Slusarczyk, 1979; Ozawa et al, 1976; Southwest Pediatric Nephrology Group, 1985; Hirsch et al, 1981). In Europe and North America, where chronic HBV antigenemia occurs infrequently in members of the general population, HBGN occurs sporadically. Many of the patients are infected during adulthood by blood transfusions or i.v. drug usage. In one study, 20% of children with membranous nephropathy had detectable HBV in the blood. However, the pathogenic role of the virus in the disease of these patients is not clear. The overall prevalence of HBGN in developing countries is not known. Clinically, HBGN commonly presents in children as the nephrotic syndrome usually unaccompanied by significant renal failure. Patients occasionally have an

associated systemic vasculitis that proves to be mediated by immune complex. The occurrence of hepatitis does not appear to be a factor influencing whether or not disease of the kidney occurs. Renal disease in younger patients customarily resolves uneventfully regardless of treatment. About 65% of children with HGBN remit spontaneously before the end of the first year following onset, and 85% are well at the end of the second year (Venkataseshan et al, 1990). Studies of adults with GN and chronic HBV antigenemia yield more ominous findings. In one report from Hong Kong, an HB V-endemic area, almost a third of the adult patients studied had progressive renal failure and 10% required long-term dialysis (Lai et al, 1991). It is likely these patients acquired the infection early in life and had chronic antigenemia over a period of many years. Membranous glomerulonephritis with or without mesangial thickening is the most common form of HBGN. Ultrastructurally, the basement membrane of the glomerular capillaries are irregularly thickened and show glandular subepithelial deposits and the socalled "spikes" (Ito et al, 1981). Mesangioproliferative and diffuse proliferative GN occurs less commonly in children and more frequently in adults. In these cases, sizable subendothelial deposits are found on both sides of the basement membrane and in the mesangium by electron microscopy. Immunohistochemistry demonstrates diffuse or segmental glomerular deposits of IgG, IgM, and IgA, as well as complement components. Abundant accumulations of HBsAg are also found along the capillary walls and occasionally in the mesangium. HBcAg is present in roughly two-thirds of cases, whereas HbeAg is not found commonly (Venkataseshan et al, 1990; Lai et al, 1996). The nature of the antigens and antibodies comprising the immune complex localizing in the glomerulus dictate the timing of the process and the site of deposition in the kidneys. Since three antigens and three types of antibodies may be involved, the pathogenesis of the glomerulitis can be complex. Clearly, the factors determining whether or not GN will develop in an individual patient, and the pathogenic basis for both the morphological features and severity of the lesions, are poorly understood. As noted above, woodchucks are naturally infected with a hepadnavirus (WHV) strikingly similar to HBV. Renal lesions identical to those seen in humans evolve in woodchucks experimentally infected with WHV. This model system provides an exceptionally useful tool for exploring the mechanisms involved in HBGN (Peters et al, 1992). Accumulating evidence suggests that the chronic viremia of HCV may result in immune complex GN. Johnson and colleagues (1993) reported eight patients

Hepatitis Viruses

with chronic HCV infections who had membranoproliferative GN associated with ultrastructurally demonstrable subendothelial and mesangial deposits and consistently accompanied by accumulations of IgM, IgG, and C3 in the glomeruli. Most of these patients had circulating immune complexes containing HCV RNA and cryoglobulins. This evidence suggests a possible immunopathological role for HCV in the GN. Since the investigators did not demonstrate HCV antigenic or molecular components in the glomeruli, the mechanism of the disease in these patients remains uncertain.

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19 Hemorrhagic Fever Viruses radically and unpredictably, often in geographical sites where modern diagnostic laboratory facilities are unavailable and the capacity to conduct the sophisticated test procedures necessary to define the basis for a coagulopathy in an individual patient do not exist. Perhaps more importantly, the physiologic status of the patient and their hemostatic system appears to change during the course of these serious, often fatal infections. Although information is fragmentary, several possible defects in the integrity of small blood vessels and coagulation mechanisms are believed to account for hemorrhagic lesions. In this chapter, I will consider the various hemorrhagic fever viruses using their traditional virological classification. The commonalities in epidemiology, as well as their clinical presentation and pathological features, will readily become evident. I also present the limited hemostatic information available in the context of pathological changes observed clinically and at autopsy (Figure 19.1). With modern transportation, the exotic viruses considered in this chapter pose a potential diagnostic and therapeutic challenge for physicians in developed countries (McFarland et al, 1997).

INTRODUCTION 277 ARENAVIRUSES 277

Argentinian and Bolivian Hemorrhagic Fevers 278 Venezuelan and Sao Paulo Hemorrhagic Fever 280 West African Hemorrhagic Fever (Lassa Virus) 280 BUNYAVIRUSES 282

Hemorrhagic Fever with Renal Disease 282 Rift Valley Fever (RVF) 285 Crimean-Congo Hemorrhagic Fever (CCHF) 286 FiLOVIRUSES 287

Marburg Virus Disease 287 Ebola Virus 287 FLAVIVIRUSES

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Yellow Fever 290 Dengue 292 Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) 293 REFERENCES

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INTRODUCTION Petechiae and hemorrhages develop on occasion in patients infected with many of the common viruses infecting humans (McKay and Margaretten, 1967; Angle and Alt, 1950; Bayer et ah, 1965; Perlman, 1934; Shershow et al, 1969; Oski and Naiman, 1966; Talley and Assumpcao, 1971; Davison et ah, 1973; Ninomiya et al, 1977; Verdonck et al, 1985; Saunders et al, 1986; Bodensteiner et al, 1992; Gerson et al, 1993). Widespread bleeding in the skin, mucus membranes, and internal organs, however, characterize infections with a small number of exotic viruses often, but not invariably, occurring in isolated ecological niches worldwide. The so-called hemorrhagic fever viruses considered in this chapter are classified into four families, each having distinct biological features. The pathophysiologic basis for bleeding in patients infected with these agents is incompletely characterized and poorly understood mechanistically. This shortfall in our knowledge can be attributed to practical considerations, for there has been a lack of critical clinical and laboratory study. In general, the hemorrhagic viral infections occur spo-

PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

ARENAVIRUSES These viruses exhibit a distinctive structural morphology, but the virions differ considerably in size (60 to 350 nm) and structural configuration. The virion is comprised of an RNA core surrounded by a dense membrane that exhibits spikes. The ultrastructural appearance of these agents suggests the appearance of sand sprinkled on a smooth surface, thus accounting for the name (a term derived from the Latin word "arenosus," referring to sand). Arenaviruses are pantropic, and cell surface receptors for the virus appear to be widely distributed in various tissues. Macrophages are unusually susceptible to infection, but a variety of epithelial organs also support virus replication. The viruses of this family replicate in the cell cytoplasm and are released from the plasma membrane by budding 277

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.V\EPAT/C

FIGURE 19.1 Interrelationships and overlapping pathophysiological features of the so-called hemorrhagic fever viruses. Adapted with permission from a cartoon prepared by P. Child, MD.

from the surface. During the course of infection, macromolecular cell functions are perturbed and their socalled luxury functions are altered, but the cells often survive. The cytopathology of cells maintained in culture, and in the infected organs of experimentally infected primates and in humans with naturally occurring infections is variable in extent, but usually quite limited. Arenaviruses do not infect lymphocytes, but CD8+ immune effector T cells are believed to play an important role in both the pathogenesis of the disease and recovery from infection. Cytokines such as interferon may contribute to the profound ''toxicity" patients experience during the acute illness. Lymphocytic choriomeningitis virus (LCMV) is the prototype agent of the 16 viruses classified in this family, but it is usually the least virulent of the recognized human pathogens and does not cause hemorrhagic fever in humans and experimental animals (see Chapter 31). Junin (Argentinian hemorrhagic fever) and Machupo (Bolivian hemorrhagic fever) viruses are highly pathogenic agents occurring in geographically defined lowland regions of South America where they are apparently transmitted to humans by aerosols emanating from the feces and urine of rodents that serve as the virus reservoir in nature. The viruses of Venezuelan hemorrhagic fever (Guanarito virus) and Sao Paulo (Brazil) hemorrhagic fever (Sabia virus) are two additional arenaviruses known to be responsible for disease in isolated niches in South America. Finally, Lassa virus, an agent occurring in tropical West Africa, causes substantial mortality among those overtly infected, but

asymptomatic subclinical infections also occur commonly in endemic areas. This infection is acquired by means of aerosols from rodent excreta, but person-toperson spread occurs with ease. Whereas LCMV infections in humans are either subclinical or result in a nonfatal meningitis, Junin, Machupo, Guanarito, Sabia, and Lassa cause a remarkably similar clinical picture, and somewhat comparable pathological features are found among those patients who do not survive (see below). The initial symptoms develop roughly 7 to 10 days after exposure and are characterized by nonspecific chills and fever, headache and myalgia, asthenia and lethargy, accompanied by petechiae and exanthema, as well as edema of the tissues of the head, neck, and thorax. In addition, the conjunctivae are inflamed and there is lymphadenopathy. To a variable extent, hypotension appears terminally. As the end of life approaches, the kidneys fail and hemorrhages occur in body organs and the lumina of the digestive tract (Figure 19.2). The mortality rate is approximately 30 to 40%. Argentinian and Bolivian Hemorrhagic Fevers These two antigenically related viruses are considered together because of their similar epidemiological and clinical features. Junin occurs primarily among agricultural workers during the summer months in relatively circumscribed regions of the pampas of north

FIGURE 19.2 Hemorrhage from the nose of a young rhesus monkey 17 days the experimental inoculation of the virus of Bolivian hemorrhagic fever. Reprinted with permission from McLeod et al. (1978).

Hemorrhagic Fever Viruses

central Argentina. Machupo first appeared in the tropical lowlands of the Bolivian Amazon, where it proved to be endemic. Both viruses are believed to be transmitted to humans through the excreta of rodents (Calomys sp.), but person-to-person secondary spread can occur upon intimate contact. Outbreaks develop when the population density of the rodent reservoirs in a community increases because of the ready availability of food. Thus, the potential exists for more intimate interactions of humans with rodents and, as a result, their excreta. Autopsy material serves as the exclusive basis for our insights into the pathology of these two infections (Eisner et al, 1973; Child et al, 1967). Overall, focal hemorrhage and petechiae on internal organs are prominent features. Lymphadenopathy with a nonspecific lymphoid hyperplasia is almost invariably present, but the histopathology of the lymphoid changes is incompletely defined. To a variable extent, morphologic evidence of a mild encephalitis is discovered by the pathologist, but it is not a prominent feature of the clinical disease. Interstitial myocarditis without necrosis of the myocardium and interstitial pneumonitis are described. While structural renal changes have been noted, they most probably reflect the results of hemoglobinuria and shock, rather than an intrinsic lesion of the nephron system of the kidney. The liver shows nonspecific foci of acute necrosis. Characteristically, necrotic or apoptotic hepatocytes are found either in the sinus or phagocytized by macrophages. Kupffer cells of the liver are a prominent microscopical feature of the liver lesion (Figure 19.3). These cells often exhibit erythrophagocytosis. Rhesus and African green monkeys have been infected with Machupo virus experimentally in an effort to understand better the pathogenetic mechanisms in-

279

volved in the disease (Kastello et al, 1976; McLeod et al, 1978). Unfortunately, the scope of this work has been limited, and critical insights into the disease and the associated coagulopathies have not accumulated. Rodent models have not proved to be useful for the study of human infections because the animals sustain a nonfatal, persistent, and low-grade infection, as no doubt is the case in the reservoir rodent species in nature. In the primate, the duration of the incubation period is related to the inoculum dosage, and the clinical syndrome remarkably simulates the picture in humans. Primates develop a neutropenia and lymphopenia a few days after virus inoculation. Viremia appears shortly thereafter and persists until death of the animal. At autopsy, the primates experimentally infected with Machupo virus exhibit lesions identical to those described in humans at autopsy (Terrell et al, 1973). Remarkably little information has accumulated on the coagulopathy of Junin and Machupo virus infections in humans and primate models. Thrombocytopenia is claimed to occur, but the decrease in platelet numbers is insufficient to account for the hemorrhagic lesions observed. Similarly, reductions in blood concentrations of coagulation proteins are reported, but the aberrations are not severe (Molinas et al, 1989). Although disseminated intravascular coagulation (DIC) has been suggested as a mechanism, laboratory studies fail to provide evidence supporting this conjecture and intravascular fibrin thrombi are rarely found at autopsy. Hemoconcentration and vascular congestion attributable to vascular and capillary leakage are prominent features as death approaches, but they do not appear to account for the hemorrhagic manifestations.

FIGURE 19.3 Prominent Kupffer cells (arrowheads) lining the liver sinuses of a hemorrhagic fever patient. This nonspecific finding is commonly observed in the livers of patients dying from infection by many of these viruses.

280

Pathology and Pathogenesis of Human Viral Disease

Venezuelan and Sao Paulo Hemorrhagic Fever Guanarito virus is the cause of an acute febrile systemic illness with hemorrhagic features occurring annually among male agricultural workers in the central plains (llanos) of Northwest Venezuela (Salas et ah, 1991). Mortality is 33%. Autopsies yield the findings of pulmonary congestion and edema, renal cortical necrosis, and hemorrhage in mucus membranes, major internal organs, and both the digestive and urinary tracts. Many fatal cases are reported to have encephalitic manifestations, but neuropathological observations on these cases have not been reported. Profound leukopenia and thrombocytopenia are consistently documented, and platelet deficiencies are believed to account for the hemorrhagic manifestations. While the epidemiology of the virus is unknown, isolation of virus from the indigenous rodents, Sigmodon hispidus and Zygo dontomys, and demonstration of antibody in a rice rat (Oryzonys sp.) suggests that these animals may serve as a source of infection for humans. The origin of Sabia virus, the presumptive cause of Sao Paulo hemorrhagic fever, is more obscure (Coimbra et ah, 1994). It developed seemingly without rural exposure in a 25-year-old office worker in Sao Paulo, Brazil. In addition to generalized systemic signs and symptoms, this patient developed neurological symptoms before lapsing into coma. Autopsy findings were comparable to those observed in the other South American arenavirus infections. The extraordinary infectivity of this virus is exemplified by the occurrence of two nonfatal laboratory infections in technicians working with the original Brazilian strain (Barry et ah, 1995).

After an incubation period of roughly 10 days, there is the insidious onset of disease with diverse constitutional symptoms evolving over an approximate 6-day period. Myositis, myocarditis, and meningitis are occasionally diagnosed clinically. Evidence of vascular incontinuity in the form of tissue edema and hemorrhage appears in roughly a third of patients. Leukopenia and thrombocytopenia are documented during this period. About a third of patients exhibit platelet counts less than 1 x 10^/mm^. Hemorrhagic complications are a poor prognostic sign, as up to 50% of patients so affected die in shock (Frame, 1989). Viremia persists for as long as 2 weeks in those who recover, but may continue unabated until death (Figure 19.4). Abrupt-onset sensorineural deafness apparently occurs commonly in infected patients with all degrees of disease severity. In one study, roughly a third of hospitalized patients manifested an acute hearing impairment, and two-thirds of the survivors experienced some degree of permanent hearing loss. The pathogenesis of the auditory problem is unclear, but inflammatory involvement of the middle ear seems likely. Interestingly enough, the prevalence of the hearing loss substantially exceeds that observed among patients in developed countries infected with mumps, measles, and varicella-zoster virus infections (Cummins et ah, 1990). A systematic study of autopsy material from patients with documented Lassa virus infections was reported by Winn and Walker (1975). Noteworthy was the consistent presence of hepatic lesions of variable extent to the exclusion of changes in other major organ

6.0

West African Hemorrhagic Fever (Lassa Virus) Lassa was first recognized by modern medicine in 1969 when it presented as a highly fatal febrile illness in a small mission hospital in Central Nigeria. The name Lassa refers to the community where the index case developed (Frame et ah, 1970). The virus has inflamed exceptional fear because of the early recognition of its extraordinary infectivity for hospital caregivers. Subsequently, outbreaks have continued to occur in Nigeria and in the small West African coastal countries of Sierra Leone, Liberia, and Guinea. Subclinical infections are relatively common in these endemic areas. As with the South American arenaviruses, Lassa virus is believed to be maintained in nature by rats of the genus Mastomys. This is a common indigenous rodent on the African continent.

5.0 O -I 4.0

J2 CD 3.0

F 2 2.0 1.0 <0.6 I

I I

I I I

5

I I I I I

10

I I

15

I I

20

25

Days After Inoculation FIGURE 19.4 Pattern of viremia in adults fatally infected with Lassa virus, in comparison to those who survive. Patients survive when viremia is aborted, presumably due to an effective immune response. Adapted with permission from Jahrling et al. (1980).

Hemorrhagic Fever Viruses

281

FIGURE 19.5 Councilman-like bodies in the liver parenchyma of an adult infected with Lassa virus. The bodies are identical to those classically found in the liver of yellov^ fever. Reprinted with permission from Winn and Walker (1975) and through the courtesy of W. Winn, MD.

systems. These authors described a focal but nonzonal necrosis of the liver with a morphologic picture that, at times, resembles the liver of yellow fever (Figure 19.5). There was a remarkable absence of inflammation. Necrotic, eosinophilic, contracted hepatocytes were found scattered in the hepatic cords and sinusoids. Necrosis frequently bridged between portal and central regions of the hepatic lobules, and confluent lobular necrosis was occasionally present. McCormick et al. (1986) described in more detail the liver pathology in 19 fatal cases. They classified six cases into an acute hepatitis stage in which less than 20% of the hepatocytes were necrotic. Eight patients fell into a second category in which randomly distributed multifocal hepatocellular necrosis was found. Eosinophilic bodies extruding into the hepatic sinusoids (i.e., the so-called Councilman bodies) were commonly seen in the liver at this stage. Finally, an early recovery stage was identified in the remaining five cases. The liver exhibited prominent accumulations of macrophages, presumably in response to the necrosis of hepatocytes. Hepatocellular mitotic activity was also present — a finding compatible with regeneration. This work shows quite clearly that the liver disease in fatally infected patients evolves through a series of stages. Electron microscopy reveals the expected ultrastructural feature of necrotic hepatocytes, but viral particles are identified in only a small proportion of the liver cells in the cases studied. At present, we have little insight into the mechanisms of hepatocyte damage in this disease. The paucity of inflammatory cells in the

lesions argue against, but does not exclude, cellular immunity in the pathogenesis of the liver disease. The severity of the morphologic changes in the liver correlate poorly with clinical biochemical parameters of liver cell damage. The clinical laboratory and pathological studies fail to establish the basis for the hemorrhagic lesions observed in the roughly one-third of patients. While thrombocytopenia is documented in some patients, it is not a consistent laboratory finding, and it is rarely so severe as to account irrefutably for the bleeding. As with Junin and Machupo infections, fibrin thrombi, the pathologic hallmark of DIC, are not found in the small blood vessels and renal glomeruli of these cases at autopsy. However, insufficient laboratory studies have been carried out to exclude a less obvious consumptive coagulopathy. Recently, Lassa viral antigen has been demonstrated by in situ hybridization in endothelial cells of infected human tissue. This finding may account for the evidence of vascular incontinuity seen clinically. The pathology of experimentally induced infections in primates have been investigated in an effort to better understand the pathogenesis of Lassa in humans (Walker, 1975a,b, 1982a,b; Calls et al, 1982; Jahrling et ah, 1980). Unfortunately, this work has yielded relatively few insights into the human disease, in part because of the differing susceptibility of the species of primates used in the studies, and the possible influence of the route of inoculation and dosage. Some, but not all, infected squirrel monkeys {Saimiri scirreus) develop necrotizing lesions in the heart, liver, kidney, and

282

Pathology and Pathogenesis of Human Viral Disease

spleen. Meningoencephalitis of limited severity was noted in a single animal. On the other hand, a necrotizing hepatitis and interstitial pneumonitis are the prominent lesions in infected rhesus monkeys {Macacjue mullata). Detailed studies of platelet and fibrinogen consumption in these monkeys failed to provide evidence of a consumptive coagulopathy (Lange et ah, 1985). Clearly, much additional work will be required to better understand the pathogenesis of the lesion developing in humans, and the primate models. Table 19.1 summarizes pathological findings in autopsies of humans dying with the three major arenavirus infections.

areas overlap. Countless names have been used to refer to illnesses that seem to be geographically isolated. The responsible viruses in many of these outbreaks remain to be characterized. Viruses of the Bunyavirus family, 80 to 120 nm in diameter, are spherical enveloped agents that utilize RNA as the genome. On the external surface, the virion exhibits glycoprotein molecular projections. Three distinct single-stranded nucleotide sequences further characterize the RNA genome; these segments serve as the basis for categorization of the viruses in the genera referred to above, although antigenic relationships between family members have also been established.

BUNYAVIRUSES

Hemorrhagic Fever with Renal Disease

This is a large and complex family comprised of four genera of viruses having health importance for humans. Members of this family are found in ecological niches worldwide, where they utilize a variety of vectors for transmission. La Crosse virus, a member of the genus Bunyavirus, causes epidemic acute encephalitis in central North America and is considered in Chapter 24. Viruses causing febrile illness with hemorrhagic manifestations are classified in the genera Hantavirus, Phlebovirus, and Nairobivirus and cause, respectively, hemorrhagic fever with renal disease. Rift Valley fever, and Crimean-Congo hemorrhagic fever. The diseases caused by these viruses are similar and their endemic TABLE 19.1 Arenavirus Infection-Associated Lesions Percentage of fatal cases Argentine hemor. fevei^

Bolivian hemor. fever^

West African hemor. fever (Lassa)^

Mortality

-

Pneumonitis and/or hyalin membranes

33

100

40

Myocarditis

33

0

33

Renal tubular necrosis

50

25

54

Lymphocytic meningitis and/or focal encephalitis

43

100

0

Liver necrosis

60

100

100

Spleen necrosis Myositis

NR = no report. ''Eisner et al (1973). ^Child et al (1967). 'Walker et al. (1982b).

20

0

0

92

NR

NR

33

This is not one, but several, clinical syndromes caused by closely related virus strains of the Hantavirus genus in scattered regions of the Eurasian continents (Gajdusek, 1962). Although long thought to have a viral etiology, the responsible agent for the Korean variant of hemorrhagic fever syndrome, Hantaan virus, was only identified in 1978 (Lee and van der Groen, 1989). Since that time, viruses of three distinct lineages based on antigenic serotype and molecular features have been associated with disease, and other agents, no doubt, remain to be identified. In addition, considerable strain-to-strain variability in antigenic composition and pathogenic potential probably exist (Gligic et al, 1992). The geographic range and ecology of these diverse viruses remain to be fully defined, and the pathogenesis of the diseases they cause requires further characterization. In addition, linkage with the pulmonary syndrome recently described in the southwestern United States and due to the Sin Nombre strain of Hantavirus requires definition (see Chapter 20). Worldwide, inapparent Hantavirus infections have been documented in rodents of several genera, and these animals are believed to serve as reservoirs for the virus in nature. As with the arenaviruses, excreta from animals most probably serve as the means of transmission among animals in the wild, and to humans. Korean and Seoul Hemorrhagic Fever (Hantaan and Seoul Viruses) The prototype Hantavirus strain, Hantaan, was recovered in Korea from the lungs of the striped field mouse {Apodemus agaricus). Its association with Korean hemorrhagic fever was established shortly thereafter. This severe, often fatal, illness gained a well-deserved reputation during the Korean conflict in the early

283

Hemorrhagic Fever Viruses

1950s, when it was believed to have caused some 250 to 300 deaths among United Nations troops. A somewhat less pathogenic serotype, termed Seoul, also causes disease on the Korean Peninsula and throughout the northeastern mainland provinces of the Peoples' Republic of China, as well as in Manchuria, eastern Russia, and Japan. Reports of illnesses similar to those caused by Hantavirus are recorded in the ancient Chinese medical literature. This virus is maintained in densely populated urban communities by Rattus rattus norwegicus and R. rattus; Apodetnus agaricus, a field mouse, serves as the principal rural reservoir for the virus (Casals, 1970). Although the prevalence of disease and its severity differs from year to year, as many as 1 X 10^ residents of Northeast China are believed to be infected on an annual basis. Mortality ranges from 5 to 15% (Chen et al, 1986). In endemic areas of Zhejing Province (China), 12% of the population possess serum

FCBRILC

antibodies, an indication that the prevalence of subclinical infection is high (Ruo et ah, 1994). During the Korean conflict in the early 1950s, American investigators conducted detailed clinical studies on United Nations soldiers infected with Hantavirus (Giles et al, 1954; Lukes, 1954; Furth, 1954; Giles and Langdon, 1954; Earle et al, 1954; Earle, 1954; Cugell, 1954; Froeb and McDowell, 1954; Hunter et al, 1954). The disease continues to occur among United States troops stationed in South Korea (Pon et al, 1990). This work has provided detailed insights into the pathophysiology of the life-threatening and fatal Korean hemorrhagic fever. Several stages of disease characterize the more severe cases (Figure 19.6). After an incubation period of 2 to 3 weeks, the infection erupts as a high fever with systemic symptoms. Hypotension can appear at this time, or later in the illness. Shock, accompanied by hemoconcentration and hemorrhage.

I T»IS?VC I ^ ^ ^ ^ " ' ^

I

OtURCTIC

RECOVCRY

FIGURE 19.6 Pathophysiological features of a Hantaan virus infection in a young soldier. The four clinical phases of the disease are illustrated. Patients die throughout the course of the infection, with irreversible shock and renal failure being critical causative factors. Reprinted with permission from Earle (1954).

284

Pathology and Pathogenesis of Human Viral D i s e a s e

follow in a small percentage of patients (about 16%) (Giles et ah, 1954). An oliguric stage accompanied by hypertension develops shortly thereafter. About 20% of patients exhibit life-threatening severe disease. Death can occur at any time during the course of the illness. A detailed evaluation of the hemorrhagic parameters of this disease was recently reported by Guang et at. (1989). The major sites of bleeding proved to be the skin, the aerodigestive tract, and the urinary tract (microscopic hematuria was invariably present). Clinical and pathologic evidence of vascular endothelial cell damage was the hallmark of the disease, but thrombocytopenia was almost invariably present early in the course of the illness. Platelet counts are less than 1 x lO^lvciim? in 90% of patients, and platelet counts of less than 30,000 have been documented in some patients. In addition, abnormalities in the structure of platelets and platelet dysfunction have been documented (Furth, 1954). The degree of thrombocytopenia appeared to correlate with the overall severity of the disease. Many patients have clinical laboratory evidence suggestive of DIG, but satisfactory laboratory documentation of this process is lacking. In addition, platelet/fibrin thrombi are not demonstrated in postmortem material. Other features that might play a role in abnormal hemostasis in these patients are the presence of circulating immune complexes and heparin-like activity in the blood of some patients. Clearly, the clinical and pathologic evidence strongly suggested that diffuse endothelial vascular lesions could account for consumptive thrombocytopenia and might easily trigger intravascular coagulation (Sheedy, 1954). Reports on pathology in the English literature are limited. A comprehensive study of more than 50 United Nations personnel dying in 1951 during the first 2 weeks of illness was conducted by Hullinghorst and Steer (1953). Of paramount interest in these cases was the evidence for diffuse capillary damage with engorgement of vessels and erythrocyte diapedesis across capillary walls. Petechiae and hemorrhages were found in the skin and internal organs, including the gut. In addition, both interstitial and retroperitoneal edema and ascites proved to be common. Fluid leakage into body compartments was a grave prognostic sign (Lukes, 1954). The kidneys were important sites of disease. Almost invariably, they were congested and swollen and increased in weight (average combined weight -525 g). Gross and microscopic evidence of tubular necrosis associated with extreme degrees of congestion and hemorrhage in the peritubular tissue were found (Figure 19.7) (Oliver and MacDowell, 1957). (The interested reader is referred to the detailed Oliver and MacDowell report, which considers patient clinical course

in relation to findings in the kidney using microdissections of individual nephron systems.) Glomerular structural lesions were not seen, although hemorrhage into the glomerular cavity is often found. In addition, sites of infarction necrosis were present in the pyramids. A surprising lesion of uncertain physiologic importance is described in the report of Hullinghorst and Steer (1953). In three-quarters of cases, there was ischemic infarction of the anterior pituitary. This finding was not recorded in the patient series published by Lukes (1954). Death in patients with hemorrhagic Hantavirus infections is usually attributable to shock or azotemia and blood electrolyte imbalances, but could pituitary failure be a contributing factor? Nephropathia Endemica (Puumala Virus) The third Hantavirus of human importance, Puumala, is endemic among rural residents of North Cen-

FIGURE 19.7 A microdissection analysis of a renal lesion in a young soldier who died in shock on the 10th day of a Hantaan virus infection. Heavy black indicates hemorrhage into glomeruli and tubules. Striped area represents sites of extravasation of blood into the kidney stroma. Cross-hatched tubules are sites of epithelial necrosis. Small circles within the tubular lumina indicate deposits of desquamated lining cells and debris. Reprinted with permission from Oliver and MacDowell (1957).

285

Hemorrhagic Fever Viruses

tral Sweden, Finland, and Northwest Russia. To a lesser extent, sporadic cases occur in Central Europe and the Balkans. In southern Europe, Puumala overlaps the endemic range of a newly described hemorrhagic fever caused by a Bunyavirus (Antoniadis et al., 1996). In the early 1930s, Swedish physicians reported for the first time an acute nonfatal febrile illness characterized by transient renal failure and occasional hemorrhagic manifestations (Myhrman, 1934; Zetterholm, 1934). Typically, the disease occurred in male forest and field workers. Outbreaks of similar illness were reported during the Second World War among Finnish and German soldiers billeted in the province of Karolia in eastern Finland (Niklasson and LeDuc, 1987). Nephropathia endemica is etiologically associated with Puumala virus infection, a Hantavirus related to, but molecularly distinct from, the Hantaviruses of east Asia and southern Europe (Antoniadis et ah, 1996; Pilaski et ah, 1994). The virus appears to be transmitted in the wild by the riverbank vole, Clethrionomys glareolus. In the endemic areas of central Sweden, the prevalence of serum antibodies in wild rodents is as high as 35% (Niklasson and LeDuc, 1987). The mode of virus transmission to human residents of these areas is presently unclear. In a serological survey of members of the general population, the incidence of antibodies was 8%, an indication of a high prevalence of subclinical infection. Nephropathia endemica resembles the disease caused by Hantaan virus in eastern Central Asia, but it is relatively mild. The acute febrile illness is said to resemble influenza without the respiratory symptoms, but it is accompanied by abdominal pain. Oliguria associated with elevated serum creatinine concentrations commonly occur several days after the onset of fever. In one series, one-third of patients had creatinine levels of >500 |imol/ml, but on only rare occasions was renal dialysis required (Settergren et ah, 1988). The oliguric phase lasts for only a few days and is followed by polyuria. During the acute stages of Puumala virus infection, 28 to 53% of patients exhibit pulmonary infiltrates or pleural effusion by X-ray. Functional studies indicate an alveolar capillary lesion characterized by reduced diffusion capacity and blood oxygen desaturation (Linderholm et al, 1997). Full recovery occurs about 2 weeks after the onset of illness (Lahdevirta, 1971; Lahdevirta et al, 1978). Saari and colleagues (1977) noted the relative high prevalence of visual complaints among patients. They documented retinal edema and acute glaucoma in 3 of the 14 studied. In addition, hemorrhagic conjunctivitis and edema of the cornea proved to be common. These authors attributed the ophthalmic problems to alterations in the capillar-

ies of the affected eyes. Neurological complications develop in only a small portion of infected patients (Alexeyev and Morozov, 1995). Overt hemorrhage into the skin and internal organs is relatively uncommon in nephropathia endemica. In one study, hematuria occurred in 73% of patients and the overall prevalence of bleeding was 10%. Thrombocytopenia (platelet counts less than 1 x 10^/mm^^) was demonstrated in 62% of patients in one series (Settergren et ah, 1988) and 20% in a second (Lahdevirta, 1971). Reductions in the concentrations of coagulation factors in the blood are not found. Pathological changes in biopsies of the kidney are well documented, but we possess few insights into the pathogenesis of the renal disease. Glomerular hypercellularity and localized thickening of the basement membrane are seen in the kidney of some, but not all, patients during the acute stages of an illness. Immunohistochemistry has demonstrated deposits of IgC, IgA, and C3, but in amounts substantially less than in chronic immune complex disease (Settergren et ah, 1997). A variety of tubular alterations of a nonspecific nature are described, and viral antigens are demonstrable in tubular lining cells (Groen et ah, 1996). To a variable extent, interstitial hemorrhage, edema, and focal infiltration of the parenchyma by a variety of inflammatory cells are often seen during the acute stages of illness (Kuhlback et ah, 1964; Lahdevirta, 1971; Saari, et ah, 1977). Studies of patients who have recovered from Puumala virus infections fail to demonstrate significant residual renal functional abnormalities. Rift Valley Fever (RVF) Rift Valley fever is a mosquito-transmitted highly fatal enzootic disease of domestic animals that pathologically manifest the infection as an extensive hepatic necrosis. The responsible virus was initially recovered in 1930 in the Rift Valley of western Kenya (Daubney et ah, 1931), but it is now known to be enzootic in many regions of sub-Saharan Africa as far south as Capetown. This virus is a member of the Phlebovirus genus of the family Bunyaviridae. Human infections are acquired from a mosquito vector, usually a species of the genus Aedes. These arthropods adapt particularly well to the human habitat. Thus, the virus is transmitted readily in areas having a high population density, such as the Nile river basin. Infection is usually subclinical, or is expressed as an influenza-like illness of 4 to 7 days duration with severe myalgias, headache, retroorbital pain, and fever (Laughlin et ah, 1979). On occasion, the infection in humans is sufficiently severe to be life-threatening. In these cases, centrilobular hepatic necrosis, meningoencephalopathy.

286

Pathology and Pathogenesis of Human Viral Disease TABLE 19.2 Observations of the Fundi of the Eyes of 22 Suspected RVF Patients Who Were Examined During Acute and Convalescent Phases of Illness % Affected Initial Examinations (during acute illness) Macular lesion Paramacular lesion Vitreous flare Discoid lesions Arteriolar narrowing and/or sheathing Peripheral lesions FoUow-Up Examinations (6 to 9 months later) Retinal microangiopathies Macular scars Optic atrophy Anterior uveitis

FIGURE 19.8 Photoophthalmologic evaluation of the retina of an Egyptian man at the time of onset of visual complaints due to Rift Valley virus infection, and during convalescence. Acutely, microangiopathies and retinal edema are present (top). In the late stages, scarring of the macula is seen (bottom). Reprinted with permission from Elwan et al. (1997).

and retinopathy are present to a variable extent (Figure 19.8, Table 19.1). Commonly, a combination of these medical problems occur. In the extensive Egyptian epidemic of 1977, several hundred thousand human infections were estimated to have occurred, but there were fewer than 100 fatalities (Laughlin et al, 1979). Hemorrhagic manifestations in infected patients were reflected as petechiae and purpura of the skin, accompanied by gingival bleeding, hematemesis, and melena. Ocular signs and symptoms due to lesions in the retina occurred in about 1% of patients with RVF. The pathogenesis of the retinopathy is unknown. Vascular occlusion by thrombosis, vasculitis, and inflammation have been suggested as possible causes based on ophthalmological evaluations. Visual complaints begin 4 to 10 days after the onset of illness and persist for variable periods of time. Fundus examination reveals a variety of changes (Figure 19.8, Table 19.2). Although visual acuity is compromised permanently, blindness does not develop. Hemorrhagic lesions were observed in only 20% of experimentally infected Macaca mulaita (Peters et al, 1988). These animals exhibit profound thrombocytopenia and a reduction in the activated partial thromboplastin time, accompanied by circulating fibrin split products and a microangiopathic hemolytic anemia. Pathological studies demonstrate focal hemorrhages in internal organs and fibrin thrombi in glomerular tufts and elsewhere in the small vessels of the kidneys

100 74 33 26 23 5 100 87 20 0

Reprinted with permission from Elwan et al. (1977).

(Peters et al, 1988). These findings are pathognomonic of Die. Centrilobular coagulation necrosis develops in the liver of some, but not all, experimentally infected rodents, dogs and cats, and subhuman primates (Mitten et al, 1970; McGavran and Easterday, 1963; Peters et al, 1988). Congo-Crimean Hemorrhagic Fever (CCHF) The virus of CCHF is a member of the genus Nairovirus of the family Bunyaviridae (Casals et al, 1970; Korolev et al, 1976). In the former Soviet Union, humans are infected by soft-shelled ixodid ticks, principally members of the genus Hyalomma. Wild and domestic mammals may serve as reservoirs for the virus since serological surveys have demonstrated a high prevalence of antibody in members of a variety of species (Swanepoel et al, 1983). In southern Ukraine and Central Eurasia, outbreaks occur on an annual basis. The virus has also been recovered at multiple sites in sub-Saharan Africa, and outbreaks in humans have been described from the Baltic countries to western China, including the countries of Syria, Iran, Iraq, Afghanistan, and Pakistan. The virus of CCHF is readily transmitted by person-to-person contact, such as commonly occurs in the hospital setting. Thus, several nosocomial outbreaks of disease in secondary and tertiary contacts are described (Burney et al, 1980; van de Wal et al, 1985). The clinical illness of CCHF is variable in severity, with mortality ranging in various reported outbreaks from 13 to 50%. It is characterized by sudden onset of headache, fever, and myalgias. After a quiescent pe-

287

Hemorrhagic Fever Viruses

riod, hemorrhagic lesions appear and death follows, with extensive hemorrhage and shock being common. During the initial phase of the disease, lymphopenia and thrombocytopenia are evident. Thus, thrombocytopenia precedes the development of hemorrhagic lesions. Although a few reported cases have exhibited evidence of DIC, it is likely this is a late manifestation and not the primary cause of the hemorrhage (Joubert et al, 1985; Burt et ah, 1997; Suleiman et al, 1980; Swanepoel et ah, 1989). In autopsy studies, the liver shows variable degrees of hepatic necrosis. Central zonal coagulative necrosis and intracanicular bile retention are seen in some cases. Extrusion of necrotic or apoptotic hepatocytes into sinusoids (so-called Councilman bodies) is observed, but it is not a prominent feature. Kupffer cell hyperplasia is also evident, but inflammatory cell infiltrates are sparse. Examination of the spleen shows necrosis of the lymphoid elements and congestion of sinusoids. Elsewhere, the lungs are edematous and, to a variable degree, hemorrhagic. Hemorrhages are also found in the myocardium, adrenals, and the mucosa of the digestive tract. Massive hemorrhage into the abdominal cavity has been described (Suleiman et al, 1980). Immunohistochemistry and in situ hybridization studies show that endothelial cells and hepatocytes, as well as mononuclear cells, are infected (Burt et a/., 1997). The prevalence of inapparent infections in humans is not well documented (Baskerville et ah, 1981), and little clinical laboratory and information is available on persons with subclinical disease.

FILOVIRUSES Members of this recently discovered family of viruses have been associated with outbreaks of a highly fatal hemorrhagic disease in native populations and nonhuman primates at scattered sites in sub-Saharan Africa. The virions are pleomorphic filamentous strands that assume a variety of forms when examined ultrastructurally The filaments that exhibit maximal infectivity are greater than 790 nm in length. The virus has seven RNA genes arranged in sequence and located within a nucleocapsid that is surrounded by a lipid bilayer envelope. At present, our knowledge of the biology of these viruses is fragmentary and the pathogenic mechanisms of disease not understood. Marburg Virus Disease The filoviruses first came to scientific attention during 1967 in Marburg, Germany, and shortly thereafter

in Belgrade, Yugoslavia, when outbreaks of disease developed among laboratory workers preparing cell cultures from the kidneys of green Vervet monkeys {Cercopithecus aethiops) imported from Uganda. There were 25 primary cases in the Marburg outbreak, but six caregivers subsequently became infected. Overall, seven patients died (23%). An additional outbreak appeared in South Africa during 1975, attributable to a traveler who was thought to have contracted the infection in Zimbabwe. He died, but two caregivers who later became ill survived. Ebola Virus Ebola virus came to the attention of virologists and the general public in 1976 when two near-simultaneous outbreaks erupted in the Democratic Republic of Congo (formerly Zaire) and southern Sudan. Mortality was high and many secondary cases developed among physicians and nurses who used limited or no barrier protection while treating the patients. Subsequent outbreaks of a much more limited scope have occurred in Gabon and Cote dTvoire, and at scattered sites elsewhere in West Africa. Of more recent interest was the well-publicized outbreak among Philippine Macaca fascicularis primates imported into a holding facility in a commercial laboratory in the eastern United States (Preston, 1994). Four animal caretakers developed serological evidence of infection but no illness. Thus, subclinical infections occur, as was amply demonstrated by serological surveys in the former Zaire after the 1977-78 outbreak. In two isolated locales, over 16% of adults possessed antibodies suggesting past infection (Heymann et ah, 1980). The viruses recovered in these outbreaks proved to be related and were assigned the name Ebola, referring to a river in the north central Democratic Republic of Congo, where the virus first appeared. However, strains from geographically isolated areas (such as southern Sudan) exhibit unique molecular characteristics, and the antigenic similarities among these different virus strains are limited. The reservoir(s) for these viruses in nature are not known, and the means by which humans acquire an infection are obscure. At least three species of Old World primates have acquired the infection in their natural habitat: Pan troglodytes in Cote dTvoire, Macaca fascicularis in the Philippines, and Ceropitecus aethiops in Uganda. Secondary cases among health workers are generally attributed to exposure to infected blood. Ebola and the primate-acquired disease that occurred in Marburg result in similar clinical illnesses even though the responsible viruses are antigenically distinct (Martini, 1971; Stille and Bohle, 1971). After an

288

Pathology and Pathogenesis of Human Viral Disease

Conjunctivitis, limb-aches ,

Leucopenia ,

Invariably present Sometimes presen

,

Enanthem Thrombocytopenia

...-J

Diarrhoea

L I

Hepatitis

\,

Exanthem

,

Renal involvement ,

,

,

Leucocytosis

,

,

Encephalitis

J

^ Haemorrhages _, Myocarditis Orchitis

Late organ phase 1 2

3

4

5

6

7

8

1 1 J \ I I 10 11 12 13 14 15 16 17 18 19 20

FIGURE 19.9 Clinical course of an adult with fatal Marburg virus infection. Reprinted with permission from Egbring et al (1971).

incubation period of 1 to 2 weeks, high fever and prostration develop accompanied by headache, a severe sore throat, myalgias, arthralgias, and abdominal pain. Vomiting and diarrhea follow. A morbilliform rash beginning on the abdomen is seen in Caucasians. About 70% of patients exhibit hemorrhagic problems, with skin and severe digestive tract bleeding being common. Customarily, about half of the infected patients with fatal disease die 1 to 3 weeks following onset of the illness (Figure 19.9). In the cases with Marburg virus infection, central nervous system depression was often followed by coma. At autopsy, densely cellular glial nodules were found to be scattered in both the white and grey matter of the cerebrum, and at scattered sites in the brainstem and cerebellum. Perivascular lymphocytic cuffs were evidenced but were not prominent (Figure 19.10) (Jacob, 1971). Reports from Germany document the pathological findings in five thoroughly studied cases {Gedigket al, 1971). Focal areas of necrosis were evident in many organs, but particularly noteworthy were the lesions of the liver, spleen, and testicles. Interstitial edema of the myocardium seemed to account for the myocarditis frequently diagnosed clinically. Renal tubular damage was consistently noted. Gedigk et al. (1971) describe "peculiar basophilic bodies" scattered in many organs, but these authors failed to characterize the structures further.

CF 55/67

R.H, 57/67

FIGURE 19.10 Diagrammatic representation of sites of glial nodules in the brains of two adults who died in coma during an infection with Marburg virus. Reprinted with permission from Jacob (1971).

Hemorrhagic Fever Viruses

The pathology of naturally occurring disease among persons infected with the Sudanese and Zairian strains of Ebola virus have been evaluated most thoroughly using autopsy material. Of obvious significance is the necrotizing hepatitis that develops early in the infection and the extensive involvement of the spleen with necrosis of lymphoid follicles. In addition, there is generalized depletion of lymphoid tissue with necrosis of cortical lymphoid follicles in lymph nodes. The liver lesions strikingly resemble those of yellow fever, a disease Ebola has often been confused with in Africa (see below). More specifically, necrotic cells in the liver exhibit eosinophilic hyalinization, while dead and dying hepatocytes frequently resemble Councilman bodies (Murphy et al, 1971). Experimental subhuman primate models have been established using the Zairian virus strains (Bowen ei al, 1978; Baskersville ei al, 1977; Murphy et al, 1971). Of great interest in experimentally infected primates is the presence of fibrin thrombi in capillaries of the glomeruli and elsewhere. This finding is compatible with the D i e suspected clinically in both the human and primate models. In Ebola-infected rhesus and vervet monkeys, no evidence of endothelial damage is found. This finding conflicts with the limited evidence accumulated in humans where vascular lesions are apparent, and the presence of endothelial cell involvement in a chimpanzee infected in the wild (GeorgesCourbot et al, 1997). Lesions of major organs in humans and experimentally infected primates are similar. The clinical evidence from the German studies of Marburg virus victims suggests that thrombopenia with platelet counts of <1 x 10^ accounts best for the hemorrhagic diathesis (Egbring et al, 1971). These observers did not detect significant defects in coagulation factors, and evidence of DIG was not found at autopsy in the form of intravascular platelet/fibrin microthrombi.

FLAVIVIRUSES The majority of the human flaviviruses cause, on occasion, meningoencephalitis; although infection is often asymptomatic or results in a relatively mild and self-limited febrile systemic illness. These conditions are considered in Ghapter 24. Infections with the cosmopolitan mosquito-transmitted viruses of yellow fever and dengue can result in hemorrhagic systemic disease accompanied to a variable extent by hepatic necrosis and liver failure. These two viral diseases will be considered below. Two additional tick-borne flavivirus infections (Kyasanur forest disease and

289

Omsk hemorrhagic fever) also can have hemorrhagic manifestations as part of a systemic illness often with encephalitis. The geographic distribution of these viruses is limited, and the basis for hemorrhagic disease is not known. More detailed information on these infectious diseases is found in the following references: Lyer et al, (1959), Kharitonova and Leonov (1985), and Kenyon et al (1992). The virions of the various species of flaviviruses differ in size, ranging from 40 to 60 nm. They are spherical and have a dense nucleocapsid, comprised of a single-stranded RNA that serves as the messenger. It codes several important structural and nonstructural proteins. Alipid-rich bilayered envelope surrounds the nucleocapsid and adheres to susceptible cells by means of the so-called E protein, which interacts with an unidentified receptor. The E protein is also the virion's major antigen, and immunoglobulins synthesized against it serve to protect the host against reinfection. The means whereby the host resolves an established infection is unknown. Infection of susceptible cells in vitro, and presumably in vivo, results from fusion of the viral membrane with the plasma membrane of the cell. The virions then enter the host cell, where uncoating occurs and RNA replication begins. The simple viral message utilizes the synthetic equipment of the cell, but this process does not measurably inhibit cell metabolism. Nonetheless, to a variable degree, infection results in cytolysis by a mechanism yet to be defined. With fabrication of new viral proteins and RNA, the virion is assembled and is then released as the cell undergoes lysis. Yellow fever and dengue viruses exhibit similar epidemiological characteristics. Both are transmitted to humans by mosquito vectors and therefore have a replicative cycle in both vertebrates and the host insect. Humans are the major source reservoir of virus in urban communities. Thus, these viruses usually have a person-to-person pattern of transmission, with the mosquito serving as an intermediate. However, yellow fever virus is also maintained in the canopies of dense tropical forests by arthropods and intermediary mammalian hosts, often subhuman primates (Figure 19.11A,B). These arboreal reservoirs serve as a means for introducing "wild" strains to humans during the clearing of jungles by foresters and agriculturalists. Dengue virus does not have an intermediate mammalian host and a sylviatic life cycle. It is maintained in human populations by continuous sequential transmission from one person to another. Outbreaks therefore reflect the density of the mosquito vector and the proportion of the population who possess serum antibodies, reflecting prior infection (so-called herd immunity) (Ehrenkranz et al, 1971).

290

Pathology and Pathogenesis of Human Viral Disease

A^ simpsoni ond others

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jt

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Mosquitoes near humon hobitottons become infected from morouding mof»Keys ond in turn infect m a n , thus initiating mon m o s q u i t o - m o n cycle

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FIGURE 19.11 Ecology of yellow fever in Africa (A) and South America (B). The virus usually maintains either an independent urban or sylviatic cycle, with the potential for crossover when humans enter the tropical forest. Reprinted with permission from Bugher (1951).

When humans are infected, the virus replicates at the site of introduction into the skin, and then enters the bloodstream, after which it undergoes growth cycles in several major organs. Most probably, macrophages and endothelial cells are initial sites of virus replication, but the hepatocytes of the liver are also a major locus of viral multiplication. Yellow Fever Yellow fever first appeared in the Western Hemisphere after the initial exploratory ventures of Europeans in Latin America. It is believed that the virus originated in Africa and was transported to the West by the slave trade. Contemporary isolates from West Africa and tropical America exhibit great genetic similarity. Yellow fever, as a human disease, is now largely limited

to certain areas of tropical Africa and America. In the past, it plagued urban areas in the tropics and occurred seasonally in temperate zones, where its major vector, Aedes aegypti, bred with seeming abandon. The notion that yellow fever virus was transmitted by mosquitoes was first advanced in 1848 and later popularized by Dr. Carlos J. Finley, a Cuban physician. Walter Reed, a U.S. Army physician and researcher, showed that the yellow fever virus was a "filterable agent" and demonstrated transmission by the mosquito. General William C. Gorgas established the feasibility of mosquito eradication as a means for control of the virus in Cuba and Panama during the first years of the twentieth century The extraordinary contribution of Reed in intuitively deciphering the life cycle of the yellow fever virus and demonstrating for the first time its nonbacterial nature was an imposing scientific ac-

Hemorrhagic Fever Viruses

complishment for that time. While yellow fever was once responsible for countless deaths in both the Western and Eastern Hemispheres, it has now been brought under control in developed countries by the widespread use of the 17-D vaccine developed by Dr. Max Theiler, and the public health mosquito eradication techniques initially established by General Gorgas. Nonetheless, yellow fever continues to be a potentially important medical problem in both Africa and tropical Latin America, largely due to burgeoning population growth in major cities, and from the widespread dispersion of settlers into isolated jungle communities. Control of the disease now largely depends upon effective implementation of vaccination programs since mosquito abatement is often difficult, if not impossible. In many indigenous areas of yellow fever transmission, the Aedes aegypti mosquito was once effectively controlled by DDT, but the mosquito has returned as it has developed resistance to the insecticide. In South America, yellow fever currently occurs sporadically among unvaccinated workers in the forests of the major river basins. In Africa, the epidemiology is much more complex and the disease is often not reported. Most probably, it is also confused clinically with Lassa fever, Ebola, and Congo-Crimean hemorrhagic fever. The clinical illness of yellow fever has three phases. During the infectious stage, viremia occurs, accompanied by systemic symptoms and fever. Recovery can follow, presumably based on a brisk immune response. Alternatively, a transient period of clinical improvement follows the initial acute illness. Subsequently,

291

however, evidence of liver disease appears, accompanied by jaundice and the later occurrence of hemorrhage in a variety of organs, including the digestive tract and the skin. The mortality rate can be as high as 50% in some outbreaks, but in others relatively few die. Africans experience a relatively low mortality rate (Klotz, 1927). Flaviviruses, in general, and more specifically yellow fever virus, have a high degree of mutability, and it is likely that "wild" strains differ with regard to their pathogenicity for humans and their ability to infect and destroy liver cells. The pathological features of the liver disease of yellow fever have been amply described in numerous publications, but our understanding of the pathogenesis of this lesion is limited (Bugher, 1951). Suffice it to state that there is an extensive coagulative necrosis of the midzonal regions of the liver between the central and peripheral zones. Classically, necrotic or apoptotic hepatocytes are found by the pathologist in the sinusoids of the hepatic radicals, where they are termed the Councilman bodies (Figure 19.12). The nucleoli of infected liver cells are often prominent (sometimes termed Torres bodies), and the Kupffer cells are frequently prominent (classically termed Villela bodies). Patients with terminal yellow fever often exhibit bradycardia and a toxic shock syndrome. Older pathological literature vaguely describes cloudy swelling and fat accumulation in myocytes of the heart. The term Zenker's degeneration is used to refer to these incompletely described and poorly understood patho-

F I G U R E 19.12 Prominent Councilman bodies in the sinuses of the liver at autopsy. Although this illustration displays considerable autolysis, necrosis in the tissue section is limited to the cells forming the Councilman bodies.

292

Pathology and Pathogenesis of Human Viral Disease

logical changes (Lloyd, 1931). Alterations of a similar nature have been noted in the hearts of experiraentally infected primates. "Cloudy swelling" of the renal tubular epithelium is also described. It is unclear whether these apparent tissue changes develop as a consequence of viral replication, or reflect agonal or artificial autolytic changes. As a result of extensive liver necrosis, there is a marked decrease in synthesis of coagulation factors by hepatic cells. Concomitantly, evidence of DIC appears in experimentally infected primates in the form of circulating fibrin split products. DIC may be an important factor in the pathogenesis of the hemorrhages that occur in yellow fever, but the evidence supporting this conclusion is limited and based largely on the demonstrated clinical improvement of some patients when heparin therapy is administered. Pathological evidence of DIC in the form of fibrin thrombi in small blood vessels is currently lacking. In a 1987 consensus conference, it was concluded that decreased synthesis of coagulation factors due to liver parenchymal damage was largely responsible for the hemorrhagic manifestations of the infection (Monath, 1987). Dengue The virus of dengue fever exhibits many of the biological and ecological characteristics of yellow fever virus. It is currently indigenous around the globe between 30° north and 20° south of the equator, where over half of the world's population resides. However, in our historical past, dengue fever ranged far more widely because its mosquito vector {Aedes aegypti) was uncontrolled and bred preferentially in and around urban areas of both the tropic and temperate climates. At the present time, up to 1 x 10^ cases of dengue are believed to occur annually on a worldwide basis, with an infection rate of as high as 10% in some endemic areas (Halstead, 1988). Persons of all ages develop clinical disease, but in most outbreaks children in the early years of their lives customarily are infected. Four distinct, but antigenically related, serotypes (I-IV) of dengue virus circulate worldwide, although there seemingly are geographic differences in the prevalence of certain types. Outbreaks of one or another type develop regionally, followed by the appearance of a second antigenic type, customarily 2 or 3 years later. Often, several serotypes of virus circulate in the same community simultaneously. Children acquire protective immunity to the endemic virus type as a result of a primary infection, but, in addition, they elaborate broadly reactive antibodies directed against antigens of other dengue virus types. These antibodies fail to neutralize the heterologous viruses, but nonetheless can

bind with them. It is hypothesized that uptake of virus by macrophages in vivo is facilitated by the coating of the virus by these heterologous antibodies. In experimental studies, the virus replicates more readily in mononuclear cells when heterologous antibody is introduced into cultures of the cells with the virus. Uncomplicated dengue in adults and most children is a relatively transient, nonfatal, but debilitating illness of finite duration, often accompanied by severe bone and joint symptoms (so-called breakbone fever). Although nonfatal myocarditis occasionally is documented by electrocardiography, there are no long-term residual cardiac problems. Pathological studies of tissues from patients with this uncomplicated form of dengue virus have not been reported. Recently, a case of dengue with splenic rupture was described by Redondo et al (1997). D e n g u e Hemorrhagic Fever (DHF) and D e n g u e Shock Syndrome (DSS) Sporadic cases of hemorrhagic dengue were reported in the early medical literature, possibly a reflection of the severe thrombocytopenia observed in some patients. However, in the 1950s, a new clinical picture evolved when large numbers of infants and young children in Thailand and the Philippines developed a severe hemorrhagic fever with variable death rates, sometimes exceeding 10% (Nelson, 1960). Moreover, a substantial proportion of the affected children were less than 1 year of age. For unknown reasons, the prevalence of the syndrome proved to be twofold greater in female than in male infants. Characteristically, two patterns of illness are associated with these clinically severe dengue virus infections. The first is seen among infants born to mothers who have serological evidence of past infection with one or more dengue virus types. Hemorrhagic disease in these youngsters develops when they undergo a primary infection early in life with a virus not previously experienced by the mother. In this set of circumstances, transplacentally acquired antibody is believed to play a key role in the pathogenesis of disease. The second pattern is observed in children that have serological evidence of a primary postnatal infection by a virus of a type antigenically different from the agent responsible for the current illness. The heterologous acquired antibodies are believed to contribute to development of the illness. Clinical observations strongly indicate that DHF develops only when a child is "sensitized" as a result of acquisition of cross-reacting serum antibodies to a dengue virus type that differs from the one responsible for the acute illness. Only a relatively small proportion of

293

Hemorrhagic Fever Viruses

the infected children in areas of dengue virus endemicity develop hemorrhagic disease, and only a few of these cases evolve into hypovolemic shock, the socalled DSS. Estimates in various outbreaks range from 0.5 to 20% of those infected. Autopsies of children with DHF and DSS demonstrate the features of hemorrhagic disease in the form of petechiae on the skin and serosal surfaces, and throughout the digestive tract. In addition, the heart (42%) and the lungs (24%) exhibit hemorrhages to a variable extent. Coagulation necrosis is found in the enlarged livers of about two-thirds of patients, and Councilman-like bodies are identified in the parenchyma of more than a third of these cases. Enlarged Kupffer cells line the liver sinusoid, and macrophages are prominent pathological features elsewhere in the body of these children (Bhamarapravati et ah, 1967). In an autopsy study of 100 fatal cases of DHF and DSS, intravascular fibrin coagula consistent with DIC were not identified. Boonpuknavig et al. (1976) documented intravascular deposits of IgC and complement in the glomeruli of renal biopsies of their cases. Skin biopsies from children with dengue demonstrate viral antigen, IgM, and fibrinogen in the lumina of capillaries. The autopsy series of Bhamarapravati et al. (1967, 1989) revealed evidence of bone marrow hyperplasia in almost half of the patients. In contrast, incompletely documented reports indicate that the bone marrow is often hypoplastic (Bierman and Nelson, 1965). These conflicting observations may only reflect the time in the course of the clinical illness when the studies were done. Experimental evidence suggests that hematopoietic progenitor cells and megakaryocytes support the growth of virus (Murgue et al, 1997). The clinical and laboratory observations briefly summarized herein provide a complex picture of interrelated hemorrhagic and shock syndromes having an uncertain pathogenesis. Depletion of coagulation factors as a result of necrotizing liver disease a n d / o r thrombocytopenia may be, in part or in whole, responsible for the hemorrhagic problems of some patients. The evidence conflicts with respect to whether or not dengue virus replicates in endothelial cells (Boonpucknavig et al, 1979). Infection of these cells could contribute to vascular leakage, and thus hemorrhage and fluid extravasation, leading to shock. Over 30 years ago, Halstead proposed that the interaction of an infecting dengue virus with heterologous cross-reacting antibodies, elaborated in response to a second antigenically distinct but related virus type, was responsible for development of hemorrhagic shock syndrome. Evidence systematically accumulated in experimental models and in humans since that time tends to support this concept (reviewed in Halstead,

1988). Studies of infants with DSS have demonstrated depletion of blood complement and presence of IgG antibody-virus complexes in the blood serum (Ward, 1973). As a result, complement-derived anaphylatoxins could alter vascular continuity, resulting in leaks (Bokisch et al 1973; Halstead, 1989). Despite the many attractive features of the hypothesis championed by Halstead, other investigators argue that the evidence supporting the concept is incomplete and that alternate theories still warrant consideration (Rosen, 1977). For example, as noted above, cross-reacting antibodies appear to facilitate the uptake of virus by macrophages, thus enhancing virus replication and the severity of the illness. The question is of more than academic importance since immunization of at-risk populations with dengue virus vaccines is a potentially feasible public health approach to controlling dengue.

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Elwan, M., Sharaf, M., Gameel, K., El-Hadi, E., and Arthur, R. (1997). Rift valley fever retinopathy: Observations in a new outbreak. Ann. Saudi Med. 17, 377-378. Frame, J. (1989). Clinical features of Lassa fever in Liberia. Rev. Infect. Dis. 11 (Suppl. 4). Frame, J., Baldwin Jr., J., Gocke, D., and Troup, J. (1970). Lassa fever, a new virus disease of man from West Africa, I: Clinical description and pathological findings. Am. J. Trop. Med. Hyg. 19, 670-676. Froeb, H., and McDowell, M. (1954). Renal function in epidemic hemorrhagic fever. Am. J. Med. 16, 671-676. Furth, F. (1954). Observations on the hemostatic defect in epidemic hemorrhagic fever. Am. ]. Med. 16, 651-653. Gajdusek, D. (1962). Virus hemorrhagic fevers. /. Pediatr. 60, 841-857. Gedigk, R, Bechtelsheimer, H., and Korb, G. (1971). Pathologic anatomy of the Marburg virus disease. In "Marburg Virus Disease" (G. Martini and R. Siegert, eds.), pp. 50-53. Springer-Verlag, New York. Georges-Courbot, M., Sanches, A., Lu, C , Baize, S., Leroy, E., Lansout-Soukate, J., Tevi-Benissan, C , Georges, A., Trappier, S., Zaki, S., Swanepoel, R., Leman, P., Rollin, P., Peters, C , Nichol, S., and Ksiazek, T. (1997). Isolation and phylogenetic characterization of Ebola viruses causing different outbreaks in Gabon. Emerging Infect. Dis. 3, 59-62. Gerson, W, Dickerman, J., Bovill, E., and Golden, E. (1993). Severe acquired protein C deficiency in purpura fulminans associated with disseminated intravascular coagulation: Treatment with protein C concentrate. Pediatrics 91, 418^22. Giles, R., and Langdon, E. (1954). Blood volume in epidemic hemorrhagic fever. Am. J. Med. 16, 654-661. Giles, R., Sheedy, J., Ekman, C , Froeb, H., Conley, C , Stockard, J., Cugell, D., Vester, J., Kiyasu, R., Entwisle, G., and Yoe, R. (1954). The sequelae of epidemic hemorrhagic fever: With a note on causes of death. Am. ]. Med. 16, 629-638. Gligic, A., Dimkovic, N., Xiao, S., Buckle, G., Jovanovic, D., Velimirovic, D., Stojanovic, R., Obradovic, M., Diglisic, G., Micic, J., Asher, D., LeDuc, J., Yanagihara, R., and Gajdusek, D. (1992). Belgrade virus: A new hantavirus causing severe hemorrhagic fever with renal syndrome in Yugoslavia. /. Infect. Dis. 166, 113120. Groen, J., Bruijn, J., Gerding, M., Jordans, J., Moll van Charante, A., and Osterhaus, A. (1996). Hantavirus antigen detection in kidney biopsies from patients with Nephropathia epidemica. Clin. Nephrol. 46, 379-383. Guang, M., Liu, G., and Cosgriff, T. (1989). Hemorrhage in hemorrhagic fever with renal syndrome in China. Rev. Infect. Dis. 11 (Suppl. 4), S884-S890. Halstead, S. (1988). Pathogenesis of Dengue: Challenges to molecular biology Science 239, 476-481. Halstead, S. (1989). Antibody, macrophages. Dengue virus infection, shock, and hemorrhage: A pathogenetic cascade. Rev. Infect. Dis. 11 (Suppl. 4), S830-S839. Heymann, D., Weisfeld, J., Webb, P., Johnson, K., Cairns, T., and Berquist, H. (1980). Ebola hemorrhagic fever: Tandala, Zaire, 1977-1978. /. Infect. Dis. Ul, 372-376. HuUinghorst, R., and Steer, A. (1953). Pathology of epidemic hemorrhagic fever. Ann. Intern. Med. 38, 77-101. Hunter, R., Yoe, R., and Knoblock, E. (1954). Electrolyte abnormalities in epidemic hemorrhagic fever. Am. J. Med. 16, 677-682. Jacob, H. (1971). The neuropathology of the Marburg disease in man. In "Marburg Virus Disease" (G. Martini and R. Siegert, eds.), pp. 54-61. Springer-Verlag, New York.

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Salas, R., de Manzione, N., Tesh, R., Rico-Hesse, R., Shope, R., Betancourt. A., Godoy, O., Bruzual, R., Pacheco, M., Ramos, B., Taibo, M., Tamayo, J., Jaimes, E., Vasquez, C , Araoz, R, and Querales, J. (1991). Venezuelan haemorrhagic fever. Lancet 338,1033-1036. Saunders, R, Reid, M., and Cohen, B. (1986). Human parvovirus-induced thrombocytopenias: A report of five cases. Br. J. Haematol. 63, 407. Settergren, B., Juto, R, Trollfors, B., Wadell, G., and Norrby, S. (1988). Hemorrhagic complications and other clinical findings in Nephropathia epidemica in Sweden: A study of 355 serologically verified cases. /. Infect. Dis. 157: 380-382. Settergren, B., Ahlm, C , Alexeyev, O., Billheden, J., and Stegmayr, B. (1997). Pathogenetic and clinical aspects of the renal involvement in hemorrhagic fever with renal syndrome. Renal Failure 19,1-14. Sheedy, J., Froeb, H., and Batson, H. (1954). The clinical course of epidemic hemorrhagic fever. Am. J. Med. 16, 619-628. Shershow, L., Ekert, H., Swanson, V, Wright Jr., H., and Gilchrist, G. (1969). Intravascular coagulation in generalized herpes simplex infection of the newborn. Acta Paediat. Scand. 58, 535-539. Stille, W., and Bohle, E. (1971). Clinical course and prognosis of Marburg virus ("Green Monkey'') disease. In "Marburg Virus Disease" (G. Martini and R. Siegert, eds.), pp. 10-18. Springer-Verlag. New York. Suleiman, M., Muscat-Baron, J., Harries, J., Satti, A., Piatt, G., Bowen, E., and Simpson, D. (1980). Congo/Crimean haemorrhagic fever in Dubai: An outbreak at the Rashid Hospital. Lancet 2, 939-941. Swanepoel, R., Struthers, J., Shepherd, A., McGillivray, G., Nel, M., and Jupp, P. (1983). Crimean-Congo hemorrhagic fever in South Africa. Am. ]. Trop. Med. Hyg. 32,1407-1415. Swanepoel, R., Gill, D., Shepherd, A., Leman, P., Mynhardt, J., and Harvey, S. (1989). The clinical pathology of Crimean-Congo hemorrhagic fever. Rev. Infect. Dis. 11 (Suppl. 4), S794-S800.

