Advances in Virus Research: v. 3

Advances in

VIRUS RESEARCH VOLUME I11

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Advances in

VIRUS

RESEARCH Edited by

KENNETH M. SMITH

MAX A. LAUFFER

Virus Research Unit Molteno Institute Cambridge, England

Department of Biophysics University of Pittsburgh Pittsburgh, Pennsylvania

VOLUME I11

1955 A C A D E M I C P R E S S INC., Publishers N E W YORK 10, N. Y.

Copyright, 1955, by ACADEMIC PRESS INC. 125 East 23rd Street, New York 10, N. Y . All Rights Reserved

NO PART OF THIS BOOK MAY B E REPRODUCED I N ANY FORM, BY PHOTOSTAT, MICROFILM,

OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION

FROM THE PUBLIBHERS.

Library of Congress Catalog Card Number, 63-11669

PRINTED IN THE UNITED STATES OF AMERICA

Contributors to Volume I11 J. W. BEARD,Department of Surgery, Duke University School of Medicine, Durham, North Carolina SEYMOUR S . COHEN,Children’s Hospital of Philadelphia (Department of Pediatrics), and Department of Physiological Chemistry, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania EDWARD A. ECKERT,Department of Surgery, Duke University School of Medicine, Durham, North Carolina HARRIETT EPHRUSSI-TAYLOR, Laboratoire de Gh4tique de la Facult4 des Sciences, Paris, France, et du Centre Nationale de la Recherche Scientifique

L. 0. KUNKEL,The Rockefeller Institute for Medical Research, New York, New York KARL MARAMOROSCH, The Rockefeller Institute for Medical Research, New York, New York R. E. F . MATTHEWS, Virus Research Unit, Agricultural Research Council, Molteno Institute, Cambridge, England D. G. SHARP,Department of Surgery, Duke University School of Medicine, Durham, North Carolina

J. D. SMITH,Virus Research Unit, Agricultural Research Council, Molteno Institute, Cambridge, England KENNETH M . SMITH,Virus Research Unit, Agricultural Research Council, Molteno Institute, Cambridge, England

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Contents CONTRIBUTORS TO VOLUME111.. . . . . . . ........................................

v

Comparative Biochemistry and Virology B Y SEYMOUR S. COHEN Children’s Hospital of Philadelphia (Department of Pediatrics), and Department of Physiological Chemistrg, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . ............. 11. On Biochemical Variability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Parasitic Patterns in Virus-Infected Cells . .... . . IV. The Form and Composition of the Viruses.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. On the Fine Structure of Viral Constituents.. . , , . . . . . . . . . . . . . . . . . . . . . . . VI. The Metabolic Equipment of the Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Some Relations of Virus Structure and Enzyme Content t o Virus Entrance and Release. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. On the Metabolic Consequence of a New Building Block: 5-(Hydroxymethy1)cytosine in the T-even Phages.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I X . Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . , . . . , , , . , , , , . , . , . , , , , , . , . , , . . . . . . . , . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 8 11 22

29 32 35 43 43

The Chemotherapy of Viruses BY R. E . F. MATTHEWS A N D J. D. SMITH Virus Research Unit, Agricultural Research Council, Molteno Institute, Cambridge, England

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . , . . . . . . . . , . . . . . . . . . . . . . 11. Methods of Testing Compounds for Virus Inhibition.. . . . . . . . . . . . . . . . . . . 111. Structure and Multiplication of Viruses in Relation to Chemotherapy.. IV. Effects of Purine and Pyrimidine Analogues on Viruses.. . . . . . . . . . . . , . . . V. Virus Inhibition by Other Types of Compounds. . . . . . . . . . . . . . . . . . . . . . . . VI. Incorporation Phenomena in Relation to Antimetabolite Action.. . . . . . . . VII. General Discussion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51 52 61 74 108 137 140 142

Tumor Viruses

BY J. W. BEARD,D. G. SHARP,A N D EDWARD A. ECKERT Departnient of Surgery, Duke University School of Medicine, Durham, North Carolina

I. Introduction. ............... . . . . . . . . . . .. . . . . . . . . . . . 11. Rabbit Papillomatosis and Avian Leukosis. . .. . . . . . . . . . . . . . . . . . . . . . . . . . 111. Purification . . . . . . . . . . . . .. _ _ . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . _ .. . . . , . . , , . . IV. Physical and Chemical Properties.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Virus Infectivity and Host Response.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

149 151 155 158 173

VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morphology and Development of Insect Viruses BY KENNETH M. SMITH Virus Research Unit, Agricultural Reaearch Council, Molten0 Institute, Cambridge, England I. Introduction.. . . . . . ..................................... 11. The Polyhedral Viruses: Nuclear Type.. ............................... 111. The Polyhedral Viruses: Cytoplasmic Type.. . . . . . IV. The Granuloses or Capsular Diseases.. . . . . . . . . . . . V. Viruses without Intracellular Inclusions. . . . . . . . . . . . . . . VI. Apparent Viruses, Insufficiently Studied.. . . . . . . . . VII. Development of Insect Viruses ..... References. ..................................................................

199 200

219

MultiplicaMon of Plant Viruses in Insect Vectors BY KARLMARAMOBOSCH The Rockefeller Znstitute for Medical Research, New York, New York I. Introduction ........................................................... 221 11. Rice Stunt Virus ....................................................... 223 111. Aster Yellows Virus.. .................................................. 225 IV. Clover Club Leaf Virus.. .............................................. 233 V. Wound Tumor Virus.. ................................................. 235 VI. Corn Stunt Virus.. .................................. . . . . . . . . . . . . . . . 237 VII. Curly Top Virus. ....................................... 238 VIII. Possible Multipli ctor Species of Leafhoppers.. . . . . . . . . . . 241 IX. Possible Multiplication in Other Groups of Arthropod Vectors. . . . . . . . . . 243 X. Conclusions.. . . . . . . . . . . . . . . . . . . . ............ . . . 245 References. .................................................................. 248

Cross Protection Between Strains of Yellows-type Viruses BY L. 0. KUNKEL The Rockefeller Institute for Medical Research, New York, New York I. Introduction.. ....................... 11. Identification of California Aster Yell 111. Cross Protection Experiments with Vinca rosea Plants.. . . . . . . . . . . . . . . . . IV. Cross Protection in Leafhoppers.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Discussion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary ................................................. References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

262 263 269 272 272

Current Status of Bacterial Transformations BY HARRIETTEPHRUSSI-TAYLOR Laboratoire de Gdndtique de la Facultd des Sciences, Paris, France, et du Centre Nationale de la Recherche Scientijique I. Introduction ........................................................... 275 11. General Biological Description of Transformation. ..................... 276 111. Chemical and Physical Properties of Transforming Agents. . . . . . . . . . . . . . 278 IV. Mechanism of Transformation. ................. . . . . . . . . . . . . . . . . . . . . . . . . 282 V. Genetic Recombination between Transforming Factors. . . . . . . . . . . . . . . . . . 293 viii

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

307

INDEX ...............................................................

309

SUBJECT INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

319

CONTENTS OF VOLUMES1-11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

339

AUTHOR

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Comparative Biochemistry and Virology’ SEYMOUR S. COHEN Children’s Hospital of Philadelphia (Department of Pediatrics), and Department of Physiological Chemistry, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 11. On Biochemical Variability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 111. Parasitic Patterns in Virus-Infected Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 SV. The Form and Composition of the Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 A. Plant Viruses.. . B. Bacterial Viruse ......................................... 14 C. Animal Viruses. . . . . . . . . . . . . . V. On the Fine Structure of Viral V I . The Metabolic Equipment of the Viruses.. . . . . . . . . VII. Some Relations of Virus Structure and Enzyme Content t o Virus Entrance and Release. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 VIII. On the Metabolic Consequence of a New Building Block: 5-(Hydroxymethy1)cytosine in the T-even Phages. . . . . . . . . . . IX. Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION

It has taken most workers in animal virology approximately ten years to realize the potentialities of the phage methods in work with the animal viruses. This realization went hand in hand with the recognition that animal virology required a more penetrating examination of in vitro systems containing infected animal cells. Within a few years, this approach culminated in the achievement of Enders and his group in the cultivation of poliomyelitis virus in tissue cultures and in the exploitation of this fact for purposes of virus diagnosis and of mass production of poliomyelitis virus for vaccines. That plaque counting techniques comparable to those employed with bacteriophages were also applicable to animal viruses in tissue cultures has been most clearly demonstrated by Dulbecco (51). Thus for the first time animal virology has approached a stage wherein biological questions concerning virus-cell interactions may be answered experimentally. Within the last decade the questions posed concerning the bacterial 1 This paper is an expanded version of a lecture given in a course of Physiology a t the Marine Biological Laboratory, Woods Hole, Massachusetts in the Summer of 1953.

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viruses and their interactions with their host bacteria have shifted from the biological to the chemical level. Many of the most important biological questions have been rephrased as chemical problems. Questions are now being posed concerning the nature of the building blocks and the pathways of their biosynthesis. The time course of infection, duplication, and virus liberation is being dissected minute by minute in terms of the molecular transformations occurring in these systems. As will be discussed in part in this paper, problems of genetic duplication and virus synthesis are being posed and tested a t molecular and intermolecular levels. Many of the biologists working on phage are now engaged in chemical work, in learning the elements of chemical manipulation, or in hiring chemically trained personnel for their research projects? Since the biological prerequisites for chemical work in the field of the animal viruses are essentially solved, i.e. systems of infected cells can be made readily available, it can be anticipated that animal virology will soon see a burst of activity in chemical directions as well. It therefore seems appropriate to scan the present status of chemical virology in the light of the impending growth of this specialization. It also seems desirable to assess some current trends in biochemistry for the purposes of orienting approaches to biochemical work in virology.

11. ON BIOCHEMICAL VARIABILITY Approximately five years ago when basic discoveries concerning the bacterial viruses T2, T4,and T6 were being made rapidly, e.g., inhibition of cell multiplication by infection, genetic recombination, the extreme redirection of cellular metabolism, etc., it was considered possible that these phenomena might prove to be of general importance among all types of virus-infected cells. At least at that time rigorous data relevant to these 1 T h e entrance of the chemist and physicist into the work on bacterial viruses was greeted by an insistent and correct demand of the biologists that the former group master the elements of the biology with which they would be concerned. The initial findings and reports of the chemists were scrutinized with caution, skepticism, and harsh criticism, a procedure which helped greatly in molding them into useful contributors. It is t o be hoped that the biologists will approach their chemical studies with a similar regard for the dangers inherent in entering an unfamiliar discipline. It is the feeling of the author that a special note of caution is important with regard t o the problem of the presentation of chemical data. The initial work on the chemistry of the plant viruses in the period from 1935 through 1942 was conducted by organic and physical biochemists in a most rigorous fashion. Publication was effected under the unrelaxed scrutiny of t h e editors of chemical journals. Today an increasing amount of unsupported “chemical data” on the bacterial viruses is being published in unedited abstracts, lectures, and reviews. It is possible that we are having too many meetings and reviews, calling for the announcement, however diffident, of at least one new fact, perhaps not too well-proven.