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C H A P T E R

20

Hantavirus Pulmonary Syndrome (HPS) May of 1993, two housemates residing in rural ew Mexico developed an acute febrile flu-like illness that rapidly evolved into acute respiratory insufficiency accompanied by shock. Death followed within a few days. Other cases in this same geographic area appeared shortly thereafter. Members of both sexes were affected and many were Native Americans. Laboratory studies demonstrated hemoconcentration accompanied by leukocytosis and thrombocytopenia. Atypical lymphocytes circulated in the blood (see Figure 9.4). Although proteinuria was detected, renal failure did not ensue until late in the clinical course; most probably it was secondary to hypotension. The initial microbiological studies of these patients and their tissues obtained at autopsy were negative, ruling out plague and more traditional viral infections. However, the demonstration of antigens in the blood that reacted with antibodies against several pathogenic hantaviruses, such as the viruses responsible for Korean hemorrhagic fever, and PCR demonstration of the molecular markers for these viruses in lung tissue established the cause (see Chapter 19). Additional studies detected similar Hantavirus markers in the tissues of the predominant local field rodent, the deer mouse (Peromyscus maniculatus) (Childs et al., 1994; Hjelle et al., 1996). Further work with the animal tissue yielded a virus termed "Sin Nombre" (without name). In the ensuing years, retrospective studies have linked this virus and two closely related agents to cases of Hantavirus pulmonary syndrome (HPS) occurring in many scattered sites in North America (Shefer et al., 1994; Mackow et al., 1995; Jay et al., 1996; Huang et al., 1996). Almost invariably~ field studies associated the illness with environmental exposure to the excreta of wild rodents, indicating a mode of transmission identical to that responsible for the Hantavirus hemorrhagic fevers of Europe and Asia (Armstrong et al., 1995). However, in a recent outbreak of HPS in Argentina,

person-to-person spread of the virus was documented (Levis et al., 1997). This South American strain of virus (termed Andes), and a second virus (termed Bayou) from a case of HPS in Louisiana, as well as a third strain from Florida (termed Black Creek), exhibit unique molecular characteristics, allowing one to differentiate them from the Sin Nombre strain (Hjelle, 1996; Wells et al., 1997; Padula et al., 1998; Morzunov et al., 1995). Interestingly enough, patients with HPS caused by the BAYOUand Black Creek strains of virus exhibited acute renal failure before the onset of clinical shock. These "new" viruses, therefore, seem to possess some of the pathobiological features of Hantavirus disease on the Eurasian continent. Hantaviruses sharing the biological features of the Sin Nombre strain are widely distributed in the Americas (Schmaljohn and Hjelle, 1997; Zaki et al., 1996). Their existence elsewhere in the world remains to be established. As members of the Bunyaviridae famil~ they are similar to the viruses of the hemorrhagic fever with renal failure syndrome discussed in Chapter 19. Antigenicall~ and from a molecular perspective, these newly discovered viruses are also closely related to Hantaviruses of Eurasia, although the viruses of HPS obviously exhibit pathogenic features that differ strikingly from those caused by the ubiquitous Hantaviruses of hemorrhagic fever and renal failure. Most notabl~ bleeding disorders are not a feature of HPS in the American cases. Human pathogens of the Bunyaviridae family possess specific tropism for the endothelial cells of small blood vessels, and their disease-causing potential appears to relate largely to the effects of the virus on the integrity of vessels. However, the virus is not cytolytic; thus, the pathogenesis of the pulmonary disease is obscure. It may reflect the effects of cytochemicals generated by infected cells on vascular continuity. Immunopathological processes seem unlikely in view of the

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Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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Pathology and Pathogenesis of Human Viral D i s e a s e

acute nature of the disease, and the lack of evidence suggesting that patients have experienced prior exposure to Hantavirus. Thus, the illness is not comparable to the dengue shock syndrome (see Chapter 19). Currently, we just don't understand the mechanistic basis for the fatal pulmonary syndrome. The clinical features of HPS are well described in several recent publications (Levy and Simpson, 1994; Duchin et a/., 1994; Butler and Peters, 1994; Moolenaar et al, 1995; Zaki et al, 1995; Hallin et al, 1996). The prodromal phase of 3 to 6 days is characterized by fever and myalgia associated with nonspecific upper digestive tract symptoms as well as headache and dizziness (Table 20.1). Patients generally deteriorate rapidly, entering a cardiopulmonary phase with cough and shortness of breath. Their symptoms announce the onset of pulmonary edema. Tachypnea and tachycardia are readily apparent, accompanied by metabolic acidosis in roughly one-third of patients. At this time.

the laboratory abnormalities summarized in Table 20.2 are evident. Fever persists and hypotension can occur. Hypoxemia and hemoconcentration are apparent at this time. Based on the evidence of norn\al pulmonary wedge pressures and the high protein content of the pulmonary fluid (>80%), one can conclude that the pulmonary edema is noncardiac. However, a shock state readily evolves in many patients with a low cardiac stroke volume and high vascular resistance. Death follows in about 75% of patients (Hallin et ah, 1996). Recovery can be rapid in patients who survive, with the prompt return of respiratory sufficiency and cardiovascular stability. No clinical evidence of intrinsic disease of the heart, liver, and kidney are observed, and hemorrhages do not appear, despite the profound thrombocytopenia detected in 50% of patients. Pathological examination (Nolle et al, 1995; Zaki et al., 1996) documents the presence of pulmonary edema with intra-airspace protein exudate, but without evi-

TABLE 20.1 Major S y m p t o m s of Young A d u l t Patients w i t h Hantavirus Pulmonary S y n d r o m e Fever Myalgia Headache Cough Nausea and Vomiting Malaise Diarrhea Shortness of breath

100% 100% 71% 71% 71% 59% 59% 53%

Reprinted with permission from Duchin et al. (1994).

B TABLE 20.2 Laboratory Findings in Young A d u l t s w i t h Hantavirus Pulmonary S y n d r o m e Leukocytosis and left shift

64%

Platelets (<150,000/mm3)

67%

Platelets (<50,000 / mm^)

50%

Elevated blood lactate dehydrogenase

100%

Elevated aspartate transaminase

83%

Hematocrit (>48%)

54%

Blood bicarbonate (<20 mEq/L)

79%

Prothrombin time (>14 sec)

17%

Partial activated thromboplastin time (>37 sec)

71%

Adapted with permission from Moolenaar et al. (1995) and Zaki et al. (1995).

FIGURE 20.1 Lung of typical case of HPS showing pulmonary edema with fibrin accumulation and slight interstitial inflammation. Note the intact alveolar pneumocytes. Reprinted with permission from Zaki et al. (1995). Interested readers are referred to the comprehensive and amply illustrated report on HPS by Dr. Zaki and his associates (''Hantavirus pulmonary syndrome: Pathogenesis of an emerging infectious disease," Am. ]. Pathol. 146, 552-579, 1995).

Hantavirus Pulmonary Syndrome

dence of cytolytic changes in the alveolar pneumocytes lining airspaces, and the endothelium of the pulmonary vasculature (Figure 20.1A,B)- Electron microscopy confirms the light microscopic findings and shows that the type I and II pneumocytes, and the vascular endothelium of the interstitium, are intact. The airspaces contain protein-rich fluid and lungs weigh two- to threefold greater than normal. While hyaline membranes are variably present, they are not a prominent feature. Interstitial edema and modest interstitial infiltrates by large immuoblastoid T cells and monocytes/macrophages are seen. Respiratory insufficiency in these patients is compounded by the universal presence of pleural effusions with combined volumes of pleural fluid from the two chest cavities ranging from 210 to 8420 ml. Lesions of the heart, liver, and kidney are not evident by light microscopy. Electron microscopy demonstrates the so-called Hantavirus inclusions and individual virions in the cytoplasm of endothelial cells of capillaries in the pulmonary interstitium (Figures 20.2 and 20.3). Virions are also found in alveolar macrophages (Figure 20.4A,B) (Zaki ei al., 1995). Immunocytochemistry establishes the antigenic specificity of virus particles in these cells (Figure 20.5A). The finding of viral antigen in promi-

FIGURE 20.2 Low-magnification electron micrograph of a capillary in the lung exhibiting a characteristic Hantavirus inclusion in the cytoplasm of an endothelial cell. Reprinted with permission from Zaki ei al. (1995).

299

nent amounts in the endothelial cells of myocardial capillaries and endocardial cells is of particular interest. As noted above, patients with HPS terminally often exhibit noncardiogenic shock; thus, viral involvement of the myocardial capillary network would appear to have little or no effect on the function of the heart. In the liver, endothelial cells appear to be involved only rarely, although antigen is found in an occasional Kupffer cell. Hepatic parenchymal cells are intact and exhibit no evidence of infection. In the kidneys, abundant amounts of viral antigen are detected in capillaries of both the glomeruli and the medullary interstitium (Figure 20.5B). Zaki ei al (1995) speculate that this finding may account for the proteinuria seen in many patients with HPS. Finally, antigen is localized in the endothelium of the sinusoids of the spleen and lymph nodes. It is also detected in dendritic cells of lymphoid

FIGURE 20.3 High-resolution electron micrograph of the inclusion shown in Figure 20.2. Note the filamentous composition (x64,000). Ultrastructural immunochemistry demonstrates viral antigen in these structures. Similar inclusions were described by Tao ei al. (1987) in the tissues of animals infected with Oriental strains of Hantavirus. Those authors described three morphological types of inclusions (granular, granulofilamentous, and filamentous) and demonstrated their association with the virus by ultrastructural immunological labeling. Although virus particles are seen by electron microscopy in scattered endothelial cells, they are not found in proximity to the inclusions. Reprinted with permission from Zaki ei al. (1995).

300

Pathology and Pathogenesis of Human Viral Disease

FIGURE 20.4 Virus-like particles in a pulmonary interstitial macrophage. (A) The virions are associated with phagolysosomes containing fragmented cellular debris. Scale: bar = 1 micron. (B) Higher magnification of boxed area of A showing virus particles, one of which is budding from a cell membrane (arrow). Reprinted with permission from Zaki et at. (1995).

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Hantavirus Pulmonary Syndrome

follicles. Immunoblastic cells commonly accumulate in these lymphoid organs (Figure 20.6A,B). References Armstrong, L., Zaki, S., Goldoft, M., Todd, R., Khan, A., RF, K., Ksiazek, T., and Peters, C. (1995). Hantavirus pulmonary syndrome associated with entering or cleaning rarely used, rodentinfested structures. /. Infect. Dis. 172,1166. Butler, J., and Peters, C. (1994). Hantaviruses and hantavirus pulmonary syndrome. Clin. Infect. Dis. 19, 387-395. Childs, J., Ksiazek, T., Spiropoulou, C , Krebs, J., Morzunov, S., Maupin, G., Gage, K., RoUin, P., Sarisky, J., Enscore, R., Frey, J., Peters, C., and Nichol, S. (1994). Serologic and genetic identification of Peromyscus maniculatus as the primary rodent reservoir for a new hantavirus in the Southwestern United States. /. Infect. Dis. 169,1271-1280. Duchin, J., Koster, R, Peters, C., Simpson, G., Tempest, B., Zaki, S., Ksiazek, T., Rollin, P., Nichol, S., Umland, E., Moolenaar, R., Reef, S., Nolte, K., Gallaher, M., Butler, J., Breiman, R., and Group, A. T. H. S. (1994). Hantavirus pulmonary syndrome: A clinical description of 17 patients with a newly recognized disease. New Engl. J. Med. 330, 949-955. Hallin, G., Simpson, S., Crowell, R., James, D., Koster, R, Mertz, G., and Levy, H. (1996). Cardiopulmonary manifestations of hantavirus pulmonary syndrome. Crit. Care Med. 24, 252-258. Hjelle, B. (1996). Hantavirus pulmonary syndrome, renal insufficiency, and myositis associated with infection by Bayou hantavirus. Clin. Infect. Dis. 23, 495-500. Hjelle, B., Torrez-Martinez, N., Koster, R, Jay, M., Ascher, M., Brown, T., Reynolds, P., Ettestad, P., Voorhees, R., Sarisky, J., Enscore, R., Sands, L., Mosley, D., Kioski, C., Bryan, R., and Sewell, C. (1996). Epidemiologic linkage of rodent and human hantavirus genomic sequences in case investigations of hantavirus pulmonary syndrome. /. Infect. Dis. 173, 781-786. Huang, C., Campbell, W., Means, R., and Ackman, D. (1996). Hantavirus S RNA sequence from a fatal case of HPS in New York. /. Med. Virol. 50, 5-8. Jay, M., Hjelle, B., Davis, R., Ascher, M., Baylies, H., Reilly, K., and Vugia, D. (1996). Occupational exposure leading to hantavirus pulmonary syndrome in a utility company employee. Clin. Infect. Dis. 22, 841-844. Levis, S., Rowe, J., Morzunov, S., Enria, D., and St. Jeor, S. (1997). New hantaviruses causing hantavirus pulmonary syndrome in central Argentina [letter]. Lancet 349, 998-999. Levy, H., and Simpson, S. (1994). Hantavirus pulmonary syndrome. Am. J. Respir. Crit. Care Med. 149,1710-1713.

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Mackow, E., Luft, B., Bosler, E., Goldgaber, D., and Gavrilovskaya, I. (1995). More on hantavirus in New England and New York. New Engl. J. Med. 332, 337-338. Moolenaar, R., Dalton, C , Lipman, H., Umland, E., Gallaher, M., Duchin, J., Chapman, L., Zaki, S., Ksiazek, T, Rollin, P., Nichol, S., Cheek, J., Butler, J., Peters, C , and Breiman, R. (1995). Clinical features that differentiate hantavirus pulmonary syndrome from three other acute respiratory illnesses. Clin. Infect. Dis. 21, 643649. Morzunov, S., Feldmann, H., Spiropoulou, C , Semenova, V., Rollin, P., Ksiazek, T, Peters, C , and Nichol, S. (1995). A newly recognized virus associated with a fatal case of hantavirus pulmonary syndrome in Louisiana. /. Virol. 69,1980-1983. Nolte, K., Feddersen, R., Foucar, K., Zaki, S., Koster, R, Madar, D., Merlin, T., McFeeley, R, Umland, E., and Zumwalt, R. (1995). Hantavirus pulmonary syndrome in the United States: A pathological description of a disease caused by a new agent. Hum. Pathol. 26,110-120. Padula, P., Edelstein, A., Miguel, S., Lopez, N., Rossi, C , and Rabinovich, R. (1998). Hantavirus pulmonary syndrome outbreak in Argentina: Molecular evidence for person-to-person transmission of Andes virus. Virology 241, 323-330. Schmaljohn, C , and Hjelle, B. (1997). Hantaviruses: A global disease problem. Emerging Infect. Dis. 3, 95-103. Shefer, A., Tappero, J., Bresee, J., Peters, C , Ascher, M., Zaki, S., Jackson, R., Werner, S., Rollin, P., Ksiazek, T., Nichol, S., Bertman, J., Parker, S., and Failing, R. (1994). Hantavirus pulmonary syndrome in California: report of two cases and investigation. Clin. Infect. Dis. 19,1105-1109. Tao, H., Semao, X., Zinyi, C , Gan, S., and Yanagihara, R. (1987). Morphology and morphogenesis of viruses of hemorrhagic fever with renal syndrome, II: Inclusion bodies — ultrastructural markers of hantavirus-infected cells. Intervirology 27, 45-52. Wells, R., Young, J., Williams, R., Armstrong, L., Busico, K., Khan, A., Ksiazek, T., Rollin, R, Zaki, S., Nichol, S., and Peters, C. (1997). Hantavirus transmission in the United States. Emerging Infect. Dis. 3, 361-365. Zaki, S., Greer, R, Coffield, L., Goldsmith, C , Nolte, K., Poucar, K., Feddersen, R., Zumwalt, R., Miller, G., Khan, A., Rollin, R, Ksiazek, T., Nichol, S., Mahy, B., and Peters, C. (1995). Hantavirus pulmonary syndrome: Pathogenesis of an emerging infectious disease. Am. J. Pathol. 146, 552-579. Zaki, S., Khan, A., Goodman, R., Armstrong, L., Greer, P., Coffield, L., Ksiazek, T., Rollin, R, Peters, C , and Khabbaz, R. (1996). Retrospective diagnosis of hantavirus pulmonary syndrome, 19781993: Implications for emerging infectious diseases. Arch. Pathol. Lab. Med. 120,134-139.

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C H A P T E R

21 Papillomaviruses INTRODUCTION 303 DISEASE OF THE SKIN 305 DISEASE OF THE FEMALE GENITAL TRACT

proved to be out of the mainstream of experimental cancer research at the time. Accordingly, an appreciation of their importance awaited the modern revolution of molecular virology during the last three decades of the twentieth century. Papillomaviruses and the papovaviruses (see Chapter 22) are classified as subfamilies of the Papovavirus family. Although the two have similarities, the viruses of these two subfamilies differ in size, and their DNA genomes are dissimilar. Papillomaviruses of humans (HPVs) are obligate parasites of epithelial cells, and their replication is intimately tied u p with host cell multiplication and differentiation. HPVs are about 55 nm in diameter. They have a capsid comprised of 72 capsomeres arranged in icosahedral symmetry. The capsomeric protein is the major antigen of the virion, but the antigenic makeup of the virus has not been utilized for classification or typing purposes. Based on molecular analysis of the viral doublestranded DNA, 70 distinct types of papillomavirus have now been identified in human tissues.^ The pathogenic importance of many of these virus types has yet to be established. While several individual types tend to be associated with distinct pathologic lesions in specific anatomic sites, considerable overlap exists. To a large extent, HPVs are not believed to be infectious for lesser species of animals, but the basis for this conclusion has not been rigorously examined. Similarly, the countless papillomaviruses of subhuman mammalian and avian species are not thought to be infectious for humans, but, for obvious reasons, experimental proof is lacking (Rowson and Mahy, 1967). Many gaps currently exist in our knowledge of the papillomaviruses and their pathogenic mechanisms. In part, these shortcomings and our lack of understanding of papillomaviruses relate to our inability to grow these agents in cultured cells in vitro. Thus, experimental work has largely been done with virus extracted from tumor tissue.

308

Vulva and Vagina 309 Cervix Uteri 311 Endometrium 314 DISEASE OF THE GLANS PENIS 314 DISEASE OF THE DIGESTIVE TRACT 315

Oropharynx 315 Esophagus 315 Anus 317 DISEASE OF THE LARYNX AND TRACHEOBRONCHIAL TREE 317 DISEASE OF THE EYE 321 DISEASE OF THE MIDDLE EAR 322 REFERENCES 322

INTRODUCTION I only heard him speak once! Elderly, but alert and engaging, Richard Shope described for his audience the fascinating story of the discovery of the rabbit papilloma virus (RPV) (Shope, 1933). He related subsequent experiments that established this common epidemic infectious agent of wild rabbits as a model for human viral carcinogenesis. RPV, once passed through a bacteria-tight filter, proved to be transmissible in serial from the skin of one animal to another of the same species, but it could not be passaged serially in domestic rabbits, although it caused benign cutaneous papillomas and squamous carcinomas in this species (Rous and Beard, 1935; Amelia et al, 1994). In these domesticated animals, the infectious virus became masked, although its genes persisted, an event now^ observed in human cervical carcinoma associated with papillomavirus infection (Kidd and Rous, 1940). Additional work (Rous and Kidd, 1936) had shown that the application of coal tar to the skin of papillomavirus-infected rabbits accelerated development of tumors at this site. These uniquely simple, but insightful, experiments

^Types are defined as <90% homologous when compared to other types, based on the DNA sequencing of the E6, E7, and LI regions of an intact 7.9-kb genome. PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

303

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

304

Pathology and Pathogenesis of Human Viral Disease

The means whereby HPVs gain access to the basal cells of the skin and mucus membranes is unknown, and the receptor(s) on the plasma membranes that allow access to the cells has not been identified. Presumably, injury to the epithelium serves to introduce the virus into the basal epithelial cells mechanically, where its growth begins. Most skin and mucocutaneous warts have been found to arise at sites where trauma is common. Replication of papillomaviruses is regulated by the differentiation status of the cells it infects (Figure 21.1). Virus growth in basal cells is dependent upon cell division, with the resulting daughter cells receiving a copy of the progeny virus. Roughly 20 to 100 copy numbers of the viral genome are found in basal cells. Vegetative growth then follows in differentiating epithelial cells, such as the strata spinosum and granulosum of the skin, where large numbers of virions can be demonstrated in the nuclei of the cells by electron microscopy or by the use of molecular probes (Jenson et ah, 1982a,b) (Figure 21.2). Concomitantly, cell division increases, resulting in the acanthosis, parakeratosis, and luxuriant hyperkeratosis that characterize HPV lesions. The HPV DNA genome has eight so-called "early" translational open reading frames, that is, genes, and two "late" genes. The "early" genes direct complex steps in viral replication, with some focusing on regulation of critical factors in cell cycling (Laimins, 1996). Of particular interest are the E6 and E7 genes. In the so-called "high-risk" types that play a critical role in

St bosole

St spinosum

St gronuiosum St corneum

KeratinizQtion Cetl proliferotion Vifot replication Maturation Mature porticles

FIGURE 21.1 Replication of HPV is linked to the orderly sequence of maturation and keratinization of the cells of the skin and mucus membranes. Viral replication is altered in disorders of keratinization. Virus production in some epithelial lesions such as verruca vulgaris is robust and virions accumulate in inclusions (Figure 21.3B), whereas in other lesions such as anogenital condylomata it is modest. Electron microscopy shows no viral particles in the cells of these lesions. As illustrated here, HPV replicates within the nucleus of basal cells and is transferred to daughter cells as the epithelium grows. As the process of keratinization evolves, virions are released into the cytoplasm associated with parakeratosis and keratosis. Maturation of the epithelium and keratinization is influenced by virus growth. Reprinted with permission from Pfister (1992).

FIGURE 21,2 HPV type 1 particles in the nucleus of a cell of the granular layer of the skin. The nucleus is largely filled by virions in a crystalline array (27,000x). Reprinted with permission from Jenson et al. (1982a).

305

Papillomaviruses TABLE 21.1 Association Between Human Papillomavirus Types and Lesions of the Skin HPV type Verruca vulgaris Verruca plantaris Verruca plana EV* flat wart EV* pityriasis-like lesion Condyloma acuminatum

3 + +++

+++ +

4

5

7

8

9

10

11

12

13

14

15

16

++ + ++ ++

+

6

++ ++

+

*Epidermodysplasia verruciformis. Adapted with permission from Pfister (1984). the pathogenesis of cervical carcinoma, the E6 gene protein product binds and inactivates the cellular p53 protein (Werness et ah, 1990) and the E7 protein interacts with a variety of replicative intermediaries, including proteins of the pRB gene complex (Dyson et al, 1989). These interactions are believed to inactivate the products of the p53 and pRB suppressor genes, thus permitting the cells to replicate with seeming abandon. The effects of the other "early" genes (E1-E5) on various cellular targets remain to be elucidated (Kubbutat and Vousden, 1996). With regard to the "high-risk" virus types, the outcome can be immortalization of target epithelial cells and carcinoma. The "potency" of the so-called E6 and E7 oncogenes differs substantially among various virus types. The "early" gene protein products of the "low-risk" virus types apparently lack the potential for initiating malignant neoplastic transformation of epithelial cells.

DISEASE OF THE SKIN In lectures on the principles and practice of surgery. Sir Astley Cooper (1835) observed that warts "frequently secrete a matter which is able to produce a similar disease in others." He described the intraoperative infection of a surgeon with a scalpel used to excise a patient's warts. A similar observation was recorded by Payne in 1891. The viral etiology of warts was suggested by the work of Ciuffo (1907), who transferred warts person to person using a cell-free filtrate of the homogenate of a warty lesion. HPV lesions of the skin assume several clinical and pathological forms. With exceptions, a small number of specific virus types have been associated with lesions having characteristic morphologic features and distributions (Table 21.1). The prototypic lesion is the common wart verruca vulgaris (Figure 21.3A,B). One to five millimeters in diameter, these circumscribed rough-surfaced lesions

occur almost anywhere on the skin, but are commonly found on the distal extremities, particularly on the palmar and plantar surfaces. HPV types 2 and 4 have been identified in the stratum granulosum (Figure 21.4), but comprehensive studies to determine the virus types in lesions from large numbers of persons have yet to be reported. Papillary hyperplasia with basal and suprabasal cell mitotic activity, as well as acanthosis, and hyperkeratosis are characteristic of the lesions. Basophilic intranuclear inclusions that contain crystalline arrays of virions are located in the cells of the stratum spinosum. Although warts of this type develop in persons of all ages, the lesions are particularly common in children and adolescents. The overall prevalence in the general population is roughly 10%, but among residents of institutions the infection rate can be as high as 25% (Kilkenny and Marks, 1996). Warts come and go. In one study, 67% of lesions disappeared over a 2-year period (Massing and Epstein, 1963). Only about 20% remained unchanged for this duration of time. Organ transplant recipients receiving immunosuppressive treatment commonly experience warts and squamous carcinoma of the skin (Shamanin et ah, 1996). Verruca plana, the so-called flat warts, typically occur in children, and less commonly in adults, as multiple slightly elevated colorless or erythematous lesions on the face, neck, and dorsum of the hands. Verruca plana microscopically exhibit acanthosis and hyperkeratosis, but lack papillomatosis. The cells of the stratum granulosum characteristically show poikilocytosis. Several different types of HPV have been found in lesions having these features, but types 3 and 10 predominate. Interestingly enough, the number of virus particles in the keratinizing cells of verruca plana are relatively small in comparison to verruca vulgaris. Accordingly, basophilic inclusions are not seen in the nuclei of cells of this lesion. The lesions of verruca plantaris are usually clustered on the soles of the feet and the palms of the hands of young persons. Transmission in athletic locker rooms

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FIGURE 21.4 Verruca vulgaris stained by immunohistochemistry to demonstrate HPV antigens in the stratum granulosum. Arrows indicate staining of koilocytic cells. Reprinted with permission from Jenson et al (1982b).

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and bath houses is common. Plantar warts exhibit full thin papillae invested with a prominent layer of cells containing keratohyalin and invested by a thick layer of keratin. The lesions are well circumscribed, allowing them to be easily removed with a curette. Plantar warts result from infection by HPVl (Figure 21.5). Large numbers of virus particles are found in the intranuclear particles in the horny layer. Epidermodysplasia verruciformis (EV) is an exceedingly rare lifelong generalized eruption of flat warts having malignant potential. There is a familial predisposition (Jablonska et a/., 1968), but the heritable mode of transmission has not been established. Described by Lewandowsky and Lutz in 1922, our understanding of the pathogenesis of these lesions is gradually increasing, even though the mode of inheritance of the predisposition to EV has not been defined. The disease represents the unique influences of both environmental (i.e., UVB irradiation) and immunological factors on the course of an HPV infection. The disease first becomes apparent as generalized crops of (colored or colorless) macular or pityriasis versicolor-like lesions occurring in the very young. It progresses over time to form multiple large flat, often confluent, lesions

307

(Figure 21.6). After some 25 years, roughly a third of patients have developed skin cancers that predominately occur on the sun-exposed skin. Persons with the disease exhibit anergy to antigens known to induce skin hypersensitivity reactions. Thus, cellular immunity appears to be defective. EV is frequently seen in patients with AIDS and in organ allograft recipients being administered immunosuppressive regimens (de Jong-Tieben et ah, 1994). More than 20 different types of HPV have been recovered from various cases, but types 3, 5, 8, 9, 10, and 12 predominate. Interestingly enough, the virus types customarily associated with EV are commonly found in skin lesions of various kinds (including squamous carcinomas of the skin), occurring in organ allotransplant recipients and in AIDS (Soler et al, 1993; Stark et al, 1994; Shamanin et a/., 1996; Leigh and Glover, 1995; Meyer et al, 1998; de Jong-Tieben et al, 1994). EV patients infected with HPV types 5 and 8 are said to be unusually predisposed to development of actinic keratoses and invasive squamous cell carcinoma on UV-exposed areas of the skin (Majewski and Jablonska, 1995). The DNA of these two types is found in more than 90% of EV-associated cancers as a nonintegrated episome (Pfister, 1992).

F I G U R E 21.5 Verruca plantaris. The lesion exhibits the features of a verruca vulgaris but is compressed deep into the subjacent dermis. The convergence of the rete ridges allows the lesion to be curretted out and removed.

308

Pathology and Pathogenesis of Human Viral D i s e a s e

FIGURE 21.6 Epidermodysplasia verrucaformis. The typical lesion exhibits hyperkeratosis and large cells with an abundant pale-staining cytoplasm. Because they resemble cells observed in well-differentiated squamous cell carcinomas, some authors have suggested, without good basis, that they represent a premalignant change.

DISEASE OF THE FEMALE GENITAL TRACT Almost 50% of sexually active college women have detectable HBV of one or more types in their genital tract tissue. Often, there is no clinical or cytopathological evidence of infection. In an exfoliative cytology study of over 1 x lO'* women with healthy cervices, almost 9% were found to have HBV infections (this is a minimal number because of the methodology used in the study) (see de Villiers et ah, 1992). The male partner of women with clinically evident HPV-associated genital warts or cervical dysplasia often has penile lesions when carefully studied by colposcopy. In some, but certainly not all, cases, the HPV type infecting these men proves to be identical to the virus of their sexual consort (Kyo et al, 1994). The relative risk of infection with some, but not all, HPV types increases in inverse relationship to the age of the woman at first intercourse and parallels the number of sexual partners (Franco et ah, 1995). Cross-sectional analyses in which population groups are surveyed measure the recently acquired infections as well as the persistent strains acquired in the past, that is, the prevalence. Thus, the results reflect the cumulative virus burden of the population under study, not the incidence of new infections per unit of time. In younger women (i.e., less than 30 years of age), the HPV seem to persist for a shorter period of time

than in older women. The "high-risk" virus types known to be associated with cancer of the cervix and vulva tend to persist longer than types thought not to be pathogenic (Hildesheim et al, 1994). The recognition of koilocytic atypia as a reflection of HPV infection in the cervical epithelium proved to be the first hint that HPV might be involved in the pathogenesis of cervical cancer (Meisels and Fortin, 1976; Purola and Savia, 1977). The term "koilocytosis" was coined by Koss and Durfee (1956) to refer to cells having irregular hyperchromatic nuclei surrounded by a clear cytoplasmic halo. These cells are now considered to be pathognomonic of HPV infection of squamous epithelium. Some investigators believe koilocytosis appears in a cell when the protein elaborated by the E4 "early" gene of the virus binds with and collapses the cytokeratin framework of the cell. Koilocytic atypia exists when the squamous epithelial cells display perinuclear halos and nuclear atypia with numerous bi- and multinucleate cells. Mitotic activity in these lesions is increased, but atypical mitotic figures are not evident (Nuovo et al, 1989). Nuovo (1990) detected HPV in 77% of condylomatous lesions with koilocytic atypia, but 20% of the lesions they studied that lacked these pathognomonic features were also positive. The etiology and possible role of HPV in the remaining condylomata is unknown.

309

Papillomaviruses

Vulva and Vagina Condylomata acuminatum involves multifocal lesions of the vulva and vagina almost invariably accompanied by infection with so-called "low-risk" HPV type 6 and 11. As would be expected, it occurs with increased frequency in women who have had intercourse with multiple sexual partners. The prevalence of these so-called genital warts seems to be increasing among sexually active young women. Relatively little clinical and biological information is available that documents sequentially the mode of appearance, the evolution, and the natural resolution of these lesions. They occur commonly in pregnant women and users of oral contraceptives and appear to regress when endocrine stimulation to the genital tissue ceases. The epithelium of the skin adjacent to condylomata and elsewhere in the perineum is commonly infected with HPV even when histological evidence of infection in the skin is lacking (Ferenczy et ah, 1985). This observation most probably accounts for the relatively high recurrence rates of condyloma after removal chemically or by cautery. Condyloma of the vagina and cervix are often found in women with these vulvar lesions. As might be expected, condyloma acuminata are epidemiologically associated with an increased risk of anogenital neoplasia, particularly cancer of the vulva. However, the lesions customarily are not due to infection by "high-risk" HPV types. Thus, they are not premalignant lesions (Friis et a/., 1997). Condyloma acuminatum generally develops on the vulvar vestibule and on the medial aspects of the labia (Figure 21.7). It must be differentiated by the pathologist from the common squamous papillomas and fibroepitheliomatous "tags." These exophytic cauliflower-like lesions exhibit a complex branching papillary configuration associated with epithelial proliferation (Figure 21.8). Koilocytotic atypia in the superficial granular layer of the epithelium is diagnostic of the lesions. Squamous carcinoma of the vulva is a relatively uncommon disease with an incidence approximately onefifth that of invasive cervical carcinoma (Ries et at., 1996). Two biologically different categories of disease are now recognized (Table 21.2). The first is represented by lesions infected with the so-called "high-risk" HPV types, 16 and 18. They tend to occur in younger women. The appearance of these cancers is preceded by vulvar intraepithelial neoplasia (VIN) of an advanced grade (Figure 21.9). As discussed in more detail below, the lesions have distinct morphological features and a better prognosis than the second category of squamous cancer (Bloss et al, 1991; Haefner et ah, 1995). The latter tumors develop in older women, often those

FIGURE 21.7 Multiple condylomata developing on the vulvar vestibule. The lesions are discrete, nonconfluent, raised "cauliflowerlike" excrescences. There is an absence of congestion and inflammation adjacent to the lesion.

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FIGURE 21.8 Condyloma acuminatum exhibit complex branching papillae with a flbrovascular core. The acanthotic folds exhibit to a varying degree koilocytosis in the upper third of the epithelium. As shown here, poikilocytosis is characterized by cytoplasmic vacuolization surrounding enlarged nuclei that show varying degrees of atypia. The clearing of the cytoplasm most probably is a fixation artefact resulting from viral-induced changes in the cytokeratin skeleton of the cell (see text).

310

Pathology and Pathogenesis of Human Viral D i s e a s e

***^W* FIGURE 21.9 Morphological spectrum of HPV type 16-associated vulvar intraepithelial neoplasia (VIN) or squamous intraepithelial lesion (VIL) exhibiting (A) warty or Boenoid features, (B) hyperkeratosis, (C) acanthosis with minimal hyperkeratosis, and (D) near-complete absence of differentiation. A consistent feature of these lesions is the nuclear atypia extending upward through at least two-thirds of the epithelial layer. Reprinted with permission and through the courtesy of J. Crum, MD.

with the chronic inflammatory and degenerative disease known as lichen sclerosis (Toki et ah, 1991). This latter condition is characterized by atrophy of the vulvar skin, but with focal hyperplasia of the epidermis to a variable degree. The presence of atypical cells in the

epithelium portend the ultimate neoplastic outcome in some patients, and Tate et al. (1997) have demonstrated the monoclonality of the epithelial cells in these lesions. These invasive squamous carcinomas have a relatively poor prognosis. In one series, the mean age of the pa-

311

Papillomaviruses TABLE 21.2 A g e of W o m e n w i t h Vulvar Carcinoma b y M o r p h o l o g i c Type Tumor type Age (years) <56 57-66 67-78 79+

Keratinizing squamous

Basaloid

Warty

8 20 37 35

46 36 14 4

86 14 0 0

Adapted with permission from Kurman et ah (1993).

small cells with a high nuclearicytoplasmic ratio. The nuclei are pleomorphic and immature, resembling parabasal cells. In contrast, the so-called warty lesions exhibit an undulating spiked surface and both parakeratosis and hyperkeratosis are prominent. The squamous cells of the lower third of the acanthotic epithelium lack maturation, and nuclear atypicalities, multinucleate cells, and mitosis are evident. Basaloid tumors (Figure 21.11) occur roughly fourfold more often the warty type. Interestingly enough, Kurman and coworkers (1993) found that 23% of women with the basaloid and warty lesions had invasive or noninvasive neoplastic lesions of the vagina or cervix.

tients with HPV-associated cancers was 41 years, whereas the lesion accompanied by lichen sclerosis occurred at a mean age of 66 years (Abell, 1965). Tumors attributable to HPV are less keratinizing than the virus-negative lesions that tend to occur in older women. In one series, 8% of the carcinomas unrelated to HPV infection were comprised of keratin pearls that made up more than 50% of the tumor mass as demonstrated by morphometric analysis of the tumor (Bloss et al, 1991). In contrast, the virus-associated preneoplastic and carcinomatous lesions exhibited one of two types of morphology: termed basaloid and warty (Figure 21.10) (Kurman et al, 1993). The basaloid lesions have a markedly thickened epithelium with evidence of acanthosis and both parakeratosis and hyperkeratosis, but of a mild degree. They are comprised of a relatively uniform population of

In many, but certainly not all, women with HPV infections of the uterine cervix, a series of cytopathological changes evolve in the epithelium at the junction between the exo- and endocervix. At this site, the squamous epithelium of the exocervix interfaces with the mucin-secreting columnar cell epithelium of the endocervix. The morphologic features that portend the biologic events in the cervix that can ultimately lead to squamous carcinoma develop over periods of 20 years or longer in most patients, but they can evolve over much shorter periods in some patients. They begin with squamous metaplasia, a lesion that is almost universally present in sexually active women and characterized by replacement of the normal columnar endocervical mucosa with squamous epithelium.

FIGURE 21.10 "Warty" pattern of VIN, grade 3. Note the disorganized proliferation of the epithelium with atypicalities and abnormal mitoses. There is an overlying parakeratosis and prominent hyperkeratosis with folding of the surface layer. Reprinted with permission from Kurman et al. (1990).

FIGURE 21.11 Basaloid vulvar carcinoma. Note how the deep tongues of this proliferating neoplasm invade the dermis. The carcinoma is comprised of relatively uniform cells with a scant cytoplasm and a somewhat monotonous regularity. Mitotic activity is evident but not prominent. Reprinted with permission from Kurman et al. (1990).

Cervix Uteri

312

Pathology and Pathogenesis of Human Viral Disease

FIGURE 21.12.

FIGURE 21.13.

FIGURES 21.12 and 21.13 Examples of cervical intraepithelial neoplasia (CIN), grade 1 or low-grade SIL, exhibiting koilocytic atypia as reflected by cellular enlargement, cytoplasmic perinuclear vacuolization, and variable degrees of nuclear pleomorphism. There is a loss of polarity and disorganization of the proliferating epithelium. The degree of maturation is inversely proportional to the grade of the lesion. Reprinted with permission from Kurman et al. (1990).

Pathologists now classify the changes that occur in the metaplastic squamous epithelium by using maturational criteria. The so-called "low-grade" lesions (i.e., mild dysplasia or cervical intraepithelial neoplasm [CIN], grade I)^ develop in relatively mature epithelium, in which cellular atypicalities are found in the most superficial layers of the epithelium. Koilocytosis is common in such lesions (Figures 21.12 and 21.13). A variety of HPV types have been demonstrated in patients with these "low-grade" lesions, but fewer than 30% are infected with the so-called "high-risk" HPV types (McLachlin et al, 1997). Higher-grade precancerous lesions (CIN II and III) show lesser degrees of maturation (Figures 21.14 and 21.15). In some studies, "high-risk" types of HPV are found in over 90% of the advanced lesions (Koutsky et al, 1992). The cells exhibit the typical features of malignancy such as a high nuclearxytoplasmic ratio and nuclear atypicalities throughout all layers of the epithelium from the basal to the keratinizing surfaces. Customarily, there are large numbers of mitoses, and polarity of the maturing

^The contemporary trend in diagnostic terminology is to term grade 1 lesions as squamous intraepithelial lesions (SILs) and grades 2 and 3 as high-grade SILs based on the Bethesda system of nomenclature (Kurman and Solomon, 1994).

cells is lost. Almost all of these premalignant cervical lesions contain a monoclonal population of aneuploid cells; the cells with a diploid karyotype ultimately disappear (Fu et al, 1983; Park et al, 1996). The epithelium that has reached this point in its evolution rarely regresses. Overall, a minority of the dyskeratotic lesions of the cervix have virions within the cells demonstrable by electron microscopy (Roman and Fife, 1989). Countless studies have now established a strong relationship of these advanced noninvasive evolutionary changes of the endocervical epithelium with infection by HBV types 16,18, 31, 33, 35, 39, 45, and 56 (Bosch et al, 1995; McLachlin et al, 1994) (Table 21.3). These highly pathogenic viruses persist in the atypical cervical lesions, and their genomic material is demonstrable in the vast majority of invasive cancers that ultimately evolve (both the adenocarcinoma and squamous carcinoma types). In one study of HPV 16-associated cancers, the full viral genome was consistently found to be present. In 70% of cases, it was episomal, whereas in all but 5% of the remaining cancers the viral DNA was integrated into the tumor cell DNA (Matsukura et al, 1989). Differences exist in the morphological features of the carcinomas associated with the various types of "high-risk" carcinomas (Figures 21.16 and 21.17). Approximately 50% of squamous carcinomas carry the

Papillomaviruses

313

FIGURE 21.14 CIN, grade 2 or high-grade SIL. Cellular proliferation in this lesion is largely confined to the lower third of the epithelium. There is loss of polarity of the cells with a high nuclear:cytoplasmic ratio. Reprinted with permission from Kurman et al. (1990).

FIGURE 21.15 CIN, grade 3 or high-grade SIL. Maturation of the epithelium is no longer present. Abnormal mitoses and atypical cells are commonly observed throughout the lesion. Reprinted with permission from Kurman et al. (1990).

HPV 16 genome, while a lesser number are associated with HPV 18 (Table 21.3). These later lesions tend to act more aggressively and occur at an earlier age (Burger et ah, 1996). HPV 18 infections are commonly found in endocervical adenocarcinomas, adenosquamous carcinomas, and the relatively rare small cell carcinomas (Figure 21.18) (Duggan et al, 1993). Some studies have suggested that different morphological variants of squamous carcinoma may be associated with specific HPV types, but the data are too few to permit conclusions (Wilczynski et al, 1988; Kadish et al, 1992). Despite the striking linkage between infection with "highrisk" types and carcinoma of the cervix, many more women are infected with "high-risk" HPV types than develop the disease. This finding has led to the contemporary view that carcinogenic influences, in addition to

infection with the "high-risk'' HPV strains, ultimately determine whether or not overt neoplastic transformation will occur. Epidemiological studies have thus far failed to establish what these factors might be, but

TABLE 21.3 Prevalence of HPV DNA in Intraepithelial and Invasive Cervical Squamous Carcinoma In situ tumors (%)

Invasive tumors (%)

Negative

29

49

Positive HPV 16 HPV 18/45 HPV 31/33/52 HPV 6/11 HPV (type unknown)

71 62 6 6 6 3

51 44 2 4 2 2

HPV, DNA

Adapted with permission from Madeleine et al. (1997).

FIGURE 21.16 Exocervix with an advanced squamous cell carcinoma eroding the posterior lip of the cervix from approximately 4:00 to 8:00 o'clock. Note the erosion and irregular nodularity of the tumor surface. Reprinted with permission from Kurman et al. (1990).

314

Pathology and Pathogenesis of Human Viral Disease

intriguing possibilities have been identified. For example, women with carcinomas of the cervix are more likely to smoke cigarettes than healthy control subjects (Brinton et al, 1986; Lyon et al, 1983), and DNA adducts of cigarette smoke carcinogens are demonstrable in the cervical tissue of smokers (Schneider et al, 1987). The possibility that herpes simplex 1 virus plays a cocarcinogenic role continues to be considered in the literature (Jones, 1995). However, there is no evidence to suggest that herpesviruses can enhance the growth of papillomaviruses. Endometrium

FIGURE 21.17 Microinvasive squamous carcinoma with overlying advanced lesions of CIN or SIL. Note the loss of polarity of the neoplastic cells and their mitotic activity. Scattered clusters of keratinizing tumor cells are seen in the dedifferentiated epithelium, a finding typical of invasive carcinoma. Reprinted with permission from Kurman et al (1990).

Information suggesting a possible association of HPV infection with various lesions of the endometrium is accumulating, but the evidence thus far is difficult to interpret. The problems relate to the diversity of endometrial lesions studied, the techniques employed by various laboratories, possible geographic differences in viral prevalence, the lack of information on the background prevalence of infection in women with a healthy endometrium, and the possibilities of viral contamination of endometrial samples in the lower genital tract and cervical tissue. Because of the extraordinary sensitivity of PCR, minor contaminants might result in misleading results. Early reports conflict. In one study from Germany, a 37% incidence of infection was reported (MildeLangosch et al, 1991), whereas in a Canadian study no evidence of HPV was found in samples from the uterine cavity (Bergeron et al, 1988). More recent work using PCR has demonstrated a high prevalence of HPV type 16 in endometrial carcinomas (Fujita et al, 1995), and HPV type 6 (but not type 16) was found in a series of cases of adenocarcinomas with squamous metaplasia (O'Leary et al, 1998). In the latter study, none of 20 normal control endometrial specimens exhibited evidence of infection. Finally, an investigation employing in situ hybridization demonstrated either HPV types 16 or 18 in 28% of the socalled glassy cell endometrial adenocarcinomas (Kenny et al, 1992). Clearly, much additional work will be required before the importance of these initial findings can be interpreted.

DISEASE OF THE G L A N S PENIS FIGURE 21.18 Adenosquamous carcinoma in situ. The focal accumulations of leukocytes in the tumor are a response to necrosis of the tissue. This intraepithelial neoplasm is found in an endocervical gland where it replaces the normal epithelium. Reprinted with permission from Kurman et al. (1990).

HPV-related lesions of the glans penis and foreskin develop commonly in men who consort with women who have flat cervical condylomata or intraepithelial

315

Papillomaviruses

only a third of the penile carcinomas studied, etiologies other than HPV may account for many tumors (Higgins et ah, 1992). However, other etiological factors causing or contributing to the development of these lesions remain to be identified. Typically, carcinoma of the penis topographically is found on the glans, the coronal sulcus, and foreskin. In situ lesions commonly are located in proximity to sites of invasive carcinoma that may be multifocal. It is claimed that the basaloid category of squamous carcinoma (Kurman et ah, 1993) has the strongest association with HPV. These are high-grade deeply invading lesions.

DISEASE OF THE DIGESTIVE TRACT Oropharynx

FIGURE 21.19 Multiple condylomata on the glans, prepuce, and shaft of the penis. Reprinted with permission from Fenner (1995).

neoplasm (Barrasso et al, 1987; Varma et al, 1991; Castellsague et al, 1997). In the 1987 study conducted by Barrasso et ah, more than 40% of the men with condylomatous lesions (Figure 21.19) had a female partner who also had condyloma, and a third of the men with histological evidence of carcinoma in situ had sexual relations with a woman having cervical carcinoma in situ. Not surprisingly, the condylomatous lesions are consistently infected with HPV type 6 and 11, whereas carcinoma in situ and invasive squamous carcinomas are infected with the "high-risk'' types 16 and 18 (Masih et ah, 1993). As in the female genital tract, the well-differentiated verrucous carcinomas consistently reveal no evidence of infection (Villa and Lopes, 1986). Virological information on the giant condylomas of Buschke and Loewenstein has not been reported (Davies, 1965; Bulkley, 1967). Many pathologists now consider this lesion to be a carcinoma when it occurs on the penis and on the vulva. Carcinoma of the penis rarely occurs in North America and Europe, but it is prevalent in Latin America, Asia, and Africa (Cubilla, 1995). In many of these latter regions, HPV infections in women are common and sexual interactions with multiple partners is frequent. Circumcision reduces the risk for carcinoma, an influence that no doubt affects the geographic distribution of the disease. Since HPV genome DNA is found in

A diverse variety of lesions attributable to papillomaviruses are found in the oropharynx (Brandsma and Abramson, 1989; Lewensohn-Fuchs et ah, 1994; Brandwein et ah, 1994). They include florid papillomatosis (Wechsler and Fisher, 1962), condyloma acuminata (Panici et ah, 1992), verrucae (Jenson et ah, 1982b), and focal epithelial hyperplasia (so-called Heck's disease). Detailed information on the virus types associated with lesions of the mouth and throat are not currently available. Heck's focal epithelial hyperplasia appears to be due to HPV type 1, and condyloma acuminata are infected with HPV types 6 and 11. The latter lesions are said to develop with particular frequency among men and women engaged in orogenital sexual relations (Panici et ah, 1992). Cells of oral squamous carcinomas commonly have integrated HPV 16 genomic material within the DNA, or carry it as a nuclear episome (Steenbergen et ah, 1995). However, systematic studies of the normal oral mucosa of seemingly healthy adults using PCR have demonstrated infection in 41% (Maitland et ah, 1987). Sinonasal papillary squamous carcinomas were evaluated by Judd et ah (1991). HPV 6 was found on only one of six cases by PCR. The "high-risk" types 16 and 18 were not identified in the lesions. Esophagus On a worldwide basis, cancer of the esophagus is relatively common, but there are substantial geographic differences in the incidence of the disease. In the developed countries of North America and Europe, esophageal cancer occurs infrequently (incidence of new cases 1.1-2.6 cases per 1 x 10^ per year).

316

Pathology and Pathogenesis of Human Viral Disease TABLE 21.4 Evidence for Papillomavirus Infection in Squamous Cell Carcinomas of the Esophagus by Geographic Regions

Author

Country

Techniques

No. pos. / no. tested (percent)

Assay types

Chang et al, 1993 Chen et al, 1994

China China

ISH PCR

23/363 24/40

16,18 6,11,16,18

Turner et al, 1997 Togawa et al, 1994

USA USA

PCR PCR

1/51 1/15

NT 16

Toh et al, 1992 Togawa et al, 1994

Japan South Africa

PCR PCR

3/4 3/18

16 16

PCR = polymerase chain reaction; ISH = in situ hybridization; NI = not identified. In contrast, in the People's Republic of China it is second only to cancer of the stomach as a cause of death among middle-aged adult males (incidence of new cases = 130 cases per 1 x 10^ per year). Yet, even in China, marked regional differences in tumor prevalence are found. The neoplasm is also common in Iran and South Africa. Cancer of the esophagus thus appears to be a disease of developing countries, possibly related to unique diets or environmental circumstances. In the United States, alcoholic beverage consumption in excess and tobacco use are important risk factors, with vitamin and mineral deficiencies and dietary factors being of possible importance. The causes of esophageal cancer have not been identified in those regions of the world where the disease is prevalent. Squamous cell carcinoma is the major, but not the exclusive, morphologic form of the disease, but in North America adenocarcinomas of the distal esophagus (occurring in association with Barrett's esophagitis) are of increasing importance. It is likely that squamous carcinomas undergo a series of premalignant changes prior to development of clinical cancer.

However, biopsy specimens useful in evaluating this question are rarely available, because premalignant lesions customarily are not detected clinically and biopsied before symptomatic neoplastic disease presents. Studies in the People's Republic of China and South Africa suggest that HPV may play a role in the pathogenesis of the neoplasm, whereas investigations in the United States and Japan fail to demonstrate an impressive association (Table 21.4) (Chang ei al, 1990, 1993; Ashworth et al, 1993; Furihata ei al, 1993; Akutsu et al, 1995). But, even in high-risk regions of China, evidence of infection is found in fewer than half of the cases. Thus, the virus could be only an opportunistic "passenger" of the tumor cells and not an etiological agent of neoplasia. Since in much of the recent work PCR has been used as the technique for detecting the virus, information on the cellular characteristics of the infection are not available. As in other organs, HPVs have been found to infect the normal or hyperplastic atypical epithelial cells of the esophagus adjacent to the tumor (Williamson et al, 1991; Benamouzig et al, 1992; Chang ^f^/., 1993) (Table 21.5).

TABLE 21.5 Morphological Changes in the Mucosa of the Esophagus Adjacent to 27 HPV DNA-Positive and DNA-Negative Cases of Esophageal Squamous Cancers, Mainland China Lesions Koilocytosis Papillomatosis Dyskeratosis Binucleation CINI II III Basal cell hyperplasia

HPV DNA+ in = 16)

%

HPV D N A (« = 11)

%

6/16 4/16 12/16 2/16 11/16 4/16 1/16 10/16

38 25 7b 13 69 25 6 63

0/11 1/11 10/11 0/11 6/11 3/11 2/11 7/11

0 9 91 0 55 27 18 64

Adapted with permission from Chen et al (1994).

317

Papillomaviruses

TABLE 21.6 Age at Onset of Symptoms of Laryngeal Papillomas

Squamous cell papillomas are benign lesions of the esophagus of unknown etiology and pathogenesis (Syrjanen et ah, 1982). They are rarely symptomatic and are generally found incidentally when endoscopy is carried out. These lesions exhibit several morphologic forms (Lewin and Appelman, 1996). A number of published studies have failed to establish an association between esophageal squamous papillomas and HPV infection. A review of the relevant published literature has been published by Carr and his associates (1994).

Age of onset (years) 0-1 year 1-2 years 2-3 years 3-4 years 4-5 years 5-6 years 6-10 years 11-15 years 16-20 years 21-30 years 31-40 years 41-50 years Over 51 years Unknown

Anus Condyloma acuminatum and carcinoma of the anus have a strong epidemiological association with receptive homosexual intercourse and HIV-1 infection (Palefsky et al, 1991; Taxy et al, 1989; Duggan et al, 1989; Forti et al, 1995). An increase in prevalence has been documented during the past several decades. In a survey of several hundred homosexual and bisexual males using swabs to collect specimens from the anus, HPV DNA was detected by PCR in 93% of HIV-1-positive and 61% of HIV-1-negative men. Infection with multiple virus types was found in 73% HIV-1-positive and 23% of HIV-1-negative subjects surveyed. HPV type 16 was the most common agent identified (Palefsky et ah, 1998). Compelling evidence now relates condylomatous disease of the anus with infection by the so-called "low-risk" HPV types 6 and 11, whereas HPV 16 is strongly associated with both in situ and invasive squamous carcinoma of the anus (Beckmann et aU 1989; Gal et al, 1987, 1989; Wolber et al, 1990; Palefsky et al, 1990; Wells et al, 1987; Yun et al, 1988; Zaki et al, 1992), and a case of HPV-associated adenocarcinoma has been reported (Koulos et al, 1991).

DISEASE OF THE LARYNX A N D TRACHEOBRONCHIAL TREE Papillomas of the larynx are benign squamous epithelial lesions. In addition to the larynx, the epiglottis and trachea are involved seemingly by contiguity. Spread of papillomas throughout the airway occurs in roughly 5 to 10% of cases of laryngeal carcinoma. These lesions are described below. Juvenile and adult forms of the disease are recognized, but their morphologic features are comparable. The juvenile lesions develop early in life and are usually multiple (Tables 21.6 and 21.7). For unknown reasons, they occur twice as often in males than in females and tend to disappear spontaneously at puberty. The papillomas can be of sufficient

No. of patients 12 8 5 4 3 5 2 8 2 15 9 14 10 5

Total

109

Adapted with permission from Holinger et al (1950).

TABLE 21.7 S i n g l e or M u l t i p l e Papilloma of A i r w a y s According to A g e Groups

Children (under 16 years of age) Adults (over 16 years of age) Total

Single

Multiple

10 35

44 20

45

64

Adapted with permission from Holinger et al. (1950).

size to obstruct airways and threaten life. Symptomatology relates to the obstructive nature of the lesions. Compelling evidence now indicts both HPV types 6 and 11, which appear to occur with roughly equal frequency, in the pathogenesis of the juvenile lesions (Costa et al, 1981; Braun et al, 1982; Shen et al, 1996). Juvenile laryngeal papillomas commonly develop in the offspring of women with condylomas of the vulva. In one study, condylomas were found clinically in the mothers of 54% of the infants with laryngeal papilloma (Majmudar and Hallden, 1985). The pathogenesis of the relatively rare adult-onset forms of laryngeal papillomatosis is less well defined. The frequency of HPV infection is somewhat lower in adults than with the juvenile forms of the disease. Orogenital sexual contact is said to be a cause, but epidemiological evidence supporting this conjecture is lacking.

318

Pathology and Pathogenesis of Human Viral D i s e a s e

Grossly the tumors are glistening, elevated, mulberry-like, nodular masses which vary in color from a whitish pink to red [see Figure 21.20]. They may occur anywhere in the larynx, but chiefly on the true and false cords and in the anterior commissure. Frequently they extend subglottally and occasionally into the trachea and bronchi or upwards on the epiglottis, pharyngeal wall, tonsil, and soft palate. They vary in size from small nodules to sessile plaques or large nodular masses the size of a cherry. The tumors are usually friable and bleed easily with slight trauma, a quality which makes difficult complete removal by a single operation. Microscopically the papilloma tissues are sessile or papillary structures composed of a vascular connective tissue core covered by stratified squamous epithelium in many layers. There are usually secondary and even tertiary stalks of vascular fibrous tissue covered by the epithelium. Cells in mitosis are frequent, indicating growth activity, but the cells are well-differentiated mature epithelial cells. The growths have no tendency to invade the stroma or submucosa. No histologic difference is recognized between the papilloma of adults and those in children.

lesions in adults were usually confined to the larynx (Table 21.9) (Holding, 1929). Ten percent of adult papillomatous lesions were considered to be malignant (Altmann et al, 1955). In comparing malignant and benign papillomatous lesions, Bjork and Hakosalo (1957) found that malignant lesions often exhibited areas of benignancy. Mitotic activity and atypical mitoses were uncommon in the benign lesions (Table 21.10). Unfortunately, virological data comparing the malignant and benign lesions have not been published. However, relevant information was recently reported by Popper et al (1994), who evaluated bronchial papillomas in adults using both PCR and in situ hybridization. Benign lesions were infected with HPV type 11, whereas malignant squamous tumors yielded exclusively either HPV type 16 or 18 (Byrne et al, 1987). These authors suggested that PCR of papillomatous lesions might prove to be a better tool than histology for assessing the prognosis of the lesions. As pathologists know only too well, the small size of biopsies from the upper airways often precludes comprehensive morphological evaluation of lesions of this type.

In a detailed analysis of juvenile and adult papillomas, Nikolaidis et al (1985) noted that the papillary pattern is almost consistently found in both "juvenile" and "adult" lesions, but the so-called acanthotic and angiokeratotic forms are present in lesions in young people, but not in the lesions of adults (Table 21.8). These authors also documented a widespread distribution of the juvenile form in the upper airway, but the

In a recent comprehensive survey, 10% of laryngeal carcinomas had evidence of infection with HPV types 6, 11, or 18 (Pou et al, 1995). None of the 11 verrucous carcinomas proved to be positive in one study of Multhaupt and colleagues (1994). These lesions that typically develop in older men are epidemiologically strongly associated with tobacco product and alcoholic beverage abuse. Should HPV infections occur commonly in these cancers, one might

TABLE 21.8 Comparison of Microscopic Criteria of Laryngeal Papillomas b y A g e

TABLE 21.9 Comparison of Anatomic Sites of Respiratory Tract Papillomata

The pathology of laryngeal papillomas is succinctly described in the publication of Holinger et al. (1950). I quote from their paper:

Microscopic findings Koilocytosis Multinucleation Exocytosis Papillary pattern Acanthomatous pattern Intracellular keratinization Atypical parabasal cells Angiokeratotic pattern Parakeratosis Increased mitoses Inflammation Atypia Hyperkeratosis Abnormal mitoses Dysplasia Carcinoma

Juvenile, % 99 97 96 93 89 79 73 73 66 59 58 56 23 21 1 0

Adapted with permission from Nikolaidis et al. (1985).

Adult, % 90 60 70 100 40 30 20 10 10 0 10 10 0 0 0 10

Sites True vocal cords False vocal cords Anterior commissure Subglottic area Ventricle Epiglottis Trachea Arytenpepiglottic Supraglottic area Bronchi Arytenoid cartilage Pharynx Posterior commissure Oral Tonsils Other

Juvenile, % 97 82 79 67 62 47 42 36 22 16 15 14 11 7 4 1

Adapted with permission from Nikolaidis et al (1985).

Adult, % 80 0 10 10 0 0 0 0 0 0 0 0 0 10 10 0

319

Papillomaviruses TABLE 21.10 B e n i g n and Malignant Papilloma of the Larynx in A d u l t s (32 Cases): Frequency of M i t o s e s and Pathological M i t o s e s Pathological mitoses No. of mitoses per 3000 cells

% of mitoses

Total cases

1-10

11-20

>20

Total cases

1%

2-6%

Benign Malignant

14 18

10 7

3 4

1 7

1 8

1 1

0 7

Total Average mitotic frequency: Benign cases 8.2 / 3000 cells Malignant cases 17.1 / 3000 cells

32

17

7

8

9

2

7

Adapted with permission from Bjork and Hakosalo (1957).

consider the possibility of a cocarcinogenic interaction between the virus and the chemical products of tobacco incineration. Papillomatosis of the tracheobronchial tree is an exceedingly uncommon lesion (Figures 21.20 and 21.21).

FIGURE 21.20 Tracheal papilloma. This 2-cm papillary tumor was resected from the trachea of a 32-year-old man. Note the polypoid nature of the lesion as well as its sessile and broad attachment to the wall of the trachea. Reprinted with permission from Carter and Eggleston (1979).

It occasionally results in death from airway obstruction or presents clinically as intrapulmonary masses (Gilbert ei al, 1949; Hitz and Oesterlin, 1932; Kirchner, 1951; Ogilvie et al, 1953; Bjork and Teir, 1957; Stein and Volk, 1959; Moore and Lattes, 1959; Kerley et al, 1989).

WPM^TiT!|iri|Miiim'iiriUMi|T|imim

:: \'* 5j

* .. .61..

'

71

'

FIGURE 21.21 Upper airway papillomata. Two of the lesions were cauterized accounting for their roughened hemorrhagic appearance. Note the tracheostomy orifice (cross). Reprinted with permission from Ogilvie (1953).

320

Pathology and Pathogenesis of Human Viral D i s e a s e

FIGURE 21.22 Tracheobronchial papillomatosis. The lungs of a 9-year-old girl who presented at age 5 with significant laryngeal papillomatosis. The lesions progressed rapidly between ages 5 and 9. At autopsy, a multitude of papillomas involved the tracheobronchial tree from the larger bronchi to the alveolar spaces. Many of the tumor masses have been converted into cysts on which the papillary excrescences have proliferated. Reprinted with permission from Kirchner (1951). Dr. }. Kirchner (New Haven, CT) provided the photograph for publication in the AFIP fascicle.

'•*fi

*• '

- <•.

•

* •% • • ^ .

'*•4v

FIGURE 21.23 Squamous papilloma in the right upper lobe bronchus of a 57-year-old smoker with a chronic cough. The lesion was obtained by lobectomy. Panel A illustrates the lesion at low microscopic resolution. Panel B shows the acanthotic squamous epithelium with koilocytic atypicality as represented in C. Panel D demonstrates the results of in situ hybridization using an HPV 6/11 probe, as illustrated by the darkly stained nuclei. Assays for HPV types 16/18 and types 31/33/51 were negative. Panels A and B reprinted with permission from Flieder (1998) and through the courtesy of D. Flieder, MD.

321

Papillomaviruses TABLE 21.11 H P V i n Bronchogenic Carcinomas HPV type Authors Syrjanen et al, 1989 Bejui-Thivolet et al, 1990 Nuorva et al, 1995 Kinoshita et al, 1995 Soini et al, 1996

Hirayasu et al, 1996 Welt et al, 1997

Bohlmeyer et al, 1998

Tumor type

No. tested

Technique

6

Squamous carcinoma Squamous carcinoma Bronchoalveolar Squamous carcinoma Squamous carcinoma Adenocarcinoma Squamous carcinoma Squamous carcinoma Small cell carcinoma Squamous carcinoma

12

IS

2

7

33

IS

1

1

3

4/33(18%)"

IS

2

2

2

1

36%^

36

PCR

ND

ND

0

3

3/36 (30%)

28

IS

5

4

6

4

4/28 (15%)^^

12

PCR

1

2

2

3

3/12(25%)^

43

PCR

11

22

22

34/43 (79%)^

32

IS

0

6

PCR

0

IS PCR

34 34

11

16

No. pos./ no. tested

18

7/12 = 58%

0 2/34 (6%)

PCR = polymerase chain reaction. "Type 11 in one case. ^19 cases positive for both HPV 19 and 22. 'HPV types 31 and 33 detected. '^HPV type 31 and 33 detected in two of these cases. ^HPV types 31 in additional five cases.

In children, papillomas in the lung (Figure 21.22) often occur in association with laryngeal lesions of the same type, whereas in adults they commonly develop as isolated tumors. Little concrete information on their association with HPV has been published, but, as might be expected, condylomatous lesions tend to be infected with HPV 6 and 11 infections. Flieder et al. (1997) reviewed the pathology of 16 papillomatous lesions of bronchial origin. They classified the lesions into (1) squamous (six cases); (2) glandular (five cases), and (3) mixed squamous/glandular papillomas (five cases). Squamous papillomatosis was found to be multifocal in some cases, and in 2 of 5 patients squamous carcinomas developed. Koilocytosis was present in a squamous carcinoma that yielded evidence of HPV 6/11 infection (Figure 21.23). A detailed account of this work and a literature review were more recently published (Flieder et al, 1998). Table 21.11 summarizes the results of studies undertaken to assess the association of traditional spontaneously developing squamous cell carcinoma of the bronchus with HPV infection. Critical evaluation of these

data is exceedingly difficult because different methodologies and criteria for assessing results were employed. It is currently difficult to draw conclusions from the evidence available, but the results suggest that differences in the properties of HPV positive tumors may relate to geographic consideration or the sensitivity of the methods used in different laboratories. The evidence does suggest that, in some cases of invasive carcinoma, the oncogenic HPV types 16 and 18 may play a role in the pathogenesis of the neoplasms. However, the possibility also exists that the viruses may be "passengers" in the tumor cells and have no oncogenic role. Clearly, much additional data must accumulate before the role of HPV in lung cancer can be satisfactorily evaluated. DISEASE OF THE EYE Papillomas of the conjunctiva are rare. Only 27 lesions of this type were diagnosed at the Mayo Clinic over a 64-year period (Erie et al, 1986). At the Wilmer Eye Clinic of Johns Hopkins University, 23 papillomas were resected between 1934 and 1986, a 52-year period

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(McDonnell et al, 1987). Over half of the lesions in the latter series occurred in children under the age of 10, an observation supporting the notion that an HPV infection is acquired at birth. Further support for this contention is provided by in situ hybridization studies that demonstrate HPV types 6 and 11 in all or the majority of these lesions. Interestingly enough, a large number of dysplastic lesions of the conjunctiva consistently proved negative. Papillomas of the eye frequently arise in the caruncula and exhibit the same morphological features as genital condylomas (Michel et al, 1996). Lacrimal gland papillomas are uncommon. Three papillomas were recognized at the Wilmer Eye Clinic over a 31-year period. All three of these lesions yielded evidence of HPV type 11 by in situ hybridization and PCR (Madreperla et al, 1993). Three of the six lacrimal sac carcinomas diagnosed during this same 31-year period yielded evidence of HPV infection, and typing indicated infection by HPV 18 in one squamous carcinoma.

DISEASE OF THE MIDDLE EAR Squamous carcinomas of the middle ear are rare; approximately one case occurs each year per 1 x 10^ members of the population (Morton et al, 1984). The neoplasm appears in the later decades of life, and most patients have a long history of chronic suppurative otitis (Tucker, 1965). No other risk factors have been identified in epidemiological studies. Using highly sensitive molecular approaches, Jin and his associates (1997) found evidence of HPV type 16 in 11 of 14 wellor moderately well-differentiated squamous carcinomas of the middle ear. In five of these cases, HPV type 18 was also identified. Over half of these lesions exhibited papillary or verrucous growth patterns. Koilocytosis was observed microscopically in four cases, three of which were shown to be infected. HPV has also been identified in the squamous elements of cholesteatoma (Bergmann et al, 1994). The significance of the finding is unknown. References Abell, M. (1965). Intraepithelial carcinoma of epidermis and squamous mucosa of vulva and perineum. Surg. Clin. North Am. 45,1179-1198. Akutsu, N., Shirasawa, H., Nakano, K., et al. (1995). Rare association of human papillomavirus DNA with esophageal cancer in Japan. /. Infect. Dis. 171, 425-428. Altmann, R, Basek, M., and Stout, A. (1955). Papillomas of the larynx with intraepithelial anaplastic changes. AMA Arch. Otolaryngol. 62, 478-485.

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Park, T, Richart, R., Sun, X., and Wright, T. (1996). Association between human papillomavirus type and clonal status of cervical squamous intraepithelial lesions. /. Natl Cancer Inst. 88, 355-358. Payne, J. (1891). Clinical notes on the contagiousness of common warts. Br. ]. Dermatol 3,185-188. Pfister, H. (1984). Biology and biochemistry of papillomaviruses. Rev. Physiol Biochem. Pharmacol 99,114-181. Pfister, H. (1992). Human papillomaviruses and skin cancer. Sem. Cancer Biol 3, 263-271. Popper, H., El-Shabrawi, Y, Wockel, W, Hofler, G., Kenner, L., Juttner-Smolle, K, and Pongratz, M. (1994). Prognostic importance of human papilloma virus typing in squamous cell papilloma of the bronchus: Comparison of in situ hybridization and the polymerase chain reaction. Hum. Pathol 25,1191-1197. Pou, A., Rimell, R, Jordan, J., Shoemaker, D., Johnson, J., Barua, P., Post, J., and Ehrlich, G. (1995). Adult respiratory papillomatosis: human papillomavirus type and viral coinfections as predictors of prognosis. Ann. Otol Rhinol Laryngol 104 (10, Pt. 1). Purola, E., and Savia, E. (1977). Cytology of gynecologic condyloma acuminatum. Acta Cytol. 21, 26. Ries, L., Kosay C , Hankey, B., Harras, A., Miller, B., and Edwards, B. (1996). "SEER Cancer Statistics Review, 1973-1993: Tables and Graphs.'' National Cancer Institute, Bethesda, MD. Roman, A., and Fife, K. (1989). Human papillomaviruses: Are we ready to type? Clin. Microbiol Rev. 2,166-190. Rous, P., and Beard, J. (1935), The progression to carcinoma of virusinduced rabbit papillomas. /. Exp. Med. 62, 523-548. Rous, P., and Kidd, J. (1936). The carcinogenic effect of a virus upon tarred skin. Science 83, 468-469. Rowson, K., and Mahy B. (1967). Human papova (wart) virus. Bacteriol Rev. 31,110-131. Schneider, A., Hotz, M., and Gissmann, L. (1987). Increased prevalence of human papillomaviruses in the lower genital tract of pregnant women. Int. ]. Cancer 40,198-201. Shamanin, V, zur Hansen, H., Lavergne, D., Proby, C , Leigh, I., Neumann, C , Hamm, H., Goos, M., Haustein, U., Jung, E., Piewig, G., Wolff, H., and de Villiers, E. (1996). Human papillomavirus infections in nonmelanoma skin cancers from renal transplant recipients and nonimmunosuppressed patients. /. Natl Cancer Inst. 88, 802-811. Shen, J., Tate, J., Crum, C , and Goodman, M. (1996). Prevalence of human papillomaviruses (HPV) in benign and malignant tumors of the upper respiratory tract. Mod. Pathol 9,15-20. Shope, R. (1933). Infectious papillomatosis of rabbits. /. Exp. Med. 58, 607-624. Soini, Y, Nuorva, K., Kamel, D., Pollanen, R., Vahakangas, K., Lehto, v., and Paakko, P. (1996). Presence of human papillomavirus DNA and abnormal p53 protein accumulation in lung carcinoma. Thorax 51, 887-893. Soler, C , Chardonnet, Y, Allibert, P., Euvrard, S., Schmitt, D., and Mandrand, B. (1993). Detection of mucosal human papillomavirus types 6/11 in cutaneous lesions from transplant recipients. /. Invest. Dermatol 101, 286-291. Stark, L., Arends, M., McLaren, K., Benton, E., ShahiduUah, H., Hunter, J., and Bird, C. (1994). Prevalence of human papillomavirus DNA in cutaneous neoplasms from renal allograft recipients supports a possible viral role in tumour promotion. Br. ]. Cancer 69, 222-229. Steenbergen, R., Hermsen, M., Walboomers, J., Joenje, H., Arwert, R, Meijer, C , and Snijders, P. (1995). Integrated human papillomavirus type 16 and loss of heterozygosity at llq22 and 18q21 in an oral carcinoma and its derivative cell line. Cancer Res. 55, 5465-5471.

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Stein, A., and Volk, B. (1959). Papillomatosis of trachea and lung: Report of a case. Arch. Pathol. 68,124-128. Syrjanen, K., Pyrhonen, S., Aukee, S., and Koskela, E. (1982). Squamous cell papilloma of the oesophagus: Tumor probably cuased by human papillomavirus (HPV). Diag. Histopathol 5,291-296. Syrjanen, K., Syrjanen, S., Kellokoski, J., Karja, }., and Mantyjarvi, R. (1989). Human papillomavirus (HPV) type 6 and 16 DNA sequences in bronchial squamous cell carcinomas demonstrated by in situ DNA hybridization. Lung. 167, 33-42. Tate, J., Mutter, G., Boynton, K., and Crum, C. (1997). Monoclonal origin of vulvar intraepithelial neoplasia and some vulvar hyperplasias. Am. J. Pathol. 150, 315-322. Taxy, J., Gupta, R, Gupta, J., and Shah, K. (1989). Anal cancer: Microscopic condyloma and tissue demonstration of human papillomavirus capsid antigen and viral DNA. Arch. Pathol Lab. Med. 113,1127-1131. Togawa, K., Jaskiewicz, K., Takahashi, H., Meltzer, S., and Rustgi, A. (1994). Human papillomavirus DNA sequences in esophagus squamous cell carcinoma. Gastroenterology 107,128-136. Toh, Y, Kuwano, H., Tanaka, S., Baba, K., Matsuda, H., Sugimachi, K., and Mori, R. (1992). Detection of human papillomavirus DNA in esophageal carcinoma in Japan by polymerase chain reaction. Cancer 70, 2234-2238. Toki, T, Kurman, R., Park, J., Kessis, T., Daniel, R., and Shah, K. (1991). Probable nonpapillomavirus etiology of squamous cell carcinoma of the vulva in older women: A clinicopathologic study using in situ hybridization and polymerase chain reaction. Int. J. Gynecol. Pathol. 10,107-125. Tucker, W. (1965). Cancer of the middle ear: A review of 89 cases. Cancer 18, 642-650. Turner, J., Shen, L., Crum, C , Dean, R, and Odze, R. (1997). Low prevalence of human papillomavirus infection in esophageal squamous cell carcinomas from North America: Analysis by a highly sensitive and specific polymerase chain reaction-based approach. Hum. Pathol. 28, 174-178. Varma, V, Sanchez-Lanier, M., Unger, E., Clark, C , Tickman, R., Hewan-Lowe, K., Chenggis, M., and Swan, D. (1991). Association

of human papillomavirus with penile carcinoma: A study using polymerase chain reaction and in situ hybridization. Hum. Pathol. 22, 908-913. Villa, L., and Lopes, A. (1986). Human papillomavirus DNA sequences in penile carcinomas in Brazil. Int. J. Cancer 37, 853-855. Wechsler, H., and Fisher, E. (1962). Oral florid papillomatosis: Clinical, pathological and electron microscopic observations. Arch. Dermatol. 86, 140-152. Wells, M., Griffiths, S., Lewis, R, and Bird, C. (1987). Demonstration of human papillomavirus types in paraffin processed tissue from human anogenital lesions by in situ DNA hybridization. /. Pathol. 152, 77-82. Welt, A., Hummel, M., Niedobitek, G., and Stein, H. (1997). Human papillomavirus infection is not associated with bronchial carcinoma: evaluation by in situ hybridization and the polymerase chain reaction. /. Pathol. 181, 276-280. Werness, B., Levine, A., and Howley, P. (1990). Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 76-79. Wilczynski, S., Walker, J., Liao, S., Bergen, S., and Berman, M. (1988). Adenocarcinoma of the cervix associated with human papillomavirus. Cancer 62,1331-1336. Williamson, A., Jaskiesicz, K., and Gunning, A. (1991). The detection of human papillomavirus in oesophageal lesions. Anticancer Res. 11, 263-266. Wolber, R., Dupuis, B., Thiyagaratnam, P., and Owen, D. (1990). Anal cloacogenic and squamous carcinomas: Comparative histologic analysis using in situ hybridization for human papillomavirus DNA. Am. /. Surg. Pathol. 14, 176-182. Yun, K., Molenaar, A., and Wilkins, R. (1988). Detection of human papillomavirus DNA in anogenital exophytic lesions by in situ hybridization to paraffin sections. Acta Pathol. Jpn. 38, 1131-1139. Zaki, S., Judd, R., Coffield, L., Greer, P, Rolston, R, and Evatt, B. (1992). Human papillomavirus infection and anal carcinoma: Retrospective analysis by in situ hybridization and the polymerase chain reaction. Am. J. Pathol. 140,1345-1355.

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22 Papovaviruses INTRODUCTION 327 PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY (PML) 329 URINARY TRACT INFECTION AND DISEASE REFERENCES 333

diverse variety of tumors when inoculated into animals. It, and a number of other animal papovaviruses, are biologically similar to the viruses of human importance and seem to have a common ancestral origin. However, SV40, an agent initially recognized as a seemingly harmless commensual in rhesus monkey {Macaca mulatto) kidney monolayer cultures is unique, for it is believed to have been present in the brain of two patients with PML (Narayan et ah, 1973; Weiner et ah, 1972) and its genome is found in the DNA of some human mesotheliomas, osteosarcomas, and choroid plexus tumors (Bastian, 1971; Bergsagel et ah, 1992; Carbone et ah, 1997). SV40 is also of exceptional interest inasmuch as roughly 98 x 10^ Americans may have been administered inactivated poliovirus vaccines containing low concentrations of infectious SV40 (Shah and Nathanson, 1976). A great deal is known about the cellular replicative sequence of BKV and JCV, the currently recognized human pathogens. In vitro, JCV growth is limited to human fetal glial cells, including astrocytes, oligodendroglial precursors, and Schwann cells, whereas BKV replicates in epithelial cells, fibroblasts, and fetal glial cells. Cells of the adult renal tubule, liver, and brain support productive infections by both viruses in humans of all ages. The intracellular replicative cycle is straightforward (Sangalang and Embil, 1982). Viruses adsorb and are taken up by susceptible cells with low efficiency. Proteins of the class I major histocompatibility complex appear to play a role in attachment of the virus to cells, but the polarity may influence this event. The virions are transported in vesicles in the cytoplasm to the nuclear membrane, where it transgresses into the nucleus, possibly through the nuclear pores. In the nucleus, the virion is uncoated, and replication begins with viral DNA generating mRNAs to, in turn, fabricate the T antigen (Tag). From this point, the complex Tag quarterbacks events involved in production of the late proteins and RNA through the mechanism of a helicase and ATPase. The papovavirus Tag has unique

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INTRODUCTION Something was up! I knew by the way June was acting, particularly when she slipped off for several secretive meetings at an undisclosed London Underground station. At the time, I was working at the Hammersmith Hospital with June Alameda, a virologist with an exceptional skill at diagnostic negative staining of virions using the electron microscope. After the story broke, it turned out that she was meeting with fellow virologists to consider images of a "new" agent found in the urine of a child recipient of a renal allograft. The virus, termed BKV, had the morphology of a papovavirus; thus, it represented a truly "new" family of infectious agents for humans. This was my introduction to this unique family of viruses, but my interest was peaked by the simultaneous reporting in 1971 of the isolation of a papovavirus, termed JCV, from a case of progressive multifocal leukoencephalopathy (PML), using cultured glial cells (Gardner et ah, 1971; Padgett ef a/., 1971). The PA/PO/VA virus family members of human importance are the "pa," or papilloma viruses (see Chapter 21), "po," or polyomaviruses (JCV and BKV are the only established human representatives), and "va" (referring to the cytological vacuolization that occurs in epithelial cells infected with SV40, an endogenous virus of several subhuman primate species). All of these viruses are small (-45 nm in diameter) nonenveloped agents having an icosahedral capsid comprised of three different proteins, and a genome made up of double-stranded DNA. The term "polyoma" refers to an exceptional mouse virus that produces a

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biological properties and is believed to be oncogenic when these viruses are injected into certain animals, such as the golden hamster. The cells of these rodents do not support viral replication. These so-called nonpermissive animal cells permit viral DN A replication to proceed, resulting in Tag elaboration, but synthesis of capsid protein does not follow. The virus-injected animals develop a variety of tumors, depending on the route of inoculation. Oncogenesis, under these circumstances, is attributed to Tag-directed elaboration of proteins that have the capacity to inactivate pl05^^ and p53, two naturally occurring cell cycle regulatory genes. In the infection of natural host cells, the progeny virions are assembled in the nucleus and transported to the cytoplasm, and then to and through the plasma membrane. Infected cells undergoing cytolysis also release infectious virus. The events briefly outlined here result in nuclear enlargement and formation of typical intranuclear inclusions (Coleman ei a/., 1977). Little is known about the early stages of infection with BKV and JCV in humans. Presumably, the virus is

transmitted by respiratory droplet means, and infection of the epithelium of the lower respiratory tract of immunocompromised patients has been documented (Figure 22.1A,B) (Vallbracht et al, 1993). The kidney is believed to be a major site of both latency and chronic active replication in healthy immunocompetent persons. As a result, chronic viruria is common (Markowitz et al, 1993; Sundsfjord et al, 1994; Ault and Stoner, 1994). The latency state is incompletely defined, but the critical DNA segments of the virions are randomly integrated into the nuclear DNA. Thus, its existence in cells can only be detected by molecular means. Immunohistochemistry locates presumptively infectious virus in the renal medullary tubules of about 10% of the population. Presumably, this finding is representative of fully assembled nonlatent virions since the capsid proteins are the major antigens of the virus. B lymphocytes in the spleen and lymph nodes are infected: these same cells in the circulation may serve as the transportation vehicle in vivo (Houff et al, 1988). The literature concerned with viremia is somewhat

FIGURE 22.1 Lung of a young man with AIDS demonstrating cytological evidence of BKV infection established by immunohistochemistry. (A) Degeneration and desquamation of alveolar pneumocytes. Many epithelial cells possess enlarged and hyperchromatic nuclei. There is a mild mononuclear infiltration of the interstitium (xl65). (B) Arrows indicate nuclear features of the infected pneumocytes (x530). Reprinted with permission from Vallbracht et al. (1993).

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confusing, since, as would be expected, results using culture techniques, negative staining, and PCR yield differing results (Arthur and Shah, 1989). In recent studies of immunocompetent persons, the incidence of viremia using PCR ranged from 8 to 29% for JCV and from 4 to 18% for BKV (Markowitz et al, 1993). Traditional teaching indicates that the proportion of persons yielding BKV and JCV in the urine increases with immunosuppression (Manz et aL, 1971; Coleman ei ah, 1973; O'Reilly et al, 1981; Sundsfjord et a/., 1994), pregnancy (Coleman et ah, 1980), and among the elderly. However, additional studies suggest that immunosuppression may increase urinary excretion of virus quantitatively, rather than affecting the actual number of individuals excreting virus (Markowitz et ah, 1993).

PROGRESSIVE MULTIFOCAL LEUKOENCEPHALOPATHY (PML) In 1971, Padgett established the virological basis of PML, a disease described more than a decade earlier by Astrom and his colleagues (1958). A viral etiology for PML was suggested by the electron microscopic finding of typical papovavirus-like particles in inclusions of oligodendroglia associated with the demyelinating white matter lesions of PML (ZuRhein and Chou, 1965). Although JCV has been repeatedly linked etiologically with PML, as noted above, an SV40-like agent was recovered from two cases by Weiner and his associates (1972). SV40 spontaneously causes a central

FIGURE 22.2 (A) Coronal section of cerebral hemisphere showing a spectrum of degenerative changes predominantly involving the white matter in the brain of a case of PML. (B) The cerebellum in PML exhibits demyelinization that is predominantly unilateral. Note the preservation of the cerebellar folia.

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B

D

FIGURE 22.3 Cytological features of oligoglia and astrocytes in PML (A-C) and the demyelinization typical of this disease (Luxol fast blue stain) (D). To a variable extent, the glia are enlarged and either exhibit intranuclear inclusions surrounded by a halo, or a homogenous ''ground-glass" granular nucleoplasm. Multinucleate cells of variable complexity are a characteristic feature, but they are only present in small numbers.

nervous system disease identical to human PML in immunologically intact rhesus monkeys (Gribble et ah, 1975; Holmberg et al, 1977). Because of the molecular and antigenic similarities of JCV and SV40, more modern approaches to viral characterization might well show that PML is caused by a family of closely related and nearly identical agents (Padgett et al, 1976; Narayan et al, 1973; Walker, 1978). In PML, multiple foci and confluent regions of demyelinization are present throughout the cerebrum, cerebellum, and brainstem, but lesions of the spinal cord are not described (Figure 22.2A,B). There appears to be an evolution of the smaller lesions to the larger, but, throughout, the areas of demyelinization blend with normal brain tissue. The diffuseness of the disease process, no doubt, reflects widespread infection of the central nervous system, and the probable seeding of the nervous system by hematogenous spread of the virus. The extensive involvement of the central nervous system accounts for the nonspecific insidious neurological findings in these patients, with mental, motor, and visual problems predominating. Despite extensive involvement of the cerebellum in most cases, clinical

signs related to this region of the brain are not prominent. The cytological features of PML are distinctive and allow the pathologist to discriminate the disease from other demyelinating processes such as multiple sclerosis. Pathognomonic of PML are the enlarged oligodendroglia with intranuclear ground-glass nuclear inclusions and the associated, but variable, numbers of atypical large astrocytes that often appear gametocytic (Figure 22.3A-C). On occasion, these latter cells are multinucleate and exhibit mitosis, some of which is atypical. For unknown reasons, these cells accumulate large amounts of cytoplasmic glial fibrils that are intimately associated with virions. Notably absent in the PML brain lesions is an inflammatory response, although microglia and fat-laden macrophages, that is, gitter cells, are found throughout the lesions (Figure 22.3D). Recent studies have elucidated the biological nature of events in the lesions of PML. Electron microscopy shows that both oligodendroglia and astrocytes are infected. Viral involvement of these cells appears to be the principal basis for demyelinization (Watanabe and

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Papovaviruses

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FIGURE 22.4 Ureter at 200x (A) and 400x (B), exhibiting diffuse cytological alterations typical of BKV infection. Note the cellular and nuclear pleomorphism and the homogenous ground-glass appearance of the intranuclear inclusions. Prepared from autopsy specimens obtained from a 48-year-old man with non-Hodgkin's lymphoma treated with repeated cycles of combined drug chemotherapy. Photomicrographs reprinted with permission and through the courtesy of K. Nagashima, MD.

Preskorn, 1976). And, immunocytochemistry has convincingly demonstrated JCV DNA, Tag antigen, and p53 protein in intranuclear inclusions of the astrocytes, often in the absence of antigenic viral protein (Aksamit et ah, 1986; Stoner et al, 1986). It would appear that p53 binding by viral Tag results in an unstable state of cellular transformation by astrocytes that may inefficiently fabricate virus (Ariza et al, 1994). In virological terminology, the astrocytes may be semipermissive because they often do not complete the replicative cycle and produce capsid proteins. This conclusion is based on studies of Aksamit et al. (1986). These investigators found that only a small proportion of infected astrocytes (as established by in situ hybridization) displayed evidence of viral capsid protein synthesis (as evaluated by immunochemistry). Usually, but not invariably, PML is a rapidly progressive disease with a survival period of 3 to 6 months. However, chronic cases have been reported (Hedley-Whyte et al, 1966). Almost invariably, it occurs in patients with immunodeficiency disorders (Bolton and Rozdilsky, 1971). Although relatively common in cases of leukemia and lymphoma, the disease assumes its most overt form in AIDS. The rapidity of disease progression seems to relate to the degree of immunosuppression of the patient. PML is now considered an indicator sentinel of AIDS, affecting roughly 4% of these patients. JCV infection in PML is a systemic process, for in rare cases typical viral inclusions are found in numerous organs at autopsy (Martin and Banker, 1969; Castaigne, 1965).

URINARY TRACT INFECTION A N D DISEASE BKV possesses the capacity to infect cells lining the renal tubules and the transitional epithelium throughout the urinary tract (Chesters et al, 1983). Involvement of these epithelia is readily apparent morphologically and can be confirmed by histochemistry (Shinohara et al, 1993) (Figures 22.4A,B and 22.5A-D). Rare cases of interstitial nephritis with evidence of BKV inclusions in tubular lining cells are reported (Figure 22.6A-C). One patient among the cases known to me was a 6-year-old immunocompromised boy who developed renal failure and died. Large amounts of viral DNA were found in the kidneys at autopsy, with substantially lesser amounts being detected in the lungs, spleen, and lymph nodes (Rosen et al, 1983). A second case is of interest. This young male with AIDS also developed an interstitial nephritis with inclusion-bearing cells in the renal tubules. In addition, he had an interstitial pneumonia and meningoencephalitis with ventriculitis attributable to the virus (Vallbracht et al, 1993). Pappo and associates (1996) found 15 reported cases of urinary tract involvement with BKV-like papovavirus among renal allograft recipients. This review brought to light the common occurrence of graft rejection in association with infection. Urinary tract strictures were a clinical problem in five cases. Whether or not these complications are a consequence of BKV infection remains to be established. Arthur and his colleagues (1986) associated BKV infection with hemorrhagic cys-

332

Pathology and Pathogenesis of Human Viral D i s e a s e

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fel FIGURE 22.5 (A) Urinary bladder epithelium displaying the widespread cytological abnormalities typical of BKV infection as established by immunohistochemistry using an anti-JCV polyclonal antibody (B). (C) This electron micrograph (3500x) of the infected urinary bladder epithelium displays the viral nucleocapsids that are illustrated in detail in (D) (40,000x). Prepared from autopsy specimens obtained from a 48-year-old man with non-Hodgkin's lymphoma treated with repeated cycles of combined drug chemotherapy. Photomicrographs provided through the courtesy of K. Nagashima, MD.

FIGURE 22.6 Kidney of patient described in Figure 22.1. (A) Degeneration and necroses of the renal tubules. Some of the epithelial cells lining the tubules have enlarged and hyperchromatic nuclei (x210). (B) Abnormal nuclei contain granular basophilic material (arrowheads). Intranuclear bodies (arrows) in degenerated epithelial cells (x800). (C) Typical intranuclear inclusion body with halo (xSOO). Reprinted with permission from Vallbracht et al. (1993).

Papovaviruses

titis in bone marrow transplant recipients. Half of the patients who received allogenic grafts were infected, and hemorrhage cystitis developed in 71%. Reports of hemorrhagic cystitis attributed to BKV have not been reported in the literature in recent years, and a causative role for the virus has not been established. References Aksamit, A., Sever, J., and Major, E. (1986). Progressive multifocal leukoencephalopathy: JC virus detection by in situ hybridization compared with immunohistochemistry. Neurology 36, 499-504. Ariza, A., Mate, J., Fernandez-Vasalo, A., Gomez-Plaza, C , PerezPiteira, J., Pujol, M., and Navas-Palacios, J. (1994). p53 and proliferating cell nuclear antigen expression in JC virus-infected cells of progressive multifocal leukoencephalopathy. Hum. Pathol. 25, 1341-1345. Arthur, R., and Shah, K. (1989). Occurrence and significance of papovavirus BK and JC in the urine. In "Progress in Medical Virology" (J. Melnick, ed.). Vol. 36, pp. 42-61. Karger, Basel. Arthur, R., Shah, K., Baust, S., Santos, C , and Saral, R. (1986). Association of BK viruria with hemorrhagic cystitis in recipients of bone marrow transplants. New Engl. J. Med. 315, 230-234. Astrom, K., Mancall, E., and Richardson, E. (1958). Progressive multifocal leukoencephalopathy. Brain 81, 93-110. Ault, G., and Stoner, G. (1994). Brain and kidney of progressive multifocal leukoencephalopathy patients contain identical rearrangements of the JC virus promoter / enhancer. /. Med. Virol. 44,298-304. Bastian, R (1971). Papova-like virus particles in a human brain tumor. Lab. Invest 25,169-175. Bergsagel, D., Finegold, M., Butel, J., Kupsky, W., and Garcea, R. (1992). DNA sequences similar to those of simian virus 40 in ependymomas and choroid plexus tumors of childhood. New Engl. J. Med. 326, 988-993. Bolton, C , and Rozdilsky, B. (1971). Primary progressive multifocal leukoencephalopathy: A case report. Neurology 21, 72-77. Carbone, M., Rizzo, P., Grimley, P., Procopio, A., Mew, D., Shridhar, v., de Bartolomeis, A., Esposito, V., Giuliano, M., Steinberg, S., Levine, A., Giordano, A., and Pass, H. (1997). Simian virus-40 large-T antigen binds p53 in human mesotheliomas. Nature Med. 3, 908-912. Castaigne, P. (1965). La leucoencephalopathie multifocale progressive. Presse Med. 73,1167-1170. Chesters, P., Heritage, J., and McCance, D. (1983). Persistance of DNA sequences of BK virus and JC virus in normal human tissues and in diseased tissues. /. Infect. Dis. 147, 676-684. Coleman, D., Gardner, S., and Field, A. (1973). Human polyomavirus infection in renal allograft recipients. Br. Med. J. 3, 371-375. Coleman, D., Russell, W., Hodgson, J., Pe, T., and Mowbray J. (1977). Human papovavirus in Papanicolaou smears of urinary sediment detected by transmission electron microscopy. /. Clin. Pathol. 30, 1015-1020. Coleman, D., Wolfendale, M., Daniel, R., Dhanjal, N., Gardner, S., Gibson, P., and Field, A. (1980). Infectious diseases: A prospective study of human polyomavirus infection in pregnancy. /. Infect. Dis. 142,1-8. Gardner, S., Field, A., Coleman, D., and Hulme, B. (1971). New human papovavirus (B.K.) isolated from urine from renal transplantation. Lancet 1,1253-1257.

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Gribble, D., Haden, C , Schwartz, L., and Henrickson, R. (1975). Spontaneous progressive multifocal leukoencephalopathy (PML) in macaques. Nature 254, 602-604. Hedley-Whyte, E., Smith, B., Tyler, H., and Peterson, W. (1966). Multifocal leukoencephalopathy with remission and five year survival. /. Neuropathol. Exp. Neurol. 25,107-116. Holmberg, C , Gribble, D., Takemoto, K., Howley, P., Espana, C , and Osburn, B. (1977). Isolation of simian virus 40 from Rhesus monkeys (Macaca mulatta) with spontaneous progressive multifocal leukoencephalopathy. /. Infect. Dis. 136, 593-596. Houff, S., Major, E., Katz, D., Kufta, C , Sever, J., Pittaluga, S., Roberts, J., Gitt, J., Saini, N., and Lux, W. (1988). Involvement of JC virusinfected mononuclear cells from the bone marrow and spleen in the pathogenesis of progressive multifocal leukoencephalopathy. New Engl. J. Med. 318, 301-305. Manz, H., Dinsdale, H., and Morrin, P. (1971). Progressive multifocal leukoencephalopathy after renal transplantation: Demonstration of papova-like virions. Ann. Intern. Med. 75, 77-81. Markowitz, R., Thompson, H., Mueller, J., Cohen, J., and Dynan, W. (1993). Incidence of BK virus and JC virus viruria in human immunodeficiency virus-infected and -uninfected subjects. /. Infect. Dis. 167, 13-20. Martin, J., and Banker, B. (1969). Subacute multifocal leukoencephalopathy with widespread intranuclear inclusions. Arch. Neurol. 21, 590-602. Narayan, O., Penney Jr., J., Johnson, R., Herndon, R., and Weiner, L. (1973). Etiology of progressive multifocal leukoencephalopathy: Identification of papovavirus. New Engl J. Med. 289,1278-1282. O'Reilly, R., Lee, F., Grossbard, E., Kapoor, N., Kirkpatrick, D., Dinsmore, R., Stutzer, C , Shah, K., and Nahmias, A. (1981). Papovavirus excretion following marrow transplantation: Incidence and association with hepatic dysfunction. Transplant. Proc. 13, 262-266. Padgett, B., Walker, D., ZuRhein, G., and Eckroade, R. (1971). Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet 1,1257-1260. Padgett, B., Walker, D., ZuRhein, G., Hodach, A., and Chou, S. (1976). JC papovavirus in progressive multifocal leukoencephalopathy. /. Infect. Dis. 133, 686-690. Pappo, O., Demetris, A., Raikow, R., and Randhawa, P. (1996). Human polyoma virus infection of renal allografts: Histopathologic diagnosis, clinical significance, and literature review. Mod. Pathol. 9,105-109. Rosen, S., Harmon, W, Krensky, A., Edelson, P., Padgett, B., Grinnell, B., Rubino, M., and Walker, D. (1983). Tubulo-interstitial nephritis associated with polyomavirus (BK type) infection. New Engl. J. Med. 308,1192-1196. Sangalang, V., and Embil, J. (1982). Recovery of papovavirus in cell culture explants of brain tissue from case of progressive multifocal leukoencephalopathy. Lancet 2, 329-330. Shah, K., and Nathanson, N. (1976). Human exposure to SV40: Review and comment. Am. J. Epidemiol. 103,1-12. Shinohara, T., Matsuda, M., Cheng, S., Marshall, J., Fujita, M., and Nagashima, K. (1993). BK virus infection of the human urinary tract. /. Med. Virol. 41, 301-305. Stoner, G., Ryschkewitsch, C , Walker, D., and Webster, H. (1986). JC papovavirus large tumor (T)-antigen expression in brain tissue of acquired immune deficiency syndrome (AIDS) and non-AIDS patients with progressive multifocal leukoencephalopathy. Proc. Natl. Acad. Sci. U.S.A. 83, 2271-2275.

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Sundsfjord, A., Flaegstad, T., Flo, R., Spein, A., Pedersen, M., Permin, H., Julsrud, J., and Traavik, T. (1994). BK and JC viruses inhuman immunodeficiency virus type 1-infected persons: Prevalence, excretion, viremia, and viral regulatory regions. /. Infect. Dis. 169,485^90. Vallbracht, A., Lohler, J., Gossmann, J., Gluck, T., Petersen, D., Gerth, H., Gencic, M., and Dorries, K. (1993). Disseminated BK type polyomavirus infection in an AIDS patient associated with central nervous system disease. Am. J. Pathol. 143, 29-39. Walker, D. (1978). Progressive multifocal leukoencephalopathy: An opportunistic viral infection of the central nervous system. In ''Handbook of Clinical Neurology" (P. Vinken and G. Bruyn, eds.). Vol. 34, pp. 307-329. North-Holland, Amsterdam.

Watanabe, I., and Preskorn, S. (1976). Virus-cell interaction in oligodendroglia, astroglia and phagocyte in progressive multifocal leukoencephalopathy: An electron microscopic study Acta Neuropath. 36,101-115. Weiner, L., Herndon, R., Narayan, O., Johnson, R., Shah, K., Rubinstein, L., Preziosi, T., and Conley, R (1972). Isolation of virus related to SV40 from patients with progressive multifocal leukoencephalopathy New Engl. J. Med. 286, 385-431. ZuRhein, G., and Chou, S. (1965). Particles resembling papovaviruses in human cerebral demyelinating disease. Science 148,1477-1479.

C H A P T E R

23 Parvoviruses INTRODUCTION 335 JOINT DISEASE 337 ERYTHROPOIETIC SYSTEMIC DISEASE INFECTIONS IN PREGNANCY 339 INFLAMMATORY LESIONS 340 TISSUE DIAGNOSIS 340 REFERENCES 340

blocks required for replication. Thus, they tend to parasitize rapidly replicating cells such as the precursors of the erythroid elements in the bone marrow. As a family, the parvoviruses infect a diversity of plants and animals, and some are parasites of other much larger viruses, such as the adenovirus-associated viruses (a virus that infects another more complex virus but is not a pathogen for humans). Only one serotype, termed B19, has thus far been found to infect humans with resulting disease, an observation first reported by Cossart et at. (1975). Adenovirus-associated viruses are poorly understood agents that were once recovered from a few patients with rheumatoid arthritis. They are believed to infect humans (Simpson et al, 1984), but there is no known accompanying disease. Other parvoviruses of lesser mammalian species go by the terms rat virus, minute virus of mice, and the virus of Aleutian mink disease (Porter, 1986). These agents have been the subject of considerable basic research, but this experimental work at present provides little insight into the pathogenesis of parvovirus disease in humans (Margolis and Kilham, 1970). Human parvovirus B19, appears to infect persons of all ages. Community outbreaks tend to erupt in late winter and spring, but infections are known to occur sporadically throughout the year. Because it has been difficult to conduct traditional virological studies on large population groups due to the unavailability (until recently) of susceptible cell cultures, detailed epidemiological information is still lacking. Seroepidemiological studies have documented infection in about 2 to 15% of infants under the age of 5 years. Roughly 15 to 60% of children and adolescents (aged 5 to 19 years) are seropositive, and 30 to 60% of adults have evidence of infection in the distant past. Thus, at any one time, abundant numbers of susceptibles of all ages are found in the general population. Much of what we know about the epidemiology of this virus is derived from studies in North America and Europe; little information is available from developing countries. Human volunteer studies at the Common Cold Research Unit in the United Kingdom have provided

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INTRODUCTION The parvovirus strain B19 is a relatively new addition to the diverse family of viruses causing erythematous rashes. Erythema infectiosum (so-called fifth disease) was first established as a clinical syndrome by Herrick in 1962. The rash appears on the cheeks, and progresses over a short period to involve the limbs and trunk. The transient skin lesions are described as lacy or reticular. In young women (but rarely in men), a symmetrical polyarthritis primarily affecting the peripheral joints develops shortly after the appearance of the rash and commonly disappears without complications within a few days. Parvoviruses would be of little further interest to those of us who do not practice office pediatrics if it were not for the unique capacity of the so-called B19 strain of the virus to infect red blood cell precursors in the bone marrow. As a result, the virus causes erythroblastic crises in patients whose erythron has been stimulated by hemolytic anemia. It is also responsible for fetal loss with hydrops fetalis when infection occurs in utero. With the advent of chemotherapy and AIDS, parvovirus B19 has also been recognized as a cause of anemia in an occasional immunocompromised patient. This chapter provides an overview of our current understanding of this interesting virus and the diseases attributed to it. The family Parvoviridae comprises a large number of nonenveloped agents made up of a single-stranded DNA genome surrounded by a capsid comprised of protein capsomeres arranged in an icosahedral symmetry. Parvoviruses are the smallest of the human DNA pathogens and lack many of the biochemical building PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

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insightful perspectives into the natural-occurring infection in the immunologically naive adult (Anderson et al, 1984). Figure 23.1 depicts the major parameters of illness in a middle-aged woman inoculated intranasally with approximately 100 infectious viruses present in a serum specimen from a naturally infected blood donor. Both the virus and its DN A were detected in the blood of this volunteer 6 days after inoculation, accompanied by mild systemic symptoms including fever, but a rash was not evident. Neutropenia, lymphopenia, and thrombocytopenia occurred shortly thereafter. Of particular note was the decrease in reticulocytes in the blood followed by a gradual reduction in the hemoglobin concentration. On day 15, itching first was noted, and shortly thereafter a maculopapillary rash developed. Symmetrical arthralgias and arthritis of the smaller joints of the extremities then appeared and persisted for a period of about 3 weeks. At this time, fever was no longer present. As might be expected, virus-

specific IgM antibodies developed during the second week after inoculation and IgG antibody was detected in the blood a few days later. This experiment reproduced dramatically the syndrome of erythema infectiosum so amply documented clinically in association with parvovirus B19 infection (Plummer et al, 1985; Anderson et al, 1984). However, the work failed to establish the pathogenesis of the salient clinical features of the illness. Assays for interferon in the blood were consistently negative, and IgM immune complexes were present transiently during the time of maximum symptoms, but they were not detected during periods when the rash and arthritis were present. Thus, symptomatology attributable to circulating interferon or immune complexes would not appear to account for the systemic symptoms, rash, and arthralgias that accompanied this parvovirus infection. The pathogenesis of the rash of erythema infectiosum is not understood. Schwarz et al (1994) detected

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FIGURE 23.1 Features of primary parvovirus infection in a seronegative volunteer inoculated intranasally on day 0. Reprinted with permission from Anderson et al. (1985).

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viral capsid proteins by immunofluorescence and by DNA in situ hybridization in cells of the stratum basalis of the erythematous skin in a biopsy from a 5-year-old tot with typical lesions. This interesting finding has not been confirmed, and its relevance to our understanding of the skin lesions is uncertain. Experiments in animals strongly suggest that endothelial cells may be infected by parvoviruses, a finding of some possible relevance to our understanding (Margolis and Kilham, 1975), but replication of parvoviruses in the endothelial cells of human tissues has yet to be established.

JOINT DISEASE During and after an episode of erythema infectiosum, a few (i.e., less than 10%) children and adolescents experience arthralgias and arthritis. More than 75% of adults have joint symptoms, but females are more commonly affected than males (Ager et al, 1966; Anderson et al, 1984). In general, the clinical syndrome in these patients does not simulate rheumatoid arthritis, and the traditional serological markers of rheumatic disease (rheumatoid factor, latex agglutinins, and antinuclear antibody) are not found in the blood, although exceptions have been reported (Luzzi et al., 1985). Surveys of patients in general rheumatology clinics have been carried out in an effort to assess the prevalence of parvovirus B19 infections among those presenting with acute-onset arthralgias and arthritis. Elevated serum concentrations of virus-specific IgM antibody were employed as a marker. In one study, 19 of some 153 patients so affected exhibited evidence of recent parvovirus infection. Most of the symptoms in these patients improved within weeks, but several had persistent complaints for more than 2 years and three patients were symptomatic for more than 4 years (White et ah, 1985). In many of these patients, a rash preceded the onset of joint symptoms (Reid et al, 1985; Naides et al, 1990). The pathogenic mechanisms involved in parvovirus arthritis are unknown. One would anticipate that the joint spaces are seeded during viremia, and virus replication might be expected to occur in synovial tissue, but we just don't know! Because of the relatively brisk antibody response to these viruses and the high concentrations of viral DNA detected in the blood, immune complexes might serve as a means whereby arthritis could develop, but as noted above, clinical and experimental evidence supporting this conjecture is lacking.

ERYTHROPOIETIC SYSTEMIC DISEASE An infectious etiology for transient aplastic crises in children with sickle cell disease was first suggested by the occurrence of outbreaks among members of families with this heritable disease (Pattison et al, 1981). Moreover, the observation that a crisis of this type only occurs in the same patient on a single occasion (Rao et al, 1992) indicated that the first attack conferred protection, which we now know is the result of acquired immunity. The discovery that these episodes of erythroid aplasia were due to an obscure parvovirus was a startling revelation to hematologists. Because of its small size and limited informational capacity of its DNA, parvovirus B19 preys upon the synthetic tools of the host cell to support its replication (Ozawa et al, 1986). No patient could be more susceptible than one with hemolytic anemia in which a high turnover of erythroid cells is due to a hemoglobinopathy or a heritable defect of the erythrocyte (such as spherocytosis or glucose-6PD deficiency). Erythroid and burst colony-forming units as well as proerythroblasts entering the S phase prove to be the target cells because they are endowed with a specific virus receptor, the P membrane globoside that confers susceptibility (Young, 1988). As a result, the replicative capability of the cell is destroyed (Figure 23.2), resulting in a transient aplastic state characterized by the abrupt onset of anemia and reticulocytopenia. Because

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Parvovirus B19 (particles/plate) F I G U R E 23.2 Erythroid colony formation is completely inhibited by parvovirus B19 infection in bone marrow specimens from normal controls with the P erythrocyte phenotype. In contrast, replication of erythroid elements with the p phenotype is not impeded. Reprinted with permission from Brown et al. (1994).

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these patients are so dependent upon the accelerated synthesis of new erythroid elements, anemia develops rapidly due to the combined effects of acute bone marrow depression and hemolysis. It has been suggested that the severity of hemolytic disease correlates with parvovirus B19 attack rate, thus accounting for the relatively high prevalence of the condition in patients with sickle cell anemia (SS hemaglobinopathy). In a study conducted over a 7-year period at a large urban hospital, 68% of children and adolescents with SS disease developed an aplastic crisis accompanied by evidence of B19 parvovirus infection (Rao et al, 1992). As noted above, the specific P blood group globoside was recently found to be the specific cell receptor for B19 parvovirus on erythrocyte precursors, and presumably on other target cells. In actuality, there are three P antigens (P, P^ and P^), but for practical purposes the dominantly inherited P is the critical factor, since P^ is extremely rare, except in Scandinavia. The erythroid precursors of the cells that lack P (that is, P genotype), are resistant to parvovirus infection, as shown in Figure 23.2. Persons lacking P consistently possess no circulating antibody to B19 parvovirus and thus appear to be completely resistant to infection (Brown et al, 1994). The temporal association of transient aplastic crises with erythema infectiosum has been documented in individual case reports. Figure 23.3 depicts the epidemic curve of a community outbreak of the acute erythematous skin disease followed temporally by aplastic crises among patients with SS disease (Chorba et al, 1986). The erythematous rash is often difficult to detect in black patients. Thus, artefactually, the aplastic crises developing in some of these patients appear to be spontaneous, and unrelated to the occurrence of

erythema infectiosum. Since those with hemolytic disease are often recipients of blood transfusions, exogenous blood is a likely source of infection in some patients (Cossart et al, 1975). The bone marrow of patients with aplastic crisis due to parvovirus is typically hypoplastic and, on rare occasions, exhibits variable degrees of necrosis. The pathognomonic morphological feature of the infection, however, is an enlarged dysplastic erythrocyte precursor cell having a diffuse amphophilic nuclear inclusion lacking a halo (Figure 23.4). These cells are said to have a megaloblastoid appearance. Krause et al (1992) suggested that formalin fixation of both the bone marrow smears and biopsies accentuates the appearance of inclusions in cells. As would be expected, the serum ironbinding capacity and saturation are increased in patients with aplastic disease (Figures 23.5 and 23.6A,B).

F I G U R E 23.4 Aspiration smear of bone marrow showing the giant proerythroblasts exhibiting an intranuclear inclusion. The marrow aspirate shows erythroid hypoplasia and these scattered bazaar erythroid precursor cells. Wright's stain. Reprinted with permission from Schwartz et al. (1991).

Months

F I G U R E 23.3 Temporal association of community outbreak of erythema infectiosum with the subsequent appearance of new cases of aplastic crises. Adapted with permission from Chorba et al. (1986).

FIGUfRE 23.5 Bone marrow biopsy specimen. Clusters of erythroid cells with distinct intranuclear inclusions are seen. Reprinted with permission from Krause et al. (1992) and through the courtesy of J. Krause, MD.

Parvoviruses

339

B

FIGURE 23.6 Formalin-fixed air-dried smear of an aspirate of bone marrow showing erythroblastic cells bearing intranuclear inclusions. Reprinted with permission from Krause et ah (1992) and through the courtesy of J. Krause, MD.

Chronic parvovirus B19 infections with varying degrees of erythroid depression have been reported in immunologically intact persons, as well as in patients with a variety of aberrations of cellular and humoral immune responsivity (Faden et ah, 1992; Kurtzman et al, 1987, 1989; Graeve et a/., 1989). Case reports document parvovirus B19 infections in patients with AIDS accompanied by chronic anemia (Frickhofen et ah, 1990; Bowman et ah, 1990; Griffin et ah, 1991). However, surveys fail to indicate that this virus is a common cause of the anemia that afflicts about 80% of those with AIDS (van Elsacker-Niele et ah, 1996; Chernak et ah, 1995). Defects in myelopoiesis and thrombocytopoiesis have been reported in some cases. Some incomplete evidence suggests that the progenitors of these blood elements may also be infected, but the clinical evidence is incomplete. It should be recalled that neutrophil depression and thrombocytopenia were noted in the human volunteer studies described above (Anderson et ah, 1985) (Figure 23.1).

INFECTIONS IN PREGNANCY Parvovirus infections of pregnant domestic and experimentally infected animals are believed to result in congenital anomalies and stillbirths (Margolis and Kilham, 1975). Hartwig and his colleagues (1989) demonstrated parvovirus B19 DNA in the tissues of a human abortus in which developmental abnormalities of the eye were noted and a vasculitis was found in several organs. Maternal B19 infections in early pregnancy are believed to result in death of the conceptus (Woernle et ah, 1987; Kinney et ah, 1988; Hall and Cohen, 1990; Garcia-Tapia et ah, 1995; Gratacos et ah, 1995). It is

somewhat surprising that intrauterine parvovirus B19 infections have not been accompanied more frequently with teratological catastrophes in view of the predisposition of parvovirus B19 for rapidly multiplying cells. The association of hydrops fetalis with fetal parvovirus infection was first demonstrated by Brown et ah (1984). The actual prevalence is low. Only two cases are known to have occurred in Scotland during a parvovirus epidemic affecting some 500,000 persons (Anand et ah, 1987), whereas an incidence of 1.7% was reported among 60 pregnant women with serologically established infections occurring in an epidemic in Spain (Gratacos et ah, 1995). On the other hand, there was a recorded incidence of 14% among 197 cases of hydrops attributed to fetal anemia (Machin, 1989). And, 5 cases of parvovirus infection were found among 32 autopsies of hydropic stillbirths (16%) at a major urban obstetrical hospital in the United States. This latter study employed the diagnostic approach of searching for inclusions in postmortem material by light microscopy (Rogers et ah, 1993). Profound anemia due to virus replication in the fetal erythrocyte progenitor cells is the fundamental pathogenic mechanism involved in the development of hydrops fetalis, but the virus apparently also grows in the fetal heart and liver. Vasculitis is also described in fetal tissue. A high-output heart failure develops as a consequence of anemia, but the process may be compounded in some cases by myocarditis and hypoalbuminemia due to liver disease. The end result is collection of massive amounts of fluid in the tissues and the placenta of the maturing fetus. At autopsy, the tissues of these stillbirths typically reveal prominent numbers of erythroblastic cells with inclusions in the vasculature of the placenta and major organs (Figures 23.7 and 23.8). The liver can show evidence of parenchymal cell

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Pathology and Pathogenesis of Human Viral Disease

FIGURE 23.7 A nucleated red blood cell within the vessel of a placental villus shows the characteristic dark-staining intranuclear inclusion characteristic of parvovirus. Reprinted with permission and through the courtesy of Brenda Waters, MD.

FIGURE 23.8 Even in autolyzed tissue such as this spleen, the dark staining cells infected with parvovirus are recognizable. Reprinted with permission and through the courtesy of Brenda Waters, MD.

involvement by the virus, but characteristically, it exhibits massive extrameduUary hematopoiesis and prominent deposits of hemosiderin in Kupffer cells and hepatocytes. The latter finding suggests that hemolysis occurs during the acute infection, but this is not documented.

istry facilitates diagnosis in tissue sections but does not appear to be more sensitive than careful microscopical examination of routinely stained tissue sections for inclusion-bearing cells. Transmission electron microscopy employing traditional methodology readily demonstrates characteristic virions in the nuclear inclusions, but the technique is time-consuming, since infected cells must be found by the microscopist. Negative stain electron microscopic detection of virions in the blood serum of infected children has been effectively employed, but considerable technical skill and painstaking effort is required. Serum assays for IgM antibody is the most useful rapid diagnostic approach for evaluation of infection in cases of aplastic crises. Rapid diagnosis for these patients is important since treatment with virus-specific immunoglobulin may prove effective. The isolation of virus from tissues is not a practical approach because sensitive cell culture methodologies applicable to routine studies have not yet been developed.

INFLAMMATORY LESIONS On rare occasions, myopericarditis, aseptic meningitis, and encephalitis are associated with acute parvovirus infections in children and adults (Enders et ah, 1998). Scattered reports claim an association of vasculitis (i.e., giant cell vasculitis, polyarteritis nodosa, and a leukoclastic vasculitis) with parvovirus B19 infections (Torok et ah, 1992; Stand and Corman, 1996; Cooper and Choudri, 1998). It is possible that immune complexes contribute to the development of the vascular lesions in such cases if they are, in fact, virus related (Garcia-Tapia et al, 1995). Thus far, detailed pathological studies are not reported, and virological evaluation of the involved organs has been limited (Cassinotti et ah, 1993; Okumura and Ichikawa, 1993; Watanabe et al., 1994; Saint-Martin et al, 1990; Koduri and Naides, 1995; Chia and Jackson, 1996).

TISSUE D I A G N O S I S Immunohistochemistry, in situ hybridization, and the polymerase chain reaction are now commonly used sensitive approaches for the detection of virus in tissues (Porter et al, 1988; Schwarz et al, 1991, 1992; Torok, 1992; Goldstein et al, 1995). Immunohistochem-

References Ager, E., Chin, T., and Poland, J. (1966). Epidemic erythema infectiosum. New Engl. J. Med. 275,1326-1331. Anand, A., Gray, E., Brown, T., Clewley J., and Cohen, B. (1987). Human parvovirus infection in pregnancy and hydrops fetalis. New Engl ]. Med. 316,183-186. Anderson, M., Lewis, E., Kidd, I., Hall, S., and Cohen, B. (1984). An outbreak of erythema infectiosum associated with human parvovirus infection. /. Hyg. (Cambridge) 93, 85-93. Anderson, M., Higgins, P., Davis, L., Willman, J., Jones, S., Kidd, I., Pattison, J., and Tyrrell, D. (1985). Experimental parvoviral infection in humans. /. Infect Dis. 152, 257-265. Bowman, C , Cohen, B., Norfolk, D., and Lacy C. (1990). Red cell aplasia associated with human parvovirus B19 and HIV infection: Failure to respond clinically to intravenous immunoglobulin. A/DS 4,1038-1039.

Parvoviruses Brown, T, Anand, A., Ritchie, L., Clewley, J., and Reid, T. (1984). Intrauterine parvovirus infection associated with hydrops fetalis. Lancet 2, 1033-1034. Brown, K., Hibbs, J., Gallinella, G., Anderson, S., Lehman, E., McCarthy, R, and Young, N. (1994). Resistance to parvovirus B19 infection due to lack of virus receptor (erythrocyte P antigen). New Engl J. Med. 330,1192-1196. Cassinotti, R, Schultze, D., Schlageter, P., Chevili, S., and Siegl, G. (1993). Persistent human parvovirus B19 infection following an acute infection with meningitis in an immunocompetent patient. Eur. J. Clin. Microhiol. Infect. Dis. 12, 701-704. Chernak, E., Dubin, G., Henry, D., Naides, S., Hodinka, R., MacGregor, R., and Friedman, H. (1995). Infection due to parvovirus B19 in patients infected with human immunodeficiency virus. Clin. Infect. Dis. 20,170-173. Chia, J., and Jackson, B. (1996). Myocarditis due to parvovirus B19 in an adult. Clin. Infect. Dis. 23, 200-201. Chorba, T., Coccia, P., Holman, R., Tattersall, P., Anderson, L., Sudman, J., Young, N., Kurczynski, E., Saarinen, U., Moir, R., Lawrence, D., Jason, J., and Evatt, B. (1986). The role of parvovirus B19 in aplastic crisis and erythema infectiosum (fifth disease). /, Infect. Dis. 154, 383-392. Cooper, C , and Choudhri, S. (1998). Diagnosis: Leukocytoclastic vasculitis secondary to parvovirus B19 infection. Clin. Infect. Dis. 26, 989. Cossart, Y, Cant, B., Field, A., and Widdows, D. (1975). Parvoviruslike particles in human sera. Lancet 1, 72-73. Enders, G., Dotsch, J., Bauer, J., Nutzenadel, W., Hengel, H., Haffner, D., Schalasta, G., Searle, K., and Brown, K. (1998). Life-threatening parvovirus B19-associated myocarditis and cardiac transplantation as possible therapy: Two case reports. Clin. Infect. Dis. 26, 355-358. Faden, H., Gary Jr., G., and Anderson, L. (1992). Chronic parvovirus infection in a presumably immunologically healthy woman. Clin. Infect. Dis. 15, 595-597. Frickhofen, N., Abkowitz, J., Safford, M., et al. (1990). Persistent B19 parvovirus infection in patients infected with human immunodeficiency virus type 1 (HIV-1): A treatable cause of anemia in AIDS. Ann. Intern. Med. 113, 926-932. Garcia-Tapia, A., del Alamo, C , Martinez-Rodriguez, A., Martin-Reina, M., Lopez-Caparros, R., Caliz, R., Caballero, M., and Bascunana, A. (1995). Spectrum of parvovirus B19 infection: Analysis of an outbreak of 43 cases in Cadiz, Spain. Clin. Infect. Dis. 21, 1424-1430. Goldstein, L., Strenger, R., King, T., Le, S., and Rogers, B. (1995). Retrospective diagnosis of sickle cell-hemoglobin C disease and parvovirus infection by molecular DNA analysis of postmortem tissue. Hum. Pathol. 26,1375-1378. Graeve, J., de Alarcon, P., and Naides, S. (1989). Parvovirus B19 infection in patients receiving cancer chemotherapy: The expanding spectrum of disease. Am. ]. Fed. Hematol.lOncol. 11, 441-444. Gratacos, E., Torres, P.-J., Vidal, J., Antolin, E., Costa, J., Jimenez de Anta, M., Cararach, V., Alonso, R, and Fortuny A. (1995). The incidence of human parvovirus B19 infection during pregnancy and its impact on perinatal outcome. /. Infect. Dis. 171,1360-1363. Griffin, T., Squires, J., Timmons, C , and Buchanan, G. (1991). Chronic human parvovirus B19-induced erythroid hypoplasia as the initial manifestation of human immunodeficiency virus infection. /. Pediatr. 118, 899-901. Hall, S. and Cohen, B. (1990). Prospective study of human parvovirus B19 infection with pregnancy. Br. Med. J. 300,1166-1170.

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Hartwig, N., Vermeij-Keers, C , Van Elsacker-Niele, A., and Fleuren, G. (1989). Embryonic malformations in a case of intrauterine parvovirus B19 infection. Teratology 39, 295-302. Herrick, T. (1962). Erythema infectiosum: Clinical report of 74 cases. Am. ]. Dis. Child. 31, 486^95. Kinney, J., Anderson, L., Farrar, J., et al. (1988). Risk of adverse outcomes of pregnancy after human parvovirus B19 infection. /. Infect. Dis. 157, 663-667. Koduri, P., and Naides, S. (1995). Aseptic meningitis caused by parvovirus B19 [brief report]. Clin. Infect. Dis. 21,1053. Krause, J., Penchansky, L., and Knisely, A. (1992). Morphological diagnosis of parvovirus B19 infection: A cytopathic effect easily recognized in air-dried, formalin-fixed bone marrow smears stained with hematoxylin-eosin or Wright-Giemsa. Arch. Pathol. Lab. Med. 116,178-180. Kurtzman, G., Ozawa, K., Cohen, B., Hanson, G., Oseas, R., and Young, N. (1987). Chronic bone marrow failure due to persistent B19 parvovirus infection. New Engl. ]. Med. 317, 287-294. Kurtzman, G., Frickhofen, N., Kimball, J., Jenkins, D., Nienhuis, A., and Young, N. (1989). Pure red-cell aplasia of 10 years' duration due to persistent parvovirus B19 infection and its cure with immunoglobulin therapy. New Engl. J. Med. 321, 519-523. Luzzi, G., Kurtz, J., and Chapel, H. (1985). Human parvovirus arthropathy and rheumatoid factor. Lancet, May 25, p. 1218. Machin, G. (1989). Hydrops revisited: Literature review of 1,414 cases published in the 1980s. Am. J. Med. Genet. 34, 366-390. Margolis, G., and Kilham, L. (1970). Parvovirus infections, vascular endothelium, and hemorrhagic encephalopathy. Lah. Invest. 22, 478-488. Margolis, G., and Kilham, L. (1975). Problems of human concern arising from animal models of intrauterine and neonatal infections due to viruses: A review, II: Pathological studies. Prog. Med. Virol. 20, 144-179. Naides, S., Scharosch, L., Foto, R, and Howard, E. (1990). Rheumatologic manifestations of human parvovirus B19 infection in adults: Initial two-year clinical experience. Arthr. and Rheum. 33, 1297-1309. Okumura, A., and Ichikawa, T. (1993). Aseptic meningitis caused by human parvovirus B19. Arch. Dis. Child. 68, 784-785. Ozawa, K., Kurtzman, G., and Young, N. (1986). Replication of the B19 parvovirus in human bone marrow cell cultures. Science 233, 883-886. Pattison, J., Jones, S., Hodgson, J., Davis, L., White, J., Stroud, C , and Murtaza, L. (1981). Parvovirus infections and hypoplastic crisis in sickle cell anemia. Lancet 1, 664-665. Plummer, R, Hammond, G., Forward, K., Sekla, L., Thompson, L., Jones, S., Kidd, I., and Anderson, M. (1985). An erythema infectiosum-like illness caused by human parvovirus infection. New Engl. J. Med. 313, 74-79. Porter, D. (1986). Aleutian disease: A persistent parvovirus infection of mink with a maximal but ineffective host humoral immune response. Prog. Med. Virol. 33, 42-60. Porter, H., Khong, T, Evans, M., et al. (1988). Parvovirus as a cause of hydrops fetalis: Detection by in situ DNA hybridization. /, Clin. Pathol. 41, 381-383. Rao, S., Miller, S., and Cohen, B. (1992). Transient aplastic crisis in patients with sickle cell disease: B19 parvovirus studies during a 7-year period. Am. ]. Dis. Child. 146,1328-1330. Reid, D., Brown, T., Reid, T., Rennie, J., and Eastmond, C. (1985). Human parvovirus-associated arthritis: A clinical and laboratory description. Lancet, February 23, pp. 422-425.

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Rogers, B., Mark, Y, and Oyer, C. (1993). Diagnosis and incidence of fetal parvovirus infection in an autopsy series, I: Histology. Ped. Pathol. 13, 371-379. Saint-Martin, J., Choulot, J., Bonnaud, E., and Marinet, R (1990). Myocarditis caused by parvovirus. /. Pediatr. 116,1007-1008. Schwarz, T., Nerlich, A., Hottentrager, B., Jager, G., Wiest, I., Kantimm, S., Roggendorf, H., Schultz, M., Cloning, K., Schramm, T., Holzgreve, W., and Roggendorf, M. (1991). Parvovirus B19 infection of the fetus: Histology and in situ hybridization. Am. J. Clin. Pathol. 96, 121-126. Schwarz, T., Jager, G., Holzgreve, W., and Roggendorf, M. (1992). Diagnosis of human parvovirus B19 infections by polymerase chain reaction. Scand. J. Infect. Dis. 24, 691-696. Schwarz, T, Wiersbitzky, S., and Pambor, M. (1994). Case Report: Detection of parvovirus B19 in a skin biopsy of a patient with erythema infectiosum. /. Med. Virol. 43,171-174. Simpson, R., McGinty, L., Simon, L., Smith, C , Godzeski, C., and Boyd, R. (1984). Association of parvoviruses with rheumatoid arthritis of humans. Science 223,1425-1428. Staud, R., and Gorman, L. (1996). Association of parvovirus B19 infection with giant cell arteritis. Clin. Infect. Dis. 22, 1123.

Torok, T. (1992). Parvovirus B19 and human disease. Adv. Int. Med. 37, 431-455. Torok, T., Wang, Q.-Y, Gary Jr., G., Yang, C.-R, Finch, T., and Anderson, L. (1992). Prenatal diagnosis of intrauterine infection with parvovirus B19 by the polymerase chain reaction technique. Clin. Infect. Dis. 14,149-155. van Elsacker-Niele, A., Kroon, R, van der Ende, M., Salimans, M., Spaan, W., and Kroes, A. (1996). Prevalence of parvovirus B19 infection in patients infected with human immunodeficiency virus. Clin. Infect. Dis. 23, 1255-1260. Watanabe, T., Satoh, M., and Oda, Y (1994). Human parvovirus B19 encephalopathy [letter]. Arch. Dis. Child. 70, 71. White, D., Mortimer, R, Blake, D., Woolf, A., Cohen, B., and Bacon, R (1985). Human parvovirus arthropathy. Lancet 1, 419-421. Woernle, C , Anderson, L., Tattersall, R, and Davison, J. (1987). Human parvovirus B19 infection during pregnancy. /, Infect. Dis. 156, 17-20. Young, N. (1988). Hematologic and hematopoietic consequences of B19 parvovirus infection. Sem. Hematol. 25,159-172.

C H A P T E R

24 Neurotropic ArthropodTransmitted Viruses INTRODUCTION

yield an enormous amount of information over time. Much of our current understanding of the ecology of mosquito-borne viruses and the diseases they cause in humans has accumulated by correlating the observations of field entomologists, who classify the mosquitos and characterize their habitat, with the outcome of these virological studies. Of course, this is only half the story, for establishing a virus isolate as the cause of a human disease proves to be an imposing challenge. As it turns out, only a small proportion of the countless different viruses recovered from wild-caught mosquitos play a role in disease. To decipher the ecology of the viruses of human importance and to relate them to the epidemiology of the disease occurring in the rare patient with meningoencephalitis is a demanding task. Subclinical infections tend to be the rule, and clinical disease the exception, thus confounding the problem of associating a virus with disease. The work in developed countries has progressed to the point where many potentially devastating outbreaks of infection are now averted, largely by interrupting the life cycle of the vector. Worldwide, arthropod-borne viruses are a significant cause of morbidity and mortality. Several dozen of these viruses proved to be the etiologic agents for specific illnesses, each with its own natural history and ecological niche. These viruses circulate in nature in biologic cycles that usually involve specific species of mosquito having unique habitat requirements, and one or more nonhuman vertebrate intermediate hosts. Thus, two viral replicative cycles occur in nature; one in the insect, and the second in a warm-blooded (e.g., a subhuman primate, rodent, or swine) or cold-blooded animal (e.g., birds or snakes). It is usually when we humans invade the natural habitat of the vector and are bitten that infection occurs. Thus, humans are an incidental "dead-end" host.

343

TOGAVIRUSES (ALPHAVIRUSES)

344

Eastern Equine Encephalitis (EEE) 346 Western Equine Encephalitis (WEE) 347 Venezuelan Equine Encephalitis (VEE) 348 FLAVIVIRUSES 349

St. Louis Encephalitis (SLE) 351 Japanese B Encephalitis (JBE) 352 Other Flavivirus Encephalitides 353 BUNYAVIRUSES 354

LaCrosse (California Encephalitis Group) 354 REOVIRUSES 354 REFERENCES 355

INTRODUCTION Pedro Galindo was a robust engaging man! Enthusiastic and energetic, he invariably radiated excitement when discussing the biology of the mosquito. He was also much more than an enthusiastic entomologist. Galindo was a "Depudado" in the national legislature of the Republic of Panama, where he lived and worked. Somehow, he balanced his life, dividing his time between politics and entomology. But, he seemed to have an enormous amount of time for his true love — the collection and speciation of the mosquitos in the canapes of the virgin forests of the Isthmus of Panama. Both Pedro and our lab benefitted. We helped to finance his expeditions and paid some of his field staff; in return, he brought us the carcasses of freshly caught mosquitos to be used in our attempts to isolate in the laboratory and then characterize the arthropod-borne viruses of the Panamanian jungles. Our work was deceptively simple. Groups of 20 or so field-caught mosquitos were homogenized in a mortar using a pestle, and the suspended tissue inoculated into the brains of newborn mice. These tiny animals were then monitored for illness, which occasionally proved to be due to a virus. This rather traditional approach employed in laboratories worldwide can PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

Members of four families of virus are responsible for the arthropod-transmitted meningoencephalitides of humans worldwide. When clinical disease occurs, the 343

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Pathology and Pathogenesis of Human Viral Disease

physician is only detecting the "tip of the iceberg/' for, beneath the "waterline," there exists a complex ecology in which humans play only a minor role. In general, these RNA viruses are highly mutable, and "wild" strains with a diversity of biological characteristics circulate in nature. Thus, the viruses humans confront may differ both in infectivity and pathogenicity over time and in different geographical locales. Based on analogies with experimental observations in laboratory animals, there is every reason to believe that the outcome of infection in humans is influenced by virological factors as well as genetically acquired and age-associated influences unique to the human host. For example, strains of wild-caught viruses of the same serotype differ substantially in their infectivity for laboratory mice, even though various virus isolates may be antigenically identical, and exhibit only the slightest dissimilarity from a molecular perspective. For unknown reasons in humans, some viruses cause devastating disease almost exclusively in children, whereas the elderly are more severely affected with others (see below). In endemic regions of virus dissemination, it is common for serum antibodies to be found in a proportion of the adult population. This indication of an earlier subclinical infection proves to be particularly the case where vectors breed with relative abandon such as in areas of Asia where Japanese B encephalitis is endemic. In this vast region, clinical infections tend to occur in children, most probably because they are highly susceptible, but also because a relatively large number of adults are immune. Contrariwise, along the Eastern Seaboard of the United States, relatively few adult members of the population possess serum antibodies to the virus of eastern equine encephalitis, most probably because it rarely circulates in an anthrophilic mosquito species. While devastating infections occur in young children, encephalitis is very uncommon in adults. Older persons may be intrinsically resistant, as proves to be the case when adult mice are inoculated experimentally with "wild" strains of many of the arthropod viruses. Three clinical forms of infection occur in exposed persons after incubation periods of usually less than a week: (a) asymptomatic subclinical infections; (b) febrile illnesses with or without systemic symptoms, such as headache, muscle aches, and pain; and (c) meningoencephalitis with or without focal neurological signs, ranging in severity from irritability to somnolence, to death. Clinical signs of meningeal inflammation are a variable component of the clinical illness, but meningitis usually is not a prominent feature of these diseases.