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questions in systems other than some bacterial virus systems did not exist and the errors inherent in extrapolation had not yet been demonstrated. More recently with the clarification of lysogenic systems and the increasing exploration of animal virus systems, it has become increasingly evident that one may not extrapolate freely from one biological system to another. It is now understood that the methods of exploring the interactions of virus and host cell may be systematized, a t least for systems in which adsorption of virus to cell precedes infection and m~ltiplication,~ but that the course and types of phenomena to be observed in the various systems may vary considerably. Infected cells may or may not divide and multiply; they may or may not grossly alter their metabolism, they may or may not lyse as a requisite for virus liberation. Within any major category, bacterial, animal, or plant virus systems, biological properties vary considerably and the problems of virology are clearly multiplied. This is not to say that the viruses as a whole will not show .certain biological similarities, e.g. the requirement of intact cells for virus multiplication to proceed. However, each virus must be approached cautiously since it may not always be possible to predict the results of an experiment with influenza virus because of some prior knowledge of the behavior of T2 bacteriophage or vaccinia virus. This proposition is now taken as self-evident and is really so obvious that the author may be accused of wasting precious space in asserting it. However, a very different theoretical position prevails in biochemical circles, wherein another proposition, known as “the unity of biochemistry” has been dominant for some years. Many biochemists do consider this to mean that the cells of all organisms contain the same basic building blocks arranged to form similar polymers and enzymes which function in similar patterns of metabolic bahavior. T o state the thesis most baldly and critically (159) “the liver cell and E . coli, the meristematic plant cell and the purple bacterium, are sisters under the skin, their biochemical differences being principally ones of minor detail.’’ I n his early discussion of “the unity in biochemistry” Kluyver (87) did not so present this sweeping generalization, which has become the guiding principle of one school of comparative biochemistry. However, van Niel, Wilson, etc., have occasionally defined this discipline in this way. Recently Kluyver has stated the view that the function of comparative biochemistry shall be the demonstration of the unitarian principle underlying the diversity of phenomena in the microbial world (88). This formulation, although considerably less explicit than that presented above, is none the less tendentious concerning the validity of the assumption that a unitarian principle does underly the diversity of the microbial world. The problem of a rigorous approach t o a time course of infection in t h e plant virus systems has not yet been solved for this reason. J

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It is a curious fact that comparative biochemistry has had a very different connotation to biologists concerned mainly with higher organisms, e.g. Needham, Baldwin, Florkin. In their hands the discipline was concerned with the search for biochemical differences among higher organisms, differences which might point to developmental and phylogenetic relationships. The latter school has almost disappeared in recent years despite the great importance of its task, and despite its several significant succems in such areas as the relations of nitrogen excretion and water balance, and the nature and distribution of the phosphagens in vertebrate and invertebrate muscle. Of course the wide acceptance of the thesis of the unity of biochemistry does not rest on intuitive reasoning alone. The thesis is pragmatically useful; it has had many successes as a guide to experimental work, and it must be considered that these successes are the best evidence for its validity. It has often been shown that biochemical information obtained from the study of a microorganism is highly relevant to structures and processes in cells of higher organisms. For example, the inability of Streplocorns jaecalis to oxidize pyruvate in the absence of an unknown nutritional factor was used as a guide to the isolation of lipoic acid from mammalian liver (61). As is well-known, lipoic acid is now recognized to be a coenzyme of pyruvic oxidase in both S. jaecalis and the mammal, and indeed probably in most organisms capable of oxidizing pyruvate. Nevertheless, having discovered lipoic acid and its wide-spread occurrence and role in the oxidative decarboxylation of pyruvate, the pace of modern biochemistry is such that many systems have already been found which catalyze the oxidative decarboxylationof pyruvate without containing or utilizing lipoic acid. The pyruvic oxidases of Proteus vuZgaris (127) and of Lactobacillus delbrueckii (62) function without this coenzyme; in addition the oxidaaes of these organisms differ markedly between themselves. Despite all efforts to apply to one organism that which has been found for another, the many organisms now being examined are sufficiently diverse to compel the discovery of new phenomena whether they are being sought or not. The exaggerated form of the thesis of the unity of biochemistry was proposed before our experience was sufficiently large to warrant such a generalization and, aa apparent aberrancies are being revealed, a number of biochemists have begun to question the validity of the generalization, at least aa a guide to work in their own fields of study. Several such inquiries have recently been published (21, 38, 159), and, it may be added, have been challenged in their turn (96). In the area of parasitic relationships and of infections the problem of a theoretical guide to biochemical study is particularly important. The

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student of host-parasite relationships not only wishes to know the nature of the process of disease production but also how to control the course of infection, and how to inhibit the development of the parasite in such a way as to permit the survival of the host. The latter is the special task of chemotherapy, and it is evident in this era of antibiotics that microbial infections of animal cells may in many instances be controlled. If a chemical can be added to a parasitic microbe and its host cell in such a manner as to damage the former far more severely than the latter, the chemical reactivities of the two kinds of cell must be quite different in key areas of structure, metabolism, or both. A successful chemotherapy then must reflect important biochemical and metabolic differences between host and parasite. It is true that an extensive knowledge of the modes of action of many of the chemotherapeutic agents is as yet unavailable. Nevertheless it must be presumed that these agents do have modes of action, that they reflect chemical differences among organisms, and that these critical differences can be found. These statements may be taken ~ 1 9articles of faith in the validity and potential existence of a “rational chemotherapy.” It is wellknown that a rational approach to chemotherapy is often rejected by many workers intimately concerned with the problem of developing a chemotherapy of infection. The latter workers bolster their approach to this problem by quoting numerous pragmatic successes in the discovery of substances such as sulfanilamide, penicillin, DDT, etc. In this atmosphere the proposition of the “unity of biochemistry” is also a theoretical support for empiricism in the laboratory. If it is believed that the same metabolic systems operate in host and parasite alike, there will be little hope to be able to select an inhibitor which will affect the parasite alone? In this light, therefore, it will be worthwhile to elaborate the thesis of biochemical variability among cells in some greater detail. Stanier has summarized recent evidence to show that the cytochemical organization of bacteria and blue-green algae differs in important respects from the organization of other microorganisms and from the cells of plants and animals (159). He has pointed to the presence of an amino acid, a,e-diamino-pimelic acid in all gram-negative true bacteria) in photosynthetic bacteria) in some gram-positive bacteria) and in 3 genera of blue-green 4 A similar conclusion derived from the concept of “the unity of biochemistry” has recently been expressed by Dubos (50), who considers that the biochemical differences among organisms, both pathogens and saprophytes, are so “trivial” that they will “escape the attention of those concerned with the general biochemical phenomena of life.” Believing this, Dubos has therefore turned away from the study of the invading agent to the study of the special environments which microorganisms find in animal tissues.

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algae. The amino acid is absent from other algae, fungi, protozoa, and all higher cells. This characteristic substance has been found to be a component of cell walls. Stanier has also summarized studies on the isolated cell walls of bacteria whose composition, antigenicity, and structure are highly characteristic of the particular organism from which they are derived. These structures differ widely from bacterium to bacterium. Within the bacteria and blue-green algae, the organization of key metabolic systems appears to differ from that observed in other cells. For example, among most photosynthetic microorganisms, photosynthetic pigments are organized in characteristic particles called chromatophores which are far smaller than the chloroplasts of other algae or higher green plants. Or in B . megaterium, from which it is possible to strip the cell wall selectively with lysozyme in media of high osmotic pressure, it appears that the entire cytochrome system of the bacterium is present in the surface structure of the protoplast, rather than in particles analogous to mitochondria. These examples should serve then to indicate that phylogenetic differences may mirror deepseated chemical differences in composition and in organized structure. Other important categories of biochemical variability may be mentioned, with the following examples: A. Variability in the content and types of chemical components. 1. Mycobacteria, such as the tubercle bacillus, not only contain a,tdiaminopimelic acid but also other distinctive constituents which are not present in the mammal. These include large amounts of D-arabinose in the polysaccharide of its cell wall, fatty acids with cyclopropane rings, mycolic acids, phosphatides with amino acids other than serine, etc. (101). 2. Sterols are absent from the Mycobacteria, E. coli, etc. 3. No microorganism has yet been found to contain 5-methylcytosine, although many bacteria and viruses have been examined for this substance. B. Variability in the structure and mode of action of enzymes affecting similar over-all reactions. 1. Many bacteria contain cytochromes but none appear to contain cytochrome a 3 , the typical cytochrome c oxidase of the mammal and yeast (157). 2. In mammalian systems, the a-keto acid oxidases contain integrated lipoic acid dehydrogenases. In bacteria in which the keto acid oxidases utilize lipoic acid, e.g. E . coli, Proteus OX-19, the lipoic dehydrogenase is structurally separated from the oxidase. Of course, as mentioned above, other bacteria may by-pass lipoic acid completely in the performance of this function. 3. In green algae and in higher plants the chloroplast contains chloro-

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phyll a and is the site for both the absorption of light and the photolysis of water. In blue-green algae, light may be absorbed by the soluble phycobilins pigments absent from higher organisms.48 The energy is then transmitted to chlorophyll present in the submicroscopic chromatophores which effect the photolysis of water and generate reducing substance (4,221. C. Variability in basic pathways leading to the synthesis of key building blocks. 1. In Neurospora, lysine is derived from m-aminoadipic acid (77). In E. coli, m,c-diaminopimelic acid appears to be the precursor t o lysine (46, 48). 2. In Neurospora, tryptophan is the sole precursor for niacin (16). I n E. coli and B. subtilis, niacin is not derived from tryptophan or indole (184). 3. In bacteria, acetate may be converted to acetyl CoA via the intermediate formation of acetyl phosphate. This compound is not found in higher organisms and is inactive in acetylation reactions. D. Variability in the formation, organization, and integration of multiple alternative pathways. At least six major pathways for glucose metabolism are known, and several may exist simultaneously in the same organism (39). In studies on carbohydrate metabolism in E. coli, two major pathways of glucose-6-phosphate metabolism were observed, namely, the phosphogluconate and glycolytic pathways. Both pathways were shown to operate in the growing bacterium. Virus infection affected the balance of these paths (34). The appropriate choice of substrate also affected the operation of these systems (37). I n other bacteria, heredity has determined that Leuconostoc mesenteroides shall lack the glycolytic pathway and operate exclusively via phosphogluconate. Conversely, Lactobacillus casei uses only the glycolytic pathway in anaerobic and aerobic growth. These pathways are also present in the mammal; differentiation has concentrated the phosphogluconate path in the liver and the Embden-Meyerhof scheme in muscle. In B. subtilis, a diet containing amino acids permits the normal operation of both paths. Deprived of exogenous amino acids, B. subtilis is deficient in key enzymes of the Embden-Meyerhof scheme. Thus the quantitative significance of these systems in a functioning cell is determined by heredity, differentiation, nutrition, virus infection, and the carbohydrate substrate offered. ‘a Recent studies on the phycocyanins and phycoerythrins have suggested that these proteins are also located in the chlorophyll-containing structures of the algae but ale readily liberated on cell disruption c. f. Thomas and De Rover, Biochint. Biop h y s . Acta 16 391 (1955); McClendon and Blinks Nature 170 577 (1952).

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It must be stressed that the number of examples presented above may be multiplied very considerably in each category. In view of the existence of a growing body of data demonstrating many types of variability in biochemical systems, this author has suggested that the study of the origin, nature, and control of such variability should constitute the special area of concentration of the discipline of comparative biochemistry (38). This chapter will attempt to describe some of the phenomena of biochemical variability which have been encountered among the viruses and will remark on some current theories in virology in the light of this variability. 111. PARASITIC PATTERNS IN VIRUS-INFECTED CELM It is now possible to describe a gamut of patterns of viral parasitism, from those in which infection leads almost exclusively to the synthesis of virus to those in which infection permits both virus and host cell to multiply together. Such a range may be observed within the group of the bacterial viruses; for these viruses one now speaks of virulent and temperate phages. The virulent phages are best exemplified by the T-even phages, T2,T4, and T6. These infect Escherichia coli, destroy its nuclear apparatus, prevent bacterial multiplication, and divert the metabolism of the host cell to the synthesis of large amounts of specific virus substance. Following the accumulation of large numbers of virus particles within the infected cell, lysis of the bacterium occurs with liberation of virus. The temperate phages are active in lysogenic systems, whose properties have been clarified in large part by Lwoffand his collaborators (107). In the case of the phage lambda (A), which multiplies in certain strains of E . coli, infection may result under defined environmental conditions in an apparent union of the genetic apparatus of the virus with the nuclear apparatus of the host. The two may continue to multiply together in apparent harmony for many generations. In certain lysogenic systems ( E . coli, strain K12 infected by A) induction by external stimuli such as ultraviolet radiation upsets this relation, leads to the uncontrolled multiplication of the virus at the expense of host synthesis, and culminates in the accumulation of virus and in cell lysis. It is of some interest that, following induction, virus multiplication does not always prevent some synthesis of cell substance in the induced cell. Several cell divisions may occur in these systems prior to lysis. The existence of this multiplicity of parasitic patterns clearly affects an approach to many biological problems. For example, the transmission and survival of specific viruses must be examined in terms of these patterns. Since cells infected by completely virulent viruses are killed and lyse, an extracellular phase is an obligate concomitant of the virus life cycle. On

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9

the other hand, the temperate phages may pass from cell to progeny without ever being exposed to the unsheltered rigors of extracellular existence. In their turn, these biological facts have chemical consequences; in the estracellular phase the hereditary determinants of the phages arc protected by a tough protein coat resistant to the enzymes, proteases and nucleases, liberated by lysing bacteria. Within the delicately balanced cell in which anarchic enzymatic degradation is not occurring, the protective coating of the phages is not required and is left behind.6 None of the known antigens of phages, virulent or temperate, appear within the cell during the vegetative stage of their development (9, 118). In the case of the T-even phages, the shedding of the antigenic protein coat and the insertion of the genetic determinants do provoke a temporary release of enzyme systems which degrade the host nucleus and begin a similar process on the virus substance. In this instance, as shall be seen below, a unique chemical configuration in the virus nucleic acid brakes the degradation of the injected virus substance until virus multiplication gets under way (36). As will be discussed below, the viral substance essential to bacterial viral multiplication appears to be characteristic of nuclear substances and indeed the relationships now delineated in bacterial virus systems may be described as differing types of parasitism of the host nucleus. This is not to say that virus multiplication does not appropriate the products of cytoplasmic synthesis. However, it would appear that within the division of labor observed in an infected cell the bacterial virus plays a role more closely resembling an aberrant nucleus or chromosome than that of an aberrant cytoplasmic constituent. The contrary pattern would seem t o prevail in a t least the tobacco mosaic virus infections in which it appears that various strains of this virus have compositions more closely resembling cytoplasmic coniponents, are found in large amounts in cytoplasm, form cytoplasmic inclusions, and oftJen affect plastic composition. These viruses are thought by some t o be synthesized in plastids (103) and even perhaps to have originated in the variegation of plastids (49, 179). Although little is known of the course of virus multiplication in these systems, the information available is not inconsistent with the picture of a predominantly cytoplasmic disease. The growth of the plant infected by some strains of tobacco mosaic virus is so little affected as to suggest that cell multiplication is not greatly impaired. On the other hand, some instances are known of plant virus infection resulting in the formation of nuclear inclusion bodies, e.g. solanaceous plants infected with severe etch virus (86). It is believed that viruses 6 Indeed this coat must be left behind if the hereditary determinants of the virus are to be able t o come to grips with the metabolic apparatus of the host, as the enzymatic deficiencies of the virus require.

10

SEYMOUR S. COHEN

such as herpes simplex in animal systems multiply in the nucleus and may be transported to the cytoplasm where they may be found at a later stage in the infection (147). Similarly the relatively rare discovery of nuclear inclusions in plant virus systems may not reflect the primary site of multiplication of the plant viruses. However, it must be pointed out that in plant virus infections in general it has not been rigorously demonstrated whether infected cells do or do not multiply, although the conclusion that they do is often inferred and indeed underlined by pointing to the effect of the plant viruses causing tumors. Among animal viruses a far more varied picture is beginning to form with respect to their composition, site of multiplication, and effect on cell multiplication. More than thirty different virus diseases of animals are known in which nuclear inclusions are found. Virus infections among insects are analogous in many respects to lysogenic systems (153), and recent electron micrographs have pointed to nuclear chromatin as the site of insect virus multiplication (154). The multiplication of herpes simplex virus perhaps shares many similarities with these patterns, and it is thought that apparently normal cells harboring these viruses may be induced, even as in the bacterial virus lysogenic systems. On the other hand, despite its content of a nuclear component, deoxyribose nucleic acid (DNA), vaccinia virus appears to grow intracytoplasmically (122). In tissue cultures vaccinia virus multiplies, kills, and lyses cells; in multiplication on the chorioallantoic membrane, cells in the vicinity of virus infection actually proliferate. Whether the two types of phenomena really reflect different patterns of virus multiplication and of host response in the two cellular situations is not clear at present. In the synthesis of nuclear material present in a virus which is found to occur in an extranuclear site, it is possible that this virus has a greater measure of independence from the enzymes of the host than do many other viruses which also contain DNA. In addition to animal viruses of these types, there are the influenza viruses and poliomyelitis viruses whose compositions more nearly resemble cytoplasmic components and which appear to be capable of multiplying in Lhe cytoplasm of nondividing cells. The precise characterization of the biology of animal virus systems, including that of the tumor viruses, is only beginning; nevertheless it would appear that the relations of these viruses with their hosts will be most varied. Since the chemical phenomena to be observed in infected cells are a reflection of their biological relations, it can be anticipated that the most varied types of biochemical phenomena will be found and that particular chemical deviations should be sought which are associated with the requirements for the survival, multiplication, and release of the specific virus under scrutiny. A rational chemical virology, rather

11

COMPARATIVE BIOCHEMISTRY A N D VIROLOGY

than a casual exploration in this area, should rest then on a sound knowledge of the special biological and chemical attributes of the cell which becomes infected, as well as on information concerning the chemical composition of the viruses and the biological courses of the interaction of cell and infecting agent. TABLE 1 THESIZE, SHAPE,A N D NUCLEICACID CONTENT OF SOME PLANT VIRUSES Virus*

Dime!sions (A)

Tobacco mosaic 3000 X 150 Cucumber 3 and 4 3000 X 150 Potato X 4300 x 98 Tomato bushy stunt 300 Tobacco necrosis 240 Southern bean mosaic 320 Turnip yellow mosaic 218 Alfalfa mosaic 165

Shape Rigid rod Rigid rod Less rigid rod Sphere Sphere Sphere Sphere Sphere

Ribose Crystal- Refernucleic acid lized encet (Per cent) 5.8 5.8 5 14$ 16.5 21 350 15

+ + 0 + + + + 0

162 162 10 161 131 119 112 142

* The isolation of tobacco ring spot virus (160), omitted from the table, has been challenged by Pirie (132). He feels that the material obtained may have been normal ribonucleoprotein which has a composition similar to that ascribed t o virus. Steere has recently crystallized the virus and has found it to contain 34% RNA, a value substantially in agreement with that presented by Stanley. R. L. Steereunpublished work. t Given for ribose nucleic acid content particularly. 1This has recently been corrected t o 16.5% (de Fremery and Knight, J . Biol. Chem. in press). 0 However, note Markham and Smith (111). IV. THEFORMAND COMPOSITION OF THE VIRUSES The recent review of Knight (91) provides an excellent survey of existing data on the composition of the viruses. Many viruses have been isolated and subjected to chemical analysis and it is tempting to feel that one may begin to deduce certain principles from the data which have been amassed. It is necessary, however, to note that in certain areas, the data may be somewhat misleading, having been obtained on viruses which were relatively easy to isolate, such as the plant viruses described in Table 1, or on nonrepresentative viruses selected by historic accident, such as the T2, T4, and T6 bacteriophages. And of course, in almost every case, analysis proceeded with preparations of those viruses which were relatively stable, relatively easily concentrated, and in general which had properties which were attractive to the chemist,.

12

SEYMOUR 8. COHEN

For example, data are available on the size, shape, and ribose nucleic acid (RNA) content of nine different plant viruses as well as of strains of these viruses, as summarized in Table 1. The plant viruses appear to contain all of their phosphorus in RNA. None have been shown to contain significant amounts of thymine or deoxyribose. Many of these viruses will crystallize easily in two- or three-dimensional array, and, in electron microscopy, preparations have revealed a high degree of particle uniformity. It has been customary to refer to the plant viruses as relatively simple ribonucleoproteins. In this light then it is somewhat cautionary to examine electron micrographs of preparations of potato yellow dwarf virus which appear to contain particles which are markedly irregular in shape and far from uniform in appearance (20). It is necessary to be concerned, therefore, with the possibility that our experience with plant viruses does not warrant undue generalization.6

A . Plant Viruses Lauffer and Bendet have recently reviewed the problem of the hydration of virus particles (100). Nevertheless, a few examples may be given to indicate the wide variability observed within a single group, the plant viruses, with regard to their affinity for water and the manner in which water may be held. The plant viruses of the tobacco mosaic type contain relatively little bound water when suspended in an aqueous medium. Tobacco mosaic virus is considered to be hydrated to the extent of approximately 0.27 g. water per gram of dry virus. It has been suggested that most if not all of this water is held in a shell of water 6 to 7 A thick surrounding the virus particle (144). In contrast to the apparently external hydration of tobacco mosaic virus, certain spherical particles offer the possibility of both external and internal hydration. Tomato bushy stunt virus is estimated to hydrate in solution to the extent of about 0.77 g. water per gram of dry virus (124), and this is sufficient to alter the size of the nucleoprotein molecule significantly in passing from the dry to wet states. Internal and external hydration have been calculated as 0.27 and 0.50 g. water per gram of virus, respectively (102). Particles of the turnip yellow mosaic virus also appear to increase in volume when wet (44). Potato yellow dwarf virus (20) shows a marked dependence of sedimentation constant on the density of solvent. The hydrated density of this vinis has been estimated from such centrifugation experiments in 6

All plant viruses analyzed contain RNA. All insect viruses analyzed contain

DNA. Some plant viruses, e.g. wound tumor virus, etc., multiply in their insect vectors. Which nucleic acid will these contain?

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

13

sucrose to be 1.17 in contrast to a density of 1.27 for tobacco mosaic virus under comparable conditions. In contrast to tobacco mosaic virus, the potato yellow dwarf virus appears to be a more highly differentiated particle containing a limiting membrane which permits this virus to act as a tiny osmometer. The relationship of RNA to protein among the plant viruses is far from clear. In each instance examined, the nucleic acid is in a configuration which protects it from the action of ribonuclease and phosphatase. Separation of nucleic acid from virus protein renders the RNA sensitive to enzymes, and also appears to expose acidic groups on the nucleic acid (128). The nucleic acid of turnip yellow mosaic virus may be readily removed as a large polymer by treatment in the cold with 33% ethanol (111). The nucleic acid may be precipitated in the form of birefringent fibers. The nucleic acid-free protein component appears to have surface properties, i.e. electrophoretic mobility as a function of pH, serological reactivity, and crystallizability, which are identical with those of the intact virus. It has therefore been inferred that the virus protein comprises the external shell of the particle and that the nucleic acid is an internal constituent. Kinetic data are lacking on the molecular weight of this nucleic acid, but an end group analysis has yielded the value of 17,000 per end group (113), which has been interpreted by Markham and Smith as the true “average molecular weight” for this nucleic acid. The nucleic acid of tobacco mosaic virus and its strains may be removed by heating the virus at 100OC. for a minute at pH 5 (25) or by treatment with detergents (150). It is of interest that both procedures produce a highly polymeric particle of similar molecular weights, estimated as 290,000 in the first instance, 250,000 in the second. More significantly, heating of the RNA obtained by the use of detergents did not appear to affect the ultraviolet absorption spectrum. It is well-known that comparable treatment of DNA sharply increases the optical density a t the absorption maximum, reflecting disruption of hydrogen bonding and depolymerization. If the particle of 290,000 were entirely uncoiled, it may be calculated from the internucleotide distance of 3.3 d that the nucleic acid of this virus is present in several molecules, each of which is just about as long as the intact virus. However, a recent study by light scattering of the size of the particle freshly liberated from heated virus has suggested a particle weight of the order of 2 X lo6 (126). In this case it is supposed that substantially the entire nucleic acid is in a single coiled molecule. Although it has been suggested that the RNA lies within a protein tube, other workers have felt that the nucleic acid is oriented at the surface of the virus (91). For example in the reaction of tobacco mosaic virus with mustards, the nucleic acid reacts as well as the protein (23). If the internal

14

SEYMOUR S. COHEN

hydration of the virus particle were negligible, as is commonly believed, it is difficult to understand how the mustards could penetrate the virus to react with nucleic acid within a protein tube.(" In the case of some other plant viruses, such as tomato bushy stunt virus, it is extremely difficult to separate the nucleic acids from the viruses. Data are, therefore, not yet available concerning the size of the RNA of these viruses and their possible structural relations with virus protein.

B . Bacterial Viruses The literature on the size and shape of the bacteriophages infecting many different,bacteria has recently been surveyed (138). As a rule these viruses are tadpole-shaped, possessing a head and a tail. Since chemical data are available in the main only for the T set of bacterial viruses which infect Escherichia coli and related bacteria, the existing data on the size, shape, and elementary compositions of these viruses alone are presented in Table 2. From this table it can be seen that among the seven phages, there are four groups with respect to size. The phage heads are hexagonal, those of the T-even phages being the longest. However, the tails of T1 and T5 are longest whereas those of T3 and T7 are so short that they were not detected until recently (57). Despite the considerable disparity in size, direct analyses of preparations of the T phages suggest similar nitrogen and phosphorus contents, T3 and "7 having a slightly lower content of these elements (138). Stent and Fuerst (163) have determined the phosphorus content of the phages by a method involving the analysis of P82-labeled phages. From the assay of active labeled phage and specific radioactivity before and after adsorption to sensitive bacterial cells, it was estimated that phages fell into 2 groups, T2 and T5 possessing phosphorus contents identical with values presented in Table 2, while T1, T3, and "7 were a half to a third as great, being 0.7,0.9,0.9 X 1O-l' g. P per infective unit, respectively. Although the rate of inactivation by radioactive decay of the temperate phage, X, labeled with Pszsuggested a phosphorus content comparable to that in the second group, a precise value could not be obtained with this virus. Phosphorus contents of the order of the low phosphorus group for a virulent X phage from E . coli and a temperate A1 phage from Salmonella typhimurium have been recorded by Lwoff (107) in reporting unpublished work of his laboratory. The radiosensitive volume of the lysogenic phage X is about one-third that of T2, a result consistent with a markedly lower DNA content in X, although X and T2 are nearly equal in size (55). Among the T-even viruses, it has been shown that at least 99% of the 68 An elegant electron microscopic study of the structure of partially fragmented tobacco mosaio virus haa revealed a central core of RNA o. f . Hart, Proc. Natl. Acad. Sci. 41 261 (1965).