TOGAVIRUSES (ALFHAVIRUSES) The virus family Togaviridae derives its name from the morphologic features of the viral envelope, which reminded electron microscopists of the togas worn by citizens of Imperial Rome. The family is comprised of the alphaviruses, which are the subject of this section, and the rubiviruses, represented solely by rubellavirus (see Chapter 28). Over 25 different alphaviruses have been recovered from nature, but only a small number are human pathogens (Table 24.1). The genus alphavirus may be further divided into two subgenera based upon nucleotide sequence analysis and the diseases they cause. The first group includes the encephalitis viruses. In the second are categorized a group of exotic agents that cause nonfatal febrile arthropathies (Table 24.2). These agents will not be considered further in this book. The diseases they cause, while highly symptomatic, are relatively uncommon and geographically restricted. In addition, pathological information from humans is nonexistent and our knowledge of the pathogenesis of the arthropathy is limited. Togaviruses have a traditional structure in which a single strand of RNA is surrounded by a protein capsid comprised of 240 capsomeres arranged in icosahedral symmetry. In turn, the capsid shell is surrounded by a membrane comprised of a lipid bilayer of host cell origin in which are embedded two viral-encoded glycoproteins. These glycoproteins, termed El and E2, project from the envelope surface. Their biochemical composition plays an important role in viral pathogenicity since amino acid substitution at critical sites dramatically influences infectivity. The El and E2 proteins are the major antigens of the alphaviruses. Circulating immunoglobulins directed against viral glycoproteins are elaborated early in the course of infection. These antibodies appear to play an important role in viral clearance and protect against reinfection. The importance of cell-mediated immunity in alphavirus infections is not known. Alphavirus attachment to target cells reflects the interaction of the viral G2 glycoprotein with the plasma membrane. The virion is taken up by the cell and uncoated promptly, after which replication of the next generation of progeny virion begins. The viral RNA serves as the messenger. Production of viral components by the infected cell is associated with cytolysis. In animals, this appears to be the basis for the destructive effects of alphaviruses in tissue since immunopathologic process seem not to play a role. However, the generation of cytokines during the process of infection of viral target cells may serve to amplify the host response to infection and injury to tissue.

345

Neurotropic Arthropod-Transmitted Viruses TABLE 24.1 Neurotropic Arthropod-Borne Viruses

Genus

Reservoir and amplifying host

Vector

Endemic area for human disease

Alphaviruses Eastern equine encephalitis (EEE)

Culiceta melanuria Aedes sp.

Passerine birds

Eastern US & Gulf Seaboard

Western equine encephalitis (WEE)

Culex sp.

Birds; snakes

US & Canada; West of Mississippi River; Argentina

Horse

Caribbean Basin

Venezuelan equine encephalitis (VEE) Flaviviruses JB Complex St. Louis Encephalitis (SLE)

Culex sp.

Passerine birds

South central US; Florida; California

Japanese B Encephalitis (JBE)

Culex sp.

Pigs; Water birds

Maritime Southeast Asia; China; Japan; India, Indochina

West Nile Encephalitis (WNE)

Culex sp.

Birds; mammals

Egypt; Israel

Murray Valley Encephalitis (MVE)

Culex sp.

Water birds; small mammals

Eastern Australia

TBE Complex Central Europe/Russian Spring-Summer Encephalitis (RSSE)

Ixodid ticks

East Central Europe; East/Central/ West former USSR

Powassan

Ixodid ticks

Small field mammals

Russia; Canada; Northern US

Aedes triseriatus

Small field mammals

North Central US

Dermacentor andersonia

Various field mammals

Rocky Mountains, US; Great Basin, US

Bunyavirus (California serogroup viruses) LaCrosse (LAV) Reoviruses Colorado Tick Fever (CTF)

TABLE 24.2 Alphavirus Febrile Arthropathies

Disease

Geographic distribution

Vector

Chikungunya

Aedes aegypti Mansonia sp. Aedes aegypti person-to-person

O'nyong-nyong

Anophales sp.

East Africa

Igbo Ora

Anophales sp.

West Africa

Ross River New Guinea

Aedes sp.

East & North Australia

Alphaviruses are highly mutable, and subtle amino acid substitutions in the capsid glycoproteins appear to influence the outcome of infection. Wie know very little about the molecular make-up of the virions transmit-

sub-Saharan Africa Indian subcontinent Indochina

ted by the insect vector to the human host. Unfortunately, most experimental studies are carried out with laboratory-adapted strains of virus that have been passaged from animal to animal before investigative work

346 is undertaken. This allows for selection of numerous subtle mutations that most probably have no relevance to disease in humans. After an arthropod "bite," virus replicates at the local site, possibly in resident cells or inflammatory cells such as macrophages. Viremia then occurs. The virus concentration of the blood probably determines, at least in part, whether or not the central nervous system is infected. In the brain, alphaviruses appear to replicate in endothelial cells before they broach the vascular barrier that precludes their access to the rich source of target neurons and glial cells in the brain. Alphaviruses appear to be primarily neurotropic and disseminate throughout the brain with apparent ease, although we understand very little about the intracerebral events associated with infection. Experimentally, some viruses of this family are only infectious for young mice when inoculated subcutaneously or directly into the brain, whereas other viruses infect the central nervous system of animals of all ages, regardless of the route of inoculation. Major organs other than the brain do not develop significant lesions in most alphavirus infections. Venezuelan equine encephalitis virus is an exception, for it uniquely infects and destroys lymphoid and myeloid cells indiscriminately in its natural host, the horse, as well as in a variety of small experimental animals. Necrosis of lymphoid tissues is also reported in humans, but the information available from autopsies is limited.

1986

1987

198S

Eastern Equine Encephalitis (EEE)

FIGURE 24.1 Annual incidence (1985-94) of cases of EEE (A) and WEE (B) reported to the Centers for Disease Control and Prevention.

The initial outbreak of encephalitis known to be caused by EEE occurred in Massachusetts during the late summer of 1938. Two-thirds of the cases were children and 74% died (Feemster, 1958). Among the few survivors, severe neurological sequelae (mental retardation, seizure disorders, emotional lability, and impaired motor activity, speech, and hearing) proved to be common. Since that time, outbreaks of limited size have occurred along the Eastern Seaboard and the Gulf Coast, but, customarily, only sporadic cases are reported. Between 1955 and 1993, the U.S. Centers for Disease Control and Prevention documented 223 human cases of EEE in the United States. The high mortality among children has continued with a substantially reduced death rate among adults (Figure 24.1 A). In the freshwater wetlands of its endemic range, EEE transmission in nature appears to go unrecognized. Culiceta melanura is the major vector, and passerine birds are the amplifying host. Many of these familiar songbirds enjoy a winter vacation in South and Central America, where they most probably overwinter the virus. When virus activity in reservoirs reaches a threshold during the summer months in North America, species of the Aedes mosquito may also serve as vectors

transmitting the viruses to horses and to upland birds, such as the exquisitely susceptible pheasant. Only on occasion do humans intervene. Mammalian intermediate hosts are not known to be important reservoirs for EEE in nature. A strain of EEE virus that is less pathogenic than the North American virus circulates in the American tropics. A small number of healthy adult residents of these endemic regions possess serological evidence of past subclinical infection with this virus. A clinically severe encephalitis is the usual outcome of an EEE virus infection in children in North America. The pathological features of the acute disease have been elegantly described by many earlier authors (Farber et al, 1940; Wesselhoeft et al, 1938; Dent, 1955; Herzon et al, 1957; Haymaker, 1958a; Moulton, 1960; Jordan et al, 1965). The outstanding histopathologic feature in these cases is the presence of luxuriant focal polymorphonuclear leukocyte infiltrates extensively involving the brain (Figure 24.2A-D). Both the cerebral cortices and midbrain structures (thalamus, basal ganglia, substantia nigra, and pars basalis) are affected. Lesions in the cerebellum, brainstem, and spinal cord

Neurotropic Arthropod-Transmitted Viruses

347

B

-*/'/»

P'X;

^';^^^«ik^^

F I G U R E 24.2 Lesions in the brain of a young child infected with, and dying of, EEE. (A) Acute destructive encephalitis. Note the diffuse infiltrates of polymorphonuclear leukocytes. The severe widespread acute inflammation seen here is typical of EEE, but not of most other types of arthropod-borne virus encephalitis. (B) Glial nodule. (C) Perivascular cuff. (D) Thrombosed small vessels. Note the endothelial cell changes in the vessels and the scattered inflammatory cells.

are not prominent. Recently, magnetic resonance imaging has been used to document destructive inflammatory changes (Figure 24.3) (Deresiewicz et ah, 1997; Piliero et ah, 1994). Meningeal infiltrates and perivascular inflammatory cell "cuffs" are prominent in acute lesions. Electron microscopy of the acutely infected brain documents profound changes in neurons and glia, alterations also readily apparent at lower levels of resolution. In the studies of Kim and colleagues (1985), the so-called tubular reticular complexes (ultrastructural cellular alterations seen in HIV-1-infected cells and lupus erythematosus) were evident in macrophages. These cells seem to support virus replication. With the passage of time, the pathological picture changes with subsidence of the polymorphonuclear response, and the appearance of lymphocytes and macrophages in areas where destructive changes and rarefaction of brain substance are apparent. Western Equine Encephalitis (WEE) WEE was first isolated in California from a horse during an equine epizootic in 1930 and from a child with encephalitis in 1938. This interesting virus pos-

sesses molecular features of EEE, and antigenic determinants of Sindbis virus, an Old World alphavirus that apparently lacks the ability to cause central nervous system disease in humans. Thus, it has been hypothesized that WEE is a recombinant, reflecting a clandestine interaction of these two disparate viruses aeons in the past (Hahn et ah, 1988). WEE is endemic throughout the Midwestern United States and California (Figure 23.1B), where its vector is Culex tarsalis, a common mosquito of agricultural lands, and its intermediate amplifying hosts are birds and small animals, including snakes. In the American Mid- and Far West, enzootics in horses are common, with roughly 22 to 100 equine cases of encephalitis occurring for each clinical case in humans (Figure 24.IB). WEE is infectious for persons of all ages, but about 20% of cases are infants and small children (Cohen et al, 1953). As with other viral encephalitides, the signs and symptoms of nervous system involvement are variable and may be so mild as to be overlooked. The initial fever is followed by headache, and as the infection progresses, drowsiness, malaise, mental symptoms, and seizures intervene. Stupor and coma, followed by death in some cases, generally occurs 4 to 7

348

Pathology and Pathogenesis of Human Viral D i s e a s e

FIGURE 24.3 Lesions of the basal ganglia and cortex in a 14-year-old boy who died of EEE. Panel A shows an MRI scan taken 3 days after the onset of neurologic symptoms. Large asymmetric lesions are present in the caudate nuclei, putamen, and thalami (arrowheads). Panel B shows a CT scan taken 7 days after the onset of neurologic symptoms. Large lesions of the basal ganglia and thalami are again evident (outlined by arrowheads). There is diffuse swelling of the brain. Panel C shows an image from the same MRI as shown in A. Lesions are present in the medial temporal lobes (arrows) and right insula (not shown). Autopsy revealed diffuse encephalomalacia, marked perivascular chronic inflammatory changes, and focal intraparenchymal perivascular hemorrhage in the caudate nucleus and putamen. Several microglial nodules were evident. A necrotic and hemorrhagic area measuring 3 by 2 cm was present in the anterior portion of the right temporal lobe. Reprinted with permission from Deresiewicz et al. (1997).

days after the onset of symptoms. A variety of neurological signs are detected, but in general, the encephalitis is not as severe as in EEE (Baker, 1958b). WEE is less pathogenic than EEE, and thus has a substantially lower case:infection ratio (Reeves et al, 1962). Subclinical infections occur in more than 1000 adults for each clinical case of encephalitis. However, as with EEE, clinically inapparent infections are uncommon in infants and devastating life-threatening encephalitis often occurs. Findings at autopsy are limited to the central nervous system, where, in order of decreasing frequency, lesions are found in the globus pallidum, central cortex, thalamus, and pontine tegmentum (Haymaker, 1958b). Histologically, pathologists find a mixed infiltrate of polymorphonuclear cell, lymphocytes, and macrophages involving the meninges and perivascular spaces. Areas of rarefaction necrosis stud the brain, where reactive gliosis and variable numbers of inflammatory cells are often present. Customarily, encephalitis persists for 10 days and then subsides. The severity of the clinical sequelae is variable and related to age (Baker, 1958b). In infants, motor involvement, behavioral retardation, and seizure disorders occur (Finley, 1958). Similar sequelae are seen in older children and adults, but residual com-

plications are uncommon. Severe destructive changes may be seen morphologically in the brains of those who linger with significant disabilities after the acute stages of illness. Venezuelan Equine Encephalitis (VEE) Epizootics of VEE in horses, and concurrent epidemics in humans, occur periodically in northern South America, primarily Colombia and Venezuela. These outbreaks seem to develop along with heavy rains and flooding during the traditional dry season. Horses are the principal amplifying hosts because they sustain a prolonged high level of viremia and attract a variety of mosquitos. In addition, horse-to-horse contact transmission occurs (Kissling et al, 1956). Epidemiologically differences in the pathogenic properties of various "wild" viral strains may be a significant factor. Molecular analyses indicate that the virus responsible for some 1.3 X 10^ human infections in northwest Venezuela in 1994 is linked to a strain responsible for another larger epidemic in the same region some 20 years earlier in 1973 (MMWR, 1995; Sidwell et al, 1967). VEE readily circulates in horses as epidemics in northern South America, and variably "spills over"

Neurotropic Arthropod-Transmitted Viruses

into humans. When humans are infected, the disease is customarily manifest as a "flu-like" syndrome with fever, headache, muscle pain, and prostration. Evidence of neurological disease occurs predominantly in children and the elderly, with an overall attack rate of about 4%, and a case fatality rate of less than 1%. Deaths occur predominantly in children. Few autopsy reports of fatal human cases are published. Deaths among VEE-infected persons occur as a result of encephalitis or a fulminating lymphocytolytic reaction with disseminated intravascular coagulation (de la Monte ei al., 1984). Meningoencephalitis is the predominant lesion, with focal accumulations of a mixed inflammatory cell infiltrate in the meninges and brain. A necrotizing vasculitis is observed rarely. In most patients, there is striking necrosis of lymphoid cells in the lymph nodes and spleen. This unique lesion is also observed in horses and animals of several different species experimentally infected in the laboratory (Victor et al., 1956). Finally, widespread hepatocellular degeneration and necrotic changes are observed in over 60% of patients at autopsy, and interstitial pneumonia is seen in most patients.

FLAVIVIRUSES The features of the viruses of this large family were summarized in Chapter 19. In brief, the flaviviruses of

349

human importance are small RNA-containing agents having a nucleocapsid surrounded by a bilayer envelope derived from the host cell. The viral E protein embedded in the membrane is the major antigen. It confers antigenic identity to the virus and provokes both humoral and cell-mediated immune responses. The role of humoral antibody in viral clearance and protection against reinfection is well established, but the importance of cellular immunity is unclear. Flaviviruses appear to replicate at the local site of the insect "bite" before disseminating to regional lymph nodes and to various solid organs (heart, liver, pancreas), where a second stage of replication occurs. The subsequent viremia carries the agent to the central nervous system, where it may multiply in endothelial cells before gaining access to the brain. The mode by which the virus subsequently spreads in the central nervous system is unclear. Flaviviruses attack both glia and neurons. The virus destroys neurons by a pathological process that morphologically appears remarkably similar to events in the poliovirus-infected anterior horn cell (see Chapter 1). The earliest changes in the cells accompany viral maturation. Virion cores accumulate in the cytoplasm and appear to acquire their membrane by budding into the internal cisternae of the cell cytoplasm (Figure 24.4). Cell dissolution occurs 2 or more days after the cell sustains the initial infection (Murphy ei al., 1968). Centers in the brain differ with regard to their susceptibility to infection, and the spinal cord is rarely involved. It may be that the virus gains

F I G U R E 24.4 Enveloped virions in the cisterna of the endoplasmic reticulum of a neuron in the brain of an infant mouse infected with SLE. The arrowheads point out ribosomes and irregular particles of approximately the same size that are believed to be precursors of viral particles. Envelopment occurs when the precursor particles pass through the wall of the endoplasmic reticulum. Reprinted with permission from Murphy ei al. (1968).

350

Pathology and Pathogenesis of Human Viral Disease

access to the brain at multiple sites as a result of viremia. During the course of infection, mixed inflammatory cell responses are discerned at scattered sites in the brain by the pathologist. The infiltrate is comprised of lymphocytes, plasma cells, and macrophages. The prominent accumulations of polymorphonuclear leukocytes seen in EEE and, to a lesser extent in WEE-infected brains, are not observed. Many gaps in our understanding of the pathogenesis of flavivirus infections of the nervous system remain to be filled by the work of future investigators. Based on both molecular and antigenic similarities, the flaviviruses of human importance are categorized

into two groups. The first group, termed JE, incorporates major endemic viruses of five continents that are transmitted by culicine mosquitos, and use a variety of birds and mammals as intermediary and overwintering hosts (Table 24.1, Figure 24.5). Each of these viruses has the potential to cause encephalitis in humans, but they are also responsible for a large number of inapparent infections throughout endemic ranges. Viruses of the second group (TBE) are transmitted by the ixodid tick but employ mammals and birds to amplify and overwinter the virus. While these viruses cause encephalitis on occasion, disease of the central nervous system is an uncommon outcome (Table 24.1).

Birds and/or Mamrrrals

Dead-end Hosts

Migratory Birds, Bats

Maintenance Mechanisms F I G U R E 24.5 Generalized transmission cycle of mosquito-borne flaviviruses causing encephalitis, showing summertime amplification and presumptive overwintering mechanisms. Humans are "dead-end" hosts and do not perpetuate virus transmission. Vector species vary, but culicine mosquitos (principally Culex spp.) are responsible for the amplification cycles. Wild birds are the most important intermediate viremic hosts for most viruses because they sustain a viremia, but in the case of Japanese encephalitis domestic swine play an important role. The pattern shown here applies to St. Louis, Japanese, Murray Valley, and West Nile encephalitis viruses and possibly other flaviviruses, but with individual variations. Reprinted with permission from Monath and Heinz (1996).

Neurotropic Arthropod-Transmitted Viruses

St. Louis Encephalitis (SLE) Several late summer outbreaks in the American Midwest during the early 1930s established SLE as a major cause of arthropod-borne encephalitis among adults in North America. The virus has continued to plague residents of the southern and central states, posing an ever-present threat when weather conditions favor multiplication of its vector, the culicine mosquito (Figure 24.6). SLE virus is widely disseminated in North and Central America. Virus activity in nature has been documented at one time or another throughout the continental United States, except for maritime New England, and from Canada to the Darien jungle of Panama.

A

j^

-. ft. \\

A 1992

K

351

factor influencing the occurrence of an epidemic. In recent years, public health mosquito eradication programs have played an important role in curtailing many of these outbreaks. Encephalitis due to SLE virus is primarily a disease of persons of advanced age, in contrast to the alphaviruses considered above, and Japanese B encephalitis discussed below. In one outbreak (Jones, 1934), the mortality rate was 335 per 1 x 10^ population among octogenarians, but less than 4 per 1 x 10^ in persons under 40 years of age — nearly a 100-fold difference! Hypertensive and atherosclerotic vascular disease are risk factors predisposing to encephalitis among those who are infected. The pathogenic basis for this observation is obscure. In middle-aged patients with hypertension, mortality is increased some ninefold (Broun, 1958). The onset of SLE is usually sudden, with high fever and the signs and symptoms of meningeal irritation accompanied by drowsiness and mental confusion. Stupor and coma evolve subsequently. About Vs to V2 of patients who recover from encephalitis of varying degrees of severity experience residual symptoms (Table 24.3) and exhibit a diversity of significant organic neurological defects (Table 24.4) (Figure 24.7).

!\

1993

FIGURE 24.6 Evidence of SLE virus epidemicity in the United States during the 10-year period 1985-94 based on the annual incidence of cases of encephalitis reported to the Centers for Disease Control and Prevention (MMWR, 1994).

The virus of SLE would be better considered a family of antigenically identical agents that differ greatly in pathogenicity (Monath ei al., 1980). The basis for the high degree of mutability of this family of viruses has not been established, but it has significant implications with regard to the epidemiology of SLE. In endemic areas of virus activity, the prevalence of seropositive adults in the general population is relatively high. According to Monath and Heinz (1996), the ratio of inapparent: apparent infection varies with age, being 806:1 in children and 85:1 in the elderly. During outbreaks, inapparent and mild infections are prevalent. One might speculate that these infections do not involve the central nervous system because the virus lacks neurotropic pathogenicity. However, host factors also play an important role. In the United States, sporadic devastating outbreaks in heavily populated areas of the country have been documented over the past half century. Climatic conditions favoring multiplication of the vector mosquito are perhaps the most important

TABLE 24.3 Outstanding General S y m p t o m s in 18 Patients w i t h Organic D e f e c t s D u r i n g Convalescence from SLE Impaired memory Nervousness Irritability Weakness Dizziness Inability to walk firmly Inability to work Sleeplessness Adapted with permission from Smith (1958).

In fatal cases, the brain reveals a mononuclear meningitis (Figure 24.8) and a diffuse perivenular and periarteriolar infiltrate, predominantly, but not exclusively, located in the grey matter. Lesions in the cord are occasionally prominent. Cranial nerves are generally not affected. Focal accumulations of microglial nodules and variable degrees of neuronolysis are observed (Gardner and Reyes, 1980). The lesions of SLE predominate (and are most severe) in the thalamus and substantia nigra. The cerebral cortices, cerebellum, hypothalamus, and brainstem are less frequently affected and the lesions are less severe (McCordock ei al., 1934;

352

Pathology and Pathogenesis of Human Viral D i s e a s e

FIGURE 24.7 Late stages in the recovery of a flavivirus-infected infant mouse. Note the destructive loss of brain parenchyma (A) and the accumulations of mononuclear cells and granulation tissue (B). Resolution of the lesion is associated with the gliosis and scarring.

Weil, 1934; Haymaker, 1958c; Shinner, 1963; Suzuki and Phillips, 1966; Gardner and Reyes, 1980) (Figure 24.8). The pathological effects appear to be largely due to viral damage to neurons. The degree and distribution of the inflammation does not necessarily correspond to the distribution of neuronal lesions, suggesting that the infection is more widespread. When patients recover, newly elaborated antibodies appear to be of paramount importance in clearing the virus from the central nervous system. Cellular immune mechanisms have not been found to be significant factors influencing viral infectivity. SLE disease in HIV-1infected patients is not inordinately severe (Okhuysen et al, 1993). TABLE 24.4 Nature of Organic Defects Found in 18 Patients 3 Years After Infection w i t h SLE Organic Defects Defects in speech Difficulty in walking Disturbances in vision Positive Romberg sign Deafness Positive Oppenheim and Babinski signs Change in acuity of smell Lateral nystagmus Paralysis of both lower extremities Partial paralysis of upper extremities Right hemiplegia Epileptiform convulsions Adapted with permission from Smith (1958).

33%

11%

6%

Japanese B Encephalitis (JBE) This virus is distributed over a vast densely inhabited region of the earth's surface. It is widespread in the Indian Subcontinent to the southwest, and in the Japanese archipelago on the northeast. Much of mainland China and Southeast Asia are also areas of endemicity. In tropical regions, the virus appears sporadically in individual cases and in small outbreaks throughout the year. Where climatic conditions are seasonal, virus outbreaks occur during and after the monsoon rains (in the summer months, June through September). The human toll of JBE is incalculable, but undoubtedly it is the world's most consequential arthropod-borne viral infection in terms of morbidity and mortality. The ecology of JBE is influenced over its broad range by a variety of environmental factors, but it appears to be disseminated to humans by various species of culicine mosquito, with birds and mammals being amplifying and overwintering hosts. In Japan, where extensive ecological studies have been done, the domestic pig and various species of heron are major intermediaries (Scherer, 1959). In contrast to SLE, JBE encephalitis commonly occurs in infants and children where the mortality rate is high. Because inapparent subclinical infections are so common in the general population in these endemic regions, young people represent a highly susceptible population. The molecular characteristics of various JBE strains differ over the geographic range of the virus, and the pathogenicity of field isolates for laboratory animals, and presumably humans, is variable (Huang and Wong, 1963).

Neurotropic Arthropod-Transmitted Viruses

353

F I G U R E 24.8 An inflammatory infiltrate in the meninges of the brain of an infant mouse experimentally infected with a flavivirus. The cells are a mixture of lymphocytes and macrophages.

The pathological features of JBE are similar to those of SLE described above. However, the reports of Japanese pathologists suggest that areas of rarefaction in the brain are frequently present (Miyake, 1964). Mortality is variable and highly influenced by the quality and availability of medical care. Among young adult members of the American military stationed in Japan, it is lower than 10%. In children, death is common and residual neurological defects are a frequent outcome. Zimmerman (1946) has provided us with a detailed description of the central nervous system disease in young Okinawa native residents dying within a 2week period after the onset of symptoms. Lesions were found throughout the brain and spinal cord. "The degree of neuronal involvement varied from case to case, and from zone to zone in each case." Injured cells were as few as two to three in some cases, whereas larger areas of disease incorporating 20 to 30 cells were also present. The cytological features ranged from chromatolysis of ganglion cells to eosinophilic pycnotic neurons. In the substantia nigra, melanin-containing cells were damaged and the melanin scattered in the interstitium. A neutrophilic infiltrate was sporadically observed, and at times the acute inflammation resembled a pyogenic abscess. "In the anterior horn of the spinal cord, the glia and leukocyte reactions duplicated the histolytic picture of acute poliomyelitis" (Zimmerman, 1946). Perivascular cuffs of lymphocytes and glial

nodules comprised of polymorphonuclear cells and mononuclear cells were scattered throughout the brain and cord.

Other Flavivirus Encephalitides Worldwide, several additional arthropod viruses cause sporadic cases of encephalitis in diverse endemic areas. These include: (1) the Central European and Russian spring/summer complex of immunologically related viruses that are tick-borne and range over a vast geographic region from south central East Europe into much of the southern reaches of the former Soviet Union; (2) Powassen, a member of the Russian spring/ summer complex that appears sporadically in eastern Canada and the Northeast United States; (3) West Nile encephalitis, a mosquito-borne agent distributed throughout the Middle East and in Africa south to the Cape; (4) Murray Valley encephalitis, a mosquito virus that is widely distributed in eastern Australia; and (5) Rocio, an uncommon cause of encephalitis in the Brazilian state of Sao Paulo. The illnesses caused by these exotic viruses generally are mild and not associated with neurological signs and symptoms. On occasion, however, meningoencephalitis of varying degrees of severity occurs. Mortality rates are customarily low. Unfortunately, the pathological features of the disease

354

Pathology and Pathogenesis of Human Viral Disease

in virologically verified cases are poorly characterized, and published reports are limited to observation in one or a small number of cases. The interested reader is referred to the relevant literature: Russian spring/summer encephalitis (Jervis and Higgins, 1953); Powassan (Smith et al., 1974), West Nile encephalitis (Pruzanski and Altman, 1962; Manuelidis, 1956; Marberg et al, 1969; Gadoth et al, 1979; Southam and Moore, 1951, 1954), Murray Valley encephalitis (Robertson, 1952).

BUNYAVIRUSES As discussed in more detail in Chapter 19, the family Bunyaviridae is comprised of four genera of viruses having human health importance. Members of the genera Hantavirus, Phlebovirus, and Nairovirus are the etiological agents of various hemorrhagic disease in humans. The pathogenic arthropod-transmitted Bunyaviruses are considered here.

50% experience seizures and exhibit focal neurological signs. Indeed, focal seizures and generalized convulsions prove to be the most distinguishing clinical features of acute LAC infection (Deering, 1983). In one study, seizures occurred during convalescence in 6% of children (Chun, 1983). Neuropathological information is limited because of the low mortality rate of LAV infections (Kalfayan, 1983). Evidence of elevated intracranial pressure is found at autopsy. In the reported cases, congestion of the leptomeninges is observed, and only minimal mononuclear cell accumulations are present. The grey matter of the cerebral cortices exhibits focal, patchy clusters of mononuclear cells, and localized areas of necrosis. Polymorphonuclear leukocytes are rarely observed in infiltrates. Perivascular lymphocytic cuffs are also a prominent feature. Additional but mild changes are seen in the basal nuclei and brainstem. The cerebral white matter, cerebellum, medulla, and spinal cord fail to exhibit lesions. The prominent involvement of the cerebral cortices in these patients no doubt accounts for the high prevalence of seizure disorders and focal neurological signs.

LaCrosse (California Encephalitis Group) (Calisher and Thompson, 1983) The classification of the viruses categorized in the genus Bunyavirus proves confusing. The genus includes almost 150 interrelated viruses that share antigenic and molecular characteristics but are not human pathogens. California encephalitis virus was initially recovered in the Golden State from a human case of encephalitis in 1943. Although it is the prototype virus of the group, infection by California encephalitis virus has been associated with neurological disease on only rare occasions since that time. On the other hand, an antigenically closely related member of the California complex, termed LaCrosse (LAC), is a major cause of encephalitis among children and adolescents in the Midwest and Eastern United States. During the past several decades, roughly 70 cases of LAC encephalitis have occurred each year in the United States, with the highest annual incidence in the Middle American states of Indiana, Kentucky, Wisconsin, Minnesota, and Iowa. The case fatality rate over this period has been 0.3%. Related viruses (Snowshoe hare and Jamestown) are etiologically associated with meningoencephalitis in humans in Canada and New York State, but they are exceedingly rare causes of disease. Most patients with meningoencephalitis due to LAC are 5 to 10 years of age. They present clinically with fever, headache, signs of meningeal irritation, and an altered sensorium (Gundersen and Brown, 1983). Over

REOVIRUSES The virus of Colorado tick fever (CTF) is classified in the genus Coltivirus of the family Reoviridae. The reoviruses are ubiquitous, with a worldwide distribution. They have been recovered from a number of arthropod species and a vast variety of vertebrates. CTF virus is biologically related to several agents of veterinary and wildlife importance, but it represents the only established human pathogen in this large genus of interesting viruses. On rare occasions, two antigenically related viruses have been recovered from humans, but their causative role in disease is not established. CTF is a febrile, rarely fatal, systemic illness that on occasion is manifest with an erythematous maculopapillary skin rash and uncommonly with hepatitis, arthritis, pneumonitis, myocarditis/pericarditis (Emmons, 1985) and meningoencephalitis (Spruance and Bailey 1973; Goodpasture et al, 1978; Tsai, 1991). It is transmitted in the field by the so-called hard-shelled tick Dermacentor andersonia. Thus, a disproportionate number of those with an infection are young adult males who acquired the virus vocationally or avocationally About 175 cases occur in the United States annually, throughout the endemic area of virus activity, which includes the Rocky Mountain and Great Basin states of America.

Neurotropic Arthropod-Transmitted Viruses

Coltivirus infections are unique, for the virus parasitizes erythrocytes and mononuclear cells in the blood as well as their precursors in the bone marrow and lymphoid tissues. In this fashion, CTL avoids the onslaught of the immune response to infection. Chronic infections of red cells have been demonstrated for as long as 4 months in animals (Philip et ah, 1975; Philipp et al, 1993). In experimentally infected animals, the virus of CTF exhibits neurotropic properties. However, studies applicable to understanding the pathogenesis of the meningoencephalitis in humans are lacking. The incidence of CNS disease (meningitis, meningoencephalitis, and encephalitis) has been estimated to be 3 to 7% (Monath and Guirakhoo, 1996). Case reports are scattered in the literature (Silver et ah, 1961; Fitz and Meiklejohn, 1958; Ecklund et al, 1959; Fraaer C , and Scheff, 1962). Since patients recover without significant neurologic residue, detailed neuropathological studies have not been carried out.

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Robertson, E. (1952). Murray Valley encephalitis: Pathological aspects. Med. J. Aust. 1,107. Rous, P., and Kidd, J. (1936). The carcinogenic effect of a virus upon tarred skin. Science 83, 468-469. Scherer, W (1959). Ecological studies of Japanese encephalitis in Japan, parts I-IX. Am. J. Trop. Med. Hyg. 8, 644-722. Shinner, J. (1963). St. Louis virus encephalomyelitis. Arch. Pathol. 75, 309-322. Sidwell, R., Gebhardt, L., and Thorpe, B. (1967). Epidemiological aspects of Venezuelan equine encephalitis virus infections. Bacteriol. Rev. 31, 65-81. Silver, H., Meiklejohn, G., and Kempe, C. (1961). Colorado tick fever. Am. J. Dis. Child. 101, 56-61. Smith, J. (1958). St. Louis encephalitis: Sequelae. Neurology 8,884-887. Smith, R., Woodall, J., Whitney E., Deibel, R., Gross, M., Smith, V, and Bast, T. (1974). Powassan virus infection: A report of three human cases of encephalitis. Am. J. Dis. Child. 127, 691-693. Southam, C , and Moore, A. (1951). West Nile, Ilheus, and Bunyamwera infections in man. Am. J. Trop. Med. 31, 724. Southam, C , and Moore, A. (1954). Induced virus infections in man by the Egypt isolates of West Nile virus. Am. J. Trop. Med. Hyg. 3, 19. Spruance, S., and Bailey, A. (1973). Colorado tick fever: A review of 115 laboratory confirmed cases. Arch. Intern. Med. 131, 288-293. Suzuki, M., and Phillips, C. (1966). St. Louis encephalitis: A histopathologic study of the fatal cases from the Houston epidemic in 1964. Arch. Pathol. 81, 47-54. Tsai, T. (1991). Arboviral infections in the United States. Infect. Dis. Clin. North Am. 5, 73-102. Victor, J., Smith, D., and Pollack, A. (1956). The comparative pathology of Venezuelan equine encephalomyelitis. /. Infect. Dis. 9, 5566. Weil, A. (1934). Histopathology of the central nervous system in epidemic encephalitis (St. Louis epidemic). Arch. Neurol. Psych. 31, 1139-1152. Wesselhoeft, C , Smith, E., and Branch, C, (1938). Human encephalitis: Eight fatal cases, with 4 due to the virus of equine encephalomyelitis. JAMA 111, 1735-1741. Zimmerman, H. (1946). The pathology of Japanese B encephalitis. Am. J. Pathol. 22, 965-975.

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25 Rabiesviruses INTRODUCTION 357 EPIDEMIOLOGY 357 CLINICAL DISEASE 358 PATHOGENESIS 359 CENTRAL NERVOUS SYSTEM DISEASE REFERENCES 362

cause they are exceedingly uncommon causes of disease and we know so little about them. The vesiculoviruses are the second genus of the family to infect vertebrates. The only virus of human importance is vesicular stomatitis virus, an agent of economic significance, for it causes outbreaks of a nonfatal vesicular disease of the mucus membranes of cattle and other hoofed animals. On rare occasions, humans who work in close contact with infected animals develop mild vesicular eruptions. The virion of rhabdoviruses are relatively large and bullet-shaped when examined by electron microscopy. They have a helical nucleocapsid enveloped by a lipid and glycoprotein bilayer membrane. Despite the virion's size, it contains a limited complement of genes in the form of a single-stranded RNA that codes viral polymerases and structural proteins. When infecting a cell, the rhabdovirus passively attaches to the plasma membrane and is taken into the cell. The nucleocapsid is then uncoated and replication begins in the cytoplasm. The viral polymerase catalyzes RNA synthesis, and structural components of the virus are fabricated under the direction of viral genes using cell metabolites. These components assemble subjacent to the plasma membrane of the cell, where they bud off as infectious virions. Much of what we know about rabiesvirus replication is drawn from analogies with its cousin, the vesicular stomatitis virus. Relatively little scientific research on this aspect of rabiesvirus biology has thus far been conducted. Vesicular stomatitis virus replicates rapidly in susceptible cells while destroying them, whereas in vitro growth of rabiesvirus is slow and cytotoxicity limited. Thus, comparisons between the two must be evaluated with caution.

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Take heed of yonder dog! Look when he fawns; he bites; and when he bites his venom tooth will rankle to the death; have not to do with him; beware of him; sin, death, and hell, have set their works on him. William Shakespeare

INTRODUCTION Rabies (Latin rahhos, meaning "to do violence") is a disease of antiquity documented by authors and historians long before the birth of Christ. The public health importance, indeed the fear of rabies, was profound among our distant forefathers. At the time of Pasteur, the French government sponsored a rabies commission, and Pasteur's accomplishments were followed with great interest by the general public as well as the scientific community. His work culminated in successful postexposure immunization of a young boy who had been extensively bitten by a rabid dog using an attenuated live-virus vaccine (Fisher, 1995). Rabiesvirus is the only member of the Rhabdoviridae family having human importance. This large family of viruses comprises five genera in which are classified viruses of both warm- and cold-blooded fauna, including insects and several species of flora. Rabies is the dominant member of the Lyssavirus {Lyssa = Greek for "madness") genus. There are several distant relatives of rabiesviruses in this family, namely, Lagos bat virus, Mokolavirus, and Duvenhago (Mebatsion et ah, 1992). They are indigenous to bats in Africa, but on rare occasions these viruses have caused encephalopathies in humans. I will not consider them further here, bePATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

EPIDEMIOLOGY Rabiesviruses are infectious for a wide variety of warm-blooded animals, and silent transmission in the wild among various species occurs largely unrecognized by humans. The virus infecting these diverse 357

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species of animals possesses in nature unique molecular and antigenic markers that allow the investigator to identify the origin of a particular virus strain (Smith et al., 1992). While infections of human importance are customarily acquired from dogs, wolves, and coyotes, insectivorous and fruit-eating bats are increasingly serving as a source of human disease. Infected bats have now been documented in all of the 48 contiguous United States, and 11 of the 21 cases of rabies occurring in the United States over the period from 1980 through 1995 are attributed to an infected bat (MMWR, 1995a, 1996). On rare occasions, the origin of the infection in humans is never established (Mrak and Young, 1993), and in other cases, direct contact and a penetrating wound inflicted by a suspect animal are not documented. For example, only 27% of the 33 human rabies deaths occurring in the United States over the period from 1977 through 1994 had clearly documented histories of an animal bite (MMWR, 1995b, 1992). Although considerable concern is voiced in the United States about the hazards of rabies epizootics among raccoons, skunks, and foxes living in close proximity to humans, these infected animals have not proven to be a substantial threat, even in densely populated communities. Apparently, these wild animals usually develop "dumb" rabies and do not exhibit the aggressive behavior of dogs and wolves. In addition, the virus strains that infect animals may be relatively lacking in pathogenicity or the virus concentrations in the salivary gland secretions may be relatively low. These mysteries remain to be solved. Worldwide, rabies is of greatest human importance in countries where the populations of stray dogs and wolves are high. In recent years, several cases of rabies imported into the United States have exhibited extraordinarily long incubation periods, that is, as long as 7 years (Smith et ah, 1991). In a comprehensive review of the literature. Dean (1963) found that almost 10% of published cases of rabies had an incubation period longer than 90 days. Thus, clinically unexplained cases of encephalopathy should trigger a consideration of rabies among physicians and pathologists. As discussed in more detail below, the neuropathologic features of rabies are sufficiently obscure (Anonymous, 1978) that the unsuspecting pathologist could easily overlook the diagnosis, while unknowingly conducting an autopsy on an unusually hazardous cadaver. This concern is illustrated by therapeutic misadventures when rabies-infected corneas have been unknowingly transplanted into susceptible patients (Houff et al, 1979).

CLINICAL DISEASE Two clinical forms of rabies occur in humans — the so-called "furious" rabies and "dumb" rabies. In the former, the brainstem, cranial nerves, and limbic system of the brain are extensively involved, whereas in "dumb" rabies the lesions are customarily restricted to the brainstem and spinal cord. In most cases, the illness is initially expressed as a nonspecific syndrome of chills and fever, myalgias and headaches, as well as vague respiratory and digestive tract complaints. Pain and paresthesias appear in proximity to the site of the animal bite in a substantial number of patients. The prodroma in the "furious" form is expressed as episodes of generalized arousal, accompanied by hydrophobia. The latter symptoms reflect an overwhelming terror of water resulting from violent contractions of the respiratory chest muscles and those of the hypopharynx. Arousal episodes are followed by agitation, confusion, and maniacal behavior interspersed with episodes of lucidity and calmness. Paralysis of the muscles of deglutition is common and accompanied by salivation. Coma and death follow, usually despite respiratory support (Anonymous, 1975). Although encephalitic rabies is customarily believed to be uniformly fatal, survival without residual neurological abnormalities has been documented. Unfortunately, in many of the cases, sound documentation of rabies was not reported. A virological substantiated childhood case was published by Hattwick et al. (1972). The 7-year-old child lapsed into a coma and a brain biopsy demonstrated encephalitis with neuronal damage and Babes nodules (see below). The patient recovered over a 3-month period without neurological disability. It was unclear whether or not cognitive capabilities were impaired. "Dumb" rabies is less familiar to clinicians, for it occurs in only about 20% of cases. Neurologic symptoms, pain, and sensory abnormalities and flaccid paralysis are confined to, or are most severe in, the bitten extremity. Transverse and ascending myelitis may follow with death resulting from paralysis of the muscles of deglutition and respiration. Demyelinization and remyelinization as well as fiber loss in peripheral nerves is frequently seen when pathological studies of teased nerves are done. Axonal loss and Wallerian degeneration is seen in some cases. Dorsal route ganglia often exhibit chronic inflammatory infiltrates (Chopra et al, 1980). Survival may be more protracted than in "furious" rabies because encephalitis does not develop and, as a result, some patients do not die (Anonymous, 1978; Pawan, 1939).

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Despite localization of the clinical disease to the central nervous systems, rabies is a systemic infection involving multiple organs. But, in contrast to most other viral diseases, the virus of rabies is spread to peripheral sites centrifugally along nerve trunks. Sensory nerves seem to be particularly important in this role, but the autonomic nervous system is also involved. Transport of virus by means of autonomic nerves may account for the interstitial myocarditis, sialoadenitis, and adrenal medullitis that commonly occurs in rabiesvirus-infected patients (Lopez-Corella et al, 1997; Ross and Armentrout, 1962; Cheetham et al, 1970). In some cases, the myocarditis is severe and may have contributed to death. Cardiac dysrhythmias commonly are observed (Bhatt et al, 1974). The diagnostic approaches currently employed are dependent upon this peripheral dissemination of the virus. Corneal impression smears and skin biopsies from body sites with a high density of hair follicles (such as the back of the neck) are commonly used techniques (Koch et al, 1975; Leach and Johnson, 1940; Smith et al, 1972). These approaches are relatively insensitive indicators of infection, even when immunohistochemistry is employed (Johnson et al, 1980). Information using PCR to increase sensitivity has not been reported.

PATHOGENESIS Over 100 years before Pasteur attempted his first immunization of a human, Morgagni noted that rabies "does not seem to be carried through the veins, but by the nerves, up to their origins." Experimentally pursuing this concept, DiVestea and Zagari (1889) induced rabies in dogs by inoculating the sciatic nerve with virus. They then prevented it by severing the nerve shortly after injection. Rabiesvirus appears to move in a retrograde fashion by axonal flow to the central nervous system (Gosztonyi, 1978; Jenson et al, 1969; Gillet et al, 1986). This process depends upon intact microtubules in the nerves, for it is inhibited by colchicine treatment (an agent possessing the capacity to disrupt microtubules) (Tsiang, 1979). At nerve synapses, rabiesvirus appears to bind to the acetylcholine receptor involved in cholinergic conductance (Lentz et al, 1984), thus facilitating neural transport. Centripetal movement of the virus in peripheral nerves occurs at a rate of about 5 to 10 cm per day. Based on this observation, it is difficult to account for the prolonged clinical latency period between the time of exposure and manifestation of infection. Perhaps rabiesvirus lies dormant

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at the site of introduction. For example, it might proceed through one or more replicative cycles locally in muscle cells before entering the nerve, as suggested by Murphy (1977) and Charlton and Casey (1979). However, in the murine model in which animals are infected by footpad inoculation, amputation of the extremity must be carried out within 4 hours to protect the animals from central nervous system disease. In a more recent study, masseter muscle inoculation was followed by the appearance of viral RNA in the trigeminal ganglion 18 hours later and in the brainstem after 24 hours (Shankar et al, 1991). Thus, entry of the virus into the peripheral nervous system appears to occur rapidly. One might conclude that local replication of the virus in muscle at the site of inoculation is of limited pathogenic importance. Clinically, infection resulting in death occurs with relative infrequency among those who are bitten on the distal extremities, but it is common in patients with facial and head wounds inflicted by a rabid animal. Neural transmission of the virus to the central nervous system may be a relatively inefficient process in humans. Passage of the wild so-called "street" strains of rabiesvirus in a susceptible host (such as the mouse) by inoculating brain suspensions intracerebrally shortens the latency period and increases the virulence of the virus. One concludes that highly pathogenic virions are selected by this process. In contrast, serial intracerebral passage of virus in relatively resistant animals results in attenuation and a relative loss of virulence, as originally demonstrated by Pasteur (Miyamoto and Matsumoto, 1967). Pathogenicity appears to relate to the make-up of the G glycoprotein on the surface of the enveloping membrane of the virion. This protein is a key factor in the interaction of the virus with cells of the central nervous system. Amino acid substitution at specific sites in the protein molecule dramatically affects the virulence of the virus (Rupprecht and Dietzschold, 1987). Much remains to be learned about the factors that influence pathogenicity and the role of G protein variability in this infectious process (Tuffereau et al, 1989; Lafay et al, 1991). Rabiesvirus G protein is highly antigenic in humans, as demonstrated by the prompt elaboration of specific immunoglobulins in response to administration of attenuated virus vaccines. Under natural circumstances of infection, the immune response to the naturally occurring "street" virus is comparatively slow, perhaps due to the relatively low concentration of virus in the inoculum and the sequestered nature of the virus in the nervous tissue. Circulating antibody is key to protection against reinfection, but probably plays little or no role in attenuating an established infection. In experi-

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mental models, cellular immune mechanisms are involved in modulating the course of the disease, but paradoxically, they may accentuate neuropathogenicity under certain circumstances (Soave et ah, 1961; Sugamata et ah, 1992). For example, administration of rabies-sensitized T cells to infected mice shortens survival time and increases the severity of the neurological disease. Pathologically, there is a prominent meningitis and perivenular infiltrate of mixed lymphocytes and macrophages in the brain of these experimental animals (Sugamata et al, 1992; Shope et al, 1979). Cellular immune mechanisms may explain the incompletely documented so-called "early death phenomena" in which previously immunized subhuman primates and

naturally infected humans experience a shortened latency period of disease when infected. CENTRAL NERVOUS SYSTEM DISEASE In 1903, Negri reported his finding of a structure reminiscent of an amoeboid parasite in the brain cells of a rabid dog. Almost simultaneously, Bosc (1903) independently discovered a similar structure associated with rabies, but credit for his observation rarely finds its way into the literature. Although the morphologically obvious Negri body led to the conclusion that rabies was due to a parasite, the concept was readily

FIGURE 25.1 Examples of Negri bodies in cells from different locations in the brain of different species of animals. (A) Neuron in the hippocampus of a rabid dog. (B) Hippocampus of a rabid skunk. Note the internal body so-called innerkorperchen. (C) Multiple Negri bodies in a neuron of the pons of an experimentally infected monkey. (D) Neuron in basal ganglion cell of skunk. Note the unusually large-sized Negri body (arrow). (E) Anterior Horn cell of spinal cord of rabid raccoon. Note the small Negri body (arrow). (F) Axon of hippocampus of human. Reprinted with permission from Perl (1975) and kindly provided by D. Perl, MD.

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FIGURE 25.2 Ultrastructure of a neuron from the caudate nucleus of an experimentally infected monkey. Several Negri bodies of differing size are seen (arrows). The smaller bodies are not demonstrable by light microscopy. Note the bullet-shaped dense-staining virions associated with the Negri bodies. Reprinted with permission from Perl (1975) and kindly provided by D. Perl, MD.

FIGURE 25.3 Ultrastructure of a Negri body exhibiting the bulletshaped virions raised at the periphery. The invagination in this cell is thought to represent the internal body demonstrated in Figure 25.IB. Reprinted with permission from Perl (1975) and kindly provided by D. Perl, MD.

disproved when it was found that the infectious agent passes through a bacterial-type filter. The biological nature of the eosinophilic intracytoplasmic body that bears Negri's name has perplexed countless investigators in part because it is inconsistently found in cells of the central nervous system and because the bodies vary greatly in their morphological features in different cells and at various sites in the brain. Moreover, Negri bodies in different infected animal species are often morphologically dissimilar. Thus, the Negri body of a cow is very large in comparison to the bodies found in the nervous system of rabbits and raccoons, where, in fact, multiple small inclusions are evident (Figure 25.1C). As one might expect, pathologists applied new names to describe some of these morphologic variants. Goodpasture (1925) described the Lyssa body as a Negri-like structure lacking the small basophilic internal body (Innerkorperchen) that typifies the classical Negri body. Some observers thought that the Negri body represented a degenerate cell, and that the innerkorperchen was the etiological agent. Others doggedly persisted in considering the Negri body a para-

site, despite its morphologic variability and the demonstrated capacity of the rabies agent to pass through an ultrafilter. Although the Negri body has been the topic of much research and discussion since its discovery in the early years of the twentieth century, we know now that these bodies not only differ morphologically, but biologically as well. Matsumoto (1963) was the first pathologist to provide insight into the fine structural features of the Negri body Figures 25.2 and 25.3 illustrate the ultrastructural features of Negri bodies studied by Perl (1975). In rabies encephalitis, the brain tissue is extensively involved, as shown by electron microscopy and immunohistochemistry, even when traditional histologic examination of the tissue reveals surprisingly few changes. Although incompletely studied, the evidence indicates that the virus is rapidly disseminated along multiple neural channels and by contiguous spread from cell to cell in the brain. Careful study reveals scattered areas of neuronophagia and microglial nodules (so-called Babe's nodules) (Figure 25.4). The latter abnormalities are observed in many forms of encepha-

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m.\

FIGURE 25.4 (A) Babe's nodule in the thoracic spinal cord of an experimentally infected monkey. The reactive inflammatory cells are accumulated around a neuron. In (B) neuronophagia is evident. Note the perivascular lymphocytic infiltrate in (A). Reprinted with permission from Perl (1975) and kindly provided by D. Perl, MD.

litis and indicate accumulations of activated microglia in localized areas of tissue damage. In addition, perivascular meningeal lymphocytic infiltrates are seen to a variable extent (Figure 25.5). Thus, the elusive and often difficult-to-detect Negri body remains a critical marker of infection, although it may often not be found even after careful histologic study and its distribution may prove highly variable. The important role of immunohistochemistry in establishing the diagnosis of rabies under these circumstances is apparent (Johnson

et ah, 1980). Indeed, the pathologist conducting postmortem studies on patients with unexplained encephalitis should carefully consider the advisability of exploratory immunolocalization studies when the pathological changes do not readily account for the clinical disease (Figure 25.6). References Anonymous (1975). Editorial: Diagnosis and management of human rabies. Br. Med. ]. 3 (5986), 721-722.

FIGURE 25.5 Extensive perivascular infiltrates in the midbrain of a human case of rabies. Reprinted with permission from Perl (1975) and kindly provided by D. Perl, MD.

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FIGURE 25.6 The more typical appearance of the cerebral cortex in a human with rabies. Negri bodies are present on close microscopical inspection of the tissue, but they could be easily missed by the pathologist. In this case, no perivascular infiltrates and Babe's nodules are observed.

Anonymous (1978). Editorial: Dumb rabies. Lancet 2,1031-1032. Bhatt, D., Hattwick, M., Gerdsen, R., Emmons, R., and Johnson, H. (1974). Human rabies. Am. J. Dis. Child. 127, 862-869. Bosc, F. (1903). C.R. Soc. Biol. 55,1284. Charlton, K., and Casey, G. (1979). Experimental rabies in skunks: Immunofluorescence light and electron microscopic studies. Lah. Invest. 41, 36-44. Cheetham, H., Hart, J., Coghill, N., and Fox, B. (1970). Rabies with myocarditis: Two cases in England. Lancet 1, 921-922. Chopra, J., Banerjee, A., Murthy, J., and Pal, S. (1980). Paralytic rabies: A clinico-pathological study. Brain 103, 789-802. Dean, D. (1963). Pathogenesis and prophylaxis of rabies in man. N.Y. State]. Med. 74, 3507-3513. DiVestea, A., and Zagari, G. (1889). Sur la transmission de la rage par voie nerveuse. Ann. Inst. Pasteur (Paris) 3, 237-248. Fisher, D. (1995). Resurgence of rabies: A historical perspective on rabies in children. Arch. Pediatr. Adolesc. Med. 149, 306-312. Gillet, J., Derer, P., and Tsiang, H. (1986). Axonal transport of rabies virus in the central nervous system of the rat. /. Neuropathol. Exp. Neurol. 45, 619. Goodpasture, E. (1925). A study of rabies with reference to a neural transmission of the virus in rabbits and the structural and significance of Negri bodies. Am. J. Pathol. 1, 547-582. Gosztonyi, G. (1978). Axonal and transsynaptic spread of viral nucleocapsids in fixed rabies encephalitis. /. Neuropathol. Exp. Neurol. 37, 618. Hattwick, M., Weis, T., Stechschulte, C , Baer, G., and Gregg, M. (1972). Recovery from rabies (a case report). Ann. Intern. Med. 76, 931-942. Houff, S., Burton, R., Wilson, R., Henson, T., London, W., Baer, G., Anderson, L., Winkler, W, Madden, D., and Sever, J. (1979). Human-to-human transmission of rabies virus by corneal transplant. New Engl. J. Med. 300, 603-604. Jenson, A., Rabin, E., Bentinck, D., and Melnick, J. (1969). Rabiesvirus neuronitis. /. Virol. 3, 265-269.

Johnson, K., Swoveland, P., and Emmons, R. (1980). Diagnosis of rabies by immunofluorescence in Trypsin-treated histologic sections. JAMA 244, 41-43. Koch, R, Sagartz, J., Davidson, D., and Lawhaswasdi, K. (1975). Diagnosis of human rabies by the corneal test. Am. J. Clin. Pathol. 63, 509-515. Lafay, F., Coulon, P., Astic, L., Saucier, D., Riche, D., Holley, A., and Flamand, A. (1991). Spread of the CVS strain of rabies virus and of the avirulent mutant AvOl along the olfactory pathways of the mouse after intranasal inoculation. Virology 183, 320-330. Leach, C , and Johnson, H. (1940). Human rabies with special reference to virus distribution and titer. Am. ]. Trop. Med. 20, 335-340. Lentz, T., Wilson, P., Hawrot, E., and Speicher, D. (1984). Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins. Science 226, 847-848. Lopez-Corella, E., Ridaura-Sanz, C , and Samayoa-Patma, J. (1997). Human rabies: Systemic pathology in 33 cases. Proc. Acad. Pathol., Orlando, FL. Matsumoto, S. (1963). Electron microscope studies of rabies virus in mouse brain. /. Cell. Biol. 19, 565-591. Mebatsion, T., Cox, J., and Frost, J. (1992). Isolation and characterization of 115 street rabies virus isolates from Ethiopia by using monoclonal antibodies: Identification of 2 isolates as Mokola and Lagos bat viruses. /. Infect. Dis. 166, 972-977. Miyamoto, K., and Matsumoto, S. (1967). Comparative studies between pathogenesis of street and fixed rabies infection. /. Exp. Med. 125, 447-475. MMWR (1992). Human rabies — California. Morb. Mortal Weekly Rep. 41, 461-463. MMWR (1995a). Human rabies — West Virginia, 1994. Morh. Mortal. Weekly Rep. 44, 86-87. MMWR (1995b). Human rabies — Alabama, Tennessee, and Texas, 1994. Morb. Mortal. Weekly Rep. 44, 269-272. MMWR (1996). Human rabies — Connecticut, 1995. Morb. Mortal. Weekly Rep. 45, 207.

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Mrak, R., and Young, L. (1993). Rabies encephalitis in a patient with no history of exposure. Hum. Pathol 24,109-110. Murphy, R (1977). Rabies pathogenesis [brief review]. Arch. Virol. 54, 279-297. Negri, A. (1903). Beitrag zum Studium der Aetiologie der Tollwuth. Z. Hyg. Infektionskr. 44, 519-540. Pawan, J. (1939). Paralysis as a clinical manifestation in human rabies. Ann. Trop. Med. Parasitol 33, 21. Perl, D. (1975). The pathology of rabies in the central nervous system. In 'The Natural History of Rabies'' (G. Baer, ed.). Vol. 1, pp. 235-272. Academic Press, New York. Ross, E., and Armentrout, S. (1962). Myocarditis associated with rabies. New Engl. J. Med. 266,1087-1089. Rupprecht, C , and Dietzschold, B. (1987). Perspectives on rabies virus pathogenesis [editorial]. Lah. Invest. 57, 603-606. Shankar, V, Dietzschold, B., and Koprowski, H. (1991). Direct entry of rabies virus into the central nervous system without prior local replication. /. Virol. 65, 2736-2738. Shope, R., Baer, G., and Allen, W. (1979). Summary of a workshop on the immunopathology of rabies. /, Infect. Dis. 140, 431^35. Smith, J., Fishbein, D., Rupprecht, C., and Clark, K. (1991). Unexplained rabies in three immigrants in the United States: A virologic investigation. New Engl. J. Med. 324, 205-211.

Smith, J., Orciari, L., Yager, R, Seidel, H., and Warner, C. (1992). Epidemiologic and historical relationships among 87 rabies virus isolates as determined by limited sequence analysis. /. Infect. Dis. 166, 296-307. Smith, W., Blenden, D.C., Fuh, T., and Hiler, L. (1972). Diagnosis of rabies by immunofluorescent staining of frozen sections of skin. /. Am. Vet. Med. Assoc. 161,1495-1501. Soave, O., Johnson, H., and Nakamura, K. (1961). Reactivation of rabies virus infection with adrenocorticotropic hormones. Science 133,1360-1361. Sugamata, M., Miyazawa, M., Mori, S., Spangrude, G., Ewalt, L., and Lodmell, D. (1992). Paralysis of street rabies virus-infected mice is dependent on T lymphocytes. /. Virol. 66,1252-1260. Tsiang, H. (1979). Evidence for an intraaxonal transport of fixed and street rabies virus. /. Neuropath. Exp. Neurol. 38, 286-299. Tuffereau, C., Leblois, H., Benejean, J., Coulon, P., Lafay, P., and Flamand, A. (1989). Arginine or lysine in position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice. Virology 172, 206-212.

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Poxviruses genera infect humans. The orthopoxviruses are: variola major and its less pathogenic variant variola minor, cowpox and its variant vaccinia, and monkeypox. These viruses cause disseminated infections of the skin and mucus membranes. Variola major and minor appear to be obligate human parasites, although they infect certain species of subhuman primates in the laboratory (Brinckerhoff et al, 1906). Monkeypox virus is indigenous to subhuman primates in West Africa, and it occasionally infects humans residing near the central west coast of that continent. Clinically, monkeypox can be confused with its much more virulent cousin, smallpox. Cowpox is an uncommon cause of vesicular disease in cattle. It occasionally infects herdsmen by direct contact. Recognized human outbreaks have been restricted to Europe. The virus appears to originate in wild rodents from which cattle are infected in the pasture. It is believed to be the "seed" from which vacciniavirus was derived. The virus of cowpox should be differentiated from the parapoxvirus responsible for milker's nodules, an infection discussed in more detail later. Once introduced into a herd, cowpox spreads rapidly, causing short-lived lesions of the teats and skin. As is well known, vaccinia is a laboratorymanipulated virus of uncertain derivation that has long been used for so-called jennerian immunization. While infection by direct inoculation causes a localized lesion, dissemination occurs to involve preexisting skin lesions, particularly in infants and the immunosuppressed patient. It is believed that an endemic poxvirus disease of the water buffalo in the Indian Subcontinent is caused by a strain of vacciniavirus initially derived from recently vaccinated humans. The parapoxviruses (milker's nodules, bovine popular stomatitis, and orf) are zoonotic causes of localized vesiculonodular lesions that are acquired from infected domestic animals. The moUuscipoxvirus genus has within it only one agent of human importance: the virus responsible for molluscum contagiosum (MC). This is customarily a childhood disease of the skin, but it is now an imposing threat to the immunosuppressed patient.

INTRODUCTION 365 ORTHOPOXVIRUSES 366

Variola (Major and Minor) 367 Vacciniavirus 371 Monkeypox 373 PARAPOXVIRUSES

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Milker's Nodules 377 Bovine Papular Stomatitis (BPS) 377 Ecthyma Contagiosum (orf) 377 MOLLUSCIPOXVIRUSES 377 REFERENCES 379

INTRODUCTION He is an impressive, quiet spoken, former Dean of the Johns Hopkins School of Hygiene, but in another era he successfully led the WHO effort to eliminate the virus of smallpox from its last haunts in obscure corners of Africa and Asia. Donald Henderson's task was conducted deliberately and with single-minded purpose. No physician has previously been privileged to serve in a leadership role to eliminate a disease from the face of the planet (Figure 26.1). Although smallpox virus is now thought to be securely sequestered in laboratory freezers in the United States and the former Soviet Union, concern mounts in this time of terrorism that, in some clandestine way, the virus will be unleashed to decimate selected populations. Now lacking immunity, our children and many young adults worldwide are highly susceptible and thus defenseless against this imposing potential threat (Breman and Henderson, 1998; Mahy ei ah, 1993). The poxviruses of humans and a wide variety of lesser animals share biological properties, and their clinical manifestations in mammals largely relate to their capacity to grow in the epithelium of the skin, and, to a variable extent, the mucus membranes of the oropharynx and eye. Poxviruses infect a number of species of animals, and several kinds of insects. The viruses of vertebrates are members of the chordopoxviridae subfamily; members of four of its eight PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

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FIGURE 26.1 The last case of smallpox within a 20-nation area of West and Central Africa (May 21, 1970). This 27-year-old patient was vaccinated about 10 days before the photographs were prepared (note the primary vaccination lesion on the arm in B). The classical features of smallpox are exhibited here. The patient appeared weak and/or fatigued and the lesion had a predominant peripheral distribution. The lesions are deep-seated and firm in appearance. Reprinted with permission from Hernon (1996) and through the courtesy of C. Hernon, MD.

B The largest of the viruses that infect humans, the virions of the poxvirus family have a complex structure. The genetic material of the various members of this large family is found in a linear double-stranded DNA approximately 180-190 kb in length. This sizable genome provides sufficient information to code roughly 150 proteins of average size. Although poxviruses have been the subject of considerable basic research, our understanding of the function of many of the proteins is limited. In addition, some are most probably not essential for viral replication and may only play a role in certain less critical aspects of the virion life cycle. Poxviruses replicate in the cell cytoplasm independent of the nucleus and are not dependent upon the cell's synthetic tools. Thus, the virus employs a panoply of enzymes of its own making to manufacture new progeny virions. The individual infectious units — known as elementary bodies, or Guarnieri bodies, in the earlier literature — can be seen in the cytoplasm of the infected cell by light microscopy, for they are enormous: 230 to 240 |Lim in diameter. Customarily, the virions accumulate in well-defined amphophilic cyto-

plasmic inclusions, although these inclusions are often not found histologically. Electron microscopy of the infected cells and the vesicle fluid from skin lesions makes possible a diagnosis based on virion morphology (Figure 26.2). The vesicles caused by an orthopoxvirus can also be differentiated histologically from similar lesions due to other common human viruses such as the herpesviruses (see Chapter 7).

ORTHOPOXVIRUSES The lesions caused by these viruses characteristically are vesicular, but there is an associated, sometimes dramatic, localized proliferation of the adjacent epithelium (Figure 26.3). Vacciniavirus elaborates an epidermal growth factor-like product during infection, and it is believed that this or similar growth factors account for the epithelial proliferation so commonly evident at the margins of poxvirus vesicles. The cytoplasm of infected cells of the superficial epidermal layers of the skin initially becomes vacuolated, that is.

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Poxviruses

FIGURE 26.2 Electron micrograph illustrating typical poxvirus morphology. The seemingly bilobate nucleosome is surrounded by an outer membrane. Illustrated here are accumulations of the virus of molluscum contagiosum in a cytoplasmic inclusion. Note the seemingly compressed nucleus of the infected cell at the right base. Reprinted with permission and through the courtesy of V. Burmeister, PhD.

the so-called "balloon" change. Surprisingly, we know very little about the mechanistic basis for these alterations in the infected cell structure. The cells also exhibit the classical poxvirus eosinophilic cytoplasmic inclusions. As the lesions evolve, suprabasal compartmentalized vesiculations develop in the epidermis. Later, as infected cells undergo necrosis, fluid accumulates and the vesicles expand. Ultimately, the basal cells lyse and the vesicle is encompassed by a cap comprised of the compressed granular and cornified layers. While the vesicular fluid initially is clear, polymorphonuclear leukocytes and cell debris gradually accumulate in the fluid, resulting in the pustule. Several pathogenic mechanisms no doubt account for cell damage, but their relative contribution to the development of lesions in humans is far from clear. In general, the orthopoxviruses inhibit cell macromolecular synthesis during the course of replication, and cytokines are generated by the cells. Early in the infection, viral antigens are introduced into the plasma membrane of the infected cell; they are the potential targets for cytolytic T cells. Studies employing the modern tools of cell biology and immunopathology remain to be focused on

the immune mechanisms involved in the formation of the vesicle and its subsequent resolution. Variola (Major and Minor) Smallpox has been a plague on humans since prehistory. Its extraordinary infectivity and its high mortality rate have long made it a feared scourge on civilization. Mortality due to smallpox ranges from 20 to 40% in various outbreaks, but the death toll is highest in infants and the elderly. Nutritional factors and the overall health of the patient no doubt influence survival. Antibacterial drug therapy also reduces the death rate. Although classical smallpox is spread in epidemic form by means of the respiratory route, histologically evident lesions in the airways and lung are not a feature of the disease at autopsy. The virus is transmitted from the respiratory tract to regional lymph nodes, where the initial phase of virus replication occurs. After about 10 to 12 days, viremia develops and persists for a short period. Fever and generalized symptoms accompany the viremic stage. The skin lesions initially develop on the mucus membranes of the oropharynx;

368

Pathology and Pathogenesis of Human Viral Disease

Inclusion bodies Ballooning degeneration

_

\ ';'^-,-;^

l $ & ^ * % ^ y ^ infiltrate Oedema of

Separation of epithelial cells

m i

j j ^ ^ Dilated and engorged vessels .Perivascular infiltrate Haemorrhages In dermis

Polymorpho nuclear cell infiltrate Reticulating degeneration

Ballooning degeneration

Nevs^ epithelium

Proliferated epithelium

Crust

Densely massed polymorphonuclear cells

rm..^:-FIGURE 26.3 Stages in the development and evolution of a typical skin lesion of smallpox. (A) The earliest change is oedema of the dermis, leading to separation of epithelial cells of the papillae and lymphocytic infiltration in the dermis, especially around small vessels. Balloon degeneration is seen in a few cells in the lower malpighian layer. (B) These changes progress, and the small vessels become dilated and engorged. Inclusion bodies are also visible adjacent to cells showing balloon degeneration. In early hemorrhagic-type smallpox, illustrated here, evidence of hemorrhage into the dermis was pronounced. (C) As the pathological process progresses, the epithelial cells break down by reticulating degeneration to produce a multilocular vesicle. (D) The vesicle formed by coalescence of the smaller cavities is infiltrated with polymorphonuclear leukocytes to form a pustule, around which are found cells containing inclusion bodies. (E) The fully developed pustule is packed with polymorphonuclear leukocytes and the epithelium on either side of the pustule has proliferated. (F) Eventually, the pustule forms a crust, beneath which new epithelium grows to repair the surface. Such lesions, in which the sebaceous glands are not involved, heal without leaving a scar. Reprinted with permission from Fenner ei a\. (1988).

369

Poxviruses

»

c

FIGURE 26.4 The characteristic palmar and plantar lesions in variola major. Reprinted with permission from Fenner et al. (1988).

24 hours later, a macular erythematous rash appears on the face and distal extremities, including the palms and soles. The rash rapidly spreads centrally in a centripetal fashion to involve the body as a whole (Figure 26.4), but lesions are often relatively sparse on the trunk (Figure 26.5). Histologically, the developing skin lesions take several forms. Initially, they evolve through vesicular and pustular stages. By the end of the second week of clinical illness, scabs form over the pustules, the fever defervesces, and the patient gradually recovers. The cause of death in patients with smallpox is unclear, despite careful autopsy study. Lesions in internal organs are customarily sparse, if they exist at all, and they are not believed to be a contributing factor in the progression or severity of the disease. Death is generally stated to be due to a generalized toxemia, but the pathophysiologic basis is obscure. Confluent and hemorrhagic disease is highly fatal (Table 26.1).

In smallpox, scarring of the face is one of the devastating long-term residual lesions among survivors. These scars appear to result from sebaceous gland involvement by the virus, ultimately resulting in accumulation of necrotic debris in the lumina of the glands. A granulation tissue response contributes to the scarring process. There is no evidence to suggest that smallpox virus invades the dermis. Although variola major is generally considered to be a uniformly severe disease, attenuated strains circulated in outbreak form in the past. The term "alastrim" refers to a virus of a relatively low order of pathogenicity that was endemic to South America. The variola minor viruses appeared sporadically elsewhere in the world (Gordon et ah, 1966). In the latter infections, patients (by definition) develop fewer than 100 pox on the face and lesser numbers elsewhere (Figures 26.6 and 26.7). The mortality rate is roughly 1%. In the laboratory, the various so-called "wild" strains act in a some-

370

Pathology and Pathogenesis of Human Viral Disease

FIGURE 26.5 Variola major in an infant. Note the relative paucity of lesions on the trunk and their predominance on the face and extremities. Reprinted with permission from Fenner et at. (1988).

FIGURE 26.6 A 17-year-old girl with variola minor who was hospitalized with the admitting diagnosis of chickenpox. Reprinted with permission from Gordon et al. (1966).

Poxviruses

371

TABLE 26.1 Mortality of Various Clinical Types of Variola Major in Unvaccinated Patients in India Clinical types of lesion

Ordinary" Confluent Semiconfluent Discrete Total

Percent of patients

Mortality rate (%)

23 24 42

62 37 9

89

30

Flat Type^

7

97

Hemorrhagic

2

96

Modified

2

_0

Overall mortality

36

Modified with permission from Fenner et al. (1988). ''Raised pustule lesions. ^Flat confluent or semiconfluent pustules.

what dissimilar fashion, but the molecular basis for differences in pathogenicity between the viruses of variola major and minor remain to be deciphered. Vacciniavirus Localized inoculation of variola virus (using infected scabs from healing lesions) into the skin of susceptibles was a well-established immunization practice before the introduction of vaccination by Jenner in 1798 (Bloch, 1993). Variolation causes an attenuated, rarely fatal, localized smallpox having a shortened incubation period. However, the virus that grew in these lesions proved to be highly contagious for contacts, thus potentially triggering outbreaks. Cowpox virus from which vacciniavirus was derived shares many molecular and biological properties with variola, including antigenicity. Perhaps it was, in fact, a smallpox virus modified by passage for centuries in field rodents and cattle. Further repeated passage of cowpox in humans and laboratory animals ultimately yielded vacciniavirus. There are n\any laboratory and vaccine strains that differ somewhat in pathogenicity for humans. Systemic Complications of Vaccination (see Figure 26.8)

Before the eradication of smallpox, immunization with vacciniavirus was a routine procedure for preschool children and travelers, but its use was a concern inasmuch as complications were an inevitable outcome in a small number of cases (Lane et al, 1969). For example, in the United States during 1968 (the last year

F I G U R E 26.7 Self-portrait by a photographer with the skin lesions of variola minor. This case of variola minor occurred in the United Kingdom in the mid-1960s. Patients during the outbreak had a prodromal illness of 3 days, but they were not seriously ill and were afebrile, or had only a slight fever. The rash appeared initially on the face and then on the extremities, including the palms and soles. Vesicles were 6 to 8 mm in diameter. Pustules became evident on day 5 and scabs formed on about day 8. The illness is described in detail by Gordon et al. (1966). Reprinted with permission from Gordon et al. (1966).

for which comprehensive survey data are published), almost 600 persons were reported to have developed vaccination complications, and there were nine deaths. Thus, approximately 74 complications, and 1 death occurred per 1X10^ primary vaccinations. Morbidity and mortality proved to be highest in infants, with 112 complications and 5 deaths per 1 x 10^ primary vaccinations. While vacciniavirus usually causes a benign inflammatory reaction at the local sites of primary inoculation into the skin (Figure 26.9), it is potentially pathogenic for (1) the fetus in utero; (2) patients with naturally occurring, or acquired immunodeficiency; and (3) those with chronic open skin lesions, particularly eczema. In addition, for unknown reasons, encephalitis develops sporadically in otherwise healthy young persons with an approximate incidence of 2 to 3 cases per 1 x 10^ immunizations. A rare patient experiences ocular vaccinia (Ruben and Lane, 1970; Ellis and Winograd, 1962). Arthritis (Silby et al, 1965), pericarditis, myocarditis (Cangemi, 1958; Finlay-Jones, 1964; Matthews and Griffiths, 1974) and nonbacterial

372

Pathology and Pathogenesis of Human Viral D i s e a s e

>.

FIGURE 26.8 Vacciniavirus infections. (A) Eczema vaccinatum in a young black child. Note the ichthyotic scaling of the skin over the trunk. (B) Progressive vaccinia in a child with an immunologic deficiency of unknown type. Note the extensive lesions with satellites. There is generalized erythema and edema of the shoulder and upper extremity. (C) Benign generalized vaccinia of 10 days duration. Note the primary lesion on the left arm. (D) Autoinoculation of the eye resulting in ocular and conjunctival vaccinia. Reprinted with permission from Fenner et al. (1988).

osteomyelitis (Sewall, 1949; Haar and Meinertz, 1954; Cochran et al, 1963; Elliot, 1959) are additional uncommon complications. Progressive vaccinia, an often fatal dreaded complication of immunization, is an infrequent outcome of the inadvertent vaccination of infants and children with congenital forms of hypogammaglobulinemia and combined immunologic deficiency (Hathaway et ah, 1965). It is also seen in children and adult recipients

of chemotherapy and those with malignant hematological disease (Neff and Lane, 1970; Freed et al, 1972). Rather than resolve over a period of weeks, the localized primary lesion continues to enlarge and the virus disseminates. In the survey conducted by Lane and his colleagues (1969), 7 of 11 patients with progressive localized lesions developed "metastatic" lesions of vaccinia, and 4 died. Clinically milder forms of generalized vaccinia are believed to be the sequelae of a pro-

373

Poxviruses

F I G U R E 26.9 Uncomplicated primary vaccination, vesicular lesion at 20 days.

tracted inadequate immune response to immunization. Such an infection eventually resolves and is customarily not fatal (Annotations, 1964). Generalized disease also develops as a complication of eczema, even when the disease is in a state of remission. Patients with other forms of chronic skin disease are also at risk. In the past, those with active dermatological conditions were rarely immunized; thus, disseminated vaccinia generally occurred inadvertently among contacts of vaccinees who had chronic skin disease. Assuming an adequate immune capability, these patients recovered, but they were often severely ill for protracted periods of time, and the infection occasionally left ugly scars in its wake. Neurological Complications of Vaccination Postvaccinial central nervous system disease typically develops spontaneously in otherwise healthy vaccinees in the absence of recognized predisposing conditions. It encompasses a diversity of neurological syndromes, ranging from febrile seizures to encephalopathy with coma. In one study, 4 of the 14 encephalitic patients died. There are no characteristic neuropathological findings. In some cases, a nonspecific encephalitis with edema and perivascular infiltrates are found in the brain at autopsy, whereas in others the nervous system exhibits demyelinating changes in a pattern consistent with postinfectious encephalopathy. It would appear that certain vaccine strains of virus more commonly caused encephalitis. The pathogenesis of the disease of the central nervous system is not understood, and it is not clear to what extent the virus directly invades the brain. Gestational Complications of Vaccination Fewer than 20 cases of generalized vaccinia acquired in utero as a complication of the primary vaccination of

a pregnant woman have been reported. In these cases, immunization was carried out as early as the third week of pregnancy, and as late as the sixth month. When the fetus was infected, parturition occurred from 4 to 12 weeks later. The fetuses from early pregnancies are invariably stillbirths, whereas more mature but infected fetuses often survive for varying periods. Characteristic skin lesions are scattered over the body at autopsy. They consist of red circular ulcers up to 2 cm in diameter with sloughed centers. The placenta and viscera exhibit numerous abscess-like lesions, some of which are umbilicated. These punctate lesions are up to 3 mm in diameter and have irregular margins. Histologically, they exhibit necrosis, but no evidence of the vacciniavirus is found (Green et al, 1966; Naidoo and Hirsch, 1963). Lymphadenitis

Secondary to Vaccination

Clinical enlargement and tenderness of regional lymph nodes commonly occurs after vaccination and persists for varying periods. This is not surprising in view of the local inflammatory response to the vaccine in the skin, and the numerous antigens of the virus generated during infection. Vacciniavirus has been demonstrated in the lymph nodes (McMaster and Kidd, 1936; Hartsock and Bellanti, 1966). Lukes and colleagues (1966), Rappaport (1966), and Hartsock (1968) have described the pathological changes in these nodes and noted the potential for confusion with lymphoma, including Hodgkin's disease. As noted by these authors, there is an apparent effacement of the architecture by a diffuse or follicular hyperplasia in which immunoblasts are a prominent cellular component. Lymphocytes, plasma cells, and eosinophils are present in variable numbers. Focal vascular changes and dilated small vessels are evident. The changes are not unlike those observed in infectious mononucleosis (see Chapter 9) and herpes zoster (see Chapter 10). Monkeypox A virus similar to variola was first isolated from subhuman primates in the late 1950s (von Magnus et al., 1959). In 1970, the so-called monkeypox was first isolated in the Democratic Republic of the Congo from a human with an illness similar to smallpox (Ladnyi etal, 1972). Additional patients with monkeypox were reported from Nigeria, Liberia, and Sierra Leone during the 1970s; a total of 55 cases were documented in West Africa during that decade. Over the ensuing 24 years, some 350 clinically recognized cases were reported. Substantially fewer cases occurred over the subsequent years until 1996, when an outbreak developed in the former Zaire (Mukinda et ah, 1997; Cohen, 1997).

374

Pathology and Pathogenesis of Human Viral Disease

The clinical illness caused by the monkeypox virus in an unvaccinated human strikingly resembles smallpox (Fenner et ah, 1988) (Figure 26.10), but the mortality is substantially lower (11%) and the secondary attack rate proves to be only about 9% (in comparison to the rate for variola, which is 25 to 30%) (Breman and Henderson, 1998). Although the infectivity and mortality resulting from the infection is substantially lower than in smallpox, concern has arisen as to whether monkeypox could replace smallpox as a pandemic scourge on humanity. Computer simulations have now provided considerable assurance that this will not prove to be the case. The model forecasts that the virus would be expected to "die out" after 14 generations of human-tohuman transmission in a nonimmune population (Breman and Henderson, 1998). Thus far, natural transmission in humans has only been documented over a series of four generations. We can conclude that this virus, although similar to variola, poses little threat as a future epidemic/pandemic virus of worldwide im-

F I G U R E 26.10 Monkeypox in a child in the Democratic Republic of Congo. Reprinted with permission from Breman and Henderson (1998).

portance. Primary cases resulting from monkey to human transmission most likely will continue to occur in west central Africa. Sporadic reports of primate-to-human transmission of unclassified poxviruses not related to variola and monkeypox have been reported (Downie et al, 1971; McNulty et al, 1968). These incompletely characterized agents have proven to be relatively nonpathogenic for humans, although they cause localized skin lesions after both natural infection and experimental inoculation into human volunteers. It would appear that numerous species of subhuman primates host poxviruses having pathogenic potential for humans. Of particular interest is a West African poxvirus (Yaba) of primate origin (unrelated to recognized human poxviruses). It produces subcutaneous tumors in its natural host (Bearcroft and Jamieson, 1958). It also causes tumors when introduced into the skin of human volunteers, and after accidental inoculation of a laboratory worker (Grace et ah, 1962). The lesions exhibit a sarcomatoid microscopi-

Poxviruses

375

cal appearance and have been classified pathologically as histiocytomas (Sproul et ah, 1962).

PARAPOXVIRUSES The three members of this genus having human importance cause localized skin lesions at inoculation sites (often the hands) in farmers and tradesmen who work with cattle and sheep, or their products (Figure 26.11). The parapoxviruses are antigenically distinct

FIGURE 26.11 Lesion of ecthyma contagiosum (orf) on the proximal phalanx of the hand of a sheepherder.

from the orthopoxviruses, and cross-protection apparently does not exist. Acquisition of immunity by the infected human presumably results in imn\une-mediated protection against reinfection, but this conclusion has not been critically tested. Indeed, our understanding of the immune mechanisms involved are limited. Several stages in the evolution of the skin lesion have been described. The first is an elevated macular papular excrescence. Shortly thereafter, the so-called "target" lesion appears. It exhibits a red papular center surrounded by concentric alternating white and red halos. A vesicular-pustular nodule then develops. Subsequently, there is a regenerative phase during which the epithelium proliferates. Finally, the lesion regresses, generally 5 to 7 weeks after inoculation (Figure 26.12). Histologically, the erythematous papule that appears within a week after inadvertent inoculation exhibits spongiotic vacuolization of the squamous cells in the upper third of the epidermis. These cells display characteristic intracytoplasmic inclusions and rarely intranuclear inclusions (Figure 26.13) (Evins et ah, 1971; Leavell et al, 1968). The vesicular papule then becomes necrotic and develops a crust. The lesion then grows centrifugally forming in its wake multilobulate vesicles. The white halo that forms around the central papule in the so-called "target" lesions consists of a

FIGURE 26.12 The complex lesion of orf shows hyperplasia of the stratum malpighii and a marked parakeratosis with interlinked vesicles of varying dimensions. While the lesions of the paravaccinia group are vesicular, hyperplasia of the epithelium predominates, resulting in the nodular lesions that characterize the clinical appearance of the lesion.

ring of vacuolated cells, and the surrounding red halo results from inflammation in the dermis with the accompanying vascular dilatation. As the skin lesion evolves, the parakeratotic crust sloughs. The subjacent dermis exhibits a prominent but relatively circumscribed, but often intense, infiltrate of both lymphocytes and macrophages. The nodular stage evolves subsequently. It is characterized by extreme degrees of acanthosis, resulting in downward projections of tongues of the epidermis into the dermis. This is one of the distinguishing histological features of the lesion. At this late stage, viral inclusions rarely are observed in epithelial cells. The papillomatous stage that evolves reflects continued epidermal proliferation. By 5 weeks, regression begins. Some pathologists have described capillary proliferation and edema at the base of these lesions. Resolution of parapoxvirus lesions appears to depend upon an immune response. In patients with altered immunity, massive tumorous lesions have been described. In a case reported by Savage and Black (1972), a lesion on the finger of a patient with lymphoma grew luxuriantly, requiring amputation (Figure 26.14). On occasion, these parapoxvirus lesions have been confused clinically with malignant tumors of the skin.

376

Pathology and Pathogenesis of Human Viral Disease

^•'- . .jf^i'x........m..\..

«--::i%

#J*--

W

.vie., ?.•.<•

^'V-

1^

if-^;^-

FIGURE 26.13 Rare intranuclear (large arrow) and typical cytoplasmic (small arrow) inclusions in the squamous cells in a lesion of Milker's nodule on the hand of a dairy farmer. Reprinted with permission from Evins etal.(1971).

FIGURE 26.14 A giant lesion of orf in an elderly man with chronic lymphogenous leukemia. Reprinted with permission from Savage and Black (1972).

377

Poxviruses

Milker's N o d u l e s Jenner (1799) opined that two types of cowpox viruses exist. One causes "classical" cowpox (the orthopoxvirus discussed above) that confers immunity to both vaccinia and variola viruses, whereas the second is the virus responsible for milker's nodules (a parapoxvirus), termed by Jenner to be "spurious cowpox" and by others as pseudocowpox. The two forms of cowpox are distinguishable clinically in both cattle and humans, and the viruses exhibit different biological characteristics in the laboratory (Friedman-Kien et al, 1963; Davis and Musil, 1970). Milker's nodules are transmitted in the cow barn by milkers, who in turn are infected. It spreads with less facility than cowpox. In regions of the United States where dairy farming is common, milker's nodules are widely recognized as an endemic disease of cattle, and are a relatively common occupational hazard for the farmer. Bovine Papular Stomatitis (BPS) This is a rare infection of cattle. Proliferative lesions develop around the mouth of the animal in the absence of systemic illness. BPS infects animal handlers, causing an erythematous raised cutaneous nodule similar to milker's nodules and ecthyma contagiosum described below (Carson et al, 1968; McEvoy and Allen, 1972; Gruneberg and Heinig, 1957; Bowman et al, 1981). Ecthyma Contagiosum (orf) This parapoxvirus infection appears to be limited to sheep and goats. The virus is acquired by these animals in the pasture, and therefore appears on the legs and on the skin, lips, and face (Walley, 1890; Broughton and

Hardy 1934; Wheeler et al, 1955; Leavell et al, 1968). Lesions of the hands and fingers commonly occur among agricultural workers in regions where sheep and goat husbandry is common. MOLLUSCIPOXVIRUSES Jenner described molluscum contagiosum (MC) in the late 18th century, a few years before he introduced the practice of vaccination. The lesions of MC initially described by Jenner customarily occur in otherwise healthy children. They are 2 to 5 mm in diameter, pearl or flesh-like in appearance, and are elevated papules that exude a creamy curd-like viscous material from the apex. Customarily, several lesions develop simultaneously on the trunk and the face. Transmission of the infection was established experimentally in the mid1800s using the exudate from the papillae. Typical lesions develop at the site of inoculation 2 to 7 weeks later. They persist thereafter for several months and gradually resolve. Clinically, the infection is conveyed person to person by direct contact or through fomites, but autoinoculation also occurs. In children, MC tends to develop more commonly when body hygiene is lacking. Thus, in tribal residents of New Guinea, an annual incidence of 6% was documented with an overall prevalence of 22%. In the United States, the prevalence is believed to be approximately 1%. In recent years, with changes in sexual practices, MC genital lesions have been recognized frequently. In immunosuppressed patients with AIDS, the clinical features of the infection have changed. The disease tends to be distributed more widely over the body, but particularly in the region of the genitals and on the face (Figure 26.15). Large lesions resembling keratoacan-

FIGURE 26.15 Lesions of molluscum contagiosum on the face of a young man with AIDS. The CD4+ T lymphocyte count was 60/mm^. Numerous similar lesions were scattered on the scalp, buttocks, and legs. Reprinted with permission from Cotell and Rohalt (1998).

378

Pathology and Pathogenesis of Human Viral Disease

thomas or carcinomas commonly develop in immunosuppressed patients with AIDS (Schwartz and Myskowski, 1992; Koopman et ah, 1992). Biopsy diagnosis becomes imperative under these circumstances. Although the biology of immunity in MC has not been extensively studied, the common occurrence of widespread severe lesions in advanced HIV-1 infections strikingly suggest that cellular immune mechanisms are involved in resolution of MC lesions (Figure 26.16). Recently, a serious MC infection in a patient with AIDS was reversed by effective anti-HIV-1 protease inhibitor therapy (Epstein, 1993; Hicks et a/., 1997). 4UU-

•

FIGURE 26.17 Typical skin lesion of molluscum contagiosum. Note the depth of invagination of the lesion into the dermis and the expanding accumulation of cells with inclusions encroaching upon the vesicular umbilicated surface. Hyperplasia of the squamous epithelium in the lesion is evident. Note the surrounding lip of normal squamous epithelium.

350-»-• c O 3

300250-

+o 200Tf Q O

150100500- 1

X

• #^ • • -e-

••t •• < •• f <12

• -

^ >12

—

Number of MC Lesions FIGURE 26.16 Molluscum contagiosum lesion counts in patients with advanced HIV-1 infection. The patients with fewer than 12 lesions tend to have lesser degrees of immunosuppression as measured by the CD4+ T lymphocyte count. Patients with disseminated molluscum contagiosum lesions exhibited extremely low CD4+ blood concentrations of T cells. Reprinted with permission from Schwartz and Myskowski (1992).

The virus of MC is the sole member of the genus Molluscipoxvirus of human importance. The viral genome encodes 182 proteins, 105 of which have counterparts in members of the orthopoxvirus genus (Senkevich et ah, 1997). We can only surmise the biologic role of the proteins elaborated during replication of MC virus by drawing comparisons with other poxviruses inasmuch as they have not been characterized. Transmission of MC virus in cultured cells has been claimed, but tissue culture is not a tool for growing virus for investigative purposes, or for isolation attempts from clinical material. MC virus has been transmitted to chimpanzees experimentally.

The individual lesion of MC is largely located in the dermis, where circumscribed pear-shaped hyperplastic clusters of hypertrophied squamous cells fill a craterlike vesicle, covered in part by a lip of normal squamous epithelium (Figure 26.17). The characteristic hypertrophied cells of the lesion contain multiple cytoplasmic inclusions known as molluscum or Henderson-Patterson bodies (Figure 26.18). In turn, the inclusions are comprised of countless vacuoles that contain virions in various stages of evolution (Figure 26.19) (Middelkamp and Munger, 1964). These lesions have a typical morphology that is instantly recognized by the pathologist. Specific virological diagnosis currently can only be established by electron microscopic evaluation of the inclusions.

FIGURE 26.18 Molluscum bodies in squamous cells of the lesion shown in Figure 26.17.

Poxviruses

379

FIGURE 26.19 Electron micrograph of the massive inclusion of a moUuscum body in which are found nonmembranous vesicles containing virions. Note the nucleus and the prominent nucleoli at the margin of the cell boundary (4:00-6:00 o'clock). The cytoplasm exhibits striped opaque accumulations of keratohyalin. Nearby nucleated cells appear normal (12:00-1:30 o'clock). This morphologic picture is diagnostic of an MC infection. Reprinted with permission and through the courtesy of Dr. V. Burmeister.

References Annotations (1964). Sequelae of smallpox vaccination. Lancet 2,1380. Bearcroft, W., and Jamieson, M. (1958). An outbreak of subcutaneous tumors in rhesus monkeys. Nature 182,196-196. Bloch, H. (1993). Edward Jenner (1749-1823) The history and effects of smallpox, inoculation, and vaccination. Am. /. Dis. Child. 147, 772-774. Bowman, K., Barbery, R., Swango, L., and Schnurrenberger, R (1981). Cutaneous form of bovine papular stomatitis in man. JAMA 246, 2813-2818. Breman, J., and Henderson, D. (1998). Poxvirus dilemmas: Monkeypox, smallpox, and biologic terrorism. New Engl. J. Med. 339, 556-559.

Brinckerhoff, W., Tyzzer, E., and Councilman, W. (1906). Studies on experimental variola and vaccinia in Quadrumana. /. Med. Res. 14, 213-259. Broughton, I., and Hardy, W. (1934). Contagious ecthyma (sore mouth) of sheep and goats. /, Am. Vet. Med. Assoc. 85,150-178. Cangemi, V. (1958). Acute pericarditis after smallpox vaccination. New Engl. ]. Med. 258,1257. Carson, C , Kerr, K., and Grumbles, L. (1968). Bovine papular stomatitis: Experimental transmission from man. Am. J. Vet. Res. 29, 1783-1790. Cochran, W., Connolly, J., and Thompson, I. (1963). Bone involvement after vaccination against smallpox. Br. Med. J. 2, 285. Cohen, J. (1997). Is an old virus up to new tricks? Science 111, 312-313.

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Cotell, S., and Rohalt, M. (1998). Molluscum contagiosum in a patient with AIDS. New Engl J. Med. 338, 888. Davis, C , and Musil, G. (1970). Milker's nodule: A clinical and electron microscopic report. Arch. Dermatol. 101, 305-311. Downie, A., Taylor-Robinson, C , Caunt, A., Nelson, G., MansonBahr, R, and Matthews, T. (1971). Tanapox: A n e w disease caused by a pox virus. Br. Med. J. 1, 363-368. Elliot, W. (1959). Vaccinial osteomyelitis. Lancet 2,1053. Ellis, P., and Winograd, L. (1962). Ocular vaccinia. Arch. Ophthalmol. 68, 600-609. Epstein, W. (1993). Molluscum contagiosum. Sem. Dermatol. 11,184189. Evins, S., Leavell, U., and Phillips, I. (1971). Intranuclear inclusions in milker's nodules. Arch. Dermatol. 103, 91-93. Fenner, R, Henderson, D., Arita, I., Jezek, Z., and Ladnyi, I. (1988). ''Smallpox and Its Eradication." World Health Organization, Geneva. Finlay-Jones, L. (1964). Fatal myocarditis after vaccination against smallpox: Report of a case. New Engl. J. Med. 270, 4 1 ^ 2 . Freed, E., Duma, R., and Escobar, M. (1972). Vaccinia necrosum and its relationship to impaired immunologic responsiveness. Am. J. Med. 52, 411-420. Friedman-Kien, A., Rowe, W., and Banfield, W. (1963). Milker's nodules: Isolation of a poxvirus from a human case. Science 140, 1335-1336. Gordon, C , Donnelly J., Fothergill, R., Ker, R, Millar, E., Flewett, T., Bedson, H., and Cruickshank, J. (1966). Variola minor: Apreliminary report from the Birmingham Hospital Region. Lancet 1,1311-1314. Grace, J., Mirand, E., Millian, S., and Metzgar, R. (1962). Experimental studies of human tumors. Fed. Proc. 21, 32-36. Green, D., Reid, S., and Rhaney, K. (1966). Generalised vaccinia in the human foetus. Lancet 1,1296-1297. Gruneberg, T., and Heinig, A. (1957). Melkerknoten nach Infektion mit dem Virus der Stomatitis papulose der rinder. Arch. Klin. Exp. Dermatol. 205,144-149. Haar, H., and Meinertz, O. (1954). Akute osteomyelitis nach pochenund typhusschutzimpfung. Munchen. Med. Wschr. 96, 87. Hartsock, R. (1968). Postvaccinial lymphadenitis: Hyperplasia of lymphoid tissue that simulates malignant lymphomas. Cancer 21, 632-649. Hartsock, R., and Bellanti, J. (1966). Comparative histologic changes of postvaccinial lymphadenitis in man, monkey and rabbit. Fed. Proc. 25, 534. Hathaway, W., Githens, J., Blackburn, W., Fulginiti, V, and Kempe, C. (1965). Aplastic anemia, histiocytosis and erythrodermia in immunologically deficient children. Probable human runt disease. New Engl. }. Med. 273, 953-958. Hernon, C. (1996). Smallpox: 26 years ago. New Engl. J. Med. 334,1304. Hicks, C., Myers, S., and Giner, J. (1997). Resolution of intractable molluscum contagiosum in a human immunodeficiency virus-infected patient after institution of antiretroviral therapy with ritonavir. Clin. Infect. Dis. 24,1023-1024. Jenner, E. (1799). "Further Observations on the Variolae Vaccine." Samson Low, London (privately printed). Koopman, R., Van Merrienboer, R, Vreden, S., et al. (1992). Molluscum contagiosum: A marker for advanced HIV infection. Br. J. Dermatol. 126, 528-529. Ladnyi, I., Ziegler, P., and Kima, A. (1972). A human infection caused by monkeypox virus in Basankusu Territory, Democratic Republic of the Congo. Bull. WHO 46, 593-597.

Lane, J., Ruben, R, Neff, J., and Millar, J. (1969). Complications of smallpox vaccination, 1968: National surveillance in the United States. New Engl. J. Med. 281,1201-1208. Leavell, U., McNamara, M., Muelling, R., Talbert, W., Rucker, R., and Dalton, A. (1968). Orf: Report of 19 human cases with clinical and pathological observations. JAMA 204, 657-664. Lukes, R., Butler, J., and Hicks, E. (1966). Natural history of Hodgkin's disease as related to its pathologic picture. Cancer 19, 317-344. Mahy, B., Almond, J., Berns, K., Chanock, R., Lvov, D., Pettersson, R., Schatzmayr, H., and Fenner, F. (1993). The remaining stocks of smallpox virus should be destroyed. Science 262,1223-1226. Matthews, A., and Griffiths, I. (1974). Post-vaccinial pericarditis and myocarditis. Br. Heart J. 36,1043-1045. McEvoy, J., and Allen, B. (1972). Isolation of bovine papular stomatitis virus from humans. Med. J. Aust. 1,1254-1256. McMaster, P., and Kidd, J. (1936). Lymph nodes as a source of neutralizing principle for vaccinia. /. Exp. Med. 62, 73-100. McNulty, W., Lobitz, W., Hu, R, Maruffo, C , and Hall, A. (1968). A pox disease in monkeys transmitted to man: Clinical and histological features. Arch. Dermatol. 97, 286-293. Middelkamp, J., and Munger, B. (1964). The ultrastructure and histogenesis of molluscum contagiosum. /. Pediatrics 64, 888-905. Mukinda, V, Mwema, G., Kilundu, M., Heymann, D., Khan, A., and Esposito, J. (1997). Re-emergence of human monkeypox in Zaire in 1996. Lancet 349, 1449-1450. Naidoo, P., and Hirsch, H. (1963). Prenatal vaccinia. Lancet 1,196-197. Neff, J., and Lane, J. (1970). Vaccinia necrosum following smallpox vaccination for chronic herpetic ulcers. JAMA 213,123-125. Rappaport, H. (1966). Tumors of the hematopoietic system. In "Atlas of Tumor Pathology," sec. Ill, fasc. 8. Armed Forces Institute of Pathology, Washington, DC. Ruben, R, and Lane, J. (1970). Ocular vaccinia: An epidemiologic analysis of 348 cases. Arch. Ophthalmol. 84, 45-^8. Savage, J., and Black, M. (1972). "Giant" orf of finger in a patient with lymphoma. Proc. Royal Soc. Med. 65, 28-29. Schwartz, J., and Myskowski, P. (1992). Molluscum contagiosum in patients with human immunodeficiency virus infection: A review of twenty-seven patients. /. Am. Acad. Dermatol. 27, 583-588. Senkevich, T, Koonin, E., Bugert, J., Darai, G., and Moss, B. (1997). The genome of molluscum contagiosum virus: Analysis and comparison with other poxviruses. Virology 233,19-42. Sewall, S. (1949). Vaccinia osteomyelitis. Bull. Hosp. Joint Dis. 10: 59. Silby H., Farber, R., O'Connell, C , Ascher, J., and Marine, E. (1965). Acute monarticular arthritis after vaccination: Report of a case with isolation of vaccinia virus from synovial fluid. Ann. Intern. Med. 62, 347-350. Sproul, E., Metzgar, R., and Grace, J. (1962). The pathogenesis of the Yaba virus tumor in the rhesus monkey. Proc. Am. Assoc. Cancer Res. 3: 363. von Magnus, P., et al. (1959). A pox-like disease in cynomolgus monkeys. Arch. Pathol. Microbiol. Scand. 46,156-176. Walley, T. (1890). Contagious dermatitis: "Orf" in sheep. /. Comp. Path. Ther. 3, 357-365. Wheeler, C , Cawley, E., and Johnson, J. (1955). Ecthyma contagiosum (Orf). Arch. Dermatol 71, 481^85.

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nities at roughly 2-year intervals with intervening periods of quiescence. As a result, in the prevaccine era, a substantial proportion of the childhood population exhibited serological evidence of past infection by the end of the first decade of life. In order for mumps virus to be sustained in a community, a sizable pool of susceptible children must be at risk. Thus, the ''herd immunity" invoked by the use of vaccines in developed countries has largely eliminated the virus from developed countries. Respiratory symptoms and parotitis to a variable degree of severity evolve after a latency period of 2 to 3 weeks from the time of exposure. Virus is shed in the respiratory tract secretions for a period of less than 1 week, its disappearance being temporally associated with the arrival of IgA. Studies by the pathologists Johnson and Goodpasture in the early 1930s established the association of mumps virus with parotitis and demonstrated the presumptive route of parotid gland involvement. These investigators injected oral pharyngeal secretions from infected patients into the Stenson's duct of monkeys and observed the appearance of inflammatory lesions in the parotids. After several passages in monkey parotid tissue, they removed and minced the glands of infected animals and sprayed an extract of the tissue on the buccal mucosa, and into the nostrils, of children. Four subjects with past histories of epidemic parotitis failed to develop parotitis, whereas 6 of 13 presumptively susceptible children had clinical mumps. Thus, these investigators fulfilled Koch's postulates by experiments that would be ethically unacceptable today. In naturally infected children, viremia potentially seeds the central nervous system, testes, pancreas, eyes (Fields, 1947; Meyer et al, 1974; Strong et al, 1974; Al-Rashid and Cress, 1987), inner ears, kidneys (Utz et al, 1964), thyroid, joint spaces, ovaries (Morrison et a/., 1975), mammary tissue, myocardium, and serous surfaces where inflammatory lesions occasionally develop (Utz et a/., 1964; Overman, 1958). Fortuitous observation of an epidemic in an immunologically "virgin" population

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INTRODUCTION Epidemic illness characterized by nonsuppurative swelling of the parotid glands and accompanied at times by swelling of the testes was first described by Hippocrates in the fifth century B.C.E. In more recent years, mumps has claimed considerable medical attention because of its protean clinical features and its involvement of many systemic organs in addition to the salivary glands and testes. For example, an 8-months pregnant 28-year-old multiparous family member of mine developed an acutely painful mastitis and lower abdominal pain believed to be due to oophoritis, concomitant with classical mumps parotitis. Clearly, mumps is a generalized disease, but it has few longterm complications, and adverse outcomes of pregnancy are not reported in the literature. To a large extent, it is a disease that has disappeared in developed countries as a result of effective immunization during early childhood. Mumps virus is classified in the genus paramyxoviruses. It shares all of the virological characteristics of this genera, but possesses unique antigenic determinants that elicit a lasting immunity after recovery from infection. Spread by respiratory droplets, the infection is manifest initially as an upper respiratory tract infection, followed by viremia and inflammatory lesions in the parotid and in a variety of organs (which differ from one patient to another). Before the time of widespread immunization, mumps virus spread in commu-

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of Native Americans on the St. Lawrence Islands of the Bering Sea has provided insights into the relative prevalence of disease in a fully susceptible population (Philip et al, 1959). Over the period of the outbreak, 27% of the population developed asymptomatic infections, whereas 65% exhibited clinical parotitis. Overall, 25% of males developed orchitis with an attack rate of almost 40% in postpubertal boys and young adults. Only 9% of older men developed symptoms of testicular inflammation.

SALIVARY G L A N D DISEASE Postmortem descriptions of the parotids of humans during the course of symptomatic disease are limited to the reports of Weller and Craig (1949), Henson et ah (1971), Donohue (1941) and Bostrom et al (1968). Because the patients described in these studies died at varying intervals after onset of the parotid disease, only glimpses of the pathological changes are recorded. However, the human observations can be supplemented by the work of Wollstein (1916) and the painstaking observations of Johnson and Goodpasture (1936) using experimentally infected Macaca mullata monkeys. As one would expect, the parotid glands grossly appear boggy and edematous. The microscopical changes are variable from lobule to lobule and consist of hydropic changes with intracytoplasmic vesicles in various acinar cells accompanied by interstitial edema and a focal lymphocytic infiltrate. The ducts also show alterations of variable severity, ranging from apparent swelling of the epithelial lining cells to desquamation of the mucosa, with the accumulation of eosinophilic necrotic debris and neutrophils in the lumina. Ultrastructural studies by Henson et al. (1971) failed to elaborate on these basic observations. Apparently, residual changes are not found in the gland after recovery. The submaxillary gland, but not the mucussecreting sublingual gland, were affected in the case studied by Henson et al (1971) (Figure 27.1A-D); however, the number of observations from postmortem studies are limited. Henson et al (1971) noted alterations of a comparable type in the submucosal glands of the tracheobronchial tree. Clinical observations have documented the rare occurrence of parotitis in influenza and parainfluenza-infected patients and those with enterovirus infections, but pathologic studies have not been done on these cases (Bloom et al, 1961; ZoUar and Mufson, 1970; Banks, 1968). In most patients, parotitis resolves over a 2-week period and no residual pathological effects have been observed.

CENTRAL NERVOUS SYSTEM DISEASE (Bang and Bang, 1943)

Viremia seeds the meninges and ependyma of the CNS in many cases of mumps virus infection. Evidence of CNS involvement in the form of aseptic meningitis or mild encephalitis develops in roughly 10% of those who are infected, but an additional 65% manifest cerebrospinal fluid pleocytosis in the absence of nuchal rigidity and obtundation or symptoms (headache) of neurological disease. Males are affected more often than females (Johnson, 1982). Mumps accounted for over 15% of cases of aseptic meningitis reported in a large series of cases by Meyer et al (1960; see also Johnstone et al, 1972). It can occur concomitantly with or after the onset of parotitis, or in the absence of systemic features of mumps virus infection. While the aseptic meningitis is generally but not always transient (Azimi et al, 1975), fatal encephalitis occasionally has been reported (Bistrain et al, 1972). In the prevaccine years of the late 1960s and early 1970s, 44 fatal cases of mumps encephalitis were reported to the Centers for Disease Control over a 9-year period. The pathological features of these cases are not known. Ependymitis appears to occur commonly when the CNS is involved by mumps virus (Johnson and Johnson, 1972; Herndon et al, 1974). Ependymal cells individually and in clusters were found in the cerebrospinal fluid of six patients with mumps aseptic meningitis by examining fluid sediments by electron microscopy (Herndon et al, 1974). Viral nucleocapsid material was found ultrastructurally in these same cells. It has been postulated that the cells were sloughed from the ependymal surface of the brain. Johnson speculates that this may account for the granular ependymitis commonly seen in normal human brains (Johnson, 1982). Aqueductal stenosis and hydrocephalus has been occasionally reported in children months after an episode of mumps (Timmons and Johnson, 1970; Paraicz, 1970; Hower et al, 1972; Bray 1972; Kilham and Margolis, 1975; Spataro et al, 1976; Lagenstein et al, 1976). Although it has not been clearly established that mumps is a cause of aqueductal obstruction resulting in hydrocephalus, a substantial body of experimental evidence has accumulated to support this claim (Johnson and Johnson, 1968; Kilham and Margolis, 1974, 1975; Wolinsky 1977; London et al, 1979). In addition to the above syndromes, transverse myelitis and polyneuritis have been reported in those infected with mumps virus (Tulloch, 1973; Benady et al, 1973).

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FIGURE 27.1 The parotids of a 5-year-old male with lymphoma treated with combined drug chemotherapy. Parotid swelling began 25 days after exposure to a sibling with clinically typical mumps. The patient's condition deteriorated, with neurological signs and evidence of intravascular coagulation. He died 41 days after exposure to the virus. Eleven days before death, the serum amylase was elevated. Mumps virus was recovered from the parotid gland at autopsy. (A,B) Ductal lining cells exhibit cytoplasmic vacuolization and degeneration. There is focal desquamation of the lining of some ducts, whereas others showed regeneration of the epithelium. Eosinophilic debris consisting of sloughed ductal lining cells is evident in the lumina. The nearby connective tissue is loosely cellular and edematous. (C,D) Acinar cells are swollen and vacuolated. The vacuoles are most prominent adjacent to the basement membrane. Focal interstitial and lymphocytic infiltrates are present. Reprinted with permission from Henson et al. (1971). Photomicrographs kindly provided by D. Henson, MD.

TESTICULAR DISEASE Orchitis develops in a significant number of postpubertal males infected with mumps virus. Rarely, it occurs in younger boys (Atkinson and Bass, 1968; Green, 1964). In only about 20 to 25% of cases are both testes involved. The basis for the unilaterality of the disease

is not known, and no specific side is preferably affected. Urologists have vacillated with regard to the comparative value of surgical decompression by incision of the tunica albuginea and antiinflammatory drug therapy Gall (1947) reported pathological studies on a large series of cases treated surgically by evaluating biopsies of the small amounts of testicular tissue

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that protruded through the surgically incised capsule of the testis. These specimens were almost invariably obtained within the first 5 days after the onset of symptoms. As described by Gall (1947), the earliest change was interstitial edema, followed by vascular dilatation and engorgement. Scant perivascular lymphatic accumulations were noted during the early stages of the disease, with lymphocytic vascular cuffing developing later. In some cases, lymphocyte accumulations were said to be sufficient to completely fill the space intervening between tubules. During the course of these events, the germinal epithelium exhibits progressively severe degenerative changes. In addition, neutrophils accumulate in the lumina of occasional seminiferous tubules. Sertoli cells were relatively undamaged and remained attached to the lamina propria of tubules (which seemed to thicken). Individual tubules appeared to be involved to a variable extent early in the development of the testicular lesions, but the changes became more diffuse in the advanced stages of the disease. At a later stage, the tubules showed few, if any, germinal epithelial cells, and the interstitium was densely packed with lymphocytes (Figure 27.2). Leydig cells in the interstitium, although compressed, did not exhibit atrophy. This latter observation is of interest, for in at least one report circulating testosterone concentrations in the blood were decreased during the acute stages of orchitis, and both follicle-stimulating and luteinizing hormones were increased, as would be expected (Adamopoulos et al, 1978).

The long-term effects of orchitis on the testicular substance is unclear, for no systematic studies of the testes of men with documented prior mumps virus infections have been carried out. It is generally believed that a substantial proportion of males experience unilateral testicular atrophy as a consequence of mumps orchitis (Figure 27.3). The adverse effects of orchitis are thought to relate to pressure necrosis during the acute stages of the disease, not to a direct effect of the virus on the testicular parenchymal cells.

PANCREATIC DISEASE Clinical evidence of pancreatitis occurring shortly after the onset of parotitis has been documented in a number of studies (Imrie et al, 1977). Usually, the disease is of mild or moderate severity, and customarily it resolves without complication. The incidence in large series ranges from 0.25 to 12% (Brahdy and Scheffer, 1931). In rare cases, pancreatitis has been associated on the basis of serological studies with mumps virus infection in the absence of parotitis (Witte and Schanzer, 1968; Naficy et al, 1973). Pathological studies have rarely been reported (Lemoine and Lapasset, 1905; Sabrazes et al, 1927; Bostrom, 1968). As with the parotid, the pancreas is boggy and swollen and the acinar cells microscopically vacuolated. Changes in the pan-

FIGURE 27.2 Acute orchitis in a patient with systemic symptoms and parotitis. Note the sloughing of the tubular cells (arrowhead). The interstitium is edematous and infiltrated by lymphocytes. Note the prominent interstitial Leydig cells. They have an abundant relatively clear cytoplasm.

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F I G U R E 27.3 Normal testis in a middle-aged man at autopsy (left). Note the fine filiform appearance of the parenchyma reflecting the seminiferous tubules. The testis on the right is shrunken and has a thickened tunica albugineae. The filiform features are no longer present. Microscopically, the individual tubules are lined by Sertoli cells and have a fibrous thickened wall. The interstitium is fibrotic.

creatic ducts have not been described in the limited pathological material available. In two reported cases, fat and acinar cell necrosis were observed (Sabrazes et al, 1927; Wood et al, 1974). The Islets of Langerhans are compressed and show degenerative changes, an observation of interest in view of the alleged association of mumps virus infection with insulin-dependent diabetes mellitus. MUMPS-ASSOCIATED DIABETES MELLITUS The notion that type 1 (insulin-dependent, so-called juvenile) diabetes mellitus could have an infectious etiology has long been suggested in the medical literature. The concept was initially based upon anecdotal case reports (Gilhespy and Holden, 1917; Patrick, 1924; Kremer, 1947; Hinden, 1962; McCrae, 1963; Kahana and Berant, 1967; Dacou-Voutetakis et al, 1974; Khakpour and Nik-Akhtar, 1975; Reddy and Crump, 1976) and rather primitive epidemiological surveys (Gundersen, 1927) suggesting that diabetes occasionally developed after mumps. As noted above, pancreatitis is a wellrecognized clinical feature of mumps virus infection, but it is often a vaguely defined condition and is not consistently evident clinically. Only a few pathological studies have been carried out on individuals with clinical mumps pancreatitis. In these cases, the pancreas exhibited interstitial edema accompanied by a pleomorphic cell infiltrate predominantly

consisting of lymphocytes. In the report of Lemoine and Lapasset, the islets of Langerhans were said to be "reduced in size and number due to compression by the glandular elements" (Lemoine and Lapasset, 1905). Sabrazes et al. (1927) noted coagulative necrosis of the pancreatic acinar tissue and parapancreatic adipose tissue associated with inflammation. The few islets of Langerhans were said to show degenerative changes. The occurrence of diabetic ketoacidosis and amylasemia in an individual with rising titers of antibody to mumps virus suggests the occurrence of both acinar and insular beta cell injury during the course of the infection. This patient recovered and demonstrated no subsequent abnormalities of carbohydrate metabolism (Block et ah, 1973). The demonstration of islet cell-specific antibodies in the blood serum of children after recovery from mumps proves of interest (Helmke et al, 1986; Helmke, 1987). However, in the cases reported by Helmke, diabetes did not evolve, despite the appearance of circulating islet cell antibodies. Pancreatic beta cells have been found to be susceptible to infection by mumps virus in vitro (Prince et al, 1978). Considerable circumstantial evidence supports the concept that insulin-dependent type 1 diabetes mellitus has an infectious etiology, although immunological factors also play an important pathogenetic role. In addition to mumps, coxsackie group B and rubella viruses have been implicated on the basis of retrospective serological surveys and epidemiological studies in

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humans. Work with experimental animal models also supports the concept (Craighead, 1975) (see Chapters 1 and 28). More recently, it was suggested that mumps virus vaccine might be causatively responsible for some cases of diabetes, but this notion is not supported by epidemiological studies. At present, some 15 years after the introduction and extensive use of mumps virus vaccines in North America and Europe, cases of type 1 diabetes continue to develop, and the prevalence appears to be increasing in Scandinavian populations, where immunization rates are high.

EAR DISEASE Loss of hearing is an uncommon complication of mumps: it occurs in roughly 0.1% of cases, although Vuori ei al. (1962) reported an incidence of 4%. Typically unilateral, 20 to 30% of affected patients experience bilateral deafness. Vertigo occasionally accompanies loss of hearing (Hyden ei al., 1979). In most patients, these problems are temporary and do not persist. The pathogenesis of mumps inner ear disease is undefined. It may result from the viremia that so often occurs in mumps virus infection. On the other hand, virus may be transported to the inner ear by way of the eustachian tubes and the middle ear. A meningeal route is a third possibility. In the few reported cases that have been studied at autopsy (Smith and Gussen, 1976; Lindsay ei al., 1960; Vuori ei al., 1962), widespread degenerative changes were described in the tentorial membrane and stria vascularis, as well as in the organ or corti and cochlea. Evidence of nerve fiber loss has also been reported. The reader is referred to the referenced sources for more complete information.

JOINT DISEASE Migratory polyarthritis affecting larger joints develops in less than 1% of those with mumps, usually during the week following the onset of parotitis (Solem, 1971; Ghosh and Reddy 1973; Bryer and Rounthwaite, 1972; Caranasos and Felker, 1967; Overman, 1958; Eyland ei al., 1957; Utz ei al, 1964; Appelbaum ei al, 1952; Lass and Shephard, 1961; Filpi and Houts, 1968). The prevalence in males is fivefold greater than in females, and concurrent orchitis is common. Peak incidence is in the third decade of life. Symptoms often occur in the

absence of clinical evidence of joint inflammation, and there is little, if any symptomatic response to nonsteroidal antiinflammatory agents. The pathogenesis is uncertain, but direct infection of the synovia of affected joints seems to be a likely explanation. Virus has not been isolated from joint fluid, and the usual serological measures of rheumatic disease are not generally elevated. Pathological studies of the synovia of the joints have not been reported.

References Adamopoulos, D., Lawrence, D., Vassilopoulos, P., Contoyiannis, P., and Swyer, G. (1978). Pituitary-testicular interrelationships in mumps orchitis and other viral infections. Br. Med. J. 1,1177-1180. Al-Rashid, R., and Cress, C. (1987). Mumps uveitis complicating the course of acute leukemia. /. Pediatr. Ophthalmol. 14,100-102. Appelbaum, E., Kohn, J., Steinman, R., and Shearn, M. (1952). Mumps arthritis. Arch. Intern. Med. 90, 217-223. Atkinson, J., and Bass, H. (1968). Mumps orchitis in a three-year-old child [letter]. JAMA 203, 172. Azimi, P., Shaban, S., Hilty, M., and Haynes, R. (1975). Mumps meningoencephalitis: Prolonged abnormality of cerebrospinal fluid. JAMA 234, 1161-1162. Bang, H., and Bang, J. (1943). Involvement of the central nervous system in mumps. Acta Med. Scand. 113, 487-505. Banks, P. (1968). Nonneoplastic parotid swellings: A review. Oral Surg. 25, 732-745. Benady, S., Ben Zvi, A., and Szabo, G. (1973). Transverse myelitis following mumps. Acta Pediatr. Scand. 62, 205-206. Bistrain, B., Phillips, C., and Kaye, I. (1972). Fatal mumps meningoencephalitis. JAMA 111, 478-479. Block, M., Berk, J., Fridhandler, L., Steiner, D., and Rubenstein, A. (1973). Diabetic ketoacidosis associated with mumps virus infection: Occurrence in a patient with macroamylasemia. Ann. Intern. Med. 78, 663-667. Bloom, H., et al. (1961). Recovery of parainfluenza viruses from adults with upper respiratory illness. Am. J. Epidemiol. 74, 50-59. Bostrom, K. (1968). Patho-anatomical findings in a case of mumps with pancreatitis, myocarditis, orchitis, epididymitis and seminal vesiculitis. Virchows Arch. Abt. A Path. Anat. 344,111-117. Brahdy M., and Scheffer, I. (1931). Pancreatitis complicating mumps. Am. J. Med. Sci. 181, 255-260. Bray, P. (1972). Mumps — A cause of hydrocephalus? Pediatrics 49, 446^49. Bryer, D., and Rounthwaite, F. (1972). Cricoarytenoid arthritis due to mumps. Laryngoscope 8, 372-375. Caranasos, G., and Felker, J. (1967). Mumps arthritis. Arch. Intern. Med. 119, 394-398. Craighead, J. (1975). The role of viruses in the pathogenesis of pancreatic diabetes and diabetes mellitus. In ''Progress in Medical Virology'' (J. Melnick, ed.). Vol. 19, pp. 161-214. Karger, Basel. Dacou-Voutetakis, C , Constantinidis, M., Moschos, A., Vlachou, C , and Matsaniotis, N. (1974). Diabetes mellitus following mumps: Insulin reserve. Am. J. Dis. Child. 127, 890-891. Donohue, W. (1941). The pathology of mumps encephalitis with report of a fatal case. /. Pediatr. 19, 42-52. Eyland, E., Zmucky, R., and Sheba, C. (1957). Mumps virus and subacute thyroiditis. Lancet 1, 1062-1063.

Mumps Fields, J. (1947). Ocular manifestations of mumps. Am. J. Ophthalmol 30, 591-595. Filpi, R., and Houts, R. (1968). Mumps arthritis. JAMA 205, 216-217. Gall, E. (1947). The histopathology of acute mumps orchitis. Am. J. Pathol. 23, 637-651. Ghosh, S., and Reddy, T. (1973). Arthralgia and myalgia in mumps. Rheumatol Rehah. 12, 97-99. Gilhespy, R, and Holden, H. (1917). Grave diabetes mellitus with pulmonary tuberculosis following mumps. Br. Med. J. 115, 403-406. Green, G. (1964). Mumps orchitis in childhood. Practitioner 192, 550552. Gundersen, E. (1927). Is diabetes of infectious origin? /. Infect. Dis. 41, 197-202. Helmke, K. (1987). Islet cell antibodies in children with mumps infection. In "Virus Infections and Diabetes Mellitus" (Y. Becker, ed.), pp. 127-142. Martinus Nijhoff, Boston. Helmke, K., Otten, A., Willems, W., Brockhaus, R., Mueller-Eckhardt, G., Stief, T., Bertrams, J., Wolf, H., and Federlin, K. (1986). Islet cell antibodies and the development of diabetes mellitus in relation to mumps infection and mumps vaccination. Diabetologia 29, 30-33. Henson, D., Siegel, S., Strano, A., Primack, A., and Fuccillo, D. (1971). Mumps virus sialoadenitis: An autopsy report. Arch. Pathol 92, 469^74. Herndon, R., Johnson, R., Davis, L., and Descalzi, L. (1974). Ependymitis in mumps virus meningitis. Arch. Neurol 30, 475^79. Hinden, E. (1962). Mumps followed by diabetes. Lancet 1,1381. Hower, J., Clar, H., and Duchting, M. (1972). Aquadukt verschluss nach Mumps-Meningitis. Dtsch. Med. Wochenschr 97, 4 3 ^ 4 . Hyden, D., Odkvist, L., and Kylen, R (1979). Vestibular symptoms in mumps deafness. Acta Otolaryngol, Suppl 360, 182-183. Imrie, C , Ferguson, J., and Sommerville, R. (1977). Coxsackie and mumps virus infection in a prospective study of acute pancreatitis. Gut 18, 53-56. Johnson, C., and Goodpasture, E. (1936). The histopathology of experimental mumps in monkey, Macacus rhesus. Am. J. Pathol 12, 495-512. Johnson, K., and Johnson, R. (1972). Granular ependymitis: Occurrence in myxovirus infected rodents and prevalence in man. Am. J. Pathol 67, 511-522. Johnson, R. (1982). ''Viral Infections of the Nervous System." Raven Press, New York. Johnson, R., and Johnson, K. (1968). Hydrocephalus following viral infection: The pathology of aqueductal stenosis developing after experimental mumps virus infection. /. Neuropathol Exp. Neurol 27, 591-606. Johnstone, J., Ross, C., and Dunn, M. (1972). Meningitis and encephalitis associated with mumps infection: A10 year survey. Arch. Dis. Child. 47, 647-651. Kahana, D., and Berant, M. (1967). Diabetes in an infant following inapparent mumps. Clin. Ped. 6,124-125. Khakpour, M., and Nik-Akhtar, B. (1975). Diabetes mellitus following a mumps epidemic. /. Trop. Med. Hyg. 78, 262-263. Kilham, L., and Margolis, G. (1974). Intrauterine infections induced by mumps virus in hamsters. Lab. Invest. 31, 34-41. Kilham, L., and Margolis, G. (1975). Induction of congenital hydrocephalus in hamsters with attenuated and natural strains of mumps virus. /. Infect Dis. 132, 462-466. Kremer, H. (1947). Juvenile diabetes as a sequel to mumps. Am. ]. Med. 3, 257-258.

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Lagenstein, I., Iffland, E., Blaschke, E., and Sieg, K. (1976). Aduaduktverschlufi als Komplikation der Mumpsmeningoenzephalitis. Klin. Padiat. 188, 445-448. Lass, R., and Shephard, E. (1961). Mumps arthritis. Br. Med. J. 2, 1613-1614. Lemoine, G., and Lapasset, R (1905). Un cas de pancreatite ourlienne avec autopsie. Bull et Mem. Soc. Med. (Paris) 22, 640-646. Lindsay, J., Davey, P., and Ward, P. (1960). Inner ear pathology in deafness due to mumps. Ann. Otol Rhinol Laryngol 69, 918-935. London, W, Kent, S., Palmer, A., Fucillo, D., Houff, S., Saini, N., and Sever, J. (1979). Induction of congenital hydrocephalus with mumps in Rhesus monkeys. /. Infect. Dis. 139, 324-328. McCrae, W. (1963). Diabetes mellitus following mumps. Lancet 1, 1300-1301. Meyer, H. J., Johnson, R., Crawford, I., Dascomb, H., and Rogers, N. (1960). Central nervous system syndromes of "viral" etiology: A study of 713 cases. Am. J. Med. 29, 334-347. Meyer, R., Sullivan, J., and Oh, J. (1974). Mumps conjunctivitis. Am. J. Ophthalmol 78,1022-1024. Morrison, J., Givens, J., Wiser, W, and Fish, S. (1975). Mumps oophoritis: a cause of premature menopause: Fertility and Sterility. 26, 655-659. Naficy, K., Nategh, R., and Ghadimi, H. (1973). Mumps pancreatitis without parotitis. Br. Med. J. 1, 529. Overman, J. (1958). Viremia in human mumps virus infection. Arch. Intern. Med. 102, 354-356. Paraicz, E. (1970). Angabem zum membranosen verschluss des aqueductus Sylvii. Acta Paediatr Acad. Sci Hung. 11, 121-133. Patrick, A. (1924). Acute diabetes following mumps. Br Med. J., 1, 802-806. Philip, R., Reinhard, K., and Lackman, D. (1959). Observations on a mumps epidemic in a "virgin" population. Am. J. Hyg. 69, 91-111. Prince, G., Jenson, A., Billups, L., and Notkins, A. (1978). Infection of human pancreatic beta cell cultures with mumps virus. Nature 271, 158-161. Reddy, C , and Crump, E. (1976). Diabetes mellitus following mumps. /. Natl Med. Assoc. 68, 459^60. Sabrazes, J., Broustet, P., and Beaudiment, R. (1927). Nephrite et pancreatitie necrosaute suivie de morte au cours d'ourlienne. Gaz. hebd. d. sc. med. de Bordeaux 48, 705. Smith, G., and Gussen, R. (1976). Inner ear pathologic features following mumps infection: Report of a case in an adult. Arch. Otolaryngol 102, 108-111. Solem, J. (1971). Mumps arthritis without parotitis. Scand. J. Infect. Dis. 3,173-175. Spataro, R., Lin, S.-R., Horner, R, Hall, C , and McDonald, J. (1976). Aqueductal stenosis and hydrocephalus: Rare sequelae of mumps virus infection. Neuroradiology 12,11-13. Strong, L., Henderson, J., and Gangitano, J. (1974). Bilateral retrobulbar neuritis secondary to mumps. Am. J. Ophthalmol 78, 331-332. Timmons, G., and Johnson, K. (1970). Aqueductal stenosis and hydrocephalus after mumps encephalitis. New Engl. J. Med. 283, 15051507. Tulloch, A. (1973). Polyneuritis — An unusual complication of mumps. Med. J. Aust. 1, 844-846. Utz, R, Houk, V, and Ailing, D. (1964). Clinical and laboratory studies of mumps, IV: Viruria and abnormal renal function. New Engl J. Med. 270,1283-1286. Vuori, M., Lahikainen, E., and Peltonen, T. (1962). Perceptive deafness in connection with mumps: A study of 298 servicemen suffering from mumps. Acta Otolaryngol. 55, 231-239.

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Weller, T., and Craig, J. (1949). The isolation of mumps virus at autopsy. Am. J. Pathol. 25,1105-1115. Witte, C , and Schanzer, B. (1968). Pancreatitis due to mumps. JAMA 203,1068-1069.

WoUstein, M. (1916). An experimental study of parotitis. /. Exp. Med. 23, 353-360. Wood, C , Bradbrook, R., and Blumgart, L. (1974). Chronic pancreatitis in childhood associated with mumps virus infection. Br. J. Clin.

Wolinsky, J. (1977). Mumps virus-induced hydrocephalus in hamsters: Ultrastructure of the chronic infection. Lah. Invest. 37, 229236,

^ ^^^^- ^°' 67-69. dollar, L., and Mufson, M. (1970). Acute parotitis associated with parainfluenza 3 virus infection. Am. J. Dis. Child. 119, 147-148.

C H A P T E R

28 Rubellavirus INTRODUCTION

CONGENITALLY ACQUIRED INFECTIONS REFERENCES

virus was adopted from the observation of early electron microscopists who compared the enveloping membrane of the virion to the loose togas worn by the Ancient Romans. The 50 to 70 mm in diameter rubella virion has a helical ribonucleoprotein core and acquires its investing membrane as it buds from the plasma membrane or into intracellular vesicles of the infected cell. The membrane exhibits surface spikes comprised of a hemagglutinin protein. The virion, including its membranes, has a simple complement of eight wellcharacterized proteins, but the importance and biological role of these proteins is largely unknown. Indeed, our understanding of rubella and the biology of the virus is relatively primitive in comparison to our knowledge of many other human pathogens. Rubellavirus was isolated in cell culture by two groups of investigators in the early 1960s (Weller and Neva 1962; Parkman et al, 1962). Prior to that time, the virus had been transmitted in subhuman primates, and in human subjects inoculated experimentally with nasal washings from ill children. In addition to providing a tool for diagnostic work, the availability of virus-susceptible cultured cells permitted studies that have yielded insights into the pathogenesis of the congenital rubella syndrome. Two types of investigations have been carried out. In the first, tissues from therapeutically aborted fetuses were cultured long term as monolayers and explants. In the second, monolayer cultures prepared from human amnions and the organs of lesser species were inoculated with virus in vitro and the infection monitored for prolonged periods. This work has shown that chronically infected cells do not undergo cytolysis in vitro, as is the case with cultured cells infected with many other viruses. Rubellavirus continues to replicate in the apparent absence of interferon production by the cells, and in the presence of antibody artificially introduced into culture media to suppress cell-to-cell transmission of the virus. However, rubellainfected cells were found to have a reduced growth rate in vitro and may have a shortened lifespan (Rawls and Melnick, 1966; Pope and Van Alstyne, 1982). This effect appeared to be due to a direct inhibition of mitosis and

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INTRODUCTION Rubella, as a childhood illness, was first described in 1752 by a German clinician named de Bergen. This was the origin of the synonym ''German measles," a term now largely out of favor. In 1866, Veale, a British physician, innovatively coined the term "rubella" to provide a more euphemistic substitute for the harsh German word "Rotheln." Initially, rubella was considered a childhood illness of passing importance, and its actual existence was doubted by some. The insightful observation of the Australian ophthalmologist Gregg reported in 1941 soon terminated the apathy of the medical community. Between 1939 and 1941, rubella in epidemic form swept Australia. Shortly thereafter, Gregg detected several children with cataracts that he associated with rubella in the mother during the first trimester of pregnancy. Two years later. Swan (1944) described deaf mutism among infants exposed in utero. During the next decade, the congenital rubella syndrome became widely recognized, and its protein teratogenic manifestations were documented. With the introduction of an effective live-virus vaccine in the late 1960s, clinical rubella and its ominous gestational complications have largely become diseases of historical importance in developed countries. But, alas, the congenital rubella syndrome continues to occur in those countries where effective vaccine programs have yet to be mounted, and when immunization fails or is refused. Rubellavirus is classified on the basis of its structural characteristics into the Togovirus family, and it is the only human pathogen in the genus Rubivirus. It has as close relatives the alphaviruses and flaviviruses, which are arboviruses exhibiting entirely different biological and ecological characteristics. The term TogoPATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

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Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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possible alterations in chromosomal structure. Plotkin and Vaheri (1967) proposed that this effect on cell growth is due to an as-of-yet uncharacterized inhibitory protein elaborated by the infected cultures. The basis for the chronicity of infection through many generations of cells is not understood, but there is no evidence that the rubella virion is defective, as proves to be the case in subacute sclerosing panencephalitis due to Rubeolavirus (see Chapter 29). Unfortunately, there have been relatively few virological studies on this important subject.

NATURALLY ACQUIRED POSTNATAL INFECTIONS Rubellavirus infection is acquired by respiratory droplet transmission. The virus is believed to replicate in the nasopharyngeal mucosa for a period of several days, but the actual cells supporting virus replication at that site have not been established. Viremia follows, and after a week characteristic cervical and postauricular adenopathy appear, accompanied by fever. The typical but transient exanthem that develops is often accompanied by coryza, cough, sore throat, conjunctivitis, and keratitis. The incubation period from exposure until development of rash is about 14 to 16 days, but roughly 25% of those who are exposed fail to develop skin lesions and may only manifest an acute febrile illness. Pathological studies of skin lesions are limited, but the typical morphological picture is a nonspecific relatively mild perivascular lymphocytic infiltrate in the dermis, without the presence of specific cytological change. The manifestations of the acute illness differ somewhat in various age groups, with adults experiencing a more severe infection than children. Arthralgias and arthritis in the extremities are a common complication of the infection in adolescents and adults but infrequently occur in children. Among adults, women more commonly develop joint symptoms. Clinically evident arthritis with joint swelling and erythema is observed in about 15% of adult cases. At times, carpel tunnel syndromic may accompany joint inflammation in the wrists. In these cases, virus is recovered from the joint fluid and chronic virus replication occurs in cells cultured from biopsies of the synovium from infected joints. Interestingly enough, the ideal temperature for maintaining the chronic rubella infection in these synovial cultures is 32°C, the approximate temperature of joints in vivo (Cunningham and Fraser, 1985; Williams et al, 1981).