15

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

phosphorus is contained in DNA (28, 33, 67) and in T7 it has been shown that 95 % of the phosphorus of the preparation was nucleic acid phosphorus (45). Base analyses of T3 (138)) T5, and T7 (183) have indicated thymine but have failed to reveal uracil, a component characteristic of RNA. Similar results have been reported for a virulent X phage from E. coli and the temperate A1 phage from Salmonella typhimurium (107). Thus it may be assumed that the sole nucleic acid of these viruses is also DNA. Early electron micrographs of the T-even phages revealed the existence of ghosts, or particles with heads which were collapsed and were no longer dense to the electron beam. It was suggested a t that time that the heads TABLE 2 SIIES, SHAPES. A Vn ~ L E M E V T A R Y C O I M p n s T m w OF THE

Virus

Head* mp

T1 T2rf

50 95 X 65

T3 T4r+

47 95 X 65

T5 T6r+

65 95 X 65

T7

47

Shape* of Head Hexagon Elongated hexagon Hexagon Elongated hexagon Hexagon Elongated hexagon Hexagon

Tail*

mp

T

%CTERTOPHAGFS

Gram N per Gram P per Particlet Particlet X 10-18 X lo-''

150 X 10 100 x 25

0.8-1.3

2.1 2.2-2.4

15 X 10 100 x 25

-

1.7 2.3-2.4

170 X 10 100 x 2.5

0.8 0.8-1.1

1.8 2.4

15 X 10

0.5

1.5-1.7

* From Williams (177).

t These values are taken from the most active preparations reported in the literature. Other lower values for T1, T3, and T7 are discussed in the text. contained the nucleic acid of the virus and that this substance was in some way released in damaged particles. The release of DNA from the heads has been shown in a variety of ways. Osmotic shock (3) has been shown to rupture the phage in such a way as to release some of the DNA and render the remainder sensitive to the action of deoxyribonuclease (DNAase). The scrubbing of shocked particles with this enzyme leaves ghosts free of nucleic acid. Ghosts of this type have been isolated (66) and have been shown to consist of protein. Fibers containing nucleic acid have been released from the virus heads in another manner. Jesaitis and Goebel have isolated a lipomucoprotein antigen from dysentery bacillus Phase I1 Sh. sonnei which will inactivate all the T phages which attack this organism (81). It is believed that the antigen is the receptor substance for the bacterium. The lipocarbohydrate

16

SEYMOUR 8. COHEN

has been separated from the protein part of the antigen and is still capable of inactivating T3, T4, and T7. Addition of the lipopolysaccharide to a suspension of T phage in buffer at pH 6.8 at 37°C. results in a rapid increase in the viscosity of the suspension (82). Examination of the inactivated particles in the electron microscope revealed the presence of ghosts surrounded by filaments which may have been extruded via the tail. A preparation of this type is presented in Fig. 1. That virus DNA is injected into the host cell after adsorption, while the outer protein coat remains at the surface of the bacterium, has been demonstrated by the well-known experi-

FIQ. 1. Electron micrographs of T4 after treatment with Phase I1 Sh. sonnei lipocarbohydrate [through courtesy of Jesaitis and Goebel (82)].

ment of Hershey and Chase (69). Whether the viral material injected into the cell is free DNA or not will be considered below. Other agents are also capable of releasing material containing DNA-P from T2 bacteriophage. These include bacteria heated to 60 to 85°C. for 10 min., and the negatively charged surface of a cation exchanger (125). Herriott has recently reported that inorganic pyrophosphate will also produce this effect above pH 7 (68). Since a multiplicity of agents are capable of inducing DNA release, it will be difficult to establish that release effected by the lipocarbohydrate represents the phenomenon occurring at the cell surface. The use of concentrated urea to disrupt the protein coat of the phage and the subsequent purification of the polymeric nucleic acid were described by the author (29). This procedure has permitted the isolation of fibrous preparations of DNA in good yields from r and r+ strains of the T2, T4,

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

17

and T 6 viruses. It is not known whether concentrated urea alters the properties of the native DNA. I t may be noted, however, that concentrated solutions of urea are reported not to alter the transforming activity of DNA preparations from Hemophilus or Pneumoc,occus. Polymeric preparations of T-even DNA have also been obtained by the use of detergents (117). There are no satisfactory data on the molecular size and shape of the isolated DNA of the T-even phages, although several contradictory numbers concerning molecular weight have been published concerning the same studies on viral DNA (73, 139). Neither report of molecular weight 10,000,000 and 6,000,000, respectively, has been accompanied by a description of the isolation of the material or by data concerning its properties. .It is well-known that a precise estimation of the particle size of polymeric DNA is a very difficult matter, owing to particle interactions even a t very low DNA concentrations. Therefore, claims concerning this value require the most careful examination if they are to be believed, and if the values are to be used in estimating the numbers of DNA threads per phage particle (74) for the purpose of understanding genetic data in biochemical terms. Volkin (169) stated in a preliminary report that material released from T-even phage by osmotic shock contains DNA associated with protein, the latter accounting for 25% of the total phage protein. He has indicated that the released protein differs from ghost protein in important respects, e.g. sensitivity to papain, amino acid composition. However, Hershey states that he is unable to find norighost protein in phage lysates prepared by osmotic shock (75). Jesaitis has studied the threadlike material liberated from T4 by the lipocarbohydrate and has compared it with phage DNA which was liberated by freezing and thawing, and isolated after deproteinization (83). He reports that the intraviral material had a higher intrinsic viscosity than did the pure nucleic acid, and showed a greater tendency to form gels a t low concentrations. The intraviral material contained 16 % of protein, and Jesaitis concluded that this phage protein might indeed be linked t o the nucleic acid. Electrophoresis revealed two components in the intraviral material of which the major component had a mobility essentially identical with that of the free phage DNA. The minor component possessed a somewhat slower mobility.

C . Animal ;Viruses Among the animal viruses, as in the parasitic patterns which they present, a far greater degree of variability is apparent. This variability is evident in size, shape, and composition from virus to virus in their extracellular phases. For some of the viruses marked variability appears in the course

18

SEYMOUR 8. COHEN

of intracellular development as well. In studies of the animal viruses it has been a most difficult matter to obtain isolated preparations for which it could be stated that the bulk of the particles present were indeed virus. The infectivities of these preparations in many cases have been so low that it has almost always been possible to wonder whether the true virus particle is not obscured by the presence of apparently abnormal characteristic particles which are elaborated concomitantly with the true virus. These problems have been discussed and studied at length by Lauffer and his collaborators, particularly for the plant viruses (63, 99) and the rigorous approach to this question by these investigators provides an important frame of reference for the evaluation of statements as to the size and shape of biologically active materials in general. For the purposes of this survey it will be assumed that characteristic particles are virus particles, although this has rarely been demonstrated rigorously. On this assumption it is possible to indicate several subgroups among the animal viruses. Data on the size and shape of animal viruses have been summarized by Sharp (148). There are some relatively small viruses which approach a spherical shape; these include the influenza viruses, equine encephalomyelitis viruses, the virus of avian erythromyeloblastic leukosis, encephalomyocarditis virus, Coxsackie virus, poliomyelitis virus, and tumor viruses such as the Rous tumor virus and rabbit papilloma virus. Variations in the shape of the influenza viruses from spherical to filamentous forms have been recorded. Henle has discussed the significance of such forms as possible stages in the liberation of virus particles (64). The analysis of this subgroup is complicated particularly by the observation that particles of approximately similar size may often be isolated from normal cells, and a virus preparation concentrated by differential centrifugation may therefore be expected to contain impurities of such normal particles. In certain instances, as in the isolation of encephalomyocarditis virus (176), specific precipitation methods have been developed to eliminate normal particles. These are rare, however, and all preparations of influenza virus (26, 90), equine encephalomyelitis virus (53), the virus of avian erythromyeloblastic leukosis (11), and the Rous tumor virus (84) which have been examined, have been found to contain antigens characteristic of normal particles typical of the tissue in which the virus was elaborated. Several explanations of the presence of antigenicity similar to normal particles have been suggested. These hypotheses range from proposals of the presence of normal particles as simple admixed contaminants in the virus preparations or as adsorbed complexes with virus to the possibility that the antigens exist as integrated portions of the virus. At present the true relations of the antigenic reactivities of these viruses have not been clarified. It is evident that the analyses of virus preparations containing

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

19

antigens characteristic of normal tissue are of greater interest in so far as something is absent rather than being present. For instance, it is observed that preparations of the influenza viruses contain considerable lipid, even as do cytoplasmic granules. It is, therefore, not possible a t present to conclude that lipid is an essential integral unit of these viruses. An interesting problem has been posed for the nucleic acid content of the influenza viruses. Taylor had reported the presence of small amounts of DNA and has failed to observe RNA (167). Knight analyzed the PR8 strain and found both DNA and RNA, the latter in excess (90). Graham, in turn, examined the PR8 strain, and observed 4.5% RNA and 0.3% DNA (60). Most recently Ada and Perry find only RNA in the small amount of 0.78-0.98% (1). It is difficult to conceive of more disparate results; however, the increasing experience in analyzing for nucleic acid plus the failure t o observe DNA assists in placing more credence in the analyses stressing the presence of small but significant amounts of RNA in these preparations. Equine encephalomyelitis virus is also reported to contain only RNA (166). Of course it is difficult to assess the significance of RNA in preparations possessing antigens of normal tissues, which in their turn are known to contain RNA. The Lansing strain of poliomyelitis virus has been prepared in a concentrate containing highly uniform particles ( 5 ) . It has recently been reported that such a concentrate of a human strain of this virus contains only RNA (146). Serological cross-reactivity with antisera to normal antigens has not yet been tested. The rabbit papilloma virus has been isolated in a high degree of homogeneity with respect to particle size and shape. These preparations, despite the presence of an occasional viscous contaminant, contain only DNA, and this virus is therefore chemically distinct from the other small, approximately spherical animal viruses. From a molecular weight of 47,000,000 (125), and a DNA content of 8.7 % (165), it can be calculated that a single molecule of particle weight 4,000,000 could comprise the entire DNA of this virus. Other animal viruses appear to be larger and more complex structures, possessing one or more enclosing protein membranes which are laid down in differentstages of the development of the virus. It had long been known that in their extracellular phases the ‘elementary bodies of the vaccinia virus and of the fowl pox viruses were brick- or cylindrical-shaped bodies containing a central dense nuclear structure (151). Recent studies with the electron microscope on the development of these viruses have revealed their existence as elliptical structures enclosed by single membranes when present within the cell. Before release from the cell, the particles appear to add still another limiting membrane (122). A similar phenomenon has been

20

SEYMOUR 8. COHEN

noted for the virus of herpes simplex which appears to multiply in the nucleus. A second outer membrane is acquired on release into the cytoplasm and the particles then appear to represent mature virus (121). Vaccinia virus contains only DNA in the amount of 5.6 %. Since DNA is strictly a nuclear constituent, it is of interest that this virus has an intracytoplasmic development. Smith (153, 154) has also considered the variability of size and shape found in the different viruses, paying particular attention to the insect viruses. Recent data on the insect viruses have been reviewed by Bergold (14). The insect viruses known to date consist of rods and spheres. The rods of this group are short and thick, and are larger than the plant viruses. The spherical insect viruses are also considerably larger than the spherical plant viruses. In both types of insect virus, the virus particle body is usually encapsulated by tough specific protein coats which may be solubilized in dilute alkali. These coats or capsules assume approximately regular geometric configurations, producing the polyhedral bodies characteristic of the polyhedroses or assume a less regular oval or elliptical shape observed in the capsular diseases or granuloses. Only a single instance is known of an insect virus which lacks capsular material and does not form inclusion bodies. This is a more-or-less regular spherical to slightly ovoid body of about 25 mp found in diseased larvae of the cosmopolitan armyworm (172). The oval-shaped capsular bodies characteristic of the granuloses contain rod-shaped viruses within adhesive developmental membranes. According to Smith (154), 2 types of polyhedral viruses are known, those containing rods which originate in the nucleus, while the relatively rare encapsulated spheres form in the cytoplasm. In the latter case each spherical particle consists of 4 or 5 very small spheres (156). However, Bergold has also described an intranuclear polyhedral inclusion body containing spherical bodies (14). In the classical silkworm virus disease, free rods appear to be generated in the clumped chromatin of the nucleus, and capsular proteins are deposited on the rods in the vicinity of the nuclear membrane. Bergold and Wellington have isolated and analyzed the proteins of the three distinct components, the virus, the virus membrane, and the polyhedral protein (15). Only the virus rods appear to contain significant amounts of nucleic acid (7.9%);as in all insect viruses so far examined, this is DNA. It should be noted that, in attempting to analyze the reproductive cycle of a nuclear polyhedral virus on the basis of electron microscopic studies of early and late virus forms, Bergold has concluded that the rod was a mature form of a genetically continuous spherical organism which multiplies