Mild arthritis is also a common side-effect of livevirus vaccine administration. Ogra and Herd (1971) isolated vaccine virus from the joint fluid of immunized children over a period of several months. An unexplained and unconfirmed but intriguing finding relates to the isolation of rubellavirus from circulating "lymphoreticular" cells of 35% of a group of children with a diversity of chronic rheumatic conditions including Still's disease, spondyloarthritis and, both poly- and pauci-articular juvenile rheumatoid arthritis. These youngsters had been immunized with a livevirus vaccine in the past. The virus isolates were not characterized by molecular means, and it is not known if they were vaccine strains (Chantler et al, 1985). The results of this study provokes skepticism, but there is currently little question that chronic and recurrent arthralgias and arthritis develop in an occasional adult recipient of vaccine (Howson et al, 1992). Interestingly enough, the likelihood of developing arthralgias was increased eightfold among females with the MHC types DRl and DR4, and more than sevenfold in those with both RR4 and DR6 (Mitchell et al, 1998). This work clearly demonstrates the importance of immunogenetic factors in the pathogenesis of the joint lesions and suggests the likelihood of an autoimmune phenomena. Encephalitis and polyneuroradiculitis (Tomlinson, 1975; Aguado et al, 1993) are rare complications of naturally occurring rubella. It affects about 1 patient among some 6000 cases. Although reported neuropathological and virological studies are limited, the central nervous system picture is said to be typical of acute postinfectious encephalomyelitis. Rubellavirus has not been recovered from tissues of the central nervous system. Detailed descriptions of the pathology of postinfectious encephalomyelitis are found in standard texts of neuropathology. Purpura is also a rare complication of postnatal rubellavirus infection. The pathogenesis is obscure, but it may relate to the thrombocytopenia that occurs sporadically in these patients or either heterozygous/homozygous factor C and S deficiencies. Since it is likely that rubellavirus replicates in the endothelial cells of many tissues, the occurrence of petechiae and purpura in an occasional patient is not surprising. Nonfatal myocarditis, pericarditis, and pleuritis are uncommon complications of rubella infection in the adult (Fujimoto et al, 1979). Thanopoulos et al (1989) reported two cases of chronic myocardial disease in children after rubella. The first patient required a pacemaker because of persistent heart block, whereas the second had chronic congestive failure.

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Age (months) 95%

Acute rubella is a systemic disease, and it is not surprising that the placenta and developing fetus are involved when the pregnant susceptible female is infected during the first trimester. Congenital infections of the embryo during the first trimester commonly result in developmental abnormalities of the cardiovascular system, eyes, and ears, whereas those occurring later in pregnancy appear to have no adverse structural effects on the fetus (with the exception of the eye; see below). Increased prevalence of spontaneous abortions and stillbirths has not been reported as a result of infections in women during advanced pregnancies. The timing of the infection in the first trimester is critical to the outcome (Table 28.1), with cataracts and retinopathy, middle ear deafness, and congenital heart disease being the trilogy of conditions typically occurring as isolated lesions, or in various combinations, when infection occurs during the initial 2 gestational months. Middle ear damage and retinopathy continue to occur sporadically in fetuses infected throughout the remainder of the first trimester, and abnormalities of the eye continue to occur in fetuses infected later in pregnancy (Ueda ei a/., 1979,1992). Tondury and Smith (1966) analyzed abnormalities in fetuses obtained by therapeutic abortion after rubeliavirus infection (Table 28.1). Overall, the risk of significant congenital disease in the fetus is greater than 50% when the maternal infection develops during the first month of pregnancy, but only 20% in the second month, even though the concepti and placentas are almost invariably infected at this time (Alford et ah, 1964; Marshall, 1975; Dudgeon, 1972; Monif et al, 1965; Cradock-Watson et ah, 1989; Sugiura, 1983; Mellinger et al, 1995). While the rate of fetal infection is not known among susceptible women inadvertently vaccinated with live-virus vaccine during pregnancy, congenital lesions and postnatal infections in the offspring of vaccinees have not been reported.

40

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FIGURE 28.1 Head circumference and weight/length curves in a child with congenital rubella encephalitis, cerebral diplegia, and normal intelligence (dots). The lines depict percentile of populations. The growth dysregulation associated with chronic infection and the accompanying neurological disease is evident. Adapted with permission from Desmond et al. (1978).

About 30% of infants born after in utero rubeliavirus infection weigh less than 2500 g, and from that time forth they often fail to thrive. The placentas of these gestations are infected, and inflammatory lesions of small blood vessels are a prominent morphologic feature. As shown in Figure 28.1, postnatal weight gain is frequently poor. The pathogenesis of growth retardation after birth is unknown, but it most probably reflects both the inhibitory effects of chronic infection on cell multiplication, as discussed above, and circulatory deprivation due to vascular lesions in the placenta

TABLE 28.1 Fetal Age at Time of Maternal Infection in Relationship to the Development of Specific Organ Lesions % having abnormality in: Age of fetus at time of maternal rubella 0-4 weeks 4-8 weeks 8-11 weeks 0-11 weeks

No. 20 31 6 57

Percent abnormal 80 58 4/6 68

Reprinted with permission from Tondury and Smith (1966).

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Lens 35 48 4/6 45

Heart

Inner ear

Skeletal muscle

65 45 3/6 53

12 13 0 11

25 16 0 18

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(Naeye and Blanc, 1965; Driscoll, 1969; Garcia et al, 1985). Infection persists for variable periods of time postnatally (Figure 28.2), but some children yield virus into adolescence, and it is likely that studies incorporating molecular approaches would reveal evidence of further long-term, although low-grade, infection. Immunological tolerance may account for the chronicity of the infection, but alterations in immune responsivity of the infant comprise one alternate explanation (Stern and Forbes, 1975). As noted above, cultured cells support a persistent rubellavirus infection even when specific antibody is added to the medium that bathes the cells. tOOr

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F I G U R E 28.2 The incidence of an active rubellavirus infection as documented by urinary excretion by age in infants with stigmata of congenital disease. Reprinted with permission from Cooper and Krugman (1967).

Abnormalities of the neonate and developing child chronically infected with rubella are found to a variable extent in a myriad of organ systems, but there is no apparent unifying pathogenic mechanism that accounts for the diversity of lesions (Gray, 1960). Rather, I envision three different pathological processes occurring to a variable extent in these unfortunate children. First, there are the abnormalities of the organs infected in early gestation at a key time during embryogenesis, that is, the heart, eye, and middle ear. Campbell (1965) documented heart malformations in 153 cases of congenital rubella syndrome; 5S% had a clinically important ductus arteriosus. Ventricular and atrial septal defects were found in 18 and 7%, respectively, and the tetralogy of Fallot occurred in 7%. Aortic coarctation and aortic valvular stenosis, transposition of the great vessels, and truncus arteriosus were found in some infants. Postmortem studies in cases with pulmonary stenosis demonstrate prominent intimal proliferative changes in the vessel accompanied by variable de-

creases in the elastic tissue and smooth muscle of the media. In the ductus arteriosus, there is also an apparent decrease in the structural elements of the media of the vessel (Campbell, 1965). In the eyes, the lens exhibits spheroplakia accompanied by laminar and zonal cataracts. Autopsies of early embryos demonstrate developmental changes in the lens (Gray, 1960). While cataracts are the pathognomonic lesions, microphthalmia (and its secondary complication, glaucoma), resulting from structural developmental abnormalities of the eye, is reported. Almost 50% of the infant survivors of a first trimester in utero infection have hearing defects (Swan, 1944; Kelemen, 1966). Other studies strongly suggest, however, that damage often occurs in the middle ear as a result of 2nd and 3rd trimester infections. Clinically, the severity of the hearing defect is variable, and the two ears may differ in acuity Some evidence suggests both a conduction deficit and a neural component to deafness. The complexities of evaluating the pathology of these lesions are profound inasmuch as the development of the middle ear evolves rapidly during early gestation. In the reported studies, saccular and cochlear degenerative changes prove to be a consistent finding, but the other major structures (stria vascularis, Reissner's membrane, the tectorial membrane) are affected to a variable extent. Vestibular function seems not to be impaired in surviving children. Some authors (Ward et al, 1968; Lindsay, 1973) suggest that the changes in the middle ear progressively develop into the postnatal period and reflect a persistent inflammatory response to a chronic infection (as discussed below) rather than structural development abnormalities. Necrotizing and inflammatory lesions are the second category of disease affecting the child with congenital rubella syndrome. Reports in the literature document inflammation in the central nervous system, eyes (uveitis) (Hara et al, 1979), middle ear, the thyroid (Nieburg and Gardner, 1976), lungs, heart, liver, pancreas, and kidneys. These diverse inflammatory lesions attest to widespread dissemination of this chronic infection in the fetus and infant, and are often difficult to delineate from a third category of lesions attributable to a vasculopathy affecting arteries and arterioles of all caliber (Esterly and Oppenheimer, 1967; Frank and Purnell, 1978). While reports in the literature are sparse and conflicting, the morphologic evidence indicates that in many cases focal intimal hyperplasia is accompanied by a variable degree of fibromuscular proliferation of vessel walls. These changes would appear to result in focal necrosis of tissues in solid organs and the classic well-described abnormalities at the costochondral junctions of developing long bones (Rudolph et al, 1965; Singer et al, 1967).

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Rubellavirus

The central nervous system of the congenitally infected infant shows, to a variable extent, features of a chronic meningoencephalitis with perivascular mononuclear cell infiltrates in the brain substances accompanied by focal gliosis. Microcephaly with intracranial calcifications is occasionally observed (Chang et ah, 1996). Nonetheless, neurological development of many infected infants is consistent with customary developmental milestones. Desmond and his colleagues (1978) provided a detailed clinical account of the evolving long-term clinical course of children with central nervous system inflammatory disease due to rubellavirus infection acquired in utero. The spectrum of neurological abnormalities is large, with over 80% of these children showing evidence of neurological disease by 18 months. The babies were found to be restless, cried excessively, fed poorly, and were lethargic. There was a poverty of movement and hypotonia of the muscles, with seizure disorders being common. With advancing age, changes in muscle tone and reflexes were found. These children exhibited difficulty in feeding and were considered to have abnormal clinical behavior. As they grew older, prominent defects in their hearing and visual loss became more apparent, and half of the children were in remedial communication education programs. Encephalographic studies were abnormal in one-third of these youngsters, and learning disabilities were prominent in one-third. Half of the children in the 9-12-year age range had behavioral abnormalities. These diverse neurological findings document the profound effect of encephalitis on the developing youngster. Townsend et al (1975, 1976) and Wolinsky (1976) described a unique syndrome of progressive neurological deterioration in older children and adolescents that clinically simulates the relatively rapid mental and physical deterioration typically observed in subacute sclerosing panencephalitis due to rubeolavirus. For example, those who are affected manifest progressive deterioration in cognition and affect, leading to dementia, myoclonic seizures, and muscle weakness. Later, quadriplegia and ataxia develop. Pathologically, the brain exhibits destruction of the white matter with neuronal loss and gliosis accompanied by a mononuclear cell infiltrate. There are associated changes in small penetrating blood vessels that are vaguely described but show deposits of unidentified material and focal necrosis in the walls. Cerebellar atrophy appears to be a unique feature of the syndrome (Townsend et al, 1976) (Figure 28.3). The central nervous system tissue from several patients with this syndrome have yielded rubellavirus (Cremer et al, 1975; Wolinsky, 1976), and high concentrations of specific viral antibody are found in the cerebrospinal fluid and blood serum. From a

F I G U R E 28.3 Gross neuropathological features of progressive rubella panencephalitis of late onset. (Top) Sagittal section of cerebellum with marked thinning of folia and dilatation of the cisterna magna posteriorly. (Middle) Coronal section of brain showing severe white matter loss and ventricular enlargement. (Bottom) Diffuse contraction of cerebral white matter, with sparing of the cortical ribbon. Reprinted with permission from Townsend et al. (1976).

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Pathology and Pathogenesis of Human Viral Disease

mechanistic perspective, the basis for this late-onset disease is unknown. Unfortunately, the virus isolates have not undergone molecular characterization, and information on the immunological status of these patients is limited. Chronic lymphocytic pancreatitis and exocrine pancreatic insufficiency are described in patients with the stigmata of congenital rubella syndrome (Bunnell and Monif, 1972; Donowitz and Gryboski, 1975) (Figure 28.4), and rubellavirus has been isolated from the pancreatic tissue of stillbirths and older congenitally infected children at autopsy It was of great interest, therefore, when Forrest (Forrest ei al., 1969, 1971; Forrest and Menser, 1975) reported cases of diabetes mellitus developing in the second and third decades of life among patients with congenital rubella syndrome. With the passage of time, at least 50 cases have been reported (De Prins ei a\., 1978; Johnson and Tudor, 1970; Schopfer ei al., 1982), and diabetes mellitus now is believed to develop in about 20% of adolescents and young adults with the congenital rubella syndrome (Menser ei al., 1978). The severity of the diabetes and the congenitally acquired disease seem to correlate. In about half the cases, hyperglycemia is controlled by diet, whereas in the remainder insulin administration is needed. Thus, the diabetes does not exhibit the typical features of the type I form of the disease. To date, autopsy observations on the pancreas of affected children have not been reported, and it is unclear whether beta cell insufficiency results from the destructive effects of pancreatic inflammation or is due to a direct effect of a chronic virus infection of the beta cells. Of

interest is the striking association of diabetes in these patients with the same HLA markers that accompany classical idiopathic type I diabetes (Rubinstein ei al., 1982; Honeyman ei al., 1975', Menser ei al, 1974; Ginsberg-Fellner ei al., 1985). It is also noteworthy that certain peptide sequences of the rubellavirus are also found in the DQ beta chain locus of the HLA complex. The importance of this finding, if any, is uncertain, but it is consistent with the possibility of an autoimmune pathogenic process based on molecular mimicry. Myocarditis as documented by electrocardiography affects approximately 20% of neonates with congenital rubella syndrome. Ainger ei al. (1966) reported a 40% mortality among patients with clinical myocarditis. Extreme myocardial necrosis is observed at autopsy, but detailed pathologic descriptions are not published. These same authors documented chronic congestive heart failure in additional children with the syndrome. A variety of alterations are described in the liver of patients with congenital rubella syndrome. These changes are nonspecific and include triaditis accompanied by variable degrees of fibrosis and cholestasis. Giant cell transformation of hepatocytes and biliary atresia have also been reported, but cirrhosis is not seen. No pathognomonic changes are found by the pathologists in the tissues from suspected cases of congenital rubella syndrome. In addition to the often cumbersome isolation of virus-using cell culture, PCR proves to be a sensitive and specific diagnostic tool (Tanemura ei al, 1996).

FIGURE 28.4 Chronic lymphocytic pancreatitis in a child dying with congenital rubellavirus infection. Reprinted with permission from Monif et al. (1965) and through the courtesy of G. Monif, MD.

Rubellavirus

References Aguado, J., Posada, I., Gonzalez, M., Lizasoain, M., Lumbreras, C , Vallejo, A., and Noriega, A. (1993). Meningoencephalitis and polyradiculoneuritis in adults: Don't forget rubella. Clin. Infect. Dis. 17, 785-786. Ainger, L., Lawyer, N., and Fitch, C. (1966). Neonatal rubella myocarditis. Br. Heart J. 28, 691-697. Alford Jr., C , Neva, F., and Weller, T. (1964). Virologic and serologic studies on human products of conception after maternal rubella. New Engl. J. Med. 271,1275-1281. Bunnell, C , and Monif, G. (1972). Interstitial pancreatitis in the congenital rubella syndrome. /. Pediatr 80, 4^65-466. Campbell, P. (1965). Vascular abnormalities following maternal rubella. Br. Hear^/. 27,134. Chang, Y, Huang, C , and Liu, C. (1996). Frequency of linear hyperechogenicity over the basal ganglia in young infants with congenital rubella syndrome. Clin. Infect. Dis. 22, 569-571. Chantler, J., Tingle, A., and Petty, R. (1985). Persistent rubella virus infection associated with chronic arthritis in children. New Engl. J. Med. 313, 1117-1123. Cooper, L., and Krugman, S. (1967). Clinical manifestations of postnatal and congenital rubella. Arch. Ophthalmol. 77, 434-439. Cradock-Watson, J., Miller, E., Ridehalgh, M., Terry, G., and Ho-Terry, L. (1989). Detection of rubella virus in fetal and placental tissues and in the throats of neonates after serologically confirmed rubella in pregnancy. Prenatal Diagn. 9, 91-96. Cremer, N., Oshiro, L., Weil, M., Lennette, E., Itabashi, H., and Carnay, L. (1975). Isolation of rubella virus from brain in chronic progressive panencephalitis. /. Gen. Virol. 29,143-153. Cunningham, A., and Eraser, J. (1985). Persistent rubella virus infection of human synovial cells cultured in vitro. /. Infect. Dis. 151, 638-645. De Prins, R, Van Assche, R, Desmyter, J., De Groote, G., and Gepts, W. (1978). Congenital rubella and diabetes mellitus [letter]. Lancet 1 (8061), 439^40. Desmond, M., Fisher, E., Vorderman, A., Schaffer, H., Andrew, L., Zion, T, and Catlin, R (1978). The longitudinal course of congenital rubella encephalitis in nonretarded children. /. Pediatr. 93,584-591. Donowitz, M., and Gryboski, J. (1975). Pancreatic insufficiency and the congenital rubella syndrome. /. Pediatr 87, 241-243. Driscoll, S. (1969). Histopathology of gestational rubella. Am. J. Dis. Child. 118, 49-53. Dudgeon, J. (1972). Congenital rubella: A preventable disease. Postgrad. Med. J. 48 (Suppl. 3), 7-11. Esterly, J., and Oppenheimer, E. (1967). Vascular lesions in infants with congenital rubella. Circulation 36, 544. Forrest, J., and Menser, M. (1975). Recent implications of intrauterine and postnatal rubella. Aust. Paediat. J. 11, 65-75. Forrest, J., Menser, M., and Harley, J. (1969). Diabetes mellitus and congenital rubella. Pediatrics 44, M5-A47. Forrest, J., Menser, M., and Burgess, J. (1971). High frequency of diabetes mellitus in young adults with congenital rubella. Lancet ii, 332-334. Frank, K., and Purnell, E. (1978). Subretinal neovascularization following rubella retinopathy. Am. J. Ophthalmol. 86, 462-466. Fujimoto, T., Katoh, C , Hayakawa, H., Yokota, M., and Kimura, E. (1979). Two cases of rubella infection with cardiac involvement. Jpn. Heart J. 20, 227-235. Garcia, A., Marques, R., Lobato, Y, Fonseca, M., and Wigg, M. (1985). Placental pathology in congenital rubella. Placenta 6, 281-295.

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Ginsberg-Fellner, R, Witt, M., Fedun, B., Taub, R, Dobersen, M., McEvoy, R., Cooper, L., Notkins, A., and Rubinstein, P. (1985). Diabetes mellitus and autoimmunity in patients with the congenital rubella syndrome. Rev. Infect. Dis. 7 (Suppl. 1), S170-S176. Gray, J. (1960). Rubella in pregnancy: A report on six embryos. Br Med. J. 1,1388-1390. Hara, J., Fujimoto, R, Ishibashi, T, Seguchi, T., and Nishimura, K. (1979). Ocular manifestations of the 1976 rubella epidemic in Japan. Am. }. Ophthalmol. 87, 642-645. Honeyman, M., Dorman, D., Menser, M., Forrest, J., Guinan, J., and Clark, P. (1975). HLA antigens in congenital rubella and the role of antigens 1 and 8 in the epidemiology of natural rubella. Tissue Antigens 5,12-18. Howson, C , Katz, M., Johnston Jr., R., and Fineberg, H. (1992). Chronic arthritis after rubella vaccination. Clin. Infect. Dis. 15, 307-312. Johnson, G., and Tudor, R. (1970). Diabetes mellitus and congenital rubella infection. Am. J. Dis. Child. 120, 453^55. Kelemen, G. (1966). Rubella and deafness. Arch. Otolaryng. 83, 520532. Lindsay, J. (1973). Histopathology of deafness due to postnatal viral disease. Arch. Otolaryngol. 98, 258-264. Marshall, W. (1975). Effects of rubella virus on the human fetus. Proc. Roy. Soc. Med. 68, 369-371. Mellinger, A., Cragan, J., Atkinson, W, Williams, W, Kleger, B., Kimber, R., and Tavris, D. (1995). High incidence of congenital rubella syndrome after a rubella outbreak. Ped. Infect. Dis. J. 14, 573-578. Menser, M., Forrest, J., Honeyman, M., and Burgess, J. (1974). Diabetes, HLA antigens, and congenital rubella. Lancet 2,1508-1509. Menser, M., Forrest, J., and Bransby, R. (1978). Rubella infection and diabetes mellitus. Lancet 1, 57-60. Mitchell, L., Tingle, A., MacWilliam, L., Home, C , Keown, R, Gaur, L., and Nepom, G. (1998). HLA-DR class II associations with rubella vaccine-induced joint manifestations. /. Infect. Dis. 177, 5-12. Monif, G., Sever, J., Schiff, G., and Traub, R. (1965). Isolation of rubella virus from products of conception. Am. J. Ohstet. Gynec. 91,11431146. Naeye, R., and Blanc, W (1965). Pathogenesis of congenital rubella. JAMA 194, 1277-1283. Nieburg, P., and Gardner, L. (1976). Letter: Thyroiditis and congenital rubella syndrome. /. Pediatr. 89, 156. Ogra, P., and Herd, J. (1971). Arthritis associated with induced rubella infection. /. Immunol. 107, 810-813. Parkman, P., Buescher, S., and Arnstein, M. (1962). Recovery of rubella virus from Army recruits. Proc. Soc. Exp. Biol. Med. I l l , 225-230. Plotkin, S., and Vaheri, A. (1967). Human fibroblasts infected with rubella virus produce a growth inhibitor. Science 156, 659-661. Pope, D., and Van Alstyne, D. (1982). Further studies on the restriction of rubella virus replication in rat glial cells in culture. Can. J. Microbiol. 28, 1404-1409. Rawls, W., and Melnick, J. (1966). Rubella virus carrier cultures derived from congenitally infected infants. /. Exp. Med. 123, 795-816. Rubinstein, P., Walker, M., Fedun, B., Witt, M., Cooper, L., and Ginsberg-Fellner, F. (1982). The HLA system in congenital rubella patients with and without diabetes. Diabetes 31,1088-1091. Rudolph, A., Singleton, E., Rosenberg, H., and Singer, D. (1965). Osseous manifestations of the congenital rubella syndrome. Am. J. Dis. Child. 110, 428-432. Schopfer, K., Matter, L., Flueler, U., and Werder, E. (1982). Diabetes mellitus, endocrine autoantibodies, and prenatal rubella infection. Lancet 2,159.

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Singer, D., Rudolph, A., Rosenberg, H., Rawls, W., and Boniuk, M. (1967). Pathology of the congenital rubella syndrome. /. Pediatr. 71, 665-675. Stern, L., and Forbes, I. (1975). Dysgammaglobulinemia and temporary immune paresis in a case of congenital rubella. Aust. Paediat. J. 11, 3 8 ^ 1 . Sugiura, K. (1983). Virological studies on the feto-maternal tissues infected with rubella virus [Japanese]. Nippon Sanka Fujinka Gakkai Zasshi [Acta Obstet. Gynaec. Jpn.] 35, 674-682. Swan, C. (1944). Congenital defects and maternal rubella. Trans. Ophthalmol Soc. Aust. 4,136. Tanemura, M., Suzumori, K., Yagami, Y, and Katow, S. (1996). Diagnosis of fetal rubella infection with reverse transcription and nested polymerase chain reaction: A study of 34 cases diagnosed in fetuses. Am. J. Obstet. Gynecol. 174, 578-582. Thanopoulos, B., Rokas, S., Frimas, C , Mantagos, S., and Beratis, N. (1989). Cardiac involvement in postnatal rubella. Acta Paediatr. Scand. 78,141-144. Tomlinson, I. (1975). Rubella polyneuropathy [case report]. Postgrad. Med. J. 51, 30-32. Tondury, G., and Smith, D. (1966). Fetal rubella pathology. /. Pediatr. 68, 867-879.

Townsend, J., Baringer, J., Wolinsky, J., Malamud, N., Mednick, J., Panitch, H., Scott, R., Oshiro, L., and Cremer, N. (1975). Progressive rubella panencephalitis: Late onset after congenital rubella. New Engl. J. Med. 292, 990-993. Townsend, J., Wolinsky, J., and Baringer, J. (1976). The neuropathology of progressive rubella panencephalitis of late onset. Brain 99, 81-90. Ueda, K., Nishida, Y, Oshima, K., and Shepard, T. (1979). Congenital rubella syndrome: Correlation of gestational age at time of maternal rubella with type of defect. /. Pediatr 94, 763-765. Ueda, K., Tokugawa, K., and Kusuhara, K. (1992). Perinatal viral infections [review]. Early Hum. Dev. 29, 131-135. Ward, P., Honrubia, V., and Moore, B. (1968). Inner ear pathology in deafness due to maternal rubella. Arch. Otolaryng. 87, 22-28. Weller, T., and Neva, F. (1962). Propagation in tissue culture of cytopathic agent from patients with rubella-like illness. Proc. Soc. Exp. Biol. I l l , 215-224. Williams, M., Brawner, T., Riggs Jr., H., and Roehrig, J. (1981). Characteristics of a persistent rubella infection in a human cell line. /. Gen. Virol. 52 (Pt. 2), 321-328. Wolinsky, J., Berg, B., and Maitland, C. (1976). Progressive rubella panencephalitis. Arch. Neurol. 33, 722-723.

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29 Rubeola (Measles) INTRODUCTION 397 RESPIRATORY TRACT DISEASE 399 ATYPICAL MEASLES SYNDROME 402 CENTRAL NERVOUS SYSTEM DISEASE

the susceptible immunosuppressed patient makes awareness of the virus' morbid potential critical for the physician (Christensen et ah, 1953). Insightful observations by Panum during an 1846 epidemic in the Faroe Islands provided medical science with a near complete understanding of the epidemiology of measles (Griffin ei al, 1994). We now know measles to have an approximate 14-day incubation period after respiratory aerosol exposure. Subsequently, the classical oral lesions first publicized by Koplik in 1896 (Brem, 1972) appear. These lesions and those of the maculopapillary skin rash that follows are now known to be the sites of virus replication in capillary endothelial cells amplified by a cellular immune response (Griffin et al, 1994; Suringa et al, 1970; Kimura et al, 1975; Olding-Stenkvist and Bjorvatn, 1976) (Figures 29.1-3). Oropharyngeal and respiratory tract replication of the virus occurs simultaneously, ultimately resulting in infection of the lymphatic system and a viremia that is lymphocyte associated. Cytological studies of mucosal cells of the aerodigestive tract document the presence of cell-associated virus in cells bearing intranuclear inclusions and in the occasional polykaryocytes that characterize the widespread involvement of these tissues by this rubeolavirus. These multinucleate cells are also found in the tonsils, lymph nodes, spleen, Peyer's patches, and appendix of the occasional child who dies during the acute stages of infection (Figure 29.4). In lymphoid tissues, the polykaryocytes are familiarly known as Warthin-Finkeldey cells in recognition of the pathologists who first described them (Warthin, 1930; Finkeldey, 1931). The presence of cytokines such as gamma-interferon and interleukins in the blood during the acute infection probably account, in part or in whole, for the malaise and high fevers so typical of the infection at this stage. Cell-mediated and humoral immune responses occur simultaneously with these events. Immune CD8+ T cells appear to be the critical elements in the destruction of virus-laden cells, and thus are probably responsible for recovery from the acute infection. The sensitized CD4+ cells elaborate cytoki-

403

Meningoencephalitis After Natural Infection 403 Meningoencephalitis in the Immunosuppressed Patient 403 Meningoencephalitis After Measles Vaccine 404 Subacute Sclerosing Panencephalitis (SSPE) 404 MIDDLE EAR DISEASE EYE DISEASE 407 PREGNANCY 407 REFERENCES 408

407

INTRODUCTION The initial description of measles is credited to Rhazes in the 10th century C.E., who considered it to be more dread than smallpox because of its frequent fatal outcome. Measles requires a population base of several hundred thousand people in order for the virus to be continuously spread among susceptibles. Thus, in history, it most probably was not an important cause of childhood illness until dense population concentrations developed in the cultures of the Tigris and Euphrates several centuries B.C.E. Epidemics of what is believed to be measles occurred in China and in the Roman Empire in the second century C.E. In addition to its devastating effects on children, epidemics have occurred among adults during wars, resulting in countless deaths. Although about 80% of the earth's children now are immunized against measles, the virus continues to be a threat to life among the younger age groups in underdeveloped countries, and among patients with unique naturally occurring and acquired immunosusceptibility. In the past, the highly contagious measles virus left few susceptible adults in the population of our densely settled urban areas. The epidemiology in Western societies has now changed. While "herd" immunity in the general population reduces the risk of exposure, the existence of the occasional nonimmunized adult and PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

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Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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FIGURE 29.2 Koplik's spots on the buccal mucosa of a child serves as the harbinger of the skin lesions that appear shortly thereafter. The spots are tiny, discrete, grey-white papules on a dull red base. Reprinted with permission from Brem (1972). FIGURE 29.1 A child with the typical maculopapillary skin rash of measles.

FIGURE 29.4 Warthin-Finkeldey giant cells with intranuclear inclusions in the tonsillar tissue of a child with fatal measles pneumo-

FIGURE 29.3 Histological features of a Koplik spot. The oral squamous epithelial cells exhibit spongiosis and parakeratosis. A few scattered lymphocytes are also present. The arrows designate typical multinucleates that are pathognomonic of measles in the squamous epithelium of the oral cavity and skin. Immunohistochemistry demonstrates large amounts of viral antigens distributed diffusely in the lesions. Reprinted with permission from Suringa et al. (1970).

nes and promote antibody formation against the virus through their helper cell function. Circulating immunoglobulins appear to play a major role in protection upon reexposure to virus, but they are not an important element in recovery from initial infection. The lack of an appropriate cellular immune response as a consequence of a heritable predisposition, chemotherapy, and AIDS is the basis for the devastating consequences of infection in these highly susceptible patients, as discussed in more detail below (Gellin and Katz, 1994). Measles virus is a member of the paramyxovirus family, classified in the genus Morbillivirus (Bellini et al, 1994). While its infectivity is restricted to humans and certain higher primates, it shares common characteristics with the viruses of canine distemper and sev-

Rubeola (Measles)

eral similar viruses of domestic and wild ruminates and cetaceans (dolphins and porpoise). Recently, a newly discovered morbillivirus of horses in Australia was found to cause a fatal measles-like disease in humans (Anonymous, 1996; Murray et al, 1995). During early 1999 in Malaysia, a virulent "new" paramyxovirus, thought to be a morbillivirus, was believed to have caused an outbreak of a fatal disease in swine that "spilled over" into the human population, more than 100 of whom died that lived near the pigs. As with other paramyxoviruses, the measles virion is comprised of a helical ribonuclear protein bound by a membrane. Glycoproteins of considerable importance are found in this membrane. These are the M proteins that line the inner aspect of the viral membrane, and the transmembrane F (fusion) and H (hemagglutinin) proteins. These latter membrane-associated proteins are critical to interaction of the virion with susceptible cells and fusion of the virus membrane with these cells during infection. The formation of the multinucleate polykarysome so characteristic of measles is fostered by the F protein. The typical nuclear and cytoplasmic inclusions of the virus-infected cell represent complex accumulations of the helical cords of RNA encompassed by the N nucleoprotein. As with other paramyxoviruses, rubeolavirus is assembled at the plasma membrane of the infected cell and buds from the surface (Nakai and Imagawa, 1969). Measles virus was first isolated in cell culture by Enders and Peebles (1954), a discovery that soon resulted in characterization of the virus and development of our contemporary attenuated live-virus vaccines. John Enders's pathfinding work, along with his earlier contribution to the isolation of poliovirus in cell culture no doubt, has saved more lives and prevented more disability than any other contribution to medical virological science in our era! A unique feature of measles virus infection is suppression of the host's cellular immunity for several weeks after acute infection. The mechanistic basis for this phenomenon is undefined, although one suspects that infection of lymphoid cells is a significant contributing factor (Karp ei ah, 1996). A superimposed profound lymphopenia also is documented (Coovadia et al., 1981), and leukocyte mobility is said to be impaired (Anderson et ah, 1976). As a result, some convalescent patients are unduly susceptible to superimposed bacterial and virus infections. This appears to be particularly the case in areas of the world where protein-calorie malnutrition is common (Kaschula et al, 1983). RESPIRATORY TRACT DISEASE Measles virus infects tissues of the nasopharynx, airways, and lung parenchyma concomitantly with the

399

onset of the rash. Measles pneumonia occurs during the acute stages of the infection and is the major lifethreatening complication. In immunologically normal persons, the virus disappears from the tissues of the respiratory tract within a period of about 1 week. At this time, antibodies to the virus are first detected in the blood. Weinstein and Franklin (1949) documented pneumonia in approximately 25% of their adult patients with measles. An incidence of >3% was reported among several thousand young adult male military recruits with measles (Gremillion and Crawford, 1981). In healthy residents of developed countries, death due to pulmonary infections occurs with a frequency of approximately 2% (Mason et ah, 1993). Studies of the pulmonary pathology in these cases have not been reported, but one is tempted to assume that the cytopathologic changes attributable to measles virus are manifest throughout the respiratory tract. The common demonstration of virus and inclusion body-bearing multinucleate cells in the nasopharynx secretions of acutely ill immunologically intact children supports this conclusion. However, recent autopsy studies by Radoycich et ah (1992) indicate that, in the absence of apparent immune deficiencies, the lungs of fatally infected children with measles pneumonia exhibit a spectrum of nonspecific changes, including interstitial pneumonia, organizing diffuse alveolar damage, and necrotizing bronchiolitis, with a resulting bronchiolitis obliterans and organizing pneumonia (BOOP) picture (Figure 29.5). No doubt, these are late changes found after the subsidence of the acute viral infection. Bacterial pneumonia is often the terminal event. Half of the patients with measles pneumonia who were studied by Weinstein and Franklin (1949) had a superimposed bacterial infection. Mortality due to measles pneumonia is substantially greater in underdeveloped countries, where malnutrition and chronic infection with malaria are common. These endemic conditions are often accompanied by a state of cellular immunosuppression that predisposes to serious infection. Chronic measles virus infections of the respiratory tract have also been documented in malnourished children by Dossetor et ah (1977) and Scheifele and Forbes (1972). In one study carried out in India, 44% of children under the age of 1 year who were infected with measles virus developed bronchopulmonary disease, and 75% of these infants died (Merchant, 1976). Often, a persistent protein-losing enteritis accompanies the chronic respiratory tract infection (Watson and Parkin, 1970; Monif and Hood, 1970; Dossetor and Whittle, 1975). Children and adults with illnesses or therapies that suppress the immune response often experience persistent rubeola infections and an increased prevalence

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FIGURE 29.5 Radiological features of diffuse bronchiolitis obliterans and organizing pneumonia (BOOP) in a case immediately before death. Reprinted with permission from Goetz and Mathisen (1995).

of serious life-threatening pneumonia (Meadow et al, 1969; Siegel et al, 1977; Lewis et al, 1978). In one study of over 400 immunocompromised children, 4% developed measles pneumonia, with an associated mortality of 36% (Kernahan et al, 1987). At autopsy, evidence of disseminated infection is found. Giant cells accompanied by necrosis to a variable extent are found in the lungs, tonsils, lymph nodes, bone marrow, spleen, appendix, pancreas, renal tubules, and liver (Breitfeld et al, 1973; Akhtar and Young, 1973; Siegel and Hirschman, 1977) (Figures 29.6 and 29.7). While measles pneumonia in health adults is rarely fatal, increasing numbers of cases have been reported in unvaccinated adults with AIDS and recipients of immunosuppressive and cytotoxic drugs. Many of these patients do not exhibit the typical rubeola rash before or at any time during the evolution of the pulmonary disease (Goetz and Mathisen, 1995; Sobonya et al, 1978). Hecht (1910) first described giant cell pneumonia. Years later, Warthin (1931) and Finkeldey (1931) noted the typical inclusion-bearing multinucleate cells in the tonsils of other lymphoid tissues, as noted above. Additional cases of giant cell pneumonia were recorded in the later literature (Moore and Gross, 1930; Karsner and Meyers, 1913; Denton, 1925). Pinkerton and his associates (1945) reported their pathological studies on eight infants with giant cell pneumonia. These authors docu-

mented the distribution of multinucleate cells with cytoplasmic and nuclear inclusions in the epithelium of the airways and acini associated with a variable interstitial mononuclear inflammatory infiltrate (Figures 29.8 and 29.9). Detailed clinical information unfortunately was lacking in these reported cases, and the critical reader must question whether or not all these cases represented rubeola infections. As pointed out elsewhere (see Chapters 4 and 5), parainfluenza viruses type 2 and 3 and respiratory syncytial virus are now known to produce multinucleate giant cells that resemble those of measles, although intranuclear inclusions are not found in cells infected with these viruses. The missing link associating Hecht pneumonia with measles was provided by Enders et al (1959). These workers recovered rubeolavirus from the lungs of three fatally infected immunocompromised children who had the typical morphological features of giant cell pneumonia at autopsy, but no history of rash. Although it was not clear when these patients were exposed to measles virus, the rash presumably failed to develop before the onset of pneumonia because of the immunosuppressed state of the patients. Numerous case reports now attest to the disastrous outcome of n\easles pneumonia in immunocompromised patients (Meadow et al, 1969; Breitfeld et al, 1973; Siegel et al, 1977; Radoycich et al, 1992).

Rubeola (Measles)

401

FIGURE 29.6 Liver parenchyma showing complex network of multinucleate giant cells associated with parenchymal necrosis in a child treated with chemotherapy. Reprinted with permission from Hashida and Yunis (1970) and through the courtesy of E. Yunis, MD.

FIGURE 29.7 Kidney exhibiting multinucleate cells of varying configuration lining the renal tubules. The patient was the recipient of chemotherapy. Reprinted with permission from Hashida and Yunis (1970) and through the courtesy of E. Yunis, MD.

The association of multinucleate cells containing nuclear and cytoplasmic inclusions with rubeolavirus infection has now been established by immunohistochemical studies and the ultrastructural demonstration of the typically assembled nucleoproteins of the parainfluenza type within the inclusions (Archibald et a/., 1971; Joliat ei a/., 1973; Rand et al, 1976). These cells, to a variable extent, line the bronchial tree, bronchioles, alveolar ducts, and alveolar lining cells of the lung in cases of giant cell pneumonia. As shown by the studies of Mitus et al. (1959), virus replications can persist in the lungs of immunosuppressed patients for periods of 3 to 4 weeks. Antibody elaboration fails to occur, thus contributing to the chronicity of the infection. The end result is a debilitating, destructive, chronic inflammatory process resulting in pulmonary insufficiency, cor pulmonale, and, all too frequently, death. Throughout the lungs, interstitial

fibrosis and epithelial cell hyperplasia with squamous metaplasia are seen (Warner and Marshall, 1976; Becroft and Osborne, 1980). As might be expected, secondary bacterial infections are common. Focal squamous metaplasia of the airway epithelium is a common finding in giant cell pneumonia. Some investigators have suggested that vitamin A deficiency is a contributing factor in its pathogenesis, particularly since measles pneumonia is so devastating in populations where chronic malnutrition is common. One should recall the morphologic observations of Wolbach and Howe (1928), who examined the respiratory tract of rats deficient in vitamin A. The mucociliary tracheal and bronchial mucosa of these animals exhibited prominent squamous metaplasia throughout (Follis, 1958). These changes are promptly reversed by vitamin A administration. Interestingly enough, in recent field trials in several developing countries where

402

Pathology and Pathogenesis of Human Viral D i s e a s e f7^

FIGURE 29.8 Lung parenchyma exhibiting multinucleate cells with distinct inclusions surrounded by a halo. The cells form from the pneumocytes lining the airspaces. There is an interstitial mononuclear cell infiltrate.

ATYPICAL MEASLES SYNDROME

FIGURE 29.9 Nodular peribronchial lesion in the lung of an immunocompromised child with chronic measles pneumonia. Note the extensive squamous metaplasia in the airspaces of the lung parenchyma. A multinucleate cell is found in the diseased interstitium of this lesion (arrow). Reprinted with permission from Becroft and Osborne (1980).

malnutrition is endemic, vitamin A supplementation has substantially reduced childhood mortality due to acute respiratory disease (Craighead, 1995). While not specifically documented, measles pneumonitis is one of the more common of these infections.

Rauh and Schmidt (1965) first described the atypical measles syndrome in persons immunized with inactivated measles vaccine. These patients failed to elaborate mucosal immune responses, and systemic immunity (in the form of circulating antibodies) waned with the passage of time. The syndrome, which occurs in about 15% of vaccinees exposed to "wild" measles virus, is characterized by a prodromal high fever of several days duration, followed by the evolution of a pruritic, polymorphic, edematous rash that is first seen on the extremities, after which it spreads centripetally The rash then can become vesicular and petechial. Respiratory symptoms appear acutely, and measles pneumonia (and occasionally encephalitis) follows (Ross, 1972). For reasons that are unclear, this exudative process often exhibits a nodular configuration by X-ray with a lobar or segmental distribution. The residual nodularity of the lesions persists for extended periods of time, and biopsies of the lung have demonstrated a granulomatous process in the late established lesions (Young et al, 1970; Wood and Bernstein, 1978; Frey and Krugman, 1981). Unfortunately, the histopathology of this disease process is incompletely described. Since patients with the atypical measles syndrome rapidly elaborate antibody in response to wild measles virus infection, it is postulated that the lesions represent a hypersensitivity reaction, accompanying an anamnestic

403

Rubeola (Measles)

immune response to the infection. Presumably, the atypical measles syndrome is a disappearing disease, eliminated by immunization with live-virus vaccines. CENTRAL NERVOUS SYSTEM DISEASE Five neurological syndromes develop after natural infection with wild-type measles virus. The first of these is the typical Guillain-Barre syndrome, which occurs uncommonly (Lidin-Janson and Strannegard, 1972). It customarily appears after the onset of rash and is usually a reversible complication. The second is postinfectious leukoencephalopathy, a rare condition of unknown pathogenesis developing within weeks after the characteristic rash of measles disappears (Johnson et al, 1984). It is believed to reflect a nonspecific host response to a rubeolavirus infection, in which the virus has not directly invaded the nervous system. The incidence of postinfectious leukoencephalopathy is 1 in 1000 cases of measles, but it occurs predominantly in older persons infected with the virus. As with other virus infections, the brain in postinfectious leukoencephalopathy exhibits periventricular demyelination and infectious virus is not recoverable from nervous system tissue and cerebrospinal fluid. One assumes the process is an aberrant autoimmune phenomenon based on sensitization to myelin basic protein. How and why this dysregulated immune response occurs during naturally occurring measles infection is unknown. The remaining three syndromes result from infection involving the central nervous system tissue: acute measles encephalitis, subacute encephalitis in the immunologically defective patient, and Dawson's disease (inclusion-body panleukoencephalitis, subacute sclerosing panencephalitis, or SSPE). Meningoencephalitis After Natural Infection Measles meningoencephalitis develops sporadically and without explanation during acute infection in an otherwise healthy child (Purdham and Batty, 1974). It has a frequency of roughly 1 in 1000 cases of typical illness, but reports differ as to the actual number of cases. Virus can occasionally be recovered from the cerebrospinal fluid and the cells of the accompanying pleocytosis, as well as brain tissue either by direct inoculation of fluid into mammalian epithelial cell cultures or by co-culture of brain explants with the cell culture. According to Miller et al. (1956): The initial (pathological) change appears to be congestion, followed by infiltration of the walls, especially of the smaller veins, with mononuclear cells, and a little later by the develop-

ment of perivenous oedema and occasional hemorrhages of similar distribution. In the course of a few days, when the disease progresses, perivenous infiltration with microglial and lymphocytic cells is seen, and demyelination follows in the same region. The removal of fat by scavenging phagocytes ensues, and leaves demyelinated areas, which appear confluent in the course of time. Nerve-cell changes are not always present, and may vary in prominence. Direct involvement of blood-vessels is frequent, and may vary from patchy infiltration of the vessel wall with lymphocytes to actual necrosis. At some stage, these changes are completely reversible. In other instances, demyelinated areas persist, without progressive extension or extending gliosis.

Adams (1968) found cells in the brain with cytoplasmic and/or nuclear inclusion bodies in 75% of his cases, and multinucleate giant cells in 25%. In his cases, perivascular vascular infiltrates were invariably present. Recovery without complications usually occurs, but the long-term effects can be severe. Tyler (1957) reported ataxia in one-third of some 67 patients under his long-term care, and myelitis in 10 additional patients. Appelbaum et al. (1949) described psychosis, retardation, and paralysis, as well as a variety of other neurological problems in his patients. A great diversity of neuropathological changes are evident in the central nervous system of these chronically ill patients, when and if death occurs, but gliosis and demyelinization are the characteristic features. Evidence of focal or localized involvement of the nervous system is commonly lacking. Retrobulbar neuritis, chorioretinitis, and optic atrophy are also reported. In an isolated case report, a 40-year-old man with no predisposing disease became comatose shortly after the onset of a typical measles rash. He had been exposed to a child with measles. After death, widespread changes were found throughout the brain and spinal cord. Plaques of demyelinated white matter were located around blood vessels, where gliosis was prominent and rare perivascular cuffs of lymphocytes were evident. Intranuclear inclusions were found in ganglion cells adjacent to the plaques. Immunofluorescence studies demonstrated cells with nuclear and cytoplasmic reactivity, and rubeolavirus was recovered in cell cultures (ter Muelen et al, 1972). While this adult case exhibited some of the pathological features of postinfectious encephalomyelopathy that is, perivenous demyelinization, the evidence of infection throughout the central nervous system implicates rubeolavirus directly in its pathogenesis. Meningoencephalitis in the Immunosuppressed Patient A severe protracted form of measles encephalitis develops occasionally in patients with immunosuppression due to a wide variety of conditions several months

404

Pathology and Pathogenesis of Human Viral Disease

after the appearance of rash. The typical morbilliform rash of acute-onset measles is often not evident, presumably because of the loss of cellular immune functions involved in genesis of the skin lesions. Thus, the duration of the latency period frequently is uncertain. Measles encephalitis has been described in patients undergoing therapeutic immunosuppression for organ transplantation (Agamanolis et ah, 1979), and cancer chemotherapy (Pedersen et ah, 1978), as well as in patients with various types of acute leukemia and lymphoma (Spalke and Eschenbach, 1979; Aicardi et ah, 1977; Haltia et ah, 1977), hypogammaglobulinema (Hanissian et al, 1972), Hodgkin's disease (Wolinsky et al, 1977), and AIDS (Budka et al, 1996). Characteristically, there is the gradual onset of impaired consciousness followed by coma, convulsive disorders, and various neurological findings attributable to localized lesions in the central nervous system. The brain and spinal cord of these patients typically display scattered neurons and oligodendroglia with eosinophilic intranuclear and cytoplasmic inclusions that ultrastructurally exhibit the typical nucleoprotein complexes of the paramyxoviruses. Measles antigen can be demonstrated in these same cells using immunohistochemistry, and in situ hybridization documents the presence of viral RNA. Inflammation is not prominent in the brain, but occasional perivascular lymphocyte cuffs and microglial nodules are found. These changes are associated with variable amounts of diffuse or focal grey matter necrosis. The white matter is not customarily affected. Meningoencephalitis After Measles Vaccine Meningoencephalitis has been reported after live measles virus immunization, but the incidence is exceedingly low (1.16 cases per million immunizations), and the role of the vaccine virus in central nervous system disease is often unclear. It is likely that most cases are coincidental, and they are believed not to be due to the vaccine (Landrigan and Witte, 1973). Scattered clinical case reports, however, document an association virologically. Pathological observations have not been reported. Subacute Sclerosing Panencephalitis (SSPE) SSPE was described by Dawson (1933), a general pathologist using traditional hematoxylin and eosinstained tissue sections of brain. In the initial report, the inclusions characteristic of the disease were described.

but the typical demyelination and gliosis of white matter were not noted. Later pathologists were impressed by the widespread involvement of the brain by an inflammatory infiltrate and the profound degree of astrocytosis (Van Bogaert, 1945; Zeman and Kolar, 1968). SSPE is a rare (<1 case per million children with measles) but fatal chronic progressive inclusion-body panencephalopathy. Its association with a virus infection came n\any years after the early descriptive studies, largely as a result of elucidation of the ultrastructural features of the inclusions in the neurons (Perier and Vanderhaeghen, 1967). This work demonstrated accumulations of the typical paramyxovirus helical ribonucleoprotein cords in the inclusions but no evidence of viral budding from the plasma membrane. The specific association with measles was established by the immunohistochemical demonstration of viral antigens in both the nuclear and cytoplasmic eosinophil inclusions that are so characteristic of the disease (Jenis et al., 1973; Day an and Stokes, 1971). Serological evidence documenting high measles virus antibody concentrations in both the blood serum and cerebrospinal fluid (Connolly et al, 1967; Resnick et al, 1968) supported this conclusion. Although free virus cannot be cultured from brain tissue and cerebrospinal fluid, atypical mutated measles virus strains are recovered from explants of the brain by coculture with susceptible mammalian epithelial cells, as will be discussed in more detail below. And, viral isolates recovered from patients cause, in experimental animals, a demyelinating encephalopathy having virological features that simulate those occurring in humans (Haase et al, 1981; Raine et al, 1974, 1975; Johnson and Swoveland, 1977). The final observation establishing the role of measles virus in SSPE was the marked reduction in new cases since the advent of live-virus immunization. The cumulative evidence thus fulfills the requirements of Koch's hypotheses, and definitively establishes measles virus causatively with SSPE. The pathogenesis of SSPE is a fascinating but continuing enigma. On average, it develops 6 years after the acute episode of measles (but can occur 10 or more years later) (Cape et al, 1973; Modlin et al, 1979), and usually, but not invariably, progresses without remission to involve the cerebrum, ultimately resulting in death. Males are more frequently affected than females (male:female ratio = 2.5:1) and there appears to be a unique but totally unexplained rural predominance of cases (Table 29.1). About half the cases of naturally occurring infection develop before the age of 2 years. The sporadic appearance of cases of SSPE and the absence of clustering of cases argues strongly against the

Rubeola (Measles)

TABLE 29.1 Predominance of Rubeola Cases by Residence Domicile at the symptoms

Incidence rate White

Black

Rural, farm

7.2

7.9

Rural, other

4.1

0.3

Urban, noncentral city

3.1

1.2

Urban, central city

1.9

0.7

-

-

3.8

0.9

Residence unknown Total

Number of cases per 10,000,000 population less than 20 years of age according to the U.S. Census of 1970.

concept of an epidemic spread of a mutant pathogenic strain of virus in the community. However, the incidence of SSPE is reported to be higher in the Southeast and the Ohio River Valley than elsewhere in the United States. To the extent that mutant SSPE strains of virus occur, they would appear to develop spontaneously in the individual patient, and persist in the central nervous system independently thereafter. The viruses recovered from the brain tissue of SSPE patients by cocultivation are defective and to one extent or another exhibit point-mutations and deletions in the genes encoding the M, F, and H proteins of the virion. No consistent pattern of alterations is observed; thus, a strain of virus recovered from one patient can differ dramatically from the virus isolated from another (Billeter and Cattaneo, 1991). The most profound and consistent molecular defects are found in the matrix M protein, which is either crippled or totally deleted (Hirano et ah, 1993). As a result, infected patients rarely elaborate specific antibody to the M protein, even though the response to other viral antigens is robust. A key function of M protein is binding of the viral ribonucleocapsid to the inner aspects of the cell membrane during replication; thus, its absence, or defective structure, paralyzes the process of viral budding from the cell surface. Although the F and H proteins of SSPE virions usually are conserved, truncations, elongations, and amino acid substitutions are often demonstrable. These, alterations most probably serve to abort viral membrane formation since the cytoplasmic "tails" of both the F and H glycoproteins interact with the M protein lining the inner surface of the cell plasma membrane (Schmid et ah, 1992). The apparent absence of viral antigens on cell surfaces in the central nervous system most probably account for the persistence of infection in the presence of a high

405

concentration of circulating antibody in the blood of the patient. However, antigen expression on the plasma membrane of infected cells could be masqueraded by antibody when immunohistochemistry is done. Oldstone et al. (1975) found that circulating antibody from SSPE patients lyse cultured brain cells. Thus, humoral immune injury to these cells may occur in vivo. Other studies have demonstrated a plethora of cytokines in the inflammatory and resident cells of the SSPE brain-associated class IIMHC antigen expression on neurons (Nagano et al., 1994). Thus, cell-mediated damage to the infected cells might also occur. At present, answers to these and other questions are lacking. For example, it is totally unclear how the virus spreads widely in the brain in the absence of complete virion assembly, and in the presence of a strong immune response. Perhaps cell fusion is a mechanism despite the aforementioned molecular defects in the fusion protein. Although limited in scope, some studies indicate that peripheral lymph node cells and the circulating leukocytes of SSPE patients are infected by defective virus (Horta-Barbosa et al, 1971; Robbins et al, 1981). Although variable in severity and the rapidity of its evolution, the clinical picture of SSPE is usually quite characteristic. Three typical stages have been described. At the outset, a gradual deterioration in cognition and intellect is noted. The patient becomes disoriented and exhibits visual disturbances, seizures, and jerking hyperkinesis. In the second stage, contraction of the extremities evolves and the repetitive, simultaneous, high-frequency myoclonal "jerks," so typical of the disease, become evident. This clinical picture is accompanied by a pathognomonic electroencephalographic pattern. Coma and a vegetative state follow, leading to death in weeks or months. To a variable extent, this progressive deterioration in cerebral function can be punctuated by remissions of variable duration. The neuropathological picture correlates well with this clinical picture (Figures 29.10-12). The involvement of the cerebral cortices is widespread, but it is often particularly prominent in the occipital lobes. This lesion results in the cortical blindness that is so common early in the clinical course of the disease. The cerebellum and spinal cord are either not affected or exhibit less extensive focal lesions. The intranuclear and cytoplasmic inclusions so characteristic of the disease are found in neurons and oligodendroglia of the cortex and brain stem or upper spinal cord (Foley and Williams, 1953). In general, the nuclear inclusions are intensely eosinophilic, being rose-pink or mauve in shade and hyaline in consistency. They vary considerably in size (3 to 10 microns in diameter). Halos are not

406

Pathology and Pathogenesis of Human Viral D i s e a s e

FIGURE 29.12 Dense collections of nucleocapsids consistent with a paramyxovirus in the nucleus and cytoplasm of a neuron from a 48-year-old immunodeficient woman with measles encephalitis. Reprinted with permission from Parker et al. (1970).

FIGURE 29.10 Coronal section of the brain of a clinical case of SSPE demonstrates cortical laminar necrosis, especially prominent in left temporal cortex. Mild cerebral edema and focal cortical hemorrhages are also evident. The extent of laminar cortical necrosis is shown under higher magnification. H&E with luxol fast blue. x3. Reprinted with permission from Parker et al (1970).

a prominent feature in the involved cells, for the altered nucleoplasm often fills the entire nucleus (Hashida and Yunis, 1970). One or more cytoplasmic inclusions exhibiting intense staining characteristics are seen in some but not all cells. To a variable extent, subtle degenerative changes are observed, but necrosis is not a prominent part of the pathological picture. Indeed, some morphometric studies (Sa et al, 1995) have failed to demonstrate a reduction in neuronal number and cell size (in comparison to controls), and cortical layer thickness is said to be normal. However, the numerical

FIGURE 29.11 Most frequently encountered inclusion bodies are small eosinophilic intranuclear structures with marginated chromatin. These are found in astrocytes, oligodendroglia, and occasional neurons throughout nervous system (left). Anterior horn cells of lumbar spinal cord often contain eosinophilic intranuclear inclusion bodies, with clear halo between inclusion and peripheral chromatin (Cowdry Type A) (right). Reprinted with permission from Parker et al. (1970).

Rubeola (Measles)

density of synaptic junctions is reduced, a finding suggesting that neuronal communication is attenuated. As noted above, inflammation with mononuclear cell infiltration and perivascular lymphocyte cuffs are a common feature of the lesion (McQuaid et al., 1993). Glia nodules and both astrocytosis and microgliosis are seen. Demyelination is spotty and appears to be largely secondary to the involvement of cerebral grey matter and infection of oligodendroglia. However, a report by Parker et al. (1970) demonstrated widespread virus involvement of the brain and spinal cord with prominent degrees of laminar demyelinization. Studies of cases with unusually long survivals document the presence of argyrophilic neuronal and glial fibrillary tangles in the central neurons and in oligodendroglia in the absence of senile amyloid plaques (Ikeda et al., 1995; McQuaid et al., 1994; Mandybur et al., 1977). These cases usually show extensive cerebral atrophy.

M I D D L E EAR DISEASE Measles is a common cause of acquired deafness. In two reports, 9% of cases of noncongenital deafness in children were attributable to measles, although in many cases the damage to the middle ear was due to bacterial suppurative superinfection (Shambaugh et al., 1928; Yersle~ 1934). Isolated nonbacterial middle ear disease due to measles virus infection is much less common. A diversity of destructive and atrophic changes are described in pathological studies of the middle ear, but no specific pattern of lesions has become apparent' Multinucleate giant cells are found in the middle ear epithelium in some cases. Since the literature documents variable findings, the interested reader is referred to published detailed pathological descriptions (Lindsay and Hemenwa~ 1954; Bordley and Kapur, 1977).

EYE DISEASE Photophobia due to keratoconjunctivitis is pathognomonic of the acute measles illness. I remember it so well! Slit-lamp examination reveals punctate corneal erosions, and Koplik spots occasionally develop on the semilunar folds. These minor lesions almost invariably resolve without complication. However, Frederique and colleagues (1969) described in detail the histopathology of two eyes with corneal keratitis believed to be due to unresolved measles keratoconjunctivitis, compounded by malnutrition.

407

Lesions of the retinae of the eye occur rarely among otherwise healthy rubeolavirus-infected children and adults (accounting for about 1% of all blind children) (Scheie and Morse, 1972), but are common in patients with SSPE. Macular degeneration is a prominent feature clinically. Pathological degeneration and atrophy of the pigmented epithelium and the neuroepithelial layer of the retina are described, and, to a variable extent, inclusions are found in these cells. Gliosis of the optic nerve is also described (Grover et al., 1970; Nelson et al., 1970; Landers and Klintworth, 1971).

PREGNANCY The possible role of measles virus as a specific agent contributing to the toll of human congenital abnormalities has been critically evaluated by Jespersen et al. (1977) in a review of 10 "virgin soil" outbreaks of measles in Greenland. These workers found a spontaneous abortion rate of 32% among mothers infected during the first trimester. Overall there was an approximate threefold increase in stillbirths (i.e., deaths occurring after 20 weeks of gestation), with a disproportionately large number occurring among concepti infected during the first trimester. Only one major malformation was documented in the offsprings of some 31 women infected during the first trimester. Thus, there is no convincing evidence of a teratogenic effect of rubeolavirus, although infection does appear to result in spontaneous abortion and stillbirths, when it occurs early in pregnancy. The postmortem observations of Renne et al. (1973) on a female rhesus monkey who aborted during the acute stages of measles is of interest in this regard. These pathologists found inclusion-bearing syncytial giant cells in the decidua of the uterine cavity, near the implantation site, and additional involvement of the cervical epithelium. An opportunity for a more contemporary evaluation of measles in pregnancy was provided by a recent urban outbreak in which more than 1700 cases occurred (Atmar et al., 1992). Twelve pregnant women became infected, all but two of them in the later half of pregnancy. One patient died with pneumonia, despite therapeutic abortion, and three other women with pneumonia gave birth prematurely. The three infants did not develop measles, but aggressive immunotherapy and maternal treatment with ribavirin were employed. In this series, nonbacterial pneumonia was the most significant maternal complication, although clinical evidence of hepatitis was documented in half the cases. Since the immunization status of many of these women was unclear, some cases may have repre-

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sented the atypical measles pneumonia syndrome developing as a consequence of prior immunization with inactivated vaccine. This incidence of fatalities during pregnancy is similar to what has been observed in other reported outbreaks (Packer, 1950). References Adams, J. (1968). Clinical pathology of measles encephalitis and sequelae. Neurology 18 (Part 2), 52-57. Agamanolis, D., Tan, J., and Parker, D. (1979). Immunosuppressive measles encephalitis in a patient with a renal transplant. Arch. Neurol. 36, 686-690. Aicardi, J., Goutieres, R, Arsenio-Nunes, M.-L., and Lebon, P. (1977). Acute measles encephalitis in children with immunosuppression. Pediatrics 59, 232-239. Akhtar, M., and Young, I. (1973). Measles giant cell pneumonia in an adult following long-term chemotherapy. Arch. Pathol. 96, 145148. Anderson, R., Rabson, A., Sher, R., and Koornhof, H. (1976). Defective neutrophil motility in children with measles. /. Pediatr. 89, 27-32. Anonymous (1996). Another human case of equine morbillivirus disease in Australia. Emerging Infect. Dis. 2, 71-72. Appelbaum, E., Dolgopol, V, and Dolgin, J. (1949). Measles encephalitis. Am. J. Dis. Child. 77, 25. Archibald, R., Weller, R., and Meadow, S. (1971). Measles pneumonia and the nature of the inclusion-bearing giant cells: A light- and electron-microscope study. /. Pathol. 103, 27-34. Atmar, R., Englund, J., and Hammill, H. (1992). Complications of measles during pregnancy. /. Infect. Dis. 14, 217-226. Becroft, D., and Osborne, D. (1980). The lungs in fatal measles infection in childhood: Pathological, radiological and immunological correlations. Histopathology 4, 401^12. Bellini, W., Rota, J., and Rota, P. (1994). Virology of measles virus. /. Infect. Dis. 170 (Suppl. 1), S15-S23. Billeter, M., and Cattaneo, R. (1991). Molecular biology of defective measles virus persisting in the human central nervous system. In 'The Paramyxoviruses'" (D. Kingsbury, ed.), pp. 323-345. Plenum Press, New York. Bordley, J., and Kapur, Y. (1977). Histopathologic changes in the temporal bone resulting from measles infection. Arch. Otolaryngol. 103, 162-168. Breitfeld, V., Hashida, Y, Sherman, R, Odagiri, K., and Yunis, E. (1973). Fatal measles infection in children with leukemia. Lab. Invest. 28, 279-291. Brem, J. (1972). Koplik spots for the record: An illustrated historical note. Clin. Pediatr. 11, 161-163. Budka, H., Urbanits, S., Liberski, P., Eichinger, S., and PopowKraupp, T. (1996). Subacute measles virus encephalitis: Anew and fatal opportunistic infection in a patient with AIDS. Neurology 46, 586-587. Cape, C , Martinez, A., Robertson, J., Hamilton, R., and Jabbour, J. (1973). Adult onset of subacute sclerosing panencephalitis. Arch. Neurol. 28, 124-127. Christensen, P., Schmidt, H., Bang, H., Andersen, V., Jordal, B., and Jensen, O. (1953). An epidemic of measles in Southern Greenland, 1951: Measles in virgin soil, II: The epidemic proper. Acta Med. Scand. 144, 430-449. Connolly, J., Allen, L, Hurwitz, L., and Millar, J. (1967). Measles-virus antibody and antigen in subacute sclerosing panencephalitis. Lancet 1, 542-544.

Coovadia, H., Wesley, A., Hammond, M., and Kiepiela, P. (1981). Measles, histocompatibility leukocyte antigen polymorphism, and natural selection in humans. /. Infect. Dis. 144,142-147. Craighead, J. (1995). Vitamin A, retinoids, and carotenoids: Their role in infection and cancer prevention. In 'The Pathology of Environmental and Occupational Disease'' (J. Craighead, ed.), pp. 164168. Mosby Year-Book, St. Louis. Dawson, J. (1933). Cellular inclusions in cerebral lesions of lethargic encephalitis. Am. J. Path. 9, 7-16. Day an. A., and Stokes, M. (1971). Immunofluorescent detection of measles-virus antigens in cerebrospinal fluid cells in subacute sclerosing panencephalitis. Lancet 1, 891-892. Denton, J. (1925). The pathology of fatal measles. Am. J. Med. Sci. 169, 531-543. Dossetor, J., and Whittle, H. (1975). Protein-losing enteropathy and malabsorption in acute measles enteritis. Br. Med. ]. 2, 592-593. Dossetor, J., Whittle, H., and Greenwood, B. (1977). Persistent measles infection in malnourished children. Br Med. J. 1,1633-1635. Enders, J., and Peebles, T. (1954). Propagation in tissue cultures of cytopathogenic agents from patients with measles. Proc. Soc. Exper Biol. Med. 86, 277-286. Enders, J., McCarthy K., Mitus, A., and Cheatham, W. (1959). Isolation of measles virus at autopsy in cases of giant-cell pneumonia without rash. New Engl }. Med. 261, 875-881. Finkeldey W. (1931). Uber Riesenzellbefunde in den Gaumenmandeln, Zugleich ein Beitrag zur Histopathologie der Mandelveranderungen im Maserninkubationsstadium. Virchows Arch. 281, 323. Foley, J., and Williams, D. (1953). Inclusion encephalitis and its relation to subacute sclerosing leucoencephalitis: A report of five cases. Quart. J. Med. New Series 22(86), 157-198. Follis Jr, R. (1958). Vitamins A. In "Deficiency Disease" (R. FoUis Jr, ed.), pp. 125-140. Charles C. Thomas Publishing, Springfield, IL. Frederique, G., Howard, R., and Boniuk, V. (1969). Corneal ulcers in rubeola. Am. J. Ophthalmol. 68, 996-1003. Frey, H., and Krugman, S. (1981). Case report: Atypical measles syndrome: Unusual hepatic, pulmonary and immunologic aspects. Am. J. Med. Sci. 281, 51-55. Gellin, B., and Katz, S. (1994). Measles: State of the art and future directions. /. Infect. Dis. 170 (Suppl. 1), S3-S14. Goetz, M., and Mathisen, G. (1995). Clinical course and treatment of adults with severe measles pneumonitis. Clin. Infect. Dis. 21, 443445. Gremillion, D., and Crawford, G. (1981). Measles pneumonia in young adults: An analysis of 106 cases. Am. }. Med. 71, 539-542. Griffin, D., Ward, B., and Esolen, L. (1994). Pathogenesis of measles virus infection: An hypothesis for altered immune responses. /. Infect. Dis. 170 (Suppl. 1), S24-S31. Grover, W., Green, W., and Pileggi, A. (1970). Ocular findings in subacute necrotizing encephalomyelitis. Am./. Ophthalmol. 70,599-603. Haase, A., Swoveland, P., Stowring, L., Ventura, P., Johnson, K., Norrby, E., and Gibbs Jr., C. (1981). Measles virus genome in infections of the central nervous system. /. Infect. Dis. 144,154-160. Haltia, M., Paetau, A., Vaheri, A., Erkkila, H., Donner, M., Kaakinen, K., and Holmstrom, T. (1977). Fatal measles encephalopathy with retinopathy during cytotoxic chemotherapy./. Neurol. Sci. 32,323-330. Hanissian, A., Jabbour, J., deLamerens, S., Garcia, J., and Horta-Barbosa, L. (1972). Subacute encephalitis and hypogammaglobulinemia. Am. J. Dis. Child. 123,151-155. Hashida, Y, and Yunis, E. (1970). Re-examination of encephalitic brains known to contain intranuclear inclusion bodies: Electronmicroscope observations following prolonged fixation in formalin. Am. J. Clin. Pathol. 53, 537-543.

Rubeola (Measles) Hecht, V. (1910). Die Riesenzellenpneumonie im Kindesalter. Beitr. z. path. Anat. u. z. dig. Path. 48, 263-310. Hirano, A., Ayata, M., Wang, A., and Wong, T. (1993). Functional analysis of matrix proteins expressed from cloned genes of measles virus variants that cause subacute sclerosing panencephalitis reveals a common defect in nucleocapsid binding. /. Virol. 67,1848-1853. Horta-Barbosa, L., Hamilton, R., Wittig, B., Fuccillo, D., and Sever, J. (1971). Subacute sclerosing panencephalitis: Isolation of suppressed measles virus from lymph node biopsies. Science 173, 840-841. Ikeda, K., Akiyama, H., Kondo, H., Arai, T., Arai, N., and Yagishita, S. (1995). Numerous glial fibrillary tangles in oligodendroglia in cases of subacute sclerosing panencephalitis with neurofibrillary tangles. Neurosci. Lett. 194, 133-135. Jenis, E., Knieser, M., Rothouse, R, Jensen, G., and Scott, R. (1973). Subacute sclerosing panencephalitis: Immunoultrastructural localization of measles-virus antigen. Arch. Pathol. 95, 81-89. Jespersen, C , Littauer, J., and Sagild, U. (1977). Measles as a cause of fetal defects: A retrospective study of ten measles epidemics in Greenland. Acta Paediatr. Scand. 66, 367-372. Johnson, K., and Swoveland, R (1977). Measles antigen distribution in brains of chronically infected hamsters: An immunoperoxidase study of experimental subacute sclerosing panencephalitis. Lah. Invest. 37, 459^65. Johnson, R., Griffin, D., Hirsch, R., Wolinsky, J., Roedenbeck, S., de Soriano, I., and Vaisberg, A. (1984). Measles encephalomyelitis — clinical and immunologic studies. New Engl. J. Med. 310,137-141. Joliat, G., Abetel, G., Schindler, A.-M., and Kapanei, Y. (1973). Measles giant cell pneumonia without rash in a case of lymphocytic lymphosarcoma: An electron microscopic study. Virch. Arch. Abt. A. Path. Anat. 358, 215-224. Karp, C., Wysocka, M., Wahl, L., Ahearn, J., Cuomo, P., Sherry, B., Trinchieri, G., and Griffin, D. (1996). Mechanism of suppression of cell-mediated immunity by measles virus. Science 273, 228-231. Karsner, H., and Meyers, A. (1913). Giant-cell pneumonia. Arch. Int. Med. 2, 534-541. Kaschula, R., Druker, J., and Kipps, A. (1983). Late morphologic consequences of measles: A lethal and debilitating lung disease among the poor. Rev. Infect. Dis. 5, 395-404. Kernahan, J., McQuillin, J., and Craft, A. (1987). Measles in children who have malignant disease. Br Med. J. 295, 15-18. Kimura, A., Tosaka, K., and Nakao, T. (1975). Measles rash, I: Light and electron microscopic study of skin eruptions. Arch. Virol. 47, 295-307. Landers III, M., and Klintworth, G. (1971). Subacute sclerosing panencephalitis (SSPE): A clinicopathologic study of the retinal lesions. Arch. Ophthalmol. 86,156-163. Landrigan, P., and Witte, J. (1973). Neurologic disorders following live measles-virus vaccination. JAMA 223, 1459-1462. Lewis, M., Cameron, A., Shah, K., Purdham, D., and Mann, J. (1978). Giant-cell pneumonia caused by measles and methotrexate in childhood leukaemia in remission. Br Med. J. 1, 330-331. Lidin-Janson, G., and Strannegard, O. (1972). Two cases of GuillainBarre syndrome and encephalitis after measles. Br Med. J. 2, 572. Lindsay, J., and Hemenway, W. (1954). Inner ear pathology due to measles. Ann. Otolaryngol. 63, 754-771. Mandybur, T., Nagpaul, A., Pappas, Z., and Niklowitz, W. (1977). Alzheimer neurofibrillary change in subacute sclerosing panencephalitis. Ann. Neurol. 1, 103-107. Mason, W, Ross, L., Lanson, J., and Wright Jr, H. (1993). Epidemic measles in the postvaccine era: Evaluation of epidemiology, clinical presentation and complications during an urban outbreak. Pediatr Infect. Dis. ]. 12, 42-i8.

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McQuaid, S., Kirk, J., Zhou, A.-L., and Allen, I. (1993). Measles virus infection of cells in perivascular infiltrates in the brain in subacute sclerosing panencephalitis: Confirmation by non-radioactive in situ hybridization, immunocytochemistry and electron microscopy Acta Neuropathol. 85,154-158. McQuaid, S., Allen, I., McMahon, J., and Kirk, J. (1994). Association of measles virus with neurofibrillary tangles in subacute sclerosing panencephalitis: A combined in situ hybridization and immunocytochemical investigation. Neuropath. Appl. Neurobiol. 20,103110. Meadow, S., Weller, R., and Archibald, R. (1969). Fatal systemic measles in a child receiving cyclophosphamide for nephrotic syndrome. Lancet 2, 876-878. Merchant, R. (1976). Bronchopulmonary complications of measles: Analysis of 100 cases. Indian Pediatr 13, 847-849. Miller, H., Stanton, J., and Gibbons, J. (1956). Para-infectious encephalomyelitis and related syndromes: A critical review of the neurological complications of certain specific fevers. Quart. J. Med. 25, 427. Mitus, A,, Enders, J., Craig, J., and Holloway, A. (1959). Persistence of measles virus and depression of antibody formation in patients with giant-cell pneumonia after measles. New Engl. J. Med. 261, 882-889. Modlin, J., Halsey, N., Eddins, D., Conrad, J., Jabbour, J., Chien, L., and Robinson, H. (1979). Epidemiology of subacute sclerosing panencephalitis. /. Pediatr 94, 231-236. Monif, G., and Hood, C. (1970). Ileocolitis associated with measles (rubeola). Am. J. Dis. Child. 120, 245-247. Moore, R., and Gross, P. (1930). Giant cells in inflammation of the lung in children. Am. J. Dis. Child. 40, 247-259. Murray K., Selleck, P, Hooper, P, Hyatt, A., Gould, A., Gleeson, L., Westbury H., Hiley, L., Selvey, L., Rodwell, B., and Ketterer, P. (1995). A morbillivirus that caused fatal disease in horses and humans. Science 268, 94-97. Nagano, L, Nakamura, S., Yoshioka, M., Onodera, J., Kogure, K., and Itoyama, Y. (1994). Expression of cytokines in brain lesions in subacute sclerosing panencephalitis. Neurology 44, 710-715. Nakai, M., and Imagawa, D. (1969). Electron n\icroscopy of measles virus replication. /. Virol. 3,187-197. Nelson, D., Weiner, A., Yanoff, M., and dePeralta, J. (1970). Retinal lesions in subacute sclerosing panencephalitis. Arch. Ophthalmol. 84, 613-621. Olding-Stenkvist, E., and Bjorvatn, B. (1976). Rapid detection of measles virus in skin rashes by immunofluorescence. /. Infect. Dis. 134, 463-469. Oldstone, M., Bokisch, V., and Dixon, F. (1975). Subacute sclerosing panencephalitis: Destruction of human brain cells by antibody and complement in an autologous system. Clin. Immunol. Immunopathol. 4, 52-58. Packer, A. (1950). The influence of maternal measles (morbilli) on the unborn child. Med. J. Aust 1, 835-838. Parker Jr., J., Klintworth, G., Graham, D., and Griffith, J. (1970). Uncommon morphologic features in subacute sclerosing panencephalitis (SSPE). Am. J. Pathol. 61, 275-291. Pedersen, R, Schiotz, P., Valerius, N., and Hertz, H. (1978). Immunosuppressive measles encephalopathy [case report]. Acta Paediatr Scand. 67, 109-112. Perier, O., and Vanderhaeghen, J. (1967). Subacute sclerosing leucoencephalitis: Electron microscopic findings in two cases with inclusion bodies. Acta Neuropathol. 8, 362-380. Pinkerton, H., Smiley, W, and Anderson, W. (1945). Giant cell pneumonia with inclusions: A lesion common to Hechfs disease, distemper and measles. Am. J. Pathol. 21,1-15.

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Purdham, D., and Batty, P. (1974). A case of acute measles meningoencephalitis with virus isolation. /. Clin. Path. 27, 994-996. Radoycich, G., Zuppan, C , Weeks, D., Krous, H., and Langston, C. (1992). Patterns of measles pneumonitis. Pediatr. Pathol. 12, 773786. Raine, C , Byington, D., and Johnson, K. (1974). Experimental subacute sclerosing panencephalitis in the hamster: Ultrastructure of the chronic disease. Lah. Invest. 31, 355-368. Raine, C , Byington, D., and Johnson, K. (1975). Subacute sclerosing panencephalitis in the hamster: Ultrastructure of the acute disease in newborns and weanlings. Lab. Invest. 33,108-116. Rand, K., Emmons, R., and Merigan, T. (1976). Measles in adults: An unforeseen consequence of immunization? JAMA 236,1028-1031. Rauh, L., and Schmidt, R. (1965). Measles immunization with killed virus vaccine. Am. }. Dis. Child. 109, 232-237. Renne, R., McLaughlin, R., and Jenson, A. (1973). Measles virus-associated endometritis, cervicitis, and abortion in a Rhesus monkey. /. Am. Vet. Med. Assoc. 162, 639-641. Resnick, J., Engel, W., and Sever, J. (1968). Subacute sclerosing panencephalitis: Spontaneous improvement in a patient with elevated measles antibody in blood and spinal fluid. New Engl. J. Med. 279, 126-129. Robbins, S., Wrzos, H., Kline, A., Tenser, R., and Rapp, R (1981). Rescue of a cytopathic paramyxovirus from peripheral blood leukocytes in subacute sclerosing panencephalitis. /. Infect. Dis. 143, 396-iOl. Ross, L. (1972). Measles encephalitis in an immunized child [letter]. /. Pediatr. 90,156-166. Sa, M., Madeira, D., Cruz, C , and Paula-Barbosa, M. (1995). Morphometric study of the frontal cortex in subacute sclerosing panencephalitis. Acta Neurol. Scand. 92, 225-230. Scheie, H., and Morse, P. (1972). Rubeola retinopathy. Arch. Ophthalmol. 88, 341-344. Scheifele, D., and Forbes, C. (1972). Prolonged giant cell excretion in severe African measles. Pediatrics 50, 867-873. Schmid, A., Spielhofer, P., Cattaneo, R., Baczko, K., ter Meulen, V., and Billeter, M. (1992). Subacute sclerosing panencephalitis is typically characterized by alterations in the fusion protein cytoplasmic domain of the persisting measles virus. Virology 188, 910-915. Shambaugh, G., Hagens, E., Holderman, J., and Watkins, R. (1928). Otitis media. Arch. Otolaryngol. 7, 424. Siegel, D., and Hirschman, S. (1977). Hepatic dysfunction in acute measles infection of adults. Arch. Intern. Med. 137, 1178-1179.

Siegel, M., Walter, T., and Ablin, A. (1977). Measles pneumonia in childhood leukemia. Pediatrics 60, 38-40. Sobonya, R., Hiller, R, Pingleton, W, and Watanabe, I. (1978). Fatal measles (rubeola) pneumonia in adults. Arch. Pathol. Lab. Med. 102, 366-371. Spalke, G., and Eschenbach, C. (1979). Infantile cortical measles inclusion body encephalitis during combined treatment of acute lymphoblastic leukemia. /. Neurol. 220, 269-277. Suringa, D., Bank, L., and Ackerman, A. (1970). Role of measles virus in skin lesions and Koplik's spots. New Engl. J. Med. 283,1139-1142. ter Meulen, V., Kackell, Y., MuUer, D., Katz, M., and Meyermann, R. (1972). Isolation of infectious measles virus in measles encephalitis. Lancet 2,1172-1175. Tyler, H. (1957). Neurological complications of rubeola (measles). Medicine 36,147. Van Bogaert, L. (1945). Uneleuco-encephalite sclerosante subaique. /. Neurol. Psych. 8,101-120. Warner, J., and Marshall, W (1976). Crippling lung disease after measles and adenovirus infection. Br. ]. Dis. Chest 70, 89-94. Warthin, A. (1931). Occurrence of numerous large giant cells in tonsils and pharyngeal mucosa in the prodromal stage of measles. Arch. Pathol. 11, 864. Watson, A., and Parkin, J. (1970). Jejunal-biopsy findings during prodromal stage of measles in a child with coeliac disease. Lancet 2, 1134-1135. Weinstein, L., and Franklin, W. (1949). The pneumonia of measles. Am. ]. Med. Sci. 217, 314-324. Wolbach, S., and Howe, P. (1928). Vitamin A deficiency in the guineapig. Arch. Pathol. 5, 239. Wolinsky, J., Swoveland, P., Johnson, K., and Baringer, J. (1977). Subacute measles encephalitis complicating Hodgkin's disease in an adult. Ann. Neurol. 1, 452^57. Wood, B., and Bernstein, R. (1978). Pulmonary nodular "pneumonia" during the acute atypical measles illness. Annates de Radiologic 21, 193-198. Yersley, M. (1934). Analysis of over four thousand cases of educational deafness studied during the past twenty-five years. Br. J. Child. Dis. 31,177. Young, L., Smith, D., and Glasgow, L. (1970). Pneumonia of atypical measles: Residual nodular lesions. Am. J. Roentgenol. 110,439-448. Zeman, W, and Kolar, O. (1968). Reflections on the etiology and pathogenesis of subacute sclerosing panencephalitis. Neurology 18 (Part 2), 1-7.

C H A P T E R

30 Transmissible Spongiform Encephalopathy so-called spongiform encephalopathy. In developed countries, brain lesions of this type are found in the neurological disorder known as Creutzfeldt-Jakob disease (CJD), an exceptionally rare progressive encephalopathy with an incidence in the United States of approximately one case per 10^ members of the population (Holman et ah, 1996). The science largely stopped at this point, when in 1959 a letter was published in The Lancet by Bill Hadlow (a veterinary pathologist), that immediately provided insight into the pathogenesis of both kuru and CJD as well as two other even more rare neurodegenerative diseases. In the letter, Hadlow pointed out the similarities between the neuropathology of kuru and scrapie, a transmissible neuromuscular disease of sheep (Hunter, 1972; Hadlow, 1959). Scrapie is widely disseminated in Europe and less so in North America. Although long believed to be limited to sheep and goats, recent outbreaks of spongiform encephalopathy in animals of several different families have showed that the scrapie "agent" has the potential to infect a variety of mammalian species (Table 30.1). The concept of "slow" viruses was championed by Bjorn Sigurdsson (1954), the late Director of the Institute of Pathology in Reykjavik, Iceland. His insightful conclusions were based on observations of chronic disease among Icelandic sheep, the most notable of which was rida, more commonly known now as scrapie. He described the features of a "slow" virus infection as having a: (1) prolonged incubation period, that is, months or years, (2) protracted clinical disease phase with serious sequelae, and (3) limitation of infectivity to a single host and a single organ system. These provocative and prophetic ideas triggered great interest among Western scientists, for the notion of a slowly evolving infectious disease (in contrast to the usual acute infection) opened our eyes to the possibility that many chronic "degenerative" conditions might have an infectious origin. Various chapters in this book attest to the broad relevance of Sigurdsson's insights.

INTRODUCTION 411 SCRAPIE 412 HUMAN SPONGIFORM ENCEPHALOPATHIES: CLINICAL FEATURES 412

Creutzfeldt-Jakob Disease (CJD) 413 Gerstmann-Straussler-Scheinker Disease (CSS) 414 Kum 414 Fatal Familial Insomnia (FFI) 415 HUMAN SPONGIFORM ENCEPHALOPATHIES: PATHOLOGICAL FEATURES 415 PRIONS (PROTEIN INFECTIOUS ORGANISMS) 417 NEW VARIANT CJD (vCJD) 421 IATROGENIC CJD 422 PRECAUTIONS FOR PATHOLOGISTS 422 ANCILLARY NONHISTOPATHOLOGICAL DL\GNOSTIC APPROACHES OF THE PRION DISEASES 424 REFERENCES 424

INTRODUCTION I first met Carlton Gajdusek in 1957 over dinner at a small Chinese restaurant in Washington, DC. He dominated the conversation with fascinating tales of his work as a medical anthropologist on isolated Pacific isles. Carlton was soon to embark on a fateful expedition to the highlands of New Guinea, where he planned to study an unusual and seemingly heritable neurodegenerative disease occurring among the Fore tribe. In retrospect, Gajdusek possessed exceptional, if not prophetic, insight into the potential importance of his work with the Fore, and his subsequent studies ultimately led to a Nobel Prize. I remember only too well his salient statement of the evening: "People once thought tuberculosis was a heritable disease." The mystery of kuru, a disease described by Gajdusek, is now familiar to many of us, for the intriguing story of its discovery has been told and retold. A disease restricted largely to women and children of this isolated tribal group, it has a distinctive neuropathology, the

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Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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Pathology and Pathogenesis of Human Viral D i s e a s e TABLE 30.1 Prion D i s e a s e in A n i m a l s Disease

Animals affected

Scrapie Transmissible mink encephalopathy Chronic wasting disease Feline spongiform encephalopathy Bovine spongiform encephalopathy Zoo spongiform encephalopathy

Sheep and goats'' Commercial mink^ Wild mule, deer, and elk Domestic cats'^ Domestic cattle'^ Exotic ruminants'^ and greater cats

"Horizontal transmission. Susceptibility is influenced by poorly defined host heritable factors. ^Animals fed sheep carcasses. Animals fed carcasses of various domestic animals. '^Animals fed commercial cat food.

SCRAPIE Scrapie has been known as a naturally occurring disease of sheep since the 18th century, when it was widely distributed in England and throughout the European continent. The notion that scrapie was a transmissible disease was suggested in 1959, when it occurred in sheep that had been inoculated with a vaccine against looping illness, a common infection of these domestic animals. The vaccine consisted of a suspension of infected central nervous system and splenic tissue of sheep that contained the looping illness agent presumably inactivated with formalin. As expected, looping illness failed to develop, but scrapie did, after a prolonged latency period. Thus, the cryptic contaminating "agent" of scrapie was not destroyed by the chemical commonly used to kill viruses. Subsequently, sheep and goats were shown to acquire scrapie by the oral route, suggesting a means whereby the virus might be transmitted under natural circumstances. Since there are significant differences in the susceptibility of various breeds of sheep, heritable factors appear to influence susceptibility. Scrapie in sheep and goats differs somewhat, but the onset is insidious, with animals becoming apprehensive and restless. They soon develop tremors of the head and fasiculations of skeletal muscles. The animals become drowsy and dull, and they acquire unusual facial features. Evidence of skin irritation is emphasized by the appellation "scrapie," referring to scratching and rubbing. This turns out to be a characteristic clinical feature, with the affected sheep rubbing against fixed objects, such as fenceposts, or scratching themselves with their horns and biting and nibbling at their skin. As a result, wool is often denuded over the rump, flanks, nose, and head (Figure 30.1). As the disease

FIGURE 30.1 Adult sheep exhibiting the typical patchy loss of fleece due to mechanical irritation provoked by scrapie. Reprinted with permission and through the courtesy of W. Pendlebury, MD.

evolves, locomotion becomes increasingly difficult and the gait is unsteady. The animals fall. In the advanced stages of the disease, the infected animals wander about in an aimless seemingly stuporous state and exhibit evidence of visual impairment. This relentlessly progressive disease process evolves to death over a period of several months. Pathological changes thus far have only been demonstrated in the central nervous system, although several different organs contain infectious material. Experimentally, the "agent" of scrapie is transported along nerves to the central nervous system after peripheral inoculation. On the other hand, naturally infected lambs reproduce the agent initially in the gut and in abdominal lymphoid tissues before the infection ultimately finds its way to the brain and spinal cord. The typical neuropathological findings of neuronal degeneration, astrocytosis, and spongiform change observed in these animals represent the theme of this chapter (Figure 30.2).

H U M A N SPONGIFORM ENCEPHALOPATHIES: CLINICAL FEATURES Humans are afflicted with four uniquely different, and mercifully rare, neurological disorders that have as their common denominator a spongiform encephalopathy (Figure 30.3). They result from either the acquisition of a transmissible, nonviral, and abnormal protein known as a prion {PrP^% or are due to one of a number of genetic codon substitutions in the prion gene, as discussed below (Haywood, 1997). The concept of the prion was introduced and popularized by a brilliant experimental neurologist, Stanley B. Prusiner,

Transmissible Spongiform Encephalopathy

FIGURE 30.2 Scrapie. (A) Section of brain of a normal sheep. (B) Section of brain of a sheep that died of scrapie. Note the extensive spongiform changes. There is no evidence of an inflammatory or immune response. Reprinted with permission and through the courtesy of J. Foster, MD.

whose pathfinding work was recently recognized when he was awarded the 1998 Novel prize in medicine. Creutzfeldt-Jakob Disease (CJD) In the early 1920s, Creutzfeldt (1920) and Jakob (1921) independently described several patients with a progressive neurodegenerative syndrome typified by the loss of cognition and varying degrees of cortical blindness, ataxia, tremors, and myoclonus. In retrospect, some of their cases were not the syndrome we

now know as CJD. The disease usually is seen in the later decades of life (mean age at onset = 62 years) and is rapidly fatal, with only a few patients surviving longer than 1 year (median duration = 4.5 months) (Figure 30.4). We now recognize four clinical forms: (1) familial, (2) sporadic, (3) iatrogenic, and (4) ''new" variant. About 10% of cases are familial and seem to have an autosomal-dominant pattern of inheritance. More than 20 different substitutions have been demonstrated

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Age Group

FIGURE 30.3 Spongiform change and gliosis in the brain of a patient with CJD. A regional difference in the relative number of vacuoles is commonly observed.

FIGURE 30.4 CJD deaths and death rates by age group in the United States, 1979-1994. Reprinted with permission from Holman et al. (1996).

414

Pathology and Pathogenesis of Human Viral Disease

in the prion genes of the various families. Although phenotypically the resulting disease is similar to sporadic CJD, it commonly develops at an earlier age, and has a more protracted clinical course. The origin of the sporadically occurring CJD remains obscure. There is

no epidemiological evidence to implicate person- or animal-to-person spread, or a common source from which an infectious agent might be derived. The natural occurrence of a spontaneous mutation in the prion gene is a possible but unproven explanation. Later sections of this chapter will discuss the iatrogenic and variant forms of CJD. Gersttnann-Straussler-Scheinker Disease (GSS) In 1936, Gerstmann and his colleagues described a familial disorder predominantly manifest as a cerebellar ataxia compounded by dementia. Affected patients are, on average, one or two decades younger than those with CJD, but they tend to survive longer. As with CJD, the neuropathology of GSS is typically spongiform with gliosis, but plaques somewhat similar to the socalled senile plaques (Figure 30.5) are found microscopically in the brain (Masters et ah, 1980). Analysis of these cases has demonstrated a specific germline mutation in the prion gene and an autosomal-dominant mode of inheritance. GSS has been successfully transmitted from the brains of humans by intracerebral inoculation of subhuman primates, thus demonstrating the transmissibility of this "heritable" factor. Kuru This syndrome was brought to the attention of the Western world by D. Carlton Gajdusek, whose subsequent experimental work established the spongiform encephalopathies as a transmissible clinical and pathological entity (Figure 30.6). It was almost invariably seen in women and children, most probably because they (but not adult males) consumed human brain during the ritualistic ceremonies of cannibalism practiced by the tribe. Inasmuch as kuru was the major cause of

FIGURE 30.5 Spongiform changes and GSS-type PrP plaques in the brain of a transgenic mouse. The plaques stained by the periodic acid-Schiff reaction (A) showed green-gold birefringence when stained with Congo red and viewed with polarized light (B), and immunostained with PrP-specific antibodies after hydrolytic autoclaving (C). Bar in A = 50 |Lim (applies to A and B); bar in C = 100 jim. Reprinted with permission from DeArmond and Prusiner (1995).

FIGURE 30.6 Drs. D. Carlton Gajdusek (right) and V Gikas (left) examining a child with kuru in the eastern highlands of Papua, New Guinea in 1957. Reprinted with permission and through the courtesy of W. Pendlebury, MD.

Transmissible S p o n g i f o r m

1957

1959

1961

1963

1965

415

Encephalopathy

1967

1969

1971

1973

1975

1977

YEAR

FIGURE 30.7 The overall incidence of kuru deaths in male and female patients by year since its discovery in 1957 through 1977. More than 2500 patients died of kuru in this 20-year period of surveillance, and there has been a slow irregular decline in the number of patients. The decline in incidence of the disease followed the cessation of cannibalism, which occurred between 1957 and 1962 in various villages. Reprinted with permission from Masters et al. (1979).

premature death among the Fore (Gajdusek, 1979), those who consumed the brains of the dead were at high risk of acquiring the infection. With the elimination of cannibalism in the 1950s, the disease gradually disappeared among the Fore (Figure 30.7). The cerebral and cerebellar cortices in patients with kuru show diffuse spongiform changes, with prominent plaques predominantly comprised of amyloid and prion protein (Figure 30.8).

disease is first manifest as increasingly severe insomnia associated with diverse autonomic symptomatology. Dementia, cerebellar ataxia, and dysarthria as well as extrapyramidal signs and myoclonus evolve. The mean age for the onset of the disease is 49 years, and the clinical duration averages 13 months. This familial disorder is typified by a specific mutational change in the PrP gene (Medori et al, 1992). The spongiform lesions of FFI are largely confined to the thalamus, where they are associated with neuronal loss and gliosis.

Fatal Familial Insomnia (FFI) First described in 1986 by Lugaresi et ah, this extremely rare (approximate prevalence = 1 per 10 x 10^)

H U M A N SPONGIFORM ENCEPHALOPATHIES: PATHOLOGICAL FEATURES (see Lampert et al, 1972)

FIGURE 30.8 Diffuse spongiform changes in the brain at autopsy from a case of kuru. Note the prominent degenerative changes in neurons. Gliosis is not prominent.

Spongiform change in the cerebral cortex (Figures 30.3, 30.4, and 30.9) and the granular layer of the cerebellum (Figures 30.10 and 30.11) are pafhopneumonic of human prion disease. Involvement of the basal ganglion and thalamus is variable (Figure 30.12), being prominent in CSS and often less evident in CJD. Spongiform change is defined as the presence of small, round, or ovoid vacuoles (2-15 \xxa in diaraeter) in the neuropil. The vacuoles are usually not located in a pericellular location. Rarely, cytoplasmic vacuoles are observed in neurons and glia (Figure

416

Pathology and Pathogenesis of Human Viral Disease

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.#• FIGURE 30.9 Mild spongiform changes and neuronal loss in the hippocampus of the brain of a patient with CJD (Luxol fast blue, hematoxylin, and eosin stain, x490). Reprinted with permission from Scully et al (1993) and through the courtesy of E. Richardson, MD.

FIGURE 30.10 Sagittal section through the cerebellar vermis of a patient with CJD showing severe atrophy. Reprinted with permission from Scully et al. (1993) and through the courtesy of E. Richardson, MD.

30.9) (Masters and Richardson, 1978). The ultrastructural features of the vacuoles have been characterized (Chou et al, 1980) (Figure 30.13). Typically, the vacuoles lack a limiting membrane, but contain seemingly redundant mono- and bilaminar membranes. Surprisingly, the mechanistic basis for the spongiform change is obscure. Status spongiosis is a second type of lesion seen in the tissue. In the CJD brain, it is characterized by the

FIGURE 30.11 Neuronal loss accompanied by two kuru-like plaques in the cerebellar granular layer of the brain of a patient with CJD (Luxol fast blue, hematoxylin, and eosin stain, x490). Reprinted with permission from Scully et al. (1993) and through the courtesy of E. Richardson, MD.

FIGURE 30.12 Coronal section of the right cerebral hemisphere and temporal lobe of a patient with CJD showing atrophy of the head of the caudate nucleus, putamen, and centrum semiovale with ventricular dilatation and thinning of the cortical ribbon. Reprinted with permission from Scully et al. (1993) and through the courtesy of E. Richardson, MD.

appearance of irregular (in shape and size) cavities situated between glial fibers (Masters and Richardson, 1978). Unfortunately, the term "status spongiosis" is employed in the neuropathologic literature to refer to diffuse vacuolar changes in the brain resulting from a diverse variety of insults including trauma, anoxia, and

Transmissible Spongiform Encephalopathy

417

F I G U R E 30.13 Electron micrograph of a vacuole in the brain of a case of CJD. Note the lack of a limiting membrane, but the presence of internal complex bilayers and monolayers within the vesicles. This ultrastructural feature is typical, but the pathogenesis of the vacuoles remains unknown. Note the proximity of the vacuole to a neuron. Reprinted with permission and through the courtesy of W. Pendlebury, MD.

toxic injury (Adornato and Lampert, 1971). Because of the confusing terminology, "spongiform change" is used exclusively herein. Astrocytosis and neural loss are the additional morphological features of the diseased brain, but evidence of inflammation is notably lacking. The dendrites of degenerating neurons are "pruned," with a reduction in the number of dendritic spines (Figure 30.14) and formation of spherical blebs in axons (Scully ei al., 1980). Noteworthy in this regard is the finding of deposits of VrV^^ on dendrites of the neuron (Kitamoto ei al., 1992). Customarily, a diffuse astrocytosis accompanies the spongiform changes. In a series of 17 cases. Masters and Richardson (1978) quantitated the three major components of the lesion. As might be expected, their severity corresponded to the duration of clinical symptomatology (Figure 30.15). Changes in the white matter of the cerebrum and demyelination are not features of the disease. In the cerebellum, spongiform changes are restricted to the molecular layer, and Purkinje cells appear unaffected. However, using the Bodian stains, "torpedoes" have been demonstrated in bulbous expansions of the

axons of Purkinje cells (Figure 30.16). The pathogenesis of these unusual neuronal lesions is unknown. Prions (Protein Infectious Organisms) (see Prusiner, 1987; Prusiner ei al, 1996)

Prion-related protein (PrP'^) is a normally occurring cell membrane protein anchored by glycophosphoinositol; it has a molecular weight of 27-30 kDa. PrP^ is coded by a single gene on the 20th chromosome and expressed as deposits in the brain and a wide variety of tissues (Muramoto et al, 1992; Pammer et al, 1998). In the central nervous system, PrP^ is synthesized by neurons (DeArmond and Prusiner, 1995; Bruce et al, 1989). Its biological function is not known. The transmissible PypSc isoform is the primary, and most probably the exclusive, infectious prion in tissue (Figure 30.17). In contrast to PrP^, it is relatively resistant to digestion by proteolytic enzymes, and is stubbornly insoluble in water. These physical characteristics permit the isolation of PrP^^ in relatively pure form from the brains of patients with prion disease (Piccardo et al, 1990) (Figure 30.18). PrP^^ is resistant to alcohols, formalin, and ionizing irradiation (agents that destroy traditional

418

Pathology and Pathogenesis of Human Viral Disease

FIGURE 30.14 Cerebral cortex from a normal human adult (rapid-Golgi technique). (A) A distal apical dendrite of a layer III pyramidal neuron. There is normal spine density. Panel B shows a distal apical dendrite of a cortical layer III pyramidal neuron in the cerebral cortex of a patient with CJD. Note the abnormal paucity of dendritic spines. Panel C shows a spherical bleb (arrow) arising from a basal dendrite of a pyramidal neuron in the same patient. The dendrites are spine-poor, attenuated, and slightly irregular in their course. Reprinted with permission from Scully et al (1986).

viruses), but is inactivated through insults by agents that disrupt proteins (detergents, phenols, pH extremes, and autoclaving). Inoculation of PrP^^ into the brain results in amplification of preexisting FrP^ by mechanisms that remain speculative at this time (Figure 30.19). It is a structurally altered form of the preexisting tissue, PrP'^. This conclusion is substantiated by the observation that knock-out mice lacking the PrP'^ gene fail to develop

spongiform changes and PrP^^. Thus, the naturally occurring protein undergoes a posttranslational configurational change to a transmissible prion that is pathogenic for mice, hamsters, subhuman primates, and, presumably, humans (Horwich and Weissman, 1997). Immunochemical studies of the brain infected with PypSc document massive accumulations of the protein isoform in the neuropil. The concentrations are often sufficiently great that they accumulate into the his-

419

Transmissible Spongiform Encephalopathy

SPONGIFORM

CHANGE

V NEURONAL

LOSS

B./*

•:

FIGURE 30.15 Pathological scores documenting the degree of spongiform change (A), neuronal loss (B), and gliosis (C) in 21 cases of CJD. The horizontal axis indicates duration of clinical illness in months, and the vertical axis indicate the pathological scoring. Reprinted with permission from Masters and Richardson (1978).

• •

-r-

FIGURE 30.16 Cerebellum with axonal "'torpedoes'' (arrows) (Bodian stain, x260). Reprinted with permission from Scully et al. (1986).

tologically recognizable plaques mentioned above (see Figures 30.5 and 30.11). In general, large numbers of plaques are seen in patients who survive for long periods, whereas substantially fewer plaques are present in the brains of short-lived survivors. Plaques are prominent in the brains of most of those with naturally occurring kuru, but are found in only about 10% of patients with CJD, attesting to the relatively short survival time after the onset of symptoms in this disease. As noted above, approximately 20 different mutations in the human PrP'^ gene have thus far been identified in patients with heritable forms of CJD. Sixty percent of the cases of the rare CSS syndrome demonstrate a mutation (Pro^^^ -^ Leu-P102L). This mutated gene can be introduced into mice by transgenic technology; these animals subsequently accumulate large amounts of the mutant protein in the brain and develop a widespread spongiform change in the neuropil. In addition, an artificially fabricated transgene produces a serially transferable neurodegenerative disease in mice. Thus, the configurational change in the prion resulting from the point mutation in its gene converts PrP^ into a pathogenic transmissible protein.

420

Pathology and Pathogenesis of Human Viral Disease

FIGURE 30.17 Hypothetical structural features of naturally occurring PrP'^ (left) and disease-causing prions (PrP^'^) (right). The conformational change transforms the a-helix to a p-pleated sheet, thus accounting for the amyloid that accumulates in plaques. Reprinted with permission from Prusiner (1997).

FIGURE 30.18 Extensively purified prion protein rods from a case of CJD negatively stained with uranyl formate (bar = 100 nm). Reprinted with permission and through the courtesy of S. Prusiner, MD.

Transmissible Spongiform Encephalopathy

421

Prpc

PrP^

Prpsc

onverted molecule PrP^ "seed"

Prpsc aggregate

v# B FIGURE 30.19 (A) One hypothesis proposed to explain how infectious prions cause disease. The abnormal protein (PrP^'^) comes in contact with the ''normal" twin (PrP% The latter then changes it to an abnormal form, eventually harming neurons. (B) Alternatively, infection by PrP^'^ initiates polymerization of the molecules, leading to self-replication of the infectious agent. Reprinted with permission from Prusiner (1997).

NEW VARIANT CJD (vCJD) During the past several years, over 25 relatively young (age range 16-25 years; mean age 29) English men and women have developed CJD currently attributed to the consumption of meat from cattle infected with the prion isoform of bovine spongiform encephalopathy (BSE). Almost 1 x 10^ cattle in the United Kingdom were estimated to be carriers of the responsible prion prior to initiation of intensive eradication programs, and 1.7 x 10*^ animals have died with an encephalopathy. The epidemic of so-called "mad cow disease" was attributed to feeding these animals a highprotein supplement containing muscle and bone pre-

pared from the carcasses of domestic animals, including cattle and sheep, that had died in the field. Since several years are required for overt disease to develop in infected cattle, humans presumptively contract the infection as a result of consumption of meat from animals with subclinical infections. There are many subtleties to this story that make speculation as to the initial source of the responsible prion hazardous, but infected carcasses from sheep with scrapie seem likely to have been the source of the disease in cattle. While analysis of the responsible prion in the brains of patients with vCJD has many pitfalls, the glycoprotein differs from the prion observed in the brain of patients with classical sporadically occurring CJD (CoUinge et ah, 1996; Pattison, 1998). In addition, none

422

Pathology and Pathogenesis of Human Viral D i s e a s e

of the characteristic genotypes of familial CJD have been identified in those infected with vCJD (Will et al, 1996). The clinical picture of vCJD is characteristic. Patients present with behavioral problems or depression and often are seen initially by a psychiatrist. Dyasthesias develop, and both ataxia and choreoathetoid movements are observed. Myoclonus is evident. Dementia is an evolving problem, but memory loss (a common presenting feature of classical CJD) develops only late in the course of the disease. While abnormal, the typical electroencephalographic picture of CJD is not observed. In contrast to classical CJD, the duration of variant disease is protracted (average 14 months) (Zeidler et al, 1997; Pattison, 1998; Will et al, 1996). Pathologically, spongiform changes are present focally throughout the cerebrum and cerebellum, but they are often prominent in the basal ganglion, thalamus, and hypothalamus. There is accompanying neuronal loss and astrocytosis. The outstanding consistent feature is the widespread presence of plaques uniquely surrounded by spongiform vacuoles, a picture that vaguely resembles the petals of a daisy encompassing • ; • • # • • # • •

1

'•••'•

#^**

• #%

• •Hi

'i

#

;:©.,

•

©•

FIGURE 30.20 vCJD. Note the vacuoles tending to surround the plaques in this photomicrograph of the cerebral cortex. Reprinted with permission and through the courtesy of P. Brown, MD.

the central seed disc of the flower (Figure 30.20). Immunochemistry reveals prion deposits in the involved areas, particularly surrounding neurons. Because this seemingly unique neuropathologic picture could relate to the relatively young age of patients in this group, neuropathological comparisons with cases of classical, sporadic, and iatrogenic CJD have been carried out. The number and types of plaques observed in the brain of vCJD patients substantially exceeded the number observed in classical CJD (approximately 10% of cases) (Will et al, 1996). The prion of vCJD exhibits molecular and biological features that are strikingly similar to

those observed in the prion of BSE (CoUinge et al, 1996; Johnson and Gibbs, 1998).

IATROGENIC CJD In 1974, CJD was diagnosed in a recipient of a corneal graft 18 months after transplantation. This case proved to be a stark warning of what was to follow. During the 1960s and 1970s, pituitary glands were collected at autopsy to prepare pituitary extracts for the treatment of children with pituitary dwarfism (Table 30.2). With considerable consternation, the pathology community later learned that spongiform encephalopathies occurred in more than 100 of these unfortunate young children. Presumably, the disease originated from pituitary glands inadvertently retained from cadavers with CJD. Rare patients undergoing stereotactic electroencephalography and neurosurgical procedures also developed spongiform encephalopathy believed to be due to contaminated instruments that had previously been used on CJD patients. Over the past 10 years, more than 80 cases of CJD have occurred as a result of infection during neurosurgery (Table 30.3) (Johnson and Gibbs, 1998). Although the blood of patients with CJD and animals inoculated with PrP^^ have been shown to be infectious, the available information thus far fails to indicate that recipients of blood transfusions and blood products develop iatrogenic CJD (Ricketts et al, 1997). Organ transplant recipients are not known to have been affected, although liver, lung, and kidney tissue from CJD patients are infectious (Brown et al, 1986a). While many gaps in our knowledge exist, the potential threat of transmission of spongiform encephalopathy by fomites and human tissue is ever present. Published anecdotal reports provide a sound basis for continued concern (Klein and Dumble, 1993; Creange et al, 1995; Manuelidis et al, 1985; Tateishi, 1985).

PRECAUTIONS FOR PATHOLOGISTS Early studies with scrapie demonstrate the extraordinary resistance of the scrapie agent to both physical irradiation and chemical decontamination. This finding initially alerted the scientific community to the fact that scrapie was not caused by a traditional DNA or RNA virus that could be inactivated by the usual virus decontaminants. When the similarity of scrapie to CJD was recognized, concern justifiably arose regarding the risks of inadvertent exposure of pathologists and labo-

423

Transmissible Spongiform Encephalopathy TABLE 30.2 Comparison of R i s k s of Contracting CJD and M e a n Incubation Periods after Treatment w i t h Pituitary Extracts in Various Countries (as of October 1998)

Country of origin

Number of patients

Treated population

Risk of CJD, %

Mean incubation period (years)

United States

26

8500

0.3

18 (±6)

United Kingdom

28

1750

1.6

14 (±3)

France

55

1700

3.2

10 (±3)

Reprinted with the permission of and through the courtesy of P. Brown, MD.

TABLE 30.3 Proven or H i g h l y Probable Cases of Iatrogenic Creutzfeldt-Jakob D i s e a s e (as of October 1998) Mode of infection

Number of patients

Agent entry into brain

Mean incubation period, range

Clinical presentation

Corneal transplant

3

Optic nerve

16, 18, 320 mos

Dementia / Cerebellar

Stereotactic EEC

2

Intracerebral

18 mos (16, 20)

Dementia / Cerebellar

Neurosurgery

4

Intracerebral

20 mos (15-28)

Visual/ Dementia/ Cerebellar

Dura mater graft

80

Cerebral surface

6 yrs (1.5-16)

Cerebellar (Visual / Dementia)

Growth hormone

111

Hematogenous

12 yrs (5-30)

Cerebellar

4

Hematogenous

13 yrs (12-16)

Cerebellar

Gonadotropin

Reprinted with the permission of and through the courtesy of P. Brown, MD.

ratory technicians to infectious tissue from patients with CJD. The problem has been the focus of proposed guidelines for health professionals (Traub et ah, 1974, 1975; Gajdusek et al, 1977; Brown et al, 1982, 1986a; Rosenberg et al, 1986). It is therefore appropriate to quote from the initial paper, which focused on guidelines for pathologists: All persons in the autopsy room should wear long-sleeved gowns, gloves, and masks. The skull and spinal canal should be opened as usual with an oscillating saw, with special care not to cut the brain and cord. All the patient's tissue must be regarded as potentially infectious, not just the central nervous system. When no autoclave is available, chemical disinfection (see below) is a satisfactory alternative. Special attention should be paid to external disinfection of the instruments and containers used in collection of autopsy tissues. Instruments should be autoclaved before being cleaned for re-use. The sink in which water from the autopsy table collects should be plugged and wash water collected. If no autoclave is available, >4 volumes of 5% hypochlorite bleach should be added to the water and left for at least 2 hr before being discarded. Before it leaves the autopsy room, the body should be sponged with 5% sodium chlorite. All tissues should be considered fully infectious, even after prolonged fixation in formalin and histologic processing. Tissues may be washed in several changes of water on a shaker, rather than in running tap water; this water, the formalin, and

subsequent aqueous or alcoholic washes should be pooled and decontaminated. Glassware, forceps, and tissue carriers can also be decontaminated by soaking in 5% sodium hypochlorite or autoclaving. Xylene, toluene, or other organic solvents should be autoclaved and discarded, rather than reused. The microtome blade used to cut such tissue can be decontaminated by flaming, autoclaving, or soaking in disinfectants. Special care must be taken to avoid cuts with potentially contaminated glassware or blades. Remains of patients dying of the disease should not be accepted for teaching of gross anatomy to students, and specimens and pathological teaching collections should be handled with caution. Morticians and mortuary workers should be warned of possible hazards posed by tissue of patients with CJD and provided with advice about proper use of disinfectants. (Gajdusek et al, 1977)

The more recent experimental evidence to date (see Brown et al, 1986a) recommends the following for decontamination of suspected infectious material on instruments: 1 hour exposure to temperature of 132°C in a steam autoclave, or to 1 N sodium hydroxide (NaOH) at room temperature; nearly complete decontamination was possible with 1-hr exposure to a temperature of 121 °C or to 2.5% sodium hypochlorite.

424

Pathology and Pathogenesis of Human Viral Disease

However, the authors warn that the recommended treatments are not the "final solution" to the problem of decontamination. Tissue for histology should be treated for 1 hour in concentrated formic acid and then immersed in a 4% formaldehyde solution for 48 or more hours (Budka et al, 1995). ANCILLARY NONHISTOPATHOLOGICAL DIAGNOSTIC APPROACHES OF THE PRION DISEASES PypSc J5 protease resistant, allowing separation from the PrP^ present in normal tissue. It is antigenic. Thus, immunohistochemical and immunochemical assays could be of diagnostic usefulness in cases of prion disease in which the clinical or pathological picture is uncertain or atypical. In several recent studies. Western Blot analysis of brain biopsies permitted a specific diagnosis when pathological diagnosis was not possible (Brown et al, 1986b; Castellani et ah, 1997). In one case report, a 16-mg fragment of grey matter provided sufficient material for assay. Of greater interest was demonstration of the usefulness of biopsy tissue from a case of vCJD to accomplish diagnosis by Western Blot analysis (Hill et al, 1997). This approach, and most probably future modification, could permit diagnosis without the necessity for brain biopsy. In familial CJD and autosomal-dominant CSS, PCR analysis of DNA from nonnervous tissue permits specific diagnosis without histopathological examination of brain tissue (CoUinge et al, 1989). Hsich and colleagues (1996) have claimed that a specific diagnostic protein is present in the cerebrospinal fluid of CJD patients. It is demonstrated by SDS-PAGE. Confirmation of this interesting observation will be required.

References Adomato, B., and Lampert, P. (1971). Status spongiosis of nervous tissue: Electron microscopic studies. Acta Neuropath. (Berlin) 19, 271-289. Brown, P. (1996). Environmental causes of human spongiform encephalopathy. In "Methods in Molecular Medicine: Prion Diseases'' (H. Baker and R. Ridley, eds.), pp. 139-154. Humana Press, Totowa, NJ. Brown, P., Gibbs, C , Amyx, H., Kingsbury, D., Rohwer, R., Sulima, M., and Gajdusek, D. (1982). Chemical disinfection of Creutzfeldt-Jakob disease virus. N. Engl J. Med. 306,1279-1282. Brown, P., Rohwer, R., and Gajdusek, D. (1986a). Newer data on the inactivation of scrapie virus or Creutzfeldt-Jakob disease virus in brain tissue. /. Infect Dis. 153,1145-1148.

Brown, P., Coker-Vann, M., Pomeroy, K., Franko, M., Asher, D., Gibbs, C , and Gajdusek, D. (1986b). Diagnosis of Creutzfeldt-Jakob disease by Western blot identification of marker protein in human brain tissue. N. Engl J. Med. 314, 547-551. Bruce, M., McBride, P., and Farquhar, C. (1989). Precise targeting of the pathology of the sialoglycoprotein, PrP, and vacuolar degeneration in mouse scrapie. Neurosci. Lett. 102,1-6. Budka, H., Aguzzi, A., Brown, P., Brucher, J., Bugiani, O., Collinge, J., Diringer, H., Gullotta, F., Haltia, M., and Hauw, J. (1995). Tissue handling in suspected Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies. Brain Pathol 5, 319-322. Castellani, R., Parchi, P., Madoff, L., Gambetti, P., and McKeever, P. (1997). Biopsy diagnosis of Creutzfeldt-Jakob disease by Western blot: A case report. Hum. Pathol 28, 623-626. Chou, S., Payne, W, Gibbs, C , and Gajdusek, D. (1980). Transmission and scanning electron microscopy of spongiform change in Creutzfeldt-Jakob disease. Brain 103, 885-904. Collinge, J., Owen, F., Lofthouse, R., Shah, T, Harding, A., Poulter, M., Boughey, A., and Crow, T. (1989). Diagnosis of GerstmannStraussler syndrome in familial dementia with prion protein gene analysis. Lancet 2,15-17. Collinge, J., Sidle, K., Meads, J., Ironside, J., and Hill, A. (1996). Molecular analysis of prion strain variation and the aetiology of "new variant" CJD. Nature 383, 685-690. Creange, A., Gray, F., Cesaro, P., Adle-Biassette, H., Duvoux, C , Cherqui, D., Bell, J., Parchi, P, Gambetti, R, and Degos, J. (1995). Creutzfeldt-Jakob disease after liver transplantation. Ann. Neurol 38, 269-272. Creutzfeldt, H. G. (1920). Uber eine eigenartige herdformige Erkrankung des Zentralnervensystems. Z, Gesamt. Neurol Psychiat. 57,1-18. DeArmond, S., and Prusiner, S. (1995). Etiology and pathogenesis of prion diseases. Am. J. Pathol 146, 785-811. Gajdusek, D. (1979). Observations on the early history of kuru investigation. In "Slow Transmissible Diseases of the Nervous System" (S. Prusiner and W. Hadlow, eds.). Vol. 1, pp. 7-35. Academic Press, New York. Gajdusek, D., Gibbs, C , Asher, D., Brown, R, Diwan, A., Hoffman, R, Nemo, G., Rohwer, R., and White, L. (1977). Precautions in medical care of, and in handling materials from, patients with transmissible virus dementia (Creutzfeldt-Jakob disease). New Engl J. Med. 297,1253-1258. Gerstmann, J., Straussler, E., and Scheinker, I. (1936). Uber eine eigenartige hereditar-familiare Erkrankung des Zentralnervensystems zugleich ein Beitrag zur frage des vorzeitigen lokalen Alterns. Z. Neurol 154, 736-762. Hadlow, W. (1959). Scrapie and kuru. Lancet 2, 289-290. Haywood, A. (1997). Transmissible spongiform encephalopathies. New Engl J. Med. 337,1821-1828. Hill, A., Zeidler, M., Ironside, J., and Collinge, J. (1997). Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy. Lancet 349, 99-100. Holman, R., Khan, A., Belay, E., and Schonberger, L. (1996). Creutzfeldt-Jakob disease in the United States, 1979-1994: Using national mortality data to assess the possible occurrence of variant cases. Emerg. Infect. Dis. 2, 333-337. Horwich, A., and Weissman, J. (1997). Deadly conformations: Protein misfolding in prion disease. Cell 89, 499-510. Hsich, G., Kenney, K., Gibbs, C , Lee, K., and Harrington, M. (1996). The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies. New Engl ]. Med. 335, 924-930.

Transmissible Spongiform Encephalopathy Hunter, G. (1972). Scrapie: A prototype slow infection. /. Infect. Dis. 125, 427-440. Jakob, A. (1921). Uber eigenartige Erkrankungen des Zentralnervensystem mit bemerkenswerten anatomischen Befunden. Z. Gesamt. Neurol. Psychiat. 61,147-228. Johnson, R., and Gibbs, C. (1998). Creutzfeldt-Jakob disease and related transmissible spongiform encephalopathies. New Engl. J. Med. 339,1994-2004. Kitamoto, T., Shin, R.-W., Doh-ura, K., Tomokane, N., Miyazono, M., Muramoto, T., and Tateishi, J. (1992). Abnormal isoform of prion proteins accumulates in the synaptic structures of the central nervous system in patients with Creutzfeldt-Jakob disease. Am. J. Pathol. 140,1285-1294. Klein, R., and Dumble, L. (1993). Transmission of Creutzfeldt-Jakob disease by blood transfusion. Lancet 341, 768. Lampert, R, Gajdusek, C , and Gibbs, C. (1972). Subacute spongiform virus encephalopathies. Am. J. Pathol. 68, 626-646. Lugaresi, E., Medori, R., Montagna, R, Baruzzi, A., Cortelli, P., and Lugaresi, A. (1986). Fatal familial insomnia and dysautonomia with selective degeneration of thalamic nuclei. New Engl. ]. Med. 315(16), 997-1003. Manuelidis, E., Kim, J., Mericangas, J., and Manuelidis, L. (1985). Transmission to animals of Creutzfeldt-Jakob disease from human blood. Lancet 2, 896-897. Masters, C , and Richardson, E. (1978). Subacute spongiform encephalopathy (Creutzfeldt-Jakob disease). Brain 101, 333-344. Masters, C , Harris, J., Gajdusek, D., Gibbs, C , Bernoulli, C , and Asher, D. (1979). Creutzfeldt-Jakob disease: Patterns of worldwide occurrence. In ''Slow Transmissible Diseases of the Nervous System" (S. Prusiner and W. Hadlow, eds.). Vol. 1, pp. 113-142. Academic Press, New York. Masters, C , Gajdusek, D., and Gibbs, C. (1980). The GerstmannStraussler syndrome and the various forms of amyloid plaques which occur in the transmissible spongiform encephalopathies [abstract]. /. Neuropathol. Exp. Neurol. 39, 374. Medori, R., Tritschler, H., LeBlanc, A., Villare, R, Manetto, V., and Chen, H. (1992). Fatal familial insomnia, a prion disease with a mutation at codon 178 of the prion protein gene. New Engl. J. Med. 326, 444-449. Muramoto, T., Kitamoto, T., Tateishi, J., and Goto, L (1992). The sequential development of abnormal prion protein accumulation in mice with Creutzfeldt-Jakob disease. Am. J. Pathol 140,1411-1420. Pammer, J., Weninger, W., and Tschachler, E. (1998). Human keratinocytes express cellular prion-related protein in vitro and during inflammatory skin diseases. Am. J. Pathol. 153,1353-1358.

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Pattison, J. (1998). The emergence of bovine spongiform encephalopathy and related diseases. Emerg. Infect. Dis. 4, 390394. Piccardo, R, Safar, J., Ceroni, M., Gajdusek, D., and Gibbs, C. (1990). Immunohistochemical localization of prion protein in spongiform encephalopathies and normal brain tissue. Neurology 40, 518-522. Prusiner, S. (1987). Prions and neurodegenerative diseases. New Engl. J. Med. 317,1571-1581. Prusiner, S. (1997). Prion diseases and the BSE crisis. Science 278, 245-251. Prusiner, S., Telling, G., Cohen, R, and DeArmond, S. (1996). Prion diseases of humans and animals. Sem. Virol. 7,159-173. Ricketts, M., Cashman, N., Stratton, E., and El Saadany S. (1997). Is Creutzfeldt-Jakob disease transmitted in blood? Emerg. Infect. Dis. 3,155-163. Rosenberg, R., White, C , Brown, P., Gajdusek, D., Volpe, J., Posner, J., and Dyck, P. (1986). Precautions in handling tissues, fluids, and other contaminated materials from patients with documented or suspected Creutzfeldt-Jakob disease. Ann. Neurol. 19, 75-77. Scully R., Galdabini, J., and McNeely B. (1980). Case records of the Massachusetts General Hospital: Case 45-1980. New Engl. ]. Med. 303,1162-1171. Scully R., Mark, E., McNeely W., and McNeely B. (1993). Case records of the Massachusetts General Hospital: Case 17-1993. New Engl. J. Med. 328, 1259-1266. Sigurdsson, B. (1954). Rida, a chronic encephalitis of sheep with general remarks on infections which develop slowly and some of their special characteristics. Br Vet. ]. 110, 341-354. Tateishi, J. (1985). Transmission of Creutzfeldt-Jakob disease from human blood and urine into mice. Lancet 2,1074. Traub, R., Gajdusek, D., and Gibbs, C. (1974). Precautions in conducting biopsies and autopsies on patients with presenile dementia. /. Neurosurg. 41, 394-395. Traub, R., Gajdusek, D., and Gibbs, C. (1975). Precautions in autopsies on Creutzfeldt-Jakob disease. Am. ]. Clin. Pathol. 64, 287. Will, R., Ironside, J., Zeidler, M., Cousens, S., Estibeiro, K., Alperovitch, A., Poser, S., Pocchiari, M., Hofman, A., and Smith, P. (1996). A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347, 921-925. Zeidler, M., Stewart, G., Barraclough, C , Bateman, D., Bates, D., Bum, D., Colchester, A., Durward, W, Fletcher, N., Hawkins, S., Mackenzie, J., and Will, R. (1997). New variant Creutzfeldt-Jakob disease: Neurological features and diagnostic tests. Lancet 350, 903-907.

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C H A P T E R

31 Lymphocytic Choriomeningitis Virus (LCMV) ^ ^ h a r l e s Armstrong was a lifelong Public Health y ^ ^ / S e r v i c e virologist. In 1933, he and his col/ 0 leagues isolated LCMV for the first time while ^ ^ evaluating a case of central nervous system disease during an outbreak of arthropod-borne encephalitis in St. Louis, Missouri (see Chapter 24) (Armstrong and Sweet, 1939). While it is improbable that the illness was due to LCMV, the finding introduced the scientific community to a new virus later to be classified in the arenavirus family. I first met Armstrong many years later, when he called me into his laboratory at the National Institutes of Health to demonstrate the convulsions that occur in mice shortly after intracerebral inoculation of LCMV. By spinning an infected animal by its tail, he could routinely induce seizures and muscular spasms. This proved to be the first evidence of a developing fatal meningitis. The origin of Dr. Armstrong's initial viral isolate is uncertain, since it was recovered during a series of passages of infectious human material through monkeys. Subsequently, LCMV was isolated from a laboratory mouse (Traub, 1935) and, shortly thereafter. Rivers and Scott (1935) established an association of LCMV with meningitis in a naturally infected human. The overall medical importance of LCM virus for humans is difficult to assess because we lack detailed comprehensive epidemiological survey information on human populations. Three studies in the past provide some insight into the prevalence of human infections, but they yield little information on the frequency of subclinical and nonspecific illness due to LCMV worldwide. Almost 20% of adults were found to possess what were believed to be serum antibodies against LCMV in a survey of prison inmates in institutions scattered around the United States (Wooley ef al., 1937). In a more recent study, 5% of inner-city Baltimore residents proved to have serological evidence of past infection with LCMV (Childs ei al,, 1991). In one survey conducted in northwest Germany, over 3% of the population was found to be positive (Ackermann ei al.,

PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

1974), whereas in southern Germany there was no serological evidence of past infection among residents (Blumenthal ei al., 1970). These data provide only meager insight into the prevalence of infection in developed countries and fail to indicate how often clinical disease occurs. Moreover, the work is subject to methodological shortcomings, both with regard to the specificity and sensitivity of assays used. Clearly, we know very little about the prevalence of naturally occurring infections in the world's population overall. About 8% of cases of aseptic meningitis in North America are due to LCMV (Baird and Rivers, 1938; Adair ei al., 1953; Meyer ei al., 1960; Farmer and Janeway, 1942). On exceedingly rare occasions, a fatal encephalomyelitis with nonspecific pathological brain changes has been described (Smadel ei al., 1942; Adair ei al., 1953; Warkel ei al, 1973). LCMV infections in humans more commonly result in a febrile systemic influenza-like syndrome replete with many generalized signs and symptoms (Table 31.1) (Baum ei al., 1966; Lewis and Utz, 1961). Based on fragmentary clinical and experimental information, in severe cases the pathologist might expect to find evidence of mononuclear cell infiltration in a variety of parenchymal organs, including the liver, pancreas, salivary gland, and testicles. Autopsy verification of these hypothesized organ lesions is lacking. The results of serological surveys indicate that clinically inapparent and nonspecific febrile illnesses occur commonly as a result of LCMV infections. LCMV is the prototype arenavirus, although it is clearly less pathogenic than many other members of the family (see Chapter 19). The virions are approximately 110 to 130 nm in diameter and membrane bound. They utilize RNA as genetic material. In viiro, the viruses "bud" from the plasma membrane surface of the infected cell but cause no noteworthy morphologically recognizable cytopathic effects. Thus, the virus can be maintained as a chronic infection for

427

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

428

Pathology and Pathogenesis of Human Viral D i s e a s e

TABLE 31.1 Features of G e n e r a l i z e d Illness D u e to LCM Virus in Ten A d u l t Laboratory Workers and A n i m a l (Hamster) Caretakers Duration of illness (median) Signs and symptoms Fever Retroorbital headache Meningeal signs and symptoms Malaise and anorexia Arthralgia Arthritis Myalgia Adenopathy Orchitis Partial alopecia Laboratory Findings Leukopenia Thrombocytopenia

10 days 10/10'' 10/10 0/10 10/10 10/10 1/10 10/10 3/10 3/9 2/10 4/4 1/4

Adapted with permission from Baum et ah (1966). "This column provides number positive/number infected.

indefinite periods of time in the laboratory. As abundant experimental and pathological evidence now indicates, the capacity of LCMV to cause disease is intimately linked to the immune system of the host: specifically, the cytolytic CD8+ T cell response that accompanies infection appears to be involved (Hotchin, 1962). Spontaneous abortions and congenital infections due to LCMV have been reported in recent years. Infected infants are often underdeveloped and, to a variable extent, manifest microcephaly, internal hydrocephalus, cerebral calcifications, and chorioretinitis (Komrower et al, 1955; Ackermann et al, 1974; Sheinbergas, 1976; Sheinbergas et al, 1977; Chastel et al, 1978; Larsen et al, 1993; Barton et al, 1996). The pathologic features of congenital infections have been briefly described in the Russian literature (Sheinbergas et al, 1977). Like other members of the arenavirus family, LCM virus is maintained in nature in rodents. However, surprisingly little data exist to indicate the diversity of small animal species that are naturally infected. Laboratory mice and hamsters have served as reservoirs for the virus responsible for many of the reported human outbreaks (Traub, 1939; Biggar et al, 1975; Smadel et al, 1942; Lewis et al, 1965; Dykewicz et al, 1992). In developed countries, most of these outbreaks have occurred among laboratorians working with infected mice and Syrian hamsters. Additional cases among pet owners have been reported (Ackermann et al, 1975). Zoo animals also appear to be potential sources of human infection. Fatal LCM hepatitis has been described in marmosets (but not in humans) (Stephensen et al, 1991).

The domestic house mouse {Mus musculus) is believed to be the common host in nature. Infection under natural conditions of transmission appears to occur in the perinatal period, when the mouse is immunologically tolerant to the virus and the thymus underdeveloped. A chronic systemic nonpathogenic infection evolves, with large amounts of virus being present in various organs and the excreta. In the laboratory, nonimmune adult mice are exceptionally susceptible when inoculated intracerebrally. These animals develop a fatal lymphocytic meningoencephalitis that, interestingly enough, can often be aborted by prophylactic irradiation or chemotherapy. As might be expected, adult nude athymic mice only develop inapparent infections (Dykewicz et al, 1992). Compelling evidence now indicates that disease in the rodent, and presumably in humans, is an immunopathologic process (Hotchin, 1962). In the countless experimental animal studies that have been carried out over the past 50 years, variability in results can best be attributed to differences in the susceptibility of the strains of mouse and the pathogenic properties of the virus variant used in the experiment. Arenaviruses exhibit a high rate of mutability during transmission in laboratory mice, and it is highly probable that so-called ''wild" strains in nature vary considerably in pathogenicity for humans. This may account for the differing outcomes of naturally acquired infections, with most being subclinical, and a rare few cases that result in meningitis, encephalitis, and death. One might ask whether the voluminous laboratory research conducted with LCMV is relevant to clinical disease caused by this virus. The mouse appears to be a natural host, and humans prove to be infected incidentally when unusually intimate contact with rodents occurs. I believe the many interesting studies carried out experimentally have provided science with unique insights into the immunopathology of virus disease, but the results do not appear to be directly translatable into an understanding of the pathogenesis of clinical illness in humans caused by LCMV.

References Ackermann, R., Korver, G., Turss, R., Wonne, R., and Hochgesand, R (1974). Prenatal infection with the lymphocytic choriomeningitis virus. Dtsch. Med. Wochenschr. 13, 629-632. Ackermann, R., Stammler, A., and Armbruster, B. (1975). Isolierung von Virus der lymphozytaren Choriomeningitis aus Abrasionsmaterial nach Kontakt der Schwangeren mit einem Syrischen Gold-hamster {Mesocricetus auratus). Infection. 3, 47-49. Adair, C., Gauld, R., and Smadel, J. (1953). Aseptic meningitis, a disease of diverse etiology: Clinical and etiologic studies on 854 cases. Ann. Intern. Med. 39, 675-704.

Lymphocytic Choriomeningitis Virus Armstrong, C , and Sweet, L. (1939). Lymphocytic choriomeningitis. Publ Health Rep. 54, 673-684. Baird, R., and Rivers, T. (1938). Relation of lymphocytic choriomeningitis to acute aseptic meningitis. Am. J. Pub. Health 28, 47. Barton, L., Peters, C , Seaver, L., and Chartrand, S. (1996). Congenital lymphocytic choriomeningitis virus infection. Arch. Pediatr. Adolesc. Med. 150, 440. Baum, S., Lewis, A., Rowe, W., and Huebner, R. (1966). Epidemic nonmeningitic lymphocytic choriomeningitis-virus infection: An outbreak in a population of laboratory personnel. New Engl. J. Med. 274, 934-936. Biggar, R., Woodall, J., Walter, R, and Haughie, G. (1975). Lymphocytic choriomeningitis outbreak associated with pet hamsters. Fifty-seven cases from New York State. JAMA 232, 494-500. Blumenthal, W., Kessler, R., and Ackermann, R. (1970). Uber die Durchseuchung der landlichen Bevolkerung in der Bundesrepublik Deutschland mit dem Virus der Lymphocytaren Choriomeningitis. Zentralbl Bakteriol. Abt. I Orig. 213, 36-48. Chastel, C , Bosshard, S., Le Goff, R, Quillien, M., Gilly, R., and Aymard, M. (1978). Infection transplacentaire par le virus de la choriomeningite lymphocytaire: Resultats d'une enquete serologique retrospective en France. Nouv. Presse Med. 7,1089-1092. Childs, J., Glass, G., Ksiazek, T., Rossi, C , Barrera Oro, J., and LeDuc, J. (1991). Human-rodent contact and infection with lymphocytic choriomeningitis and Seoul viruses in an inner-city population. Am. J. Trop. Med. Hyg. 44,117-121. Dykewicz, C , Dato, V., Fisher-Hoch, S., et al. (1992). Lymphocytic choriomeningitis outbreak associated with nude mice in a research laboratory. ]AMA 1&7,1349-1353. Farmer, T., and Janeway, C. (1942). Infections with the virus of lymphocytic choriomeningitis. Medicine 21,1-64. Hotchin, J. (1962). The biology of lymphocytic choriomeningitis infection: virus-induced immune disease. Cold Spring Harbor Symp. Quant. Biol. 27, 479^99. Komrower, G., Williams, B., and Stone, R (1955). Lymphocytic choriomeningitis in the newborn, probable transplacental infection. Lancet 1, 697-698.