COMPARATIVE BIOCHEMISTRY A N D VIROLOGY

21

by growth and fission. On the other hand Smith and Xeros (155) disagree with this conclusion and have presented evidence that there are not in fact spherical forms but rather two types of rods, one of which is about half the length of the most frequent rod. Both may occur in the same nucleus and polyhedron and no evidence is available to show that the shorter rod is really infectious. They believe that the insect viruses are differentiated products of altered cell metabolism rather than independent organisms. Nevertheless these workers conclude that “there is no a priori reason to suppose all viruses are identical in their general nature and developmental properties.” The possibility of an increasing independence of growth and multiplication is posed more sharply in considering the form and composition of the viruses of the psittacosis group and of the rickettsiae. As noted above, in a given polyhedrosis within a single cell several sizes of rods appear to be generated. Although intermediate stages of virus synthesis are being encountered in studies of the plant viruses, the phages, vaccinia, etc., the narrow size range observed in preparations of these viruses isolated at terminal stages of infection has been used to argue that they do not reproduce by a process of growth and division, similar to that observed in bacteria. However, among the organisms of the psittacosis group, the size ranges and developmental forms have been interpreted as evidence for a mode of reproduction more nearly resembling that of a cell (12,123). The rickettsiae are regarded by most workers as more akin to bacteria than to the viruses, and electron micrographs of these organisms reveal bacillary forms which are often indistinguishable from true bacteria (133). I n contrast to the “true” viruses, all of which appear to contain but a single type of nucleic acid, the data on the psittacosis group and on rickettsiae suggest the presence of both RNA and DNA. This chemical characteristic would align these intracellular parasites with cells, all of which would also appear to contain both nucleic acids. Zahler and Moulder have reported the presence of both RNA and DNA in purified preparations of feline pneumonitis virus (185)) a virus of the psittacosis group. Analysis of a preparation of meningo-pneumonitis virus revealed only DNA (183); however, a careful search for RNA was not made in the small amount of material available. Moulder reports a personal communication of Crocker to the effect that meningo-pneumonitis virus does contain both nucleic acids (123). Only DNA was revealed in analysis of purified preparations of killed Rickettsia prowazeki isolated after prolonged periods of incubation in commercial typhus vaccine. This nucleic acid was isolated (32). However, in cytochemical studies of freshly isolated rickettsiae it was observed that

22

SEYMOUR S. COHEN

the cells stained with pyronine; this has been interpreted to signify the presence of ribose nucleic acid in R. prowazeki (141). Smith and Stoker have found both RNA and DNA in Rickettsia burneti (152). With regard to the criterion of the presence of enzymatic equipment it would appear that at least the rickettsiae also more nearly resemble cells, a conclusion which is held widely for both groups as a result of the morphological evidence, despite the fact that a t present no member of either group has been observed to multiply in the absence of intact cells. The viruses, therefore, present many kinds of morphological and developmental complexity; these range from particles such as the plant viruses, of apparently predominantly cytoplasmic origin and a nucleic acid more nearly characteristic of cytoplasm, i.e. RNA, to viruses such as the phages or insect viruses which appear to behave as nuclear parasites, and possess a typically nuclear constituent, DNA. In addition, there is vaccinia virus of cytoplasmic origin containing DNA, or herpes simplex virus which has many morphological similarities to vaccinia, e.g. the double external membrane and central nuclear body, but multiplies in the nucleus and perhaps matures in the cytoplasm. Finally, we may point to intracellular parasites such as members of the psittacosis group and rickettsiae which are probably more complex than the foregoing. These resemble cells in certain stages of their life cycle and perhaps possess the two nucleic acids characteristic of both cytoplasm and nucleus.

V. ON THE FINESTRUCTURE OF VIRALCONSTITUENTS The evidence is poor that virus particles contain essential structural components other than proteins or nucleic acids, e.g. lipids or polysaccharides, as intrinsic units of the infectious particle [see Knight (91)]. This problem arises and is unresolved in cases such as the discovery of traces of lipid in vaccinia virus or in rabbit papilloma virus. A more serious question is posed for particles such as influenza virus or equine encephalomyelitis virus, which contain large amounts of a complex lipid mixture. As noted earlier, the presence of normal tissue components in virus preparations obscures the significance of the presence of the lipids or of the extranucleic acid carbohydrate discovered in influenza virus by Knight. In the absence of clarity on this question, therefore, this discussion will be confined in the main to aspects of the structure of substances shown to be present in all viruses, protein and nucleic acid. In recent years data on the role of DNA in the multiplication of and parasitism of the phages and in bacterial transformations have been of exceptional interest and have relegated to the background certain facts concerning the plant viruses. Knight (91) has been almost alone in re-

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

23

turning to these facts; this author agrees that it is necessary to underline and develop certain points in this area. The plant viruses contain RNA and the total weight of such RNA in a virus particle (<3,00O,OOO)is less than that frequently ascribed to a single molecule of DNA. The analyses performed by such workers as Knight, Markham and Smith, Loring, etc., would easily reveal DNA in the amount of 5 % of this amount of RNA, but have not succeeded in doing so. It has quite properly been concluded that the plant viruses do not contain DNA. Nevertheless, it is apparently seriously posed (74) that plant virologists ought to make sure of the absence of DNA in the plant viruses on the ground that DNA in the model of Watson and Crick (173) is obviously the genetic material of choice. It has been remarked, perhaps facetiously, that this problem of the role of DNA is now of ideological importance (74), and it would appear that this can be pressed to the point of ignoring a considerable body of excellent data on the structure of the plant viruses. It is of course a consequence of the existence of such data that chemical virology must contend with at least two different mechanisms of duplication and genetic determination, if nucleic acid is to be considered as of primary interest in genetic determination, and if the multiplication of the viruses can be shown to be associated with differences in their content and composition of the nucleic acids. Finally in the problem presented by the plant viruses the situation is even more serious than this, since if we did not possess preconceived notions concerning the role of nucleic acid, the existing data would tend to support the view that the proteins are of primary interest in genetic determination. The well-known amino acid analyses of Knight and his collaborators in the comparison of strains of tobacco mosaic virus and of cucumber mosaic virus have revealed marked differences in amino acid composition among these viruses (91). It has not been possible to state that a distinct chemical change accompanies a definable mutation of the virus, but only that genetically distinct strains possess the observed differences. Nor has it been possible as yet to correlate differences in protein composition with specific biological properties. In any case, these differences in protein composition are considerable, as in the possession of the HR strain of TMV of two amino acids, methionine and histidine, not found in the other strains, or in considerable quantitative differences in the amounts of various amino acids. Indeed only three amino acids, cysteine, leucine, and proline appear to be constant in all strains of TMV. Wang and Commoner (171) have recently found that in plants with the tobacco mosaic disease there are present infectious nucleoproteins of comparable size, shape, serological specificity, and RNA content, of which

24

SEYMOUR 8. COHEN

one was the classical tobacco mosaic virus (TMV). The second nucleoprotein was distinguished from TMV by solubility properties, electrophoretic mobility, and amino acid composition. By contrast the RNAs of the strains of TMV possess the same base compositions, within the limits of the sensitivity of the analytical procedures employed (91). The data of Knight on this score have recently been confirmed (43). The nucleic acids of two strains of potato virus X are also very similar in composition (114) as are the RNAs of 3 strains of tomato bushy stunt virus (91). Of course the analytical procedures do

Yeast

Turnip Yellow Mosaic Virus

Tobacco Mosaic Virus

FIG.2. The types of end groups present in RNA derived from different sources.

not permit the assertion of absolute identity in percentage of the bases n a molecule since a 1 % error in the estimation of any base of TMV would amount to about 50 to 60 residues of purine or pyrimidine. Most important, identical base contents are irrelevant to the problem of the order of the bases, and at present this possibility of position isomerism provides the thread held by proponents of a primary role of RNA as a genetic determinant of the plant viruses. In analyzing the nucleic acids of turnip yellow mosaic virus, Markham and Smith have demonstrated that the relative positions of the bases may be determined in a polynucleotide chain of 53 ribose nucleotides (113). In addition, a number of different types of end groups on polynucleotide chains have been detected (115) as presented in Fig. 2. In

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

25

the RNA of turnip yellow mosaic virus, an end group has been identified as a cyclic adenine nucleotide. In view of the availability of methods for the estimation of such fine structure of RNA,’ the hypothesis that the ribose nucleic acids of strains of a given virus are position isomers, should be susceptible of test. Knight has presented data on the base compositions of all the plant viruses for which data is available (91). A qualitative representation of these differences is presented in Fig. 3. Similarly extensive studies have not been performed with strains of bacterial or animal viruses. Amino acid analyses have been reported only for T3 (56) and two T-even phages, T2 and T4 (105), among the bacterial viruses. I t is a matter of some interest that despite the known presence of a,c-diaminopimelic acid in the bacteriai host, E. coli, the pres-

FIG.3. The nucleotide composition of ribonucleic acids of (1) potato virus B, (2) potato virus XL, (3) tomato mosaic virus, (4) cucumber virus 4, (5) tobacco mosaic

virus, (6) tomato bushy stunt virus, (7) turnip yellow mosaic virus, and (8) yeast nucleic acid. The sectors are proportional in area to the molar ratios of nucleotides G = guanine, A = adenine, C = cytosine, U = uracil lthrough courtesy of Markham (11411.

ence of this amino acid in these phages is not recorded, nor is its absence commented upon. I n the analyses presented by Luria, it appears possible that T2 differs from T4 a t least with respect to their content of glycine, serine, arginine, lysine, and histidine. Amino acid analyses have been recorded of the virus producing polyhedral disease of the silkworm, of the intimate developmental membrane, and of the capsular proteins (15). It has been shown that each of these structures produced in virus-infected cells may be distinguished by their content of amino acids. From this it may be inferred that the specific protein metabolism of the infected cell may be expected to differ as a function of the particular stage of infection under examination, and of viral structure being produced. Within a single cell, nuclear polyhedra 7 It must be noted, however, that the use of enzymes in this analysis assumes that the enzymes do not create new polynucleotides by transfer reactions. Ribonuclease is capable of such reactions, although it is used in the analytical procedure.

26

SEYMOUR 5. COHEN

appear to be formed simultaneously after elaboration of the free virus rods. The relative lack of data on the proteins of the bacterial and animal viruses appears to be related Lo the difficulties of purification of some of these (33), but perhaps in the first place to the current emphasis on nucleic acid. Extensive data are available on the base compositions of the DNA of a number of bacterial viruses and of animal viruses, particularly of the insect viruses. In the former instance, as in the case of the plant viruses, despite marked differences in amino acid compositions the base compositions of the nucleic acids of the strains of the T-even viruses are indisTABLE 3 O F DNAn O F SOMEBACTERIAL VIRUSES BASECOMPOSITION Moles/100 moles estimated bases ~~

Virus* T2r+ T2r T4r+ T4r T6rC T6r T5 T3

Adenine 32 .O 32.3 32.3 32.2 32.5 32.3 30.3 22.8

Thymine 33.3 33.4 33.1 33.5 33.5 33.4 30.8 27.8

Guanine

5(Hydroxymethylcytosinet

18.0 17.6 18.3 18.0 17.8 17.7 19.5 23.5

16.8 16.7 16.3 16.3 16.3 16.6 19.5 26.3

-

* The data for all viruses except T3 are taken from (183); that for T3 is taken from (138). t T3 and T5 contain cytosine, unlike T2, T4, and T6. tinguishable (183). The DNAs of these viruses contain a base, 5-hydroxymethylcytosine, not as yet observed in any other virus or in the nucleic acids of any cell, including the bacterial host, E. coli. The base compositions of these viruses, and of T3 and T5 which contain cytosine, are presented in Table 3. The T-even phages are clearly distinguishable from T3 and T5 in their contents of bases other than hydroxymethylcytosine. All of these differ markedly from the composition of the DNA of the host bacterium, E. coli, in which the four bases are approximately equivalent in amount. The determination of the order of nucleotides in R polynucleotide of DNA has not yet been solved. Wyatt has presented analyses on the base compositions of eleven insect viruses (180). Selected data on the insect viruses are given in Table 4. From these data it can be seen that polyhedral viruses contain nucleic acids which may vary widely from those of the adenine-thymine type

27

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

(silkworm virus) to that of the guanine-cytosine type (gypsy moth virus), with a representabive approaching an intermediate statistical tetranueleotide as well (spruce budworm virus). In addition, different viruses may present indistinguishable DNA contents as in the case of the polyhedral viruses infecting the forest tent caterpillar and that parasitic on the silkworm, or the polyhedral virus of the pine sawfly and the capsule virus of Cacoecia murinana Hb. It is evident that the base compositions of these viruses may not be correlated at the present time with their forms or host ranges. TABLE 4 BASECOMPOSITION OF DNAs OF SOMEINSECT VIRUSES /Ioles/100 moles estimated bases Virus type

Polyhedral

Host species

Gypsy moth Spruce budworm Forest tent caterpillar Silkworm Pine sawfly

Capsule

Host order and family

Lepidoptera Lymantriidae Tortricidae Lasiocampidae

Bombycidae Hymenoptera Tenthredinidae Cacoecia muri- Lepidoptera Tortricidae nana Hb. Tortricidae Spruce budworm

AdeThyGuanine nine mine -21.2 20.05 30.5

Cytosine 28.25

24.8

24.0

26.7

24.5

29.2

28.5

21.9

20.3

29.3 32.3

28.0 30.3

22.5 19.5

20.2 17.9

32.1

30.5

19.7

17.8

32.8

32.4

18.4

16.4

The data on the nucleic acids of other animal viruses are sparse (Table 5), although some information is available on rickettsia1 DNA. An apparent similarity of composition of the DNA of R . burneti grown in embryonated eggs t o that of the DNA of chick embryos had been noted, the only significant difference being a lack of 5-methylcytosine from the former. On the basis of this similarity, it has been suggested by Smith and Stoker (152) that rickettsiae may incorporate intact nucleic acid directly from their host. -4s can be seen in Table 5, this is unlikely since the DNAs of different rickettsiae grown in the same host (134, 181) are markedly different in base composition. The base compositions of DNA, as summarized in Tables 3 to 5, in most instances tend to support the generalization that the content of adenine approximates that of thymine, and that of guanine approaches

28

SEYMOUR S. COHEN

that of cytosine or of a cytosine equivalent such as hydroxymethylcytosine. An exact equivalence between two bases is almost never observed but it is difficult to be certain of the analytical significance of the small differences obtained. It is possibly meaningful that in analyses performed in the same laboratory among six T-even viruses, the content of thymine always exceeds that of adenine, whereas these values are closer in T5 and vaccinia. Adenine is slightly greater than thymine in the DNA of R . prowazeki. In any case, the analyses of DNA do not contravert the requirements of the Watson-Crick two-strand model for DNA, a model also supported in many particulars by the existing X-ray evidence. The suggestion of Watson and Crick that pairing of bases plays a key role in the duplication of DNA (173) is therefore also not invalidated by these data. TABLE 5 OF SOME RICKETTSIAE AND

THE BASECOMPOSITIONS OF THE DNAs

OF

VACCINIA

VIRUS Moles/100 moles estimated bases DNA anslysed

Adenine

Thymine

Guanine

Chick embryo Rickettsia burneti Rickettsia prowazeki Rickettsia rickellsii Vaccinia virus

29.3 29.5 35.1 33.4 29.5

26.5 26.0 34.1 29.2 29.9

24.0 22.5 15.7 19.4 20.6

Cyto- 5-Methyl- Refersine cytosine ences 20.5 22.0 15.1 18.1 20.0

0.93 <0.2 -

152 152 181 134 183

However, an examination of the base ratios of the RNAs of plant viruses (91) (Fig. 3) demonstrates unequivocally that paired bases of this type do not exist in these viruses. For example there is more than twice as much cytosine as guanine in the RNA of turnip yellow mosaic virus. X-ray analyses of preparations of RNA have suggested that this substance does not exist as a double-stranded coil (140), and we are led to conclude that the structure of RNA possesses an organization and geometry different from that of DNA, i.e. RNA is not merely a DNA containing ribose. If duplication of DNA in the DNA viruses truly involves the separation of strands and the complementary pairing of new nucleotides to a single strand, as postulated by Watson and Crick, it follows that the duplication of RNA in an RNA virus must be a different kind of process. Thus, the analytical evidence suggests that several modes of polymer synthesis are to be anticipated, depending on whether we are concerned with an RNA or DNA virus.

COMPARATIVE BIOCHEWSTRY AND VIROLOGY

VI. THE METABOLIC EQUIPMENT

29

VIRUSES The mechanisms of the synthesis of polymeric nucleic acids and proteins are unknown at the present time. Further, although it is conceivable that an isolated polymer may catalyze its own duplication when supplied with the appropriate intermediary metabolites, it is not known what these metabolites may be, nor how to test for such synthetic capabilities. It is not possible, therefore, to assert that a virus does or does not contain the enzymes for the catalysis of polymer synthesis. However, it is probably significant that an insect virus develops in the nuclear chromatin, unlike vaccinia virus which develops in the cytoplasm. The former may require the special polymer-synthesizing capacities of the host for making deoxyribonucleoprotein; the latter may be capable of this feat by itself. Until the present, biochemistry has been concerned in the main with the problem of the origin of the intermediary metabolites and of the fate of the polymers, rather than of the fate of metabolites in the origin of the polymers. Concerning the presence of the metabolites and the enzymes which catalyze their formation and reactions a t levels other than that of polymer synthesis, it may be said that the plant and bacterial viruses appear to lack these substances. Thus tobacco mosaic virus does not contain significant amounts of biotin, riboflavin, and pantothenate (158) and the equivalence of total phosphorus and nucleic acid phosphorus excludes the presence of phosphorylated intermediates from the plant viruses. Among the bacterial viruses, the acid-soluble phosphorus is similarly so low (1% or lower) as to raise doubts concerning its reality as non-nucleic acid phosphorus (138). It has been suggested, therefore, that the cell supplies the metabolites essential for polymer synthesis. Consistent with this proposition is the fact that these viruses have not been found to be capable of any reaction which will provide essential metabolites or energy-rich compounds important in the fashioning of essential metabolites. It has been concluded that viruses utilize the enzymatic equipment of the cell for their own synthesis and multiplication, and that that requirement for this complex equipment defines the dependence of virus multiplication on the presence of intact cells. Among the animal viruses, several enzymatic activities have been observed. Thus adenosinetriphosphatase (ATPase) has been found in concentrates of avian erythromyeloblastic leucosis. The activity goes handin-hand with virus activity. However, since these concentrates contain normal host antigens and t,here is no hint of a possible role for this enzyme, the significance of the presence of the enzyme is obscure ( 1 1 ) . On the other hand, a mucinase has been found in preparations of influenza virus (58). Although this virus also contains normal host antigens, OF THE

30

SEYMOUR 8. COHEN

it is believed that the enzyme may possess a role in the penetration of the virus into the cell or in its release. There is no well-supported evidence that the mucinase plays a synthetic role in the assembly and duplication of the virus. Although several enzymes have been reported for preparations of vaccinia virus, it is believed that these are adsorbed to virus from the tissue of origin (76). Despite in vitro phosphatase activity, the virus particles do not show this activity when tested by a histochemical technique in situ (178). Similar contamination of tumor viruses by adsorbed hemin has been observed (24). A variety of other substances of metabolic interest has been observed in vaccinia virus. These include the coenzyme flavine adenine dinucleotide (FAD), copper, and biotin. The concentration of these substances on virus under conditions in which other trace materials are eliminated has led to the view that these substances may well be intrinsic components of virus (151). Nevertheless, an enzymatic activity associated with the presence of these coenzymes has not yet been detected. The metabolic role of biotin is still unclear, and a method of testing for an enzyme using biotin is still unknown. Nevertheless, more recent data on the relations of biotin suggest numerous exploratory tests, e.g. the possible existence of biotin in vaccinia as the sulfoxide or in the peptide, biocytin or biotinyl lysine, or the effect of analogues of biotin on virus multiplication in tissue cultures. Similarly, the increased knowledge of the last fifteen years on the activities of FAD in a variety of oxidases (168) suggest many additional tests for particular enzymes containing this coenzyme, and indeed for enzymes in which both FAD and copper or other metals act as prosthetic groups in the same enzyme (109). Although vaccinia virus does not appear to be able to respire independently in the presence of hexose phosphate or p-phenylenediamine or to contain cytochromes or some common dehydrogenases, there is no indication that suitable substrates, e.g. butyryl-coenzyme A, requiring a metallo-flavoprotein catalyst, have been offered. In the case of vaccinia virus, which appears not to require thc host’s nuclear apparatus to synthesize its own nuclear apparatus, and contains metabolically active units at the coenzyme level, it would seem safer to operate on the assumption that the deficiency lies with the biochemists rather than with the virus. Recently it has been shown that herpes simplex virus is inactivated by tissue phosphatases, and that virus survival is promoted by phosphatase inhibitors and substrates (2). Such a result suggests the presence in or on this virus of an essential phosphorylated compound other than internally oriented nucleic acid. Among viruses of the psittacosis group and the rickettsiae, the sensi-

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

31

tivity of infections by these agents to antibiotics and chemotherapeutic agents, such a s the sulfonamides or p-aminobenzoic acid, respectively, has been suggested as being due to the presence of enzymes sensitive to these agents. However, unlike rickettsiae, a suspension of psittacosis virus does not catalyze oxygen uptake in the presence of glutamic acid, casein hydrolyzate, and succinic or pyruvic acids (130). The multiplication of feline pneumonitis virus is inhibited by metabolic poisons such as fluoride, azide, and dinitrophenol, but these agents may act by inhibiting the growth of host cells rather than by a direct effect upon virus enzymes (123). Feline pneumonitis virus lacked hexokinase and systems for glycolysis as well as a system for oxidation of glutamate. Direct evidence of enzymatic activity is available only for rickettsiae, which consume oxygen with glutamate or other compounds as a substrate (17). Glucose is not metabolized by rickettsia1 suspensions. Enzymes such a s esterase, ATPase, and catalase could be eliminated from R. prowazeki by treatment with antisera to host materials, without affecting glutamate oxidation (85). It appears that rickettsiae may possess a Krebs cycle (18). Fumarate, malate, oxalacetate, citrate, and butyrate are not metabolized by R. prowazeki (18); this is possibly solely a matter of the lack of penetration of these substrates into this organism. However, Price has observed the oxidation of glutamate, a-ketoglutarate, succinate, pyruvate, oxalacetate, fumarate, and malate by suspensions of R. rickettsiae (134). I n addition, it has been observed that diphosphopyridine nucleotide and coenzyme A restore infectivity to rickettsiae inactivated by freezing. Recent analyses have indicated that these coenzymes are normally within the. rickettsiae and are lost when the cell membrane is damaged by freezing (19). In view of these evidences for metabolic systems among viruses infecting animals, culminating in the unequivocal possession of glutamate oxidation among the rickettsiae, it is perhaps justified to imagine that the increasing structural complexity among the viruses is perhaps paralleled by a comparable content of metabolic equipment and a relative metabolic independence. The dearth of reliable data on composition and enzyme content of the viruses, however, precludes more than a most tentative offering of this hypothesis at the present time. It would seem to be of considerable importance to fill out these data, not merely by a random test for the presence of known enzymes, but particularly by testing for those metabolic systems which may be related to the particular pattern and site of development of a given virus. How does a virus enter a cell, what are the special properties of the cell surface, and how may this surface be breached? Does virus multiplication occur in the nucleus, and does the virus contain enzymes which can handle substrates characteris-

32

SEYMOUR 8 . COHEN

tically present in the nucleus? Nuclei have a predominantly anaerobic metabolism and do not appear to possess flavoprotein enzymes; will viruses other than vaccinia, e.g. herpes simplex or the insect viruses, contain FAD? What enzymes are normally associated with the host antigens present in preparations of influenza virus, etc.? Are these enzymes also associated with the virus, and if so, is the virus inhibited by inhibitors of these enzymes? And so forth. Existing data are sufficiently diverse to permit more penetrating questions to be asked than have commonly been raised concerning the metabolic requirements of a virus, and the parts of the cell in which they multiply. It may be suggested that we are glutted with experiments on the effect of cyanide or dinitrophenol on virus multiplication in the chick. The known differences among the viruses should improve the design of our questions and increase the proportion of significant experiments. If, for example, we wished to know if isolated nuclei, supplied with many essential metabolites, can support the multiplication of virus, it would be important to choose a virus known to multiply in the nucleus and to know enough about the metabolism of the nucleus to permit a reasonable guess concerning metabolites which ought to be supplied. It would not be anticipated, for example, that this could be done with TMV or that oxygen would be a useful or necessary addition.8 RELATIONS OF VIRUSSTRUCTURE AND ENZYME CONTENT TO VII. SOME VIRUS ENTRANCE AND RELEASE Plant viruses must be placed inside a cell for multiplication to proceed. The injection procedure in nature can be effected by the mouth parts of insects or perhaps by some penetrating parasite, such as dodder. Transfer from cell to cell is often effected by intracytoplasmic threads or plasmodesmata, thereby obviating the requirement of virus liberation and cell reinfection. Virus particles may enter abraded areas of the plant and the use of abrasives on plant leaves in facilitating infection is widely employed in experimental work. These viruses do not possess enzymes for the degradation of the very tough cell walls and outer coverings. The plant viruses are naked particles and do not possess dissociable membranes which are left outside of the infected cell. The random mechanism of the entrance of a plant virus into a susceptible cell appears to be paralleled by the observed chemical simplicity of these infectious agents. The cell wall of a bacterium is always a rigid complex structure. It has been remarked by Stanier that the cell wall appears to serve as a cor8 If a virus multiplied in the nucleus and required 0 2 for this, it would be probable that the virus provided its own systems for 0 0consumption. It may be mentioned that R . rickettsiae multiplies intranuclearly.

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

33

set which prevents the rupture of bacteria which may result from osmotic differences between internal and external environments (159). The transfer of the essential materials of a bacterial virus into a tiny bacterium, therefore, requires a specialized mechanism for the penetration of the tough bacterial cell wall and possibly of the membrane as well [for a discussion of the differentiation of these structures see (15911. The differentiation of bacterial viruses into adsorbable microsyringe and essential innards is a consequence of the requirements of this phase of the life cycle. Those viruses possessing an extracellular phase have survived only if they have solved the problem of injection by evolving the characteristic microsyringe structure. The bacterial viruses, therefore, must possess a greater degree of chemical differentiation than do the plant viruses. The properties of the adsorbable microsyringe include a single adsorption site which is a t the tail of the phage. This adsorption site is believed to be serologically distinct from the remainder of the phage (97), and reacts specifically with a unique receptor on the bacterium, perhaps comparable to the lipocarbohydrate described by Jesaitis and Goebel. The number of such attachment sites on the bacterium is limited, about 200 (47, 143), and they are presumably localized a t parts of the cell which may be breached in the course of injection. The primary attachment is electrostatic, facilitated by certain ionic environments, and is readily reversible. Studies by Puck and Tolmach have indicated that positively charged amino groups on T2 bond to carboxyls on the cell surface (137). The attachment of T1 is considered to involve both amino and carboxyl groups of this virus. A second irreversible step follows having properties of an enzymatic reaction. Following adsorption of phage particles to isolated cell walls, Weidel observed tiny holes in these cell walls, suggestive of an eating away of the wall substance by a n enzyme of the virus (175). It has been reported that T1 or T2 infection of E. coli does induce a marked initial change in cell permeability resulting in leakage; the leakage is subsequently blocked by a repair mechanism (136) presumed to require the synthetic mechanisms of the host. Puck and his collaborators believe that the degradation of cell wall producing this leakage is affected by cell enzymes triggered by viral amino groups. Such an effect leading to cell lysis may be caused by certain amino-containing substances in the absence of infection (135). It has been reported that nitrogen-containing compounds of the isolated cell walls of E. coli may he solubilized by T2r+ adsorption, the amount of nitrogen soluhilized being approximately proportional to the numbers of T2rt particles adsorbed (8). This result is interpreted to suggest the existence of a viral enzyme active in penetration. However, another laboratory has been unable to confirm this result (68). It is not certain,

34

SEYMOUR 6. COHEN

therefore, whether the effect of virus adsorption and penetration on the permeability of host involves an enzyme in the cell, in the virus, or in both. The nature of the precise stage in this complex series of events at which release of the internal virus components occurs is unknown. The mechanisms of this release and of transfer to the interior of the bacterium are similarly obscure. In contrast to the plant and bacterial viruses which have no or one adsorption site, respectively, many animal viruses, such as influenza or vaccinia viruses have at least two adsorption sites. Influenza virus, when present in sufficiently high ratio to sensitive cells, will agglutinate these cells; the phenomenon of hemagglutination requires multiple attachment sites on virus and cell. Despite the far larger size of the chicken erythrocyte, the maximal number of receptor sites on this cell for influenza virus is approximately similar to that of the number on E. coli for T2 phage (143). In the latter case, the number of sites is approximately equal to the number that can fit in close packed array on the area provided by the bacterial surface. Thus there is a striking difference in the per cent of cell surface area occupied by virus receptor sites in the two kinds of systems. The primary attachment of influenza virus t o the erythrocyte is comparable in mechanism to the adsorption of T2 to bacterium; the attachment similarly possesses a low temperature coefficient and is markedly dependent on salt concentration. On the red cell at least, influenza virus is not induced to liberate nucleic acid; the virus may be released in essentially unchanged active condition. Such release can be effected by an enzyme of the virus which destroys the receptor sites of the red cell. On the allantoic membrane of the chick adsorbed virus soon disappears, as determined by efforts to release infectious units or by attempts to detect viral antigens by serological methods (78, 80). Thus an external coat comparable to the external coat of T2 does not appear to be left at the cell surface. The host cell in this instance possesses a limiting membrane which is perhaps more like that of an enveloping amoeba than the tough cell wall of a bacterium. Even virus heated to destroy the eluting enzyme disappears from view in these cells, as detected by the inability of adsorbed heated virus to absorb antibodies (80). This result implies a lack of function for the eluting enzyme in penetration. Another marked difference between bacterial viruses and influenza virus resides in the fact that the liberation of all phages in a single cell occurs simultaneously as a result of cell lysis whereas liberation of various animal viruses from a given cell is a slow process taking many hours (52, 65). Influenza virus is liberated initially within long filaments derived from the cell surface; it is thought that these are subsequently subdivided to the size of characteristic virus particles, Indeed Hoyle has concluded that a

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

35

particle of influenza virus is an aggregated mass of virus protein enclosed in cell membrane (79). It is quite conceivable that the wrapping of the cell membrane about virus is responsible for the presence of host antigens in preparations of this virus. It is possible that this manner of liberation from an infected cell is related to the phenomenon of liberation or of elution of influenza virus subsequent to adsorption on the red cell, even though the eluting enzyme appears to play no role in the penetration or in the engulfment of the virus particle. Several workers (104, 145) have reported cyclical changes in the concentration of intracellular receptor substance, with marked decreases during active virus multiplication; the significance of these findings is obscure a t present. The functional role of the receptor-destroying enzyme of influenza virus is not known. However, the nature of its activity on its substrate is being clarified. The enzyme is a mucinase, and a mucoprotein which can readily be isolated from human urine (164) is an excellent substrate. Dialyzable carbohydrate is released as a result of enzyme action. The electrophoretically and ultracentrifugally homogeneous mucoprotein from human urine has a molecular weight of about 7 million and yields 33% reducing sugar and 9 % glucosamine on hydrolysis. The carbohydrate of the mucoprotein contains galactose, mannose, fucose, and a nitrogen-containing substance which is converted t o 2-carboxypyrrole on treatment with alkali (58, 59). Gottschalk has suggested that the enzyme is an amidase cleaving the linkage between the carboxyl group of the nitrogen-containing compound and the amino group of hexosamine (58). It would appear that despite the common problem of entering into their respective host cells, tobacco mosaic virus, the T2 phage, and influenza virus have solved their problems differently, being compelled in each case to take into account the special properties of the different host cells. Each solution has left its mark on virus form and structure. It will be of considerable interest to compare various animal viruses active on a single type of cell t o see whether these mechanisms of adsorption, penetration, and exit are as superficially similar as they appear to be in the bacterial virus systems or whether their patterns of behavior will exhibit greater variability.

VIII. ON THE METABOLIC CONSEQUENCES OF A NEW BUILDING BLOCK: PHAGES 5-HYDROXYMETHYLCYTOSINE I N THE T-EVEN It has been remarked by Luria (106) in discussing “the unity of biochemistry” that the basic unity is a reflection of the existence of common building blocks employed by all organisms, i.e. about 20 amino acids, 5 or 6 nucleotides, etc. As in the development of an enormously diversified

36

SEYMOUR S. COHEN

vocabulary from a limited alphabet, so the organic world develops its innumerable diversified metabolic mechanisms and patterns. The gross similarities formerly called the “unity of biochemistry” are imposed on these possible diversifications by natural selection, which permits the survival of only those organisms possessing metabolic patterns which do not reduce the efficiency of the organism in which they are present. Although this author will agree with Luria in general in this formulation, Luria has neglected to mention the effect of occasional additions and substitutions in the alphabet itself; these result in fundamentally new phenomena. The limited distribution of certain unique building blocks has been noted in the introductory section. Such a new building block of extremely limited distribution is 5-hydroxymethylcytosine (HMC) and the new kinds of phenomena consequent upon its presence in the T-even phages will be considered below. Early efforts to obtain a stoichiometric relation of bases to phosphorus in the DNA of the T-even phages failed (116). An apparent deficiency was revealed in the cytosine content of these viruses. In systematic efforts to improve analyses on the DNA of T2, T4, and T6, it was observed that older methods, e.g. hydrolysis with 72 % perchloric acid, resulted in the degradation of a base (182, 183). Hydrolysis with formic acid permitted the detection of a new compound which possessed some properties similar to those of cytosine. The new base contained the same ionizing groups as did cytosine and 5-methylcytosine, but its ultraviolet absorption spectrum at various pHs was intermediate between the spectra of these bases. In addition, the new base possessed a hydrophilic group which permitted its separation from cytosine and 5-methylcytosine in paper chromatography. These properties suggested that the new base was cytosine with a hydroxymethyl group a t the 5 position. Several other possibilities were tested and excluded. DNA was prepared from a large amount of T613 and the new base was isolated by making use of the properties described above. A crystalline picrate was obtained. This was decomposed and the free base was isolated in the crystalline form. The analyses of this compound were consistent with the elementary composition of 5-hydroxymethylcytosine. A synthetic sample of HMC became available and proved to be identical with the isolated material (182, 183). I n analyses of the T-even phages, summarized in Table 3, it was demonstrated that the maximal cytosine content was less than 0.2% of its content of HMC. In Table 6 are given estimates of the maximal content of HMC in various biological materials, including the host bacterium, E . coli. In attempting to isolate a nucleoside of HMC for purposes of metabolic investigation, it was found that the relations of HMC in virus DNA were

37

COMPARATIVE BIOCHEMISTRY A N D VIROLOGY

unlike those of the other bases (36). The release of inorganic phosphorus and of deoxyribosides, including cytosine deoxyriboside, from most DNA samples is readily effected by hydrolysis by pancreatic deoxyribonuclease followed by treatment with alkaline phosphatase. With the DNA of virus T6r+ it was found that only 70 to 80% of the total phosphorus could be released by such treatment (36). About 90% of the original HMC remained in the nucleotide fraction. HMC was therefore greatly concentrated in the enzyme-resistant fraction and was present in about three times the concentration of guanine in such materials. Virus DNA could be hydrolyzed in 10% perchloric acid for an hour and this hydrolyzate was then found to be sensitive to alkaline phosphatase. TABLE 6 MAXIMAL CONTENTS

O F 5-(HYDROXYMETHYL)CYTOSINE

(HMC)

IN

VARIOUS

BTOLOGTAL MATERIALS

Material Examined

E coli, strain B DNA from E . coli Ox spleen DNA Phage T5 Phage T7 DNA from T7 Polyhedral virus DNA from vaccinia virus Meningo-pneumonitis virus

Maximal HMC as Wt. Hydro- percentage of cytosine lyzed (mg.) (molar proportion) 270 18 2.1 3.8 18 10.6 1.2 14.8

0.2 0.6 0.2 1 .o 2.7 0.5 2.4 0.6 3.9

After cleavage of the phosphate in this way it was possible to separate an HMC nucleoside by paper chromatography and to isolate this material in 90% yield (36). The author has recently demonstrated that this nucleoside is a deoxyriboside, giving a ratio of deoxyribose to base of 1.0 when analyzed by the perchloric acid-tryptophan reaction (27, 42). It has since been found that virus DNA is hydrolyzed more slowly by deoxyribonuclease than is thymus DNA (170). Treatment of this hydrolyzate with a diesterase permits the isolation of mononucleotides of HMC, in the amount of 17% of the total HMC (170)) and two types of HMC mononucleotides derived from this small yield have been separated by ion exchange chromatography (149). I t had been reported in 1946 that T2rt virus contained hexose in the amount of 5.4 % (28). Within the past year three workers have independently demonstrated the presence of glucose in the DNA of the T-even

38

SEYMOUR 8. COHEN

phages (83, 149, 170). These nucleic acids are unique then in their content of this hexose, as well as in the presence of HMC. Nucleotides and polynucleotides containing HMC contained equivalent amounts of glucose. Hydrolysis in normal acid a t 100°C. for 1 hr. did not free HMC from nucleotides of T4 (170). Of the two types of HMC mononucleotides isolated, one was shown to possess a mole of glucose per mole of base; the other was free of hexose. The glucose-containing HMC mononucleotide is far more resistant to phosphatase than is the normal HMC nucleotide (150). N-C-NHz O=C

I

I I

N-CH

N-C-NHz

I

C-CHIOH

I1

HC-0

O=C

I

I I

N-C

I

C-CHZO

I1

I I HCOH I HC-0

HC-0

HO CH HCOH

I HC I

CHZOH

5-Hydroxymethyl cytosine deoxyri boside

H COH

H COH

I HC I

HC-

CHzOPOSH

I I

CHzOH

5-Glucosyl hydroxymethyl cytosine deoxynucleotide

FIG. 4. A nucleoside and nucleotide containing 5-hydroxymethyl cytosine.

The isolation of a HMC deoxyriboside stable to acid indicates the existence of the usual pyrimidine-N-deoxyriboside, whereas the lability to acid of the linkage holding glucose to the HMC nucleotide implies that this moiety exists as an 0-glycoside. Since the internucleotide linkages substitute the hydroxyls at CI and Cg of the sugar, the only available hydroxyl for the formation of the glucoside is that of the hydroxymethyl group of the base. Thus the glucose moiety is tentatively allocated to this position, as presented in Fig. 4. A model of this structure shows clearly that the glucose may be sufficiently close to the phosphate to hinder its enzymatic hydrolysis. On T6 infection of E . coli, cell deoxyribonuclease is activated and host DNA is degraded. The pieces of this host substance are refashioned, in part at least at the level of deoxyribosides,and reassembled in the synthesis of virus DNA. Since more prelabeled host N than host P is incorporated into virus nucleic acid, it is considered that host DNA is hydrolyzed past

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

39

the nucleotide level to nucleosides (93). The transfer of purines from host DNA has been demonstrated (92), as has the transfer of host thymine (41, 94, 174). The cytosine but not the thymine of host DNA has been shown to be converted to the HMC of virus DNA (41). It also appears that comparable amounts of the deoxyribose of host DNA are also transferred to virus DNA (98). Since the complete degradation of host DNA is effected rapidly in the infected cell (72), it may be asked why injected virus nucleic acid is not similarly degraded, how the essential genetic material of the phage manages to survive the action of nucleases and phosphatases of the cell, and how the infection is able to continue at all. Since virus DNA is more resistant to deoxyribonuclease and phosphatase than are other DNAs and this resistance is a function of the presence of the HMC-glucoside in the DNA, it appears possible that the structural relations of this base provide a key element in the survival of virus DNA. In the course of infection there is a transfer of only 40 to 50% of the DNA of the infecting virus particle to virus progeny. The efficiency of transfer during successive cycles of growth is about the same (108). The hypothesis presented above. for a role of HMC in virus survival suggests that in the transfer of substance from infecting virus to the viral progeny, there should be a disproportionation between the transfer of HMC and of t,he other viral bases. If a large unit of viral nucleic acid which would be presumed to be concentrated with respect to HMC is not utilized to form progeny, e.g. the model or template is not incorporated into virus, it would be expected that more guanine derived from the infecting particle would appear in the progeny than would HMC. If such large units are utilized, more HMC would be incorporated in progeny than would guanine. Hershey has reported, without presenting supporting details or data, that using C14-labeled T2, the purines and pyrimidines in the progeny DNA are labeled in the same ratios as they are in the parental DNA (71). The point is of considerable theoretical importance, and requires careful examination of the experimental data. A s a consequence of infection of E. coli by a T-even phage, synthesis of RNA essentially ceases, as has been shown in several laboratories (30, 31, 110). The data of the author reveal that no more than 2 % of the phosphorus of RNA could be replaced by newly assimilated phosphorus during virus multiplication (31), in contradiction to Hershey’s curious restatement of the values presented (74). Furthermore, the data published by Hershey purporting to show a rapid assimilation of P32into RNA early in infection (70) are unsupported by a satisfactory demonstration that the radioactivity was in fact present in the nucleotides of RNA. I t would be of particular theoretical importance to reveal the synthesis of cytosine

40

SEYMOUR 8. COHEN

ribonucleotide in infected cells, especially since Hershey has already stated that no C14 enters DNA cytosine in infected cells (74). At present then, the bulk of the evidence continues to support the conclusion that the synthesis of host nucleic acids does not proceed a t a significant rate. The utilization of exogenous phosphorus in the infected cell continues at a rate which is of the same order as that occurring in growing cells. This phosphorus is now shunted into the synthesis of virus DNA and away from the host nucleic acids. The host cell appears t o lose the ability to synthesize enzymes (28) or to be induced to synthesize new enzymes in response to the presence of a new substrate (120). It is reasonable to suppose that the inability of the infected cell to grow, reproduce, and synthesize new enzymes is a concomitant of the inability t o make the host nucleic acids essential to the construction of the appropriate cell structures. At the present time it appears possible to formulate the mechanism of the shunt in phosphorus utilization as a consequence of the synthesis of HMC. If the infected cell were compelled t o synthesize HMC and were unable to make or utilize cytosine, such a cell would be unable to synthesize host RNA or host DNA. What are the metabolic relations of HMC which bear on this hypothesis? Free HMC or hydroxymethyluracil (HMU) does not support the growth of a cytosine- or uracil-requiring strain of E . coli. The hydroxymethyl group, once added, appears to be irreversibly attached to the pyrimidine ring. The free base does not support the growth of a thymine-requiring strain of E. coZi. The pyrimidine-bound hydroxymethyl group could not be converted to a methyl group in this organism. Similar inactivities in supporting pyrimidine requirements were observed for the deoxyribosides of HMC or HMU. On incubation of HMC or HMU with growing E. coli or with T2-infected bacteria, the bases did not disappear from the media. On the other hand, infected E. coli does assimilate the HMC deoxyriboside to a certain extent. Infected cells also slowly deaminate this compound to the, HMU deoxyriboside from which the base is no longer assimilated. I n studies with organisms possessing a growth requirement for deoxyriboside it was found that the HMC deoxyriboside could support growth. The HMU deoxyriboside however was completely inert in this respect. Thus the metabolism of the hydroxymethyl pyrimidines does not occur via the free base but rather a s the deoxyriboside (36). Once the hydroxymethylated pyrimidine deoxyribosides are formed they are incapable of generating cytosine or the deoxyribosides of uracil, or to put it another way, hydroxymethylation may trap the deoxycytidine or deoxyuridine from the main pathways of host metabolism.

COMPARATIVE BIOCHEMISTRY A N D VIROLOGY

41

The cytosine deaminase of E . colz or of yeast is incapable of deaminating HMC, although the yeast deaminase is active on both cytosine and 5-methylcytosine (36). Normal E. coli contain a very active deoxycytidine deaminase which has been purified (36). This enzyme possesses about I to 3 % of its deaminating activity on the deoxyriboside of HMC and generates the HMU deoxyriboside, which has been isolated following this treatment (36). Thus hydroxymethylation also helps t o preserve the amino group on deoxycytidine. The hydroxymethyl group may be readily derived from the P-carbon of serine (41). Thus carbon is an active source for one-carbon fragments at the oxidation level of formaldehyde. As noted earlier, cytosine may be converted to HMC presumably as the deoxyriboside. In view of the presence of the active deoxycytidine deaminase in E. coli, it is not certain whether the hydroxymethylation proceeds on the deaminated derivative, deoxyuridine, and the HMU deoxyriboside is then aminated or whether hydroxymethylation occurs on deoxycytidine directly. These possible relations are presented below. deoxycytidine

i

deoxyuridine

HMC deoxyriboside

T?

-& HMU deoxyriboside

Superimposed upon the irreversible character of hydroxymethylation and the lowered reactivity to deamination, it will be recalled that the HMC nucleotide is glucosylated, thereby protecting the nucleotide from hydrolysis by phosphatase and lowering the sensitivity of the completed DNA to deoxyribonuclease. This DNA in its turn is finally enclosed within an enzyme-proof protein coat and is thereby further removed from the metabolism of the cell. It is a remarkable fact that all of the reactions and relations of the HMC in virus facilitate its survival within E. coli. All the known relations of HMC facilitate the winning out of phage products, without requiring any additional inhibitory action on the cytosine metabolism of the host, other than that imposed by the one-way removal of cytosine or uracil derivatives to their hydroxymethyl derivatives. How does it happen that normal E . coli can exist and accumulate cytosine nucleotides? As a working hypothesis, it may be assumed that a pathway to hydroxymethylation of deoxycytidine does not normally occur since this substance might be trapped as a hydroxymethyl derivative. If this is so, does the virus add a component, such as an enzyme or coenzyme essential to this hydroxymethylation, or does it liberate a normally inhibited enzyme? Some slight evidence may be offered in support of the latter possibility.

42

SEYMOUR S. COHEN

It is known that growing E. coli can make a hydroxymethylated derivative of cytosine. This is the pyrimidine of thiamine (see Fig. 5). Cleavage of thiamine by thiaminase yields 2-methyl-5-hydroxymethyl-6-aminopyrimidine (95). Thus a compound much like HMC is a normal intermediate in the bacterium, albeit in trace amounts. If the same hydroxymethylating system is a t work in infection, there would have to be an enormous expansion of its activity. In studies on a thymineless strain of E. coli, strain 15T-, it was observed that on infection with T2 this organism synthesized a great deal of thymine in addition to HMC in the absence of exogenous thymine (7, 36, 40). It was then demonstrated by an isotopic method that strain 15T- was N=C-NH2

N=C-NH*

I I O=C C-CHIOH I II

I I O=C CH I It

HN-CH 5-Hydroxymethylcytosine (HMC)

HN-CH Cytosine N=C-OH O=C

I I CH I II

HN-CH Uracil

N=C-OH O=C

I I C-CHnOH II II

HN-CH 5-(Hydroxymethyl)uracil (HMU)

N=C-NH2 CHj-C

I 1 C-CHzOH II II

N-CH

Pyrimidine of thiamine N=C-OH O=C

I I C-CH, I II

HN-CH Thymine

Fro. 5. The structures of some key pyrimidines.

capable of synthesizing 2 to 4 % of its normal growth requirement of this substance. However, the synthesis displayed on infection represented more than a hundredfold expansion of this low level of thymine synthesis. The existence of any activities in this respect in the normal cell suggests that the effect of virus infection permits the expansion of a system already present. One possibility is the uncovering of an inhibited system. An analogy for the phenomenon of release of an inhibition by T 2 infection exists in the release of the inhibition of deoxyribonuclease in E. coli by virus infection (93,129). Of course the proof of such a hypothesis will require the demonstration of the presence of the enzymes for these syntheses in the necessary amounts in uninfected cells. If it is supposed that the extreme parasitism observed in infections by the T-even phages is a consequence of the presence of HMC in these viruses, what can we expect of the parasitic patterns of infections by viruses possessing cytosine? If the only possible manner in which a virus might

COMPARATIVE BIOCHEMISTRY AND VIROLOGY

43

establish its hegemony in a cell involved the reactions described above, all viruses capable of parasitic existence and inducing cellular pathology would contain HMC. This is obviously not so and it must be assumed that a virus containing cytosine possesses other mechanisms for diverting the cellular enzymes to effecting syntheses for the virus. On the other hand it may be expected that a parasitic pattern in which the cell may survive, grow, and multiply despite the presence of a virus, e.g. a lysogenic system, can not exist with a virus containing HMC. Thus it was predicted that temperate phages would contain cytosine rather than HMC, as indeed has recently been reported (107). This can scarcely be considered to be proof of the hypothesis, since all other viruses also appear to contain cytosine. It is possible however, that conditions may be obtained in which some apparently virulent phages such as T3 or T7 which contain cytosine may be induced to show more temperate characteristics, if the conditions for their virulence can be specifically inhibited.

IX. CONCLUSION It is usually true that a review and survey of the state of a discipline will be most useful to the extent to which it discovers and presents common aspects of various phenomena and systems, and generalizes the experience of many workers. In