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Larsen, P., Chartrand, S., Tomashek, K., Hauser, L., and Ksiazek, T. (1993). Hydrocephalus complicating lymphocytic choriomeningitis virus infection. Pediatr Infect. Dis. J. 12, 528-531. Lewis, J., and Utz, J. (1961). Orchitis, parotitis and meningoencephalitis due to lymphocytic choriomeningitis virus. New Engl. J. Med. 265, 776-780. Lewis, A., Rowe, W, Turner, H., and Huebner, R. (1965). Lymphocytic choriomeningitis virus in Syrian hamster tumor. Science 150, 363. Meyer, H,, Johnson, R., Crawford, I., Dascomb, H., and Rogers, N. (1960). Central nervous system syndromes of 'ViraF' etiology: A study of 713 cases. Am. J. Med. 29, 334-347. Rivers, T., and Scott, T. (1935). Meningitis in man caused by a filtrable virus. Science 81, 439. Sheinbergas, M. (1976). Hydrocephalus due to prenatal infection with the lymphocytic choriomeningitis virus. Infection 4,185-191. Sheinbergas, M., Pmashekas, R., Pikelite, R., et al. (1977). Clinical and pathomorphological data on hydrocephalus caused by prenatal infection by the lymphocytic choriomeningitis virus. Zh. Nevropatol. Psikhiatr 77,1004-1007. Smadel, J., Green, R., Paltauf, R., and Gonzales, T. (1942). Lymphocytic choriomeningitis: Two human fatalities following an unusual febrile illness. Proc. Soc. Exp. Biol. Med. 49, 683-686. Stephensen, C , Jacob, J., Montali, R., et al. (1991). Isolation of an arenavirus from a marmoset with Callitrichid hepatitis and its serologic association with disease. /. Virol. 65, 3995-4000. Traub, E. (1935). A filtrable virus recovered from white mice. Science 81, 439. Traub, E. (1939). Epidemiology of lymphocytic choriomeningitis in a mouse stock observed for four years. /. Exp. Med. 69, 801-817. Warkel, R., Rinaldi, C , Bancroft, W, Cardiff, R., Holmes, G., and Wilsnack, R. (1973). Fatal acute meningoencephalitis due to lymphocytic choriomeningitis virus. Neurology. 23,198-203. Wooley, J., Armstrong, C , and Onstott, R. (1937). The occurrence in the sera of man and monkeys of protective antibodies against the virus of lymphocytic choriomeningitis as determined by the serum-virus protection test in mice. Publ. Health Rep. 52,1105-1115.

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C H A P T E R

32 Enteric Viral Disease INTRODUCTION 431 NORWALK-LiKE VIRUSES (NLVs) 432 ROTAVIRUSES (RVs) 433 ADDITIONAL ENTERIC VIRUSES 438 PATHOPHYSIOLOGY OF VIRAL ENTERITIS REFERENCES 439

diarrhea. When pathogenic bacteria are excluded, roughly 80% of the episodes of diarrhea (>3 nonsolid stools per day) are either of unknown etiology or are due to viruses. With the discovery of the enteroviruses (see Chapter 1) and the demonstration of their chronic presence in the stools of many of those who are infected, it was thought that the search for the cause of nonbacterial diarrhea would soon be over. But despite the presence of high concentrations of enteroviruses in the stools of both ill and healthy children, it shortly became apparent that these viruses were not common etiologic agents of enteritis, but merely nonpathogenic "passengers'' in our digestive tracts. Other viruses such as members of many of the common serotypes of the adenoviruses similarly can often be found in the gut, but they too usually fail to cause disease. As an outgrowth of an enormous amount of laboratory work, it was ultimately concluded that the elusive viruses of childhood enteritis are sufficiently fastidious that they cannot be easily grown in tissue culture and in laboratory animals. Thus, in the early 1970s, investigators initiated attempts to identify viral particles in stool extracts using electron microscopy. To accomplish this, the background was stained (so-called negative staining), with the virions in startling contrast, that is, like stars in a dark sky. Should the virions be present in sufficient number, and should their morphology be sufficiently distinctive, infection could be established. By adding specific antibody (or serum from a previously infected patient) to the suspension, the virions would clump with the antibody and an antigenic identification of the virus accomplished. This also proved a means for assaying the relative concentration of antibody in the blood of those who were infected. The technique of negative-staining electron microscopy using clinical enteric specimens is an arduous art form that requires exceptional skill and attention to detail, clearly not a characteristic of many of our species. But, this painstaking approach has now yielded evidence to indicate that viruses of at least six families may contribute to enteric illness in children and in adult citizens

439

INTRODUCTION Al Kapikian learned immune electron microscopy from the talented British investigator June Alameda, who had perfected the technique into an art form, as much as a skill. In the early 1970s, Kapikian focused on this approach to the search for the elusive viruses believed to be responsible for nonbacterial gastroenteritis. It was not long before he identified the virions of the so-called Norwalk agent in a diarrheal stool from an adult volunteer who had been challenged with a fecal sample from a child ill during an outbreak of diarrhea. This virus and its soon-to-be-discovered close relatives (the so-called Norwalk-like viruses [NLVs]) proved to be important causes of explosive outbreaks of diarrhea in both children and adults. NLVs were first reported in 1972 (Kapikian et al, 1972; Kapikian, 1994). Only a year later, Ruth Bishop and her colleagues (1973) found virions of a different size and appearance in cells of the duodenal mucosa of infants with gastroenteritis. These agents proved to be the prototype for a new genus, the rotaviruses (RVs), members of which are infectious for humans and a wide variety of domestic animals. Rotaviruses, classified into Group A, are now recognized to be the major cause of severe diarrheal disease in infants and young children worldwide. Cholera morbus has plagued and threatened the lives of infants and children since the beginning of recorded history. Morbidity is universal, and the resulting mortality, particularly in developing areas of the world, continues to be tragic. Overall, as many as a third of the deaths in children under the age of 5 in the less-developed countries of the world are attributed to

PATHOLOGY AND PATHOGENESIS OF HUMAN VIRAL DISEASE

431

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

432

Pathology and Pathogenesis of Human Viral Disease

whose immunity has waned (Figure 32.1, Table 32.1). Studies in domestic animals have also shown that diarrheal disease of economic importance is caused by members of these same virus families, possibly by strains of virus that are host-specific. With the development of refined rnolecular and immunological tools, more sensitive and less laborious diagnostic approaches fortunately are now replacing negative-staining electron microscopy of stool specimens.

Q Bacteria 0

Parasites

•

Rotavirus

H Enteric Adenovirus

TABLE 32.1 Enteric Viruses D e f i n i t e l y or Possibly Causing Gastroenteritis i n H u m a n s Endemic disease Rotaviruses Group A Groups B and C

Epidemic outbreaks

Worldwide importance

+

++++ +

Calciviruses Norwalk-like viruses Unclassified

+ +

Enteric adenoviruses

+

Toroviruses

+

Astroviruses

+?

Coronaviruses

++ +? ?

TABLE 32.2 G e n o t y p e s of Norwalk-Like Viruses Based o n Molecular A n a l y s e s

i l Astrovirus •

Other viruses

n

Unknown

FIGURE 32.1 Estimated median percentage of diarrheal episodes associated with specific viruses and categories of enteropathogens in developed countries. Reprinted with permission from Kapikian (1994).

NORWALK-LIKE VIRUSES (NLVs) A number of antigenically distinct, nonenveloped, round, 27- to 32-nm RNA viruses classified into a newly proposed genus of the calcivirus family have been found to cause sporadic outbreaks of transient severe enteritis in both children and adults. The etiological role of these viruses as a cause of intestinal disease was established by demonstrating a temporal association of naturally occurring infections (as demonstrated by stool examination using electron microscopy) with illness and by experimental induction of disease in both human volunteers and experimental animals (Hall et al, 1984). The NLVs are provisionally divided for classification purposes into two distinct groups based on genetic analysis (Table 32.2). The relative clinical importance of the viruses of the various groups listed in Table 32.2 has yet to be established, but they are believed to be responsible for a substantial proportion of the nonbacterial outbreaks of acute vomiting and diarrhea that occur in families and institutions.

Genotype I Norwalk" Southampton Cruise ship Desert Shield Genotype II Gwynedd Toronto Lardsdale Snow Mountain White River Hawaii "The geographic name customarily refers to the site where the virus was initially isolated.

Although survey information is incomplete, infections with NLV are thought to occur worldwide. In developing countries, the majority of children are infected during the first 10 years of life (Figure 32.2), whereas in North America infection is relatively uncommon in children, and by the fifth decade of life only 50% of the population possess serological evidence of past infection. It is not clear what proportion of these infections are accompanied by clinical illness, and we possess only limited information on the persistence of serum antibodies during convalescence from infection. Thus, NLV-related disease may occur more commonly than serological surveys of the population now suggest. Outbreaks of illness develop as a result of a contaminated common source, such as a water supply or food, or as a consequence of person-to-person transmission (Fankhauser et ah, 1998). The mean incubation period is approximately 40 hours, and symptoms persist for 12

433

Enteric Viral Disease

100 r

TABLE 32.3 Symptoms of Norwalk Virus Infections among Children during a Naturally Occurring Outbreak

(12)

(3) o

/fSiO^ 90

-

Percent \ .

Q O

80h

m z

<

Fever Nausea Vomiting Abdominal cramps Lethargy Diarrhea

(20)

tm

(20)

(83)

70

O 60

^yw>\

L

32 85 84 62 bl 44

Reprinted with permission from Kapikian (1994). mi)

50 \-

40

h

g 30

l

I

<

(66)

( ) No. Tested o Ecuador Indians • Bangladesh A Yugoslavia • U.S.A. o Ecuador (Gabaro)

/

20 h (43)

(2) D

0-5

<6) Q 6-10

(4) a 11-15

J

AGE IN YEARS

FIGURE 32.2 Age-related prevalence of serum antibodies indicative of past infection by Norwalk virus in various countries. Reprinted with permission from Greenberg ei al. (1979).

to 24 hours (Table 32.3). The clinical course observed in two volunteers is shown in Figure 32.3) (Dolin ei al., 1971). Volunteer studies have yielded important histological and ultrastructural documentation of the profound but relatively transient changes that occur in the mucosa of the small intestine during the course of infections with NLVs (Agus ei al, 1973; Schreiber ei al, 1973, 1974; Dolin ei al, 1975). Unfortunately, biopsy material from these experimental subjects is, by necessity, limited; thus, the extent and distribution of lesions in the digestive tract is unknown. Of obvious significance is the reduction in the height of the intestinal villi, accompanied by blunting of the villi and evidence of crypt hypertrophy. The individual lining cells of the small intestine exhibit vacuolization of the cytoplasm with loss of polarity. These findings indicate a marked increase in enterocyte replacement as documented by mitotic counts in the epithelium of the crypts. Although cell infiltrates are found in the lamina propria and the core of the villi, inflammation is customarily not a prominent feature of the lesion. Ultrastructurally, the mucosal cells remain intact, but intercellular edema

is prominent and the microvilli of the brush border are significantly shortened as shown by electron microscopy. Dilatation of both the endoplasmic reticulum and mitochondria of the mucosal cells is seen, and endocytotic vesicles are evident. Ultrastructural studies thus far have not defined the cell site of replication of the virus, most probably because the virions are relatively small, that is, in a size range that would not readily allow one to distinguish them from ribosomes. Immunohistochemistry documents a substantial reduction in disaccharides and alkaline phosphatase in the epithelium of the gut. Intestinal biopsies obtained from volunteers 5 to 6 days after the ingestion of NLVs (i.e., 2-4 days after clinical recovery) continue to show shortening of villi and crypt hypertrophy, but the inflammatory response in the lamina propria and submucosa appear to be reduced. At this time, there remains a significant reduction in the surface area of the gut, and a striking increase in mitotic activity of the epithelium is seen.

ROTAVIRUSES (RVs) RVs are the most important cause of severe and often life-threatening diarrheal disease in infants and young children worldwide (Moulton ei al, 1998). Although adults are infected, their symptoms are customarily mild, with asymptomatic infections being common. However, severe infections tend to occur in older persons (Hrdy, 1987; Wenman ei al, 1979; Abbas and Denton, 1987; Lewis ei al, 1989) and in persons with attenuated immunity (Kaljot ei al, 1989; Dryden and Shanson, 1988; Eiden ei al, 1985; Wood ei al, 1988), but RVs do not appear to be a major contributing factor to the diarrheal disease that so commonly afflicts those with AIDS. RVs are members of a genus classified in the reovirus family. The Latin roia refers to the spoke-wheel

434

Pathology and Pathogenesis of Human Viral D i s e a s e

102 1 A

Volunteer 1

^

1

B \/olunteer 2

? 101! m cr ^

100

LJJ

1

I

a.

5

K < O

^

98

^

/ •

97 Ooys offer challenge Diarrhea Vomiting Abdominol cramps Nauseo Malaise Heodoche Myalgia Anorexia

WBC

Fo 0 0

1 0

3 0 >#xK 0 r/fUM'W/nrms. 0 0 fozm 5^77?v 2 0

°^ L °0,*!

'iiwiiumWWilM. 0

1 0

i ^ I 0

tTTU

w})Whi

A\

0 0

4

5 i 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0

im/if7//////M. VK 8,400 18,450 16,600 14,100 11.200

-

3

4

0 0 0 0 0 ^SMMMjit^ 0 (^^ 0

0 0 0 0 0 0 0 0

1

2

_^ia!0Stmn^ 0 0

^acri^^Jjfeh.

MK

0

j0Mim^

[8.200 8.100 8,700 8,200 8,300

5 0 0 0 0 0 0 0 0 -

FIGURE 32.3 Response of two volunteers to oral administration of Norwalk virus. The height of the curve is directly proportional to the severity of the sign or symptom. Volunteer 1 had severe vomiting without diarrhea, and volunteer 2 had diarrhea without vomiting, although both received the same inoculum. Reprinted with permission from Dolin et ah (1971).

appearance of these nonenveloped 70-nm RNA virions in negatively stained electron micrographs. Based on antigenic analysis of the virion coat proteins, five groups (A-E) have been established. The majority of human infections are due to viruses of group A in regions of the world where intensive studies have thus far been conducted. Overall, the prevalence of group B and C infections is low, and they appear only sporadically in developed countries. Widespread epidemics of group B viruses have been reported from the People's Republic of China; however, overall, infection by group B and C viruses in China as measured by presence of antibodies in the blood serum of members of the general population remains low, that is, -10% (Qui et al, 1986). Because these viruses are highly mutable, stool specimens from individuals may yield a diversity of variants that differ one from another on the basis of minor changes in the amino-acid makeup of the virion coat protein. The relative pathogenicity of these genetically diverse agents is unknown. Although RVs from domestic animals have been shown experimentally to

infect children, there is little evidence to indicate that animals are a significant reservoir for human infections. In developed countries, RV infections typically occur between the ages of 6 and 24 months. In one study conducted in North America, 40% of children were infected with at least one strain of RV during the first year of life (Ward and Bernstein, 1994) (Figure 32.4). However, fewer than a third of these infections were symptomatic (Wenman et al, 1979). In developing countries and in persons residing under poor socioeconomic conditions, as many as 30% of children are infected by 6 months of age (Moulton et al, 1998), and many infants experience their first encounter with RVs in the newborn nursery during the first week of life (Bishop, 1994). This high prevalence of infection during early life doubtlessly represents both the common occurrence of RV infections in persons residing in these environments (thus facilitating person-to-person spread) and the lack of acquired secretory IgA to the virus in the gut. The clinical features of RV infections — fever, vomiting and diarrhea — are known to all mothers. Yet,

435

Enteric Viral Disease

I

c£»

I HUMAN ROTAVIRUS

c/)| • 1

NORWALK AGENT

o <. ^

MONTGOMERY COUNTY AND ARLINGTON

CHILDREN'S HOSPITAL PATIENTS

49-60

AGE, MONTHS STUDIED

19

21

22

18

16

61 84 85-108 109-132133 180 181 248 MONTHS 13 18 17 10 7

17-26 YEARS 63

20-29

30-39

40-49

30

30

30

15

60-67 YEARS 3

FIGURE 32.4 Prevalence of antibody to rotaviruses and Norwalk virus by age utilizing three different study populations near Washington, DC. Note the evidence of infection with rotavirus early in life, and their persistence throughout adulthood. In comparison, the Norwalk virus infections occur later in life, and a much smaller proportion of the population shows evidence of past infection. Reprinted with permission from Kapikian (1994).

because symptoms usually are m^ercifuUy short-lived (24 to 48 hr), the maintenance of fluid and electrolyte balance is rarely a problem in otherwise healthy infants (Table 32.4). In developing countries, however, the threat to life is more imposing, particularly since infections so frequently occur among the very young. Illness in adults is less severe (Table 32.5), if it occurs at all. Indeed, 60 to 80% of infections in adults are asymptomatic. This may reflect age-related resistance (as documented in animals) (Ciarlet et al, 1998) or the acquisition of a spectrum of serotype-specific IgA antibodies in the gut resulting from previous natural infection. As the years pass, immunity may wane among the elderly, in part due to less frequent encounters with the virus in their home setting. Older folks more often develop severe diarrheal illnesses when infected with RVs, and deaths are reported (Hrdy 1987). RVs appear to replicate exclusively in the mucosal lining cells of the villi of the small intestine and not elsewhere in the gut (Figures 32.5 and 32.6). Up to lO^^ virions can be found in a gram of feces during the acute illness! This extraordinarily high concentration of virus

in diarrheal stools no doubt accounts in part for the ease of transmissibility of these viruses among close contacts. Since the virions are also relatively resistant to environmental stresses, they can persist in water supplies and food. Infection occurs exclusively by the oral route. As might be expected, relatively few morphologic studies have been carried out on enteric biopsies from infants and children with documented RV infections. Davidson and Barnes (1979) examined duodenal biopsies obtained 24 to 120 hours after the onset of symptoms from children 2 to 33 months of age. Abnormalities were found in 40% of the biopsies. There was blunting of villi, along with an increase in crypt depth and flattening of the epithelial cells lining the villi. Inflammatory cells were present in the lamina propria in 2 of the 17 infants studied. The pathological changes were said to resemble those seen in celiac disease with loss of villi, prominent crypt hypertrophy, and an infiltrate of inflammatory cells. RV particles were found in the enterocytes of these patients by electron microscopy. Histochemical evaluation of the gut mucosa

436

Pathology and Pathogenesis of Human Viral Disease

TABLE 32.4 Clinical Features of Children under A g e 15 w i t h Rotavirus Infections Percentage of hospitalized Diarrhea Diarrhea (>10 stools/day) Vomiting Vomiting (>5 x/day) Fever Temperature >39°C Respiratory symptoms Dehydration

94 28 92

sr

86 35 32 72«

Percentage of nonhospitalized 100 17 83 28 83 47 34 30

Reprinted with permission from Uhnoo et al. (1986). "Statistically significant (p > 0.05) difference between hospitalized and nonhospitalized patients.

TABLE 32.5 S y m p t o m s i n North American A d u l t s w i t h Familially Acquired Rotavirus Infections Symptoms

Percent

Diarrhea Abdominal cramps Vomiting Respiratory Fever

32 24 10 7 6

Reprinted with permission from Wenman et al. (1979).

FIGURE 32.5 Experimentally induced rotavirus infection in gnotobiotic piglets. Villous epithelium in the top panel shows a viroplasm with viral particles budding into the distended cisternae of endoplasmic reticulum. In the middle panel, enveloped virus particles are situated within (arrow) and at the edge of convoluted smooth membrane. The bottom panel displays the fine structural features of the viroplasm shown in Figure 32.6. Reprinted with permission from Saif et al. (1978).

Enteric Viral Disease

437

FIGURE 32.6 Experimentally induced rotavirus infection in gnotobiotic piglets. Electron micrographs of villous epithelium. Note distended cisternae of the rough endoplasmic reticulum and rarefaction of cytoplasm in panel A. The affected cell is situated between two seemingly normal cells. Panel B shows cells with shortened irregular microvilli and a break in the viroplasm-VP border (arrow) (see Figure 32.5). Reprinted with permission from Saif et al. (1978).

§ o

^^ 10'

0

I

n

I Immunofluorescence

i L

•

40 60 80 100 hours after infection

2 3 weeks after infection

FIGURE 32.7 Experimentally induced rotavirus infections in gnotobiotic piglets. Temporal evidence of infectivity as based on assays of bowel content and immunofluorescence of mucosal lining cells for the presence of virus. The associated changes in the villus lengthicrypt length ratio is shown for infected (solid black) and control (broken line) animals. Note the protracted recovery time. Reprinted with permission from Crouch and Woode (1978).

showed that disaccharidase concentrations were reduced in the epithelium. Repeat biopsies were done on several children during convalescence (3 to 8 weeks), and regeneration of the mucosa was found. As noted above, a wide variety of wild and domesticated mammals and poultry (Domermuth and Gross, 1985) acquire RV infections naturally. Experimental studies in calves (Reynolds et al, 1985), lambs (Snodgrass et al, 1977, 1979), and piglets (Theil et al, 1978; Crouch and Woode, 1978) have provided insight into the location of viral replication in the gut and the associated histologic changes (Figure 32.7). To a large extent, the changes observed in children (Davidson and Barnes, 1979) were found in these animals. In calves, the lesions tended to be more prominent in the proximal small intestine, whereas in lambs the distal ileum was more severely affected. Table 32.6 summarizes the results of immunohistochemical studies that document viral replication in the gut of lambs after experimental infection per os. In general, the sites of RV replication correlate with the pathological changes found in the intestinal mucosa (Snodgrass et al, 1977). As might be expected, the concentrations of virus in the gut of these piglets were highest before lesions in the mucosa appeared (Crouch and Woode, 1978).

438

Pathology and Pathogenesis of Human Viral D i s e a s e TABLE 32.6 Immunofluorescent Staining of the Intestine of an Experimentally Infected Lamb for Rotavirus Antigen

Time killed (hours p.i.) 12 18 27 42 48 72 96 144 Control Control

Small intestine

Large intestine

Anterior

Middle

Posterior

_

++++ +++

++++ +++ ++

++

-

+

-

++ +

++

+ +

-

+

•

-

+

Colon

Caecum

++ +

++ ++ ++

-

+

Reprinted with permission from Snodgrass et al. (1977). ++++ = Continuous fluorescent epithelial cells present over at least distal half of the villi. +++ = Continuous fluorescent epithelial cells present over tip or distal third of the villi. ++ = Sporadic fluorescent epithelial cells present in most villi. + = Sporadic fluorescent epithelial cells present in a few villi.

A D D I T I O N A L ENTERIC VIRUSES

FIGURE 32.8 Photomicrographs of histological cross-sections from the small intestines of two piglets. (Top) Observe the long slender villi with lightly stained epithelium that dominate the mucosa in the normal animal. There is a narrow band of crypts with a darkly stained epithelium around the base of the mucosa. (Bottom) Severe villous atrophy caused by the swine coronavirus responsible for transmissible gastroenteritis. Reprinted with permission from Moon (1994).

Viruses of several additional families have been implicated in human enteritis, but, in general, the disease is relatively mild and not life threatening (Table 32.1). Information concerned with the pathological effects of these viruses on the gut mucosa is lacking. In many studies, the common occurrence of asymptomatic enteric infections often makes it difficult to establish, on epidemiological grounds, an etiological relationship between the infection and disease; yet, under certain circumstances, these viruses may be pathogenic for humans. The enteric adenoviruses types 40 and 41 are recognized causes of enteritis, but they account for fewer than 10% of cases (see Chapter 14) (Brandt et al, 1985). In two recent studies, toroviruses were associated with enteritis manifest in children as both vomiting and diarrhea. Although not severe, the stools commonly were bloody and disease persisted for several days (Koopmans et ah, 1997). Enteritis due to toroviruses also occurs in cattle and horses (Weiss and Horzinek, 1987; Woode et al, 1982; Jamieson et al, 1998). Astroviruses and coronaviruses have been implicated as causes of diarrheal disease in humans, but the evidence supporting a cause-and-effect association is weak (Phillips et al, 1982; Lew et al, 1990). These latter viruses are etiologically responsible for disease in young domestic animals (Mebus et al, 1973; Thake et al, 1973; Gray et al, 1980; Kurtz et al, 1979) (Figures 32.8 and 32.9).

439

Enteric Viral Disease

FIGURE 32.9 Degeneration of mucosal lining cell in the midgut of a lamb infected with an astrovirus. Note the changes in the microvilli (arrows) and virions in a lysosome (V). Reprinted with permission from Gray et al. (1980).

PATHOPHYSIOLOGY OF VIRAL ENTERITIS Pathological observations provide insight into the physiological basis for enteritis in those infected with enteropathic viruses. At present, relatively few clinical studies have addressed these issues in detail. Involvement of the upper intestinal tract (duodenum and jejunum) may account for the vomiting that so frequently occurs in NLV infections. Diarrheal disease no doubt is consequent to the profound changes in the intestinal mucosa that occur during the acute illness. Reduction in the surface area of the gut mucosa and functional alterations in the individual enterocytes that line villi reduce absorption of fluids and solids beyond the capacity of the colon to compensate. The loss of disaccharidases in the mucosal cells of the small intestine increases the carbohydrate concentrations of the large bowel content, resulting in the generation of fermentation products.

References Abbas, A., and Denton, M. (1987). An outbreak of rotavirus infection in a geriatric hospital. /. Hosp. Infect. 9, 76-80. Agus, S., Dolin, R., Wyatt, R., Tousimis, A., and Northrup, R. (1973). Acute infectious nonbacterial gastroenteritis: Intestinal histopathology. Histologic and enzymatic alterations during illness produced by the Norwalk agent in man. Ann. Intern. Med. 79,18-25. Bishop, R. (1994). Natural history of human rotavirus infections. In ''Viral Infections of the Gastrointestinal Tract,'' 2nd ed. (A. Kapikian, ed.), pp. 131-167. Marcel Dekker, New York. Bishop, R., Davidson, G., Holmes, I., and Ruck, B. (1973). Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet 2,1281-1283. Brandt, C., Kim, H., Rodriguez, W., et al. (1985). Adenoviruses and pediatric gastroenteritis. /. Infect. Dis. 151, 437-443, Ciarlet, M., Gilger, M., Barone, C., MaArthur, M., Estes, M., and Cormer, M. (1998). Rotavirus disease, but not infection and development of intestinal histopathological lesions, is age restricted in rabbits. Virolog]/ 251, 343-360. Crouch, C., and Woode, G. (1978). Serial studies of virus multiplication and intestinal damage in gnotobiotic piglets infected with rotavirus. /. Med. Microbiol. 11, 325-334.

440

Pathology and Pathogenesis of Human Viral D i s e a s e

Davidson, G., and Barnes, G. (1979). Structural and functional abnormalities of the small intestine in infants and young children with rotavirus enteritis. Acta Paediatr. Scand. 68,181-186. Dolin, R., Blacklow, N., DuPont, H., Formal, S., Buscho, R., Kasel, J., Chames, R., Hornick, R., and Chanock, R. (1971). Transmission of acute infectious nonbacterial gastroenteritis to volunteers by oral administration of stool filtrates. /. Infect. Dis. 123, 307-312. Dolin, R., Levy, A., Wyatt, R., Thornhill, T., and Gardner, J. (1975). Viral gastroenteritis induced by the Hawaii agent: Jejunal histopathology and serologic response. Am. J. Med. 59, 761-768. Domermuth, C., and Gross, W. (1985). Hemorrhagic enteritis of turkeys. In ""Animal Models for Intestinal Disease'' (C. Pfeiffer, ed.), chap. 22. CRC Press, Boca Raton, FL. Dryden, M., and Shanson, D. (1988). The microbial causes of diarrhoea in patients infected with the human immunodeficiency virus. /. Infect. 17,107-114. Eiden, J., Losonsky, G., Johnson, V, and Yolken, R. (1985). Rotavirus RNA variation during chronic infection of immunocompromised children. Pediatr. Infect. Dis. 4, 632-637. Fankhauser, R., Noel, J., Monroe, S., Ando, T., and Glass, R. (1998). Molecular epidemiology of "Norwalk-like viruses'' in outbreaks of gastroenteritis in the United States. /. Infect. Dis. 178,1571-1578. Gray, E., Angus, K., and Snodgrass, D. (1980). Ultrastructure of the small intestine in astrovirus-infected lambs. /. Gen. Virol. 49,71-82. Greenberg, H., Valdesuso, J., Kapikian, A., Chanock, R., Wyatt, R., Szmuness, W., Larrick, J., Kaplan, J., Gilman, R., and Sack, D. (1979). Prevalence of antibody to the Norwalk virus in various countries. Infect. Immunol 26, 270-273. Hall, G., Bridger, J., Brooker, B., Parsons, K., and Ormerod, E. (1984). Lesions of gnotobiotic calves experimentally infected with a calcivirus-like (Newbury) agent. Vet. Pathol. 21, 208-215. Hrdy, D. (1987). Epidemiology of rotaviral infection in adults. Rev. Infect. Dis. 9, 461-^69. Jamieson, F., Wang, E., Bain, C., Good, J., Duckmanton, L., and Petric, M. (1998). Human torovirus: A new nosocomial gastrointestinal pathogen. /. Infect. Dis. 178, 1263-1269. Kaljot, K., Ling, J., Gold, J., Laughon, B., Bartlett, J., Kotler, D., Oshiro, L., and Greenberg, H. (1989). Prevalence of acute enteric viral pathogens in acquired immunodeficiency syndrome patients with diarrhea. Gastroenterology 97,1031-1032. Kapikian, A. (1994). Norwalk and Norwalk-like viruses. In "Viral Infections of the Gastrointestinal Tract," 2nd ed. (A. Kapikian, ed.), pp. 471-518. Marcel Dekker, New York. Kapikian, A., Wyatt, R., Dolin, R., Thornhill, T, Kalica, A., and Chanock, R. (1972). Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. /. Virol. 10,1075-1081. Koopmans, M., Goosen, E., Lima, A., et at. (1997). Association of torovirus with acute and persistent diarrhea in children. Pediatr. Infect. Dis. 16, 504-507. Kurtz, J., Lee, T, Craig, J., and Reed, S. (1979). Astrovirus infection in volunteers. /. Med. Virol. 3, 221-230. Lew, J., Glass, R., Petric, M., Levaron, C , Hammond, G., Miller, S., Robinson, C , Boutilier, J., Riepenhoff-Talty, M., Payne, C , Franklin, R., Oshiro, L., and Jaqua, M. (1990). Six-year retrospective surveillance of gastroenteritis viruses identified at ten electron microscopy centers in the United States and Canada. Pediatr. Infect. Dis. ]. 9, 709-714.

Lewis, D., Lightfoot, N., Cubitt, W, and Wilson, S. (1989). Outbreaks of astrovirus type 1 and rotavirus gastroenteritis in a geriatric inpatient population. /. Hosp. Infect. 14, 9-14. Mebus, C , Stair, E., Rhodes, M., and Twiehaus, M. (1973). Pathology of neonatal calf diarrhea induced by a coronavirus-like agent. Vet. Path. 10, 45-64. Moon, H. (1994). Pathophysiology of viral diarrhea. In "Viral Infections of the Gastrointestinal Tract," 2nd ed. (E. Kapikian, ed.), pp. 27-52. Marcel Dekker, New York. Moulton, L., Staat, M., Santosham, M., and Ward, R. (1998). The protective effectiveness of natural rotavirus infection in an American Indian population. /. Infect. Dis. 178,1562-1566. Phillips, A., Rice, S., and Walker-Smith, J. (1982). Astrovirus within human small intestinal mucosa. Gut. 23, A923-A924. Qui, F, Tian, Y, Liu, J., Zhang, X., and Hao, Y (1986). Antibody against adult diarrhoea rotavirus among healthy adult population in China. /. Virol. Methods 14,133-140. Reynolds, D., Hall, G., Debney, T, and Parsons, K. (1985). Pathology of natural rotavirus infection in clinically normal calves. Res. Vet. Sci. 38, 264-269. Saif, L., Theil, K. W, and Bohl, E. H. (1978). Morphogenesis of porcine rotavirus in porcine kidney cell cultures and intestinal epithelial cells. /. Gen. Virol. 39, 205-217. Schreiber, D., Blacklow, N., and Trier, J. (1973). The mucosal lesion of the proximal small intestine in acute infectious nonbacterial gastroenteritis. New Engl. ]. Med. 288, 1318-1323. Schreiber, D., Blacklow, N., and Trier, J. (1974). The small intestinal lesion induced by Hawaii agent acute infectious nonbacterial gastroenteritis. /. Infect. Dis. 129, 705-708. Snodgrass, D., Angus, K., and Gray, E. (1977). Rotavirus infection in lambs: Pathogenesis and pathology. Arch. Virol. 55, 263-274. Snodgrass, D., Ferguson, A., Allan, F., Angus, K., and Mitchell, B. (1979). Small intestinal morphology and epithelial cell kinetics in lamb rotavirus infections. Gastroenterology 76, 477^81. Thake, D., Moon, H., and Lambert, G. (1973). Epithelial cell dynamics in transmissible gastroenteritis of neonatal pigs. Vet. Path. 10, 330-341. Theil, K., Bohl, E., Cross, R., Kohler, E., and Agnes, A. (1978). Pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic pigs. Am. ]. Vet. Res. 39, 213-220. Uhnoo, I., Olding-Stenkvist, E., and Kreuger, A. (1986). Clinical features of acute gastroenteritis associated with rotavirus, enteric adenoviruses, and bacteria. Arch. Dis. Child. 61, 732-738. Ward, R., and Bernstein, D. (1994). Protection against rotavirus disease after natural rotavirus infection. /. Infect. Dis. 169, 900-904. Weiss, M., and Horzinek, M. (1987). The proposed family, Toroviridae: agents of enteric infections. Arch. Virol. 92,1-15. Wenman, W, Hinde, D., Feltham, S., and Gurwith, M. (1979). Rotavirus infection in adults: Results of a prospective family study. New Engl. J. Med. 301, 303-306. Wood, D., David, T, Chrystie, L, and Totterdell, B. (1988). Chronic enteric virus infection in two T-cell immunodeficient children. /. Med. Virol. 24, 435^44. Woode, G., Reed, D., Runnels, P, Herrig, M., and Hill, T. (1982). Studies with an unclassified virus isolated from diarrheic calves. Vet. Microbiol. 7, 221-240.

Index

Acquired immunodeficiency syndrome, see also Human immunodeficiency virus cervical cancer association, 234 clinical course of HIV-1 infection adults, 207-209 children and infants, 210-211 epidemiology, 205-206 history 205-206 Kaposi's sarcoma AIDS association, 171-172, 232-233 classification, 174-175 clinical features, 171-172, 233 lymph node involvement, 177-178 pathogenesis, 174-175,177-178 staging, 175 tissue distribution, 233-234 opportunistic infections adenovirus, disseminated disease, 195-196 bacterial pneumonias, 206, 225-226 chronic enterovirus meningoencephalitis, 8-10 cytomegalovirus, see Cytomegalovirus digestive tract, 206 human herpesvirus type 6,167-168 measles, 403-404 moUuscum contagiosum, 377-378 overview, 206 Adenovirus central nervous system disease, 197-198 classification, 189-190 digestive tract disease, 197 discovery, 189 disseminated disease in immunocompromised patients, 195-196 enteric virus, 438 epidemiology, 189-191 eye disease, 198 genitourinary tract disease, 196-197 heart disease, 197 immune response, 191

respiratory tract infection chronic infection, 194 inclusion-body pneumonia, 192 late complications, 192-193 serotypes, 191,194-195 transmission, 191,194 structure, 189-190 AIDS, see Acquired immunodeficiency syndrome Alphaviruses classification, 344 eastern equine encephalitis, 346-347 infection cycle, 344, 346 strains, 345-346 structure, 344 Venezuelan equine encephalitis, 348-349 western equine encephalitis, 347-348 Angiofollicular lymph node hyperplasia, Kaposi's sarcoma-associated herpesvirus as cause, 180-181 Angiosarcoma, Kaposi's sarcomaassociated herpesvirus as cause, 178 Arenavirus, see also Lymphocytic choriomeningitis virus Guanarito virus, 280 Junin virus, 278-279 Lassa virus, 280-292 Machupo virus, 278-279 Sabia virus, 280 structure and classification, 277-278 Argentinian hemorrhagic fever, see Junin virus Arthropod-transmitted viruses alphaviruses classification, 344 eastern equine encephalitis, 346-347 infection cycle, 344, 346 strains, 345-346 structure, 344 Venezuelan equine encephalitis, 348-349 western equine encephalitis, 347-348 Colorado tick fever, 354-355

441

flaviviruses classification, 350 infection cycle, 349-350 Japanese B encephalitis, 352-353 miscellaneous encephalitides, 353-354 overview, 349 St. Louis encephalitis, 351-352 LaCrosse, 354 overview of viruses and diseases, 343-344 Atherosclerosis, cytomegalovirus role, 107-109 Autoimmune hepatitis, features, 270

B BK virus, see also Papovavirus discovery, 327 urinary tract infection and disease, 331, 333 viremia, 328-329 Bolivian hemorrhagic fever, see Machupo virus Bornholm disease. Coxsackievirus infection, 17 Bovine papular stomatitis, features, 377 Bunyaviruses Congo-Crimean hemorrhagic fever virus, 286-287 Hantavirus, hemorrhagic fever with renal disease epidemiology, 282 Hantaan virus and Korean hemorrhagic fever, 282-284 Puumala virus and nephropathia endemica, 284-285 Seoul virus, 282-284 LaCrosse, 354 overview, 282, 354 Rift Valley fever virus, 285-286 Burkitt's lymphoma AIDS-associated disease, 232 chromosome translocations, 127 discovery of Epstein-Barr virus association, 117,125 epidemiology, 125-126 pathogenesis, 127

442

Castleman's disease, see AngiofoUicular lymph node hyperplasia Cervical cancer AIDS association, 234 human papillomavirus association, 311-314 Chickenpox clinical features, 147-148 course, 148-149 immune response, 148 transmission, 148 Chronic enterovirus meningoencephalitis, enterovirus disease in immune deficiency, 8-10 CJD, see Creutzfeldt-Jacob disease CMV, see Cytomegalovirus Coxsackievirus, see also Enterovirus Bomholm disease, 17 classification, 1 diabetes mellitus type I, group B infection role, 21-23 group B myocarditis epidemiology, 13 natural history, 13-14 pathogenesis, 14-16 Colorado tick fever, features, 354-355 Common cold, see Rhinovirus Congo-Crimean hemorrhagic fever virus, hemorrhagic fever, 286-287 Cowpox virus, features and diseases, 365, 371, 377 Creutzfeldt-Jacob disease, see also Transmissible spongiform encephalopathy clinical features, 413-414 iatrogeruc Creutzfeldt-Jacob disease, 422 new variant Creutzfeldt-Jacob disease (vCJD), 421-422 pathological features, 415-417 Cylindrical confronting cisternae, human immunodeficiency virus-infected cells, 231 Cytomegalovirus, see also Herpesvirus atherosclerosis role, 107-109 congenital infection and disease, 90, 92 digestive tract disease, 100-101 discovery, 87 ear disease, 107 epidemiology and natural history of infection, 89-90 eye disease, 106-107 genital disease, 103-105 heart disease, 105-106 immunologically intact patient infection, 92-93 inclusion body cells, 87-89 kidney disease, 103 liver disease, 101-102 lung disease course, 97-99 immunocompromised patients, 96, 99, 206 lesions, 99-100 pneumonia models, 99 mononucleosis, 93

Index nervous system disease adults, 94-95 children, 93-94 immunocompromised patients, 95-96, 221-222 pancreas disease, 102 placental disease, 92 posttransfusion syndrome, 93 transplant recipient infection, 88, 96 urinary tract disease, 102-103

D Dengue virus epidemiology, 292 hemorrhagic fever, 292-293 shock syndrome, 292-293 Diabetes mellitus type I autoimmunity in pathogenesis, 21 mumps role, 385-386 virus infection role, 21-23 Diffuse alveolar damage, AIDS-associated disease, 222 Digestive tract adenovirus disease, 197 AIDS-associated diseases, 230-231 cytomegalovirus disease, 100-101 herpes simplex virus disease, 76-77 human papillomavirus diseases anus, 317 esophagus, 315-317 oropharynx, 315 varicella-zoster virus disease, 159 Duncan's disease, see X-linked lymphoproliferative disease

Ear cytomegalovirus disease, 107 human papillomavirus and middle ear disease, 322 mumps and middle ear disease, 407 mumps manifestations, 386 varicella-zoster virus disease, 158 Eastern equine encephalitis, features, 346-347 Ebola virus clinical features of disease, 287-289 outbreaks, 287 pathology 288-289 EBV, see Epstein-Barr virus Echovirus, see Enterovirus Ecthyma contagiosum, features, 377 Encephalitis, see also Nervous system disease cytomegalovirus adults, 94-95 children, 93-94 immunocompromised patients, 95-96 enterovirus disease, 4 flaviviruses Japanese B encephalitis, 352-353 miscellaneous encephalitides, 353-354 St. Louis encephalitis, 351-352

herpes simplex virus diagnosis, 71-72 distribution in brain, 72-73 epidemiology, 71 immunocompromised patients, 74 seizures, 74-75 treatment, 71-72 rabies, 360-362 Enteric viral disease adenoviruses, 438 astroviruses, 438 coronaviruses, 438 diagnosis, 431^32 mortality, 431 Norwalk-like viruses clinical course of infection, 432-433 discovery, 431 epidemiology, 432 pathology, 433 structure and classification, 432 pathophysiology, 439 rotaviruses clinical features of infection, 434-435 epidemiology, 433^34 pathology 435, 437 structure and classification, 434 toroviruses, 438 Enterovirus, see also Coxsackievirus; Poliovirus Bomholm disease from Coxsackievirus infection, 17 classification, 1 diabetes mellitus type I, virus infection role, 21-23 heart disease Coxsackievirus group B myocarditis, 13-16, see Coxsackievirus diagnostic criteria, 10-13 epidemiology, 10 infection route, 2 kidney disease, 19 liver disease, 19 lung disease, 18 mutability, 1 neurological disease aseptic meningitis, 3-4 chronic enterovirus meningoencephalitis in immune deficiency, 8-10 encephalitis, 4 poliomyelitis, 4-6 post-poliomyelitis syndrome, 6, 8 placental disease, 19-20 receptors, 2 skin lesions, 20-21 striated muscle disease, 16-17 structure, 2 testicular disease, 18-19 transmission, 2-3 virulence factors, 3 Epstein-Barr virus, see also Herpesvirus Burkitt's lymphoma chromosome translocations, 127 discovery 117,125 epidemiology, 125-126 pathogenesis, 127

Index discovery, 117 hairy leukoplakia association, 136-138 hemophagocytic syndrome, 138-139 Hodgkin's disease role, 130-131 infectious mononucleosis clinical features, 120 discovery of viral etiology, 117 female genital tract, 123 kidney disease, 123 lymph node pathology, 120-121 myocarditis, 123 neuromuscular disease, 122-123 pancreatitis, 123 pericarditis, 123 inflammatory pseudotumor association, 135 lung disease, 134-135 lymphoepitheliomatous gastric carcinoma role, 133 lymphomatoid granulomatosis association, 135 lymphoproliferative disorders associated with immunosuppression, 127-129 nasopharyngeal carcinoma role, 131-133 non-Hodgkin's lymphoma role, 129-130 receptors, 118 replication, 118 sinusoidal tumor role, 133-134 Sjogren's syndrome, infection in salivary gland tumors, 135-136 strains, 118 X-linked lymphoproliferative disease, 123-124 Exanthema subitum, human herpesvirus type 6 infection, 167 Eye adenovirus disease, 198 cytomegalovirus disease, 106-107 herpes simplex virus disease, 78-81 human papillomavirus disease, 321-322 mumps disease, 407 varicella-zoster virus disease, 156-15

Fatal familial insomnia, see also Transmissible spongiform encephalopathy clinical features, 415 pathological features, 415-417 FBB, see Follicular bronchitis/bronchiolitis FFI, see Fatal familial insomnia Filoviruses Ebola virus clinical features of disease, 287-289 outbreaks, 287 pathology 288-289 Marburg virus disease, 287 overview, 287 Flaviviruses classification, 350 dengue virus epidemiology, 292 hemorrhagic fever, 292-293 shock syndrome, 292-293

infection cycle, 349-350 Japanese B encephalitis, 352-353 miscellaneous encephalitides, 353-354 overview, 289-290, 349 St. Louis encephalitis, 351-352 yellow fever virus clinical phases of illness, 291 epidemiology, 290-291 history of study 290-291 pathology 291-292 Follicular bronchitis /bronchiolitis, AIDS-associated disease, 222-223

GB virus, discovery and features, 262 Genitals, see also Testicles adenovirus disease, 196-197 cytomegalovirus disease, 103-105 Epstein-Barr virus and infectious mononucleosis of female genital tract, 123 herpes simplex virus disease females, 66-68 males, 69 human papillomavirus diseases cervix uteri, 311-314 endometrium, 314 epidemiology, 308 glans penis, 314-315 vulva and vagina, 309-311 German measles, see Rubella Gerstmann-Straussler-Scheinker disease, see also Transmissible spongiform encephalopathy clinical features, 414 pathological features, 415-417 Gianotti-Crosti syndrome, features, 271-272 Giant cell pneumonia mumps virus, 400^02 parainfluenza virus, 49-50 respiratory syncytial virus, 55 Gingivostomatitis, herpes simplex virus, 65 Glomerulonephritis, hepatitis virus association, 272-273 GSS, see Gerstmann-Straussler-Scheinker disease Guanarito virus, hemorrhagic fever, 280

H Hairy leukoplakia, Epstein-Barr virus association, 136-138 Hantaan virus, Korean hemorrhagic fever, 282-284 Hantavirus hemorrhagic fever with renal disease epidemiology, 282 Hantaan virus and Korean hemorrhagic fever, 282-284 Puumala virus and nephropathia endemica, 284-285 Seoul virus, 282-284 strains, 297

443 Hantavirus pulmonary syndrome clinical features, 298 epidemiology, 297 origins, 297 pathology 298-299, 301 Heart adenovirus disease, 197 AIDS-associated disease, 226-227 congenital rubella effects, 394 cytomegalovirus disease, 105-106 enterovirus diseases Coxsackievirus group B myocarditis, 13-16, see Coxsackievirus diagnostic criteria, 10-13 epidemiology, 10 Epstein-Barr virus and infectious mononucleosis, 123 influenza virus disease, 41-44 Hematopoietic system, human immunodeficiency virus infection effects, 215 Hemophagocytic syndrome, Epstein-Barr virus, 138-139 Hemorrhagic fever viruses, see also specific viruses arenaviruses, 277-282 bunyaviruses, 282-287 tiloviruses, 287-289 flaviviruses, 289-293 overview, 277 Hepatitis virus autoimmune hepatitis features, 270 chronic hepatitis diagnosis, 262 pathology 263-264 Gianotti-Crosti syndrome features, 271-272 glomerulonephritis association, 272-273 hepatitis A virus clinical features of disease, 254-255 epidemiology 254 pathogenesis, 255 structure, 254 hepatitis B virus discovery, 257 pathogenesis, 257-260 structure, 257 transmission, 258 hepatitis C virus clinical features of disease, 261-262 epidemiology, 261 immune response, 260-261 liver transplantation in treatment, 262 mixed cryoglobulinemia association, 262 structure, 260 hepatitis D virus, replication and clinical features of infection, 260 hepatitis E virus clinical features of disease, 256 epidemiology, 256 structure, 255 hepatocellular carcinoma association, 264, 267, 269 history of study 253-254

444 Hepatocellular carcinoma, hepatitis virus association, 264, 267, 269 Herpes simplex virus, see also Herpesvirus digestive tract disease, 76-77 encephalitis diagnosis, 71-72 distribution in brain, 72-73 epidemiology, 71 immunocompromised patients, 74 seizures, 74-75 treatment, 71-72 eye disease, 78-81 generalized systemic disease, 69-71 genital disease females, 66-68 males, 69 genome, 61 gingivostomatitis, 65 liver disease, 77-78 lymph node disease, 78 respiratory tract disease, 75-76 skin lesions, 65-66 Herpesvirus, see also Cytomegalovirus; Epstein-Barr virus; Herpes simplex virus; Human herpesvirus type 6; Kaposi's sarcoma-associated herpesvirus; Varicella-zoster virus classification, 61 cytopathology, 63 herpes B, 187-188 latency 62-63 replication, 61-62 structure, 61 Herpes zoster epidemiology 151-152 latency, 152 pathology 153-154 HHV, see Human herpesvirus HIV, see Human immunodeficiency virus Hodgkin's disease AIDS-associated disease, 232 Epstein-Barr virus role, 130-131 HPV, see Human papillomavirus HPS, see Hantavirus pulmonary syndrome HSV, see Herpes simplex virus HTLV, see Human T cell leukemia / lymphoma virus Human herpesvirus type 6 exanthema subitum, 167 immunocompromised patient infection, 167-168 pathogenicity, 167 Human herpesvirus type 8, see Kaposi's sarcoma-associated herpesvirus Human immunodeficiency virus, see also Acquired immunodeficiency syndrome; Retrovirus clinical course of HIV-1 infection adults, 207-209 children and infants, 210-211 comparison of types, 207 digestive tract diseases, 230-231 discovery, 205 epidemiology, 205-206 genome, 204, 206 heart disease, 226-227

Index hematopoietic system effects, 215 kidney disease, 228-229 lymphoma association Burkitt's lymphoma, 232 Hodgkin's disease, 232 non-Hodgkin's lymphoma, 231-232 myositis, 220-221 nervous system diseases acute meningitis, 216 dementia complex, 217-219 encephalopathy, 216-219 mechanisms of neuronal damage, 218-219 myelopathy and myelitis, 219 neuropathy, 219 origins, 205 pancreatitis, 230-231 persistent generalized lymphadenopathy nonprogressive disease, 214-215 pathogenesis, 212 stages, 213-214 receptor, 207 respiratory tract diseases diffuse alveolar damage, 222 follicular bronchitis /bronchiolitis, 222-223 lymphoid interstitial pneumonia, 222-223 nonspecific interstitial pneumonia, 222-223 pulmonary hypertension and vascular-occlusive disease, 223, 225 structure, 206-207 testicular disease, 229 ultrastructure of infected cells, tubuloreticular structures and cylindrical confronting cisternae, 231 vasculature disease, 227-228 Human papillomavirus classification, 303 digestive tract disease anus, 317 esophagus, 315-317 oropharynx, 315 E6 and E7 proteins in pathogenesis, 304-305 eye disease, 321-322 genital diseases cervix uteri, 311-314 endometrium, 314 epidemiology, 308 glans penis, 314-315 vulva and vagina, 309-311 infection cycle, 304 laryngeal papillomas, 317-319 middle ear disease, 322 skin lesions epidermodysplasia verruciformis, 307 verruca plana, 305 verruca plantaris, 305, 307 verruca vulgaris, 305 structure, 303 tracheobronchial tree papillomas, 319, 321 Human T cell leukemia/lymphoma virus, see also Retrovirus cell transformation mechanism, 245, 247

comparison between types, 243, 249 epidemiology, 243, 250 genome, 204, 243, 245, 247 HTLV-2 distribution and diseases, 243, 249-250 inflammatory conditions associated with HTLV-1 infection, 248-249 T-cell leukemia/lymphoma syndrome, HTLV-1 infection clinical course, 244-245 pathogenesis, 245, 247 transmission, 243-244 tropical spastic paraparesis, HTLV-1 infection, 247-248

I Inclusion body cells, cytomegalovirus association, 87-89 Infectious mononucleosis, see Mononucleosis Inflammatory pseudotumor, Epstein-Barr virus association, 135 Influenza virus central nervous system disease, 43 classification, 35-36 course and features of infection adults, 38-39 children, 38 epidemics, 36-38 heart disease, 4 1 ^ 4 infection route, 35-36 lung disease, 40-41 muscle disease, 43 receptors, 35 Reye-Johnson syndrome, 43-44 risk factors for infection, 39-40 salivary gland disease, 43

J Japanese B encephalitis, features, 352-353 JC virus, see also Papovavirus progressive multifocal leukoencephalopathy clinical course, 331 pathology, 330-331 viruses, 329-330 viremia, 328-329 Joint disease mumps manifestations, 386 parvovirus B19, 337 varicella-zoster virus, 162 Junin virus, hemorrhagic fever, 278-279

K Kaposi's sarcoma AIDS association, 171-172, 232-233 classification, 174-175 clinical features, 171-172, 233 lymph node involvement, 177-178

445

Index pathogenesis, 174-175,177-178 staging, 175 tissue distribution, 233-234 Kaposi's sarcoma-associated herpesvirus angiofollicular lymph node hyperplasia, 180-181 angiosarcomas and vascular lesions, 178 body cavity-based non-Hodgkin's lymphoma, 178 discovery 172-173 epidemiology 173-174, 233 Kidney AIDS-associated disease, 228-229 cytomegalovirus disease, 103 enterovirus disease, 19 Epstein-Barr virus and infectious mononucleosis, 123 glomerulonephritis, hepatitis virus association, 272-273 Hantavirus, hemorrhagic fever with renal disease epidemiology, 282 Hantaan virus and Korean hemorrhagic fever, 282-284 Puumala virus and nephropathia endemica, 284-285 Seoul virus, 282-284 varicella-zoster virus disease, 160-162 KSHV, see Kaposi's sarcoma-associated herpesvirus Kuru, see also Transmissible spongiform encephalopathy clinical features, 414^15 pathological features, 415-417

LaCrosse, features, 354 Laryngeal papillomas, human papillomavirus and cancer role, 317-319 Lassa virus, hemorrhagic fever, 280-292 Liver, see also Hepatitis virus congenital rubella effects, 394 cytomegalovirus disease, 101-102 enterovirus disease, 19 herpes simplex virus disease, 77-78 Lassa virus lesions, 280-282 varicella-zoster virus disease, 159-160 Lung adenovirus infection chronic infection, 194 disseminated disease in immunocompromised patients, 195-196 inclusion-body pneumonia, 192 late complications, 192-193 serotypes, 191,194-195 transmission, 191, 194 AIDS-associated diseases diffuse alveolar damage, 222 follicular bronchitis / bronchiolitis, 222-223 lymphoid interstitial pneumonia, 222-223 nonspecific interstitial pneumonia, 222-223

pulmonary hypertension and vascular-occlusive disease, 223, 225 cytomegalovirus disease course, 97-99 immunocompromised patients, 96, 99 lesions, 99-100 pneumonia models, 99 enterovirus disease, 18 Epstein-Barr virus disease, 134-135 herpes simplex virus disease, 75-76 influenza virus disease, 4 0 ^ 1 mumps giant cell pneumonia, 400^02 mortality 399^00 persistence, 399, 401 parainfluenza virus disease, 47-50 respiratory syncytial virus disease, 53-58 rhinovirus disease, 31-32 varicella-zoster virus disease, 158-159 Lymph node, see also Angiofollicular lymph node hyperplasia; Persistent generalized lymphadenopathy herpes simplex virus disease, 78 Kaposi's sarcoma involvement, 177-178 lymphadenitis in vaccination by vacciniavirus, 373 Lymphocytic choriomeningitis virus aseptic meningitis, 427 isolation, 427 pathogenicity in humans, 427-428 rodent hosts, 428 structure, 427 Lymphoepitheliomatous gastric carcinoma, Epstein-Barr virus role, 133 Lymphoid interstitial pneumonia, AIDS-associated disease, 222-223 Lymphomatoid granulomatosis, Epstein-Barr virus association, 135

M Machupo virus, hemorrhagic fever, 278-279 Marburg virus disease, hemorrhagic fever, 287 Measles virus atypical measles syndrome following vaccination, 402-403 central nervous system disease, see Meningoencephalitis meningoencephalitis, 404-404 overview of syndromes, 403 subacute sclerosing panencephalitis, 404-407 classification, 398-399 epidemiology, 397 eye disease, 407 immune response, 397-398 immunosuppression, 399 isolation, 399 lesions, 397 middle ear disease, 407 pregnancy effects, 407-408 respiratory tract disease giant cell pneumonia, 400-402 mortality 399-400 persistence, 399, 401 structure, 399

Meningoencephalitis, 403-404 immunized patients, 404 immunocompromised patients, 403-404 natural infection, 403 Meningitis enterovirus disease, 3-4 human immunodeficiency virus association, 216 lymphocytic choriomeningitis virus, 427 Milker's nodules, cowpox, 377 Molluscum contagiosum, features, 377-378 Monkeypox virus discovery, 373 epidemiology, 365, 374 modern threat, 374 Mononucleosis cytomegalovirus association, 93 Epstein-Barr virus and infectious mononucleosis clinical features, 120 discovery of viral etiology, 117 female genital tract, 123 kidney disease, 123 lymph node pathology, 120-121 myocarditis, 123 neuromuscular disease, 122-123 pancreatitis, 123 pericarditis, 123 Mumps central nervous system disease, 382 clinical course, 381-382 diabetes mellitus, role in type 1 disease, 385-386 ear disease, 386 history 381 joint disease, 386 pancreatic disease, 384-385 salivary gland disease, 382 testicular disease, 383-384 virus features, 381 Muscle influenza virus disease, 43 myositis in AIDS, 220-221 striated muscle disease, enterovirus, 16-17 varicella-zoster virus disease, 162 Myocarditis, see Heart

N Nasopharyngeal carcinoma, Epstein-Barr virus role, 131-133 Nervous system disease, see also Encephalitis; Transmissible spongiform encephalopathy adenovirus, central nervous system disease, 197-198 congenital rubella effects on central nervous system, 393-394 cytomegalovirus disease adults, 94-95 children, 93-94 immunocompromised patients, 95-96 enterovirus diseases aseptic meningitis, 3-4

446 chronic enterovirus meningoencephalitis in immune deficiency, 8-10 encephalitis, 4 poliomyelitis, 4-6 post-poliomyelitis syndrome, 6, 8 Epstein-Barr virus and infectious mononucleosis, 122-123 human immunodeficiency virus association acute meningitis, 216 dementia complex, 217-219 encephalopathy, 216-219 mechanisms of neuronal damage, 218-219 myelopathy and myelitis, 219 neuropathy, 219 influeiLza virus, central nervous system disease, 43 measles, see Meningoencephalitis meningoencephalitis, 403-404 overview of syndromes, 403 subacute sclerosing panencephalitis, 404-407 mumps manifestations, 382 rabies, 360-362 varicella-zoster virus diseases encephalopathies, 145-156 herpes zoster, 151-154 NHL, see Non-Hodgkin's lymphoma NLVs, see Norwalk-like viruses Non-Hodgkin's lymphoma AIDS-associated disease, 231-232 Epstein-Barr virus role, 129-130 Kaposi's sarcoma-associated herpesvirus and body cavity-based lymphomas, 178 Nonspecific interstitial pneumonia, AIDS-associated disease, 222-223 Norwalk-like viruses clinical course of infection, 432-433 discovery, 431 epidemiology, 432 pathology, 433 structure and classification, 432 NPC, see Nasopharyngeal carcinoma

Pancreas AIDS-associated disease, 230-231 congenital rubella effects, 394 cytomegalovirus disease, 102 Epstein-Barr virus and infectious mononucleosis, 123 mumps manifestations, 384-385 Papillary acrodermatitis, see Gianotti-Crosti syndrome Papillomavirus, see Human papillomavirus Papovavirus, see also BK virus; JC virus; SV40 infection cycle, 327-328 structure, 327 Parainfluenza virus classification, 47 discovery, 47

Index giant cell pneumonia, 49-50 respiratory infections, 47-48 Sendai virus infection in mice, 48 Parvovirus B19 epidemiology, 335 erythropoietic systemic disease, 337-339 fifth disease, 335-337 infection course, 336 inflammatory lesions, 340 joint disease, 337 pregnancy infections, 339-340 structure, 335 tissue diagnosis of infection, 340 Penis, see Genitals Pericarditis, Epstein-Barr virus and infectious mononucleosis, 123 Persistent generalized lymphadenopathy nonprogressive disease, 214-215 pathogenesis, 212 stages, 213-214 PGL, see Persistent generalized lymphadenopathy Placenta cytomegalovirus disease, 92 enterovirus disease, 19-20 PML, see Progressive multifocal leukoencephalopathy Poliovirus, see also Enterovirus classification, 1 poliomyelitis, 4-6 post-poliomyelitis syndrome, 6, 8 receptor, 2 Post-poliomyelitis syndrome, see Poliovirus Posttransfusion syndrome, cytomegalovirus association, 93 Poxvirus cowpox virus, 365, 371, 377 molluscum contagiosum, 377-378 monkeypox virus discovery, 373 epidemiology, 365, 374 modern threat, 374 orthopoxviruses, 365-367, 369, 371-375 parapoxviruses, 375, 377 replication, 366 smallpox clinical course, 367, 369 modem threat, 365 mortality 367, 369, 371 transmission, 367 structure, 366 vacciniavirus complications of vaccination, 371-373 development of virus, 371 dissemination, 365 epidermal proliferation, 366 Pregnancy complications in vaccination by vacciniavirus, 373 congenital infection cytomegalovirus, 90, 92 rubella, 391-394, see Rubella varicella-zoster virus, 162-163 measles effects, 407-408 parvovirus B19 infection, 339-340 Prion disease, see Transmissible spongiform encephalopathy

Progressive multifocal leukoencephalopathy clinical course, 331 JC virus role, 329-330 pathology 330-331 SV40 role, 327, 329-330 Pulmonary hypertension, vascular-occlusive disease in AIDS, 223, 225 Puumala virus, nephropathia endemica, 284-285

R Rabies central nervous system disease, 360-362 clinical forms, 358-359 epidemiology, 357-358 immunization, 357 pathogenesis, 359-360 viruses classification, 357 structure, 357 Respiratory syncytial virus classification, 53 epidemiology, 53, 57 immune-mediated disease, 54-55 risk factors for infection, 53, 55-56 symptoms of infection, 57-58 Retrovirus, see also Human immunodeficiency virus; Human T cell leukemia/lymphoma virus classification, 203 genomes, 204 Reye-Johnson syndrome, influenza virus association, 43-44 Rhinovirus cell distribution, 29-30, 32 classification, 29 discovery, 29 diseases, 31-32 receptors, 29 symptoms of infection, 31 Rift Valley fever virus, hemorrhagic fever, 285-286 Rotavirus clinical features of infection, 434-435 epidemiology, 433-434 pathology 435, 437 structure and classification, 434 RSV, see Respiratory syncytial virus Rubella congenital infection central nervous system effects, 393-394 fetal growth effects, 391 heart effects, 394 liver effects, 394 organ malformations, 392 pancreas effects, 394 persistence, 392 timing and organ abnormalities, 391 history of study 389 postnatal infection features, 390 virus classification and structure, 389 isolation and culture, 389-390 Rubeola, see Measles virus

447

Index

Sabia virus, hemorrhagic fever, 280 St. Louis encephalitis, features, 351-352 Salivary gland influenza virus disease, 43 mumps manifestations, 382 Sjogren's syndrome, Epstein-Barr virus infection in tumors, 135-136 Scrapie, features, 412 Sendai virus, see Parainfluenza virus Seoul virus, hemorrhagic fever, 282-284 Shingles, see Herpes zoster Sinusoidal tumor, Epstein-Barr virus role, 133-134 Sixth disease, see Exanthema subitum Sjogren's syndrome, Epstein-Barr virus infection in salivary gland tumors, 135-136 Skin, see also Measles virus; Poxvirus enterovirus lesions, 20-21 herpes simplex virus lesions, 65-66 human papillomavirus lesions epidermodysplasia verruciformis, 307 verruca plana, 305 verruca plantaris, 305, 307 verruca vulgaris, 305 parvovirus B19 and fifth disease, 335-337 varicella-zoster virus infection chickenpox (see Chickenpox), 147-148 chronic skin infections, 150-151 hemorrhagic skin infections, 149-150 Smallpox clinical course, 367, 369 modern threat, 365 mortality, 367, 369, 371 transmission, 367 Striated muscle, enterovirus disease, 16-17 Subacute sclerosing panencephalitis, mumps association, 404-407 SV40, human infection, 327, 329-330

T-cell leukemia/lymphoma syndrome, HTLV-1 infection clinical course, 244-245 pathogenesis, 245, 247 Testicles AIDS-associated disease, 229 enterovirus disease, 18-19 mumps manifestations, 383-384 varicella-zoster virus disease, 162 Tracheobronchial tree, human papillomavirus papillomas, 319, 321

Transmissible spongiform encephalopathy clinical features Creutzfeldt-Jacob disease, 413^14 fatal familial insomnia, 415 Gerstmann-Straussler-Scheinker disease, 414 kuru, 414^15 diagnosis by nonhistopathological methods, 424 history of study, 411 iatrogenic Creutzfeldt-Jacob disease, 422 new variant Creutzfeldt-Jacob disease, 421^22 pathological features, 415^17 prion-related protein accumulation in plaques, 418-419 mutations, 419 physical properties, 417-418, 422 structure, 417 properties, 411 safety precautions for pathologists, 422-424 scrapie, 412 species specificity, 411-412 Transplant recipient cytomegalovirus infection, 88, 96 liver transplantation in hepatitis B treatment, 262 Tropical spastic paraparesis, HTLV-1 infection, 247-248 TSP, see Tropical spastic paraparesis Tubuloreticular structures, human immunodeficiency virus-infected cells, 231

Varicella-zoster virus, see also Herpesvirus chickenpox clinical features, 147-148 course, 148-149 immune response, 148 transmission, 148 chronic skin infections, 150-151 congenital infection, 162-163 digestive tract disease, 159 ear disease, 158 eye disease, 156-158 hemorrhagic skin infections, 149-150 historical overview, 147 joint disease, 162 kidney disease, 160-162 liver disease, 159-160 lung disease, 158-159 muscle disease, 162 nervous system diseases encephalopathies, 154-156 herpes zoster, 151-154 testicular disease, 162 Variola, see Smallpox Vasculature, AIDS-associated disease, 227-228 vCJD (new variant Creutzfeldt-Jacob disease), 421-422 Venezuelan equine encephalitis, features, 348-349 Venezuelan hemorrhagic fever, see Guanarito virus

w

u Urinary tract, see also Kidney adenovirus disease, 196-197 BK virus infection and disease, 331, 333 cytomegalovirus disease, 102-103

West African hemorrhagic fever, see Lassa virus Western equine encephalitis, features, 347-348

X-linked lymphoproliferative disease, Epstein-Barr virus infection, 123-124 Vacciniavirus con\plications of vaccination central nervous system, 373 lymphadenitis, 373 pregnancy, 373 systemic complications, 371-373 development, 371 dissemination, 365 epidermal proliferation, 366

Yellow fever virus clinical phases of illness, 291 epidemiology, 290-291 history of study 290-291 pathology, 291-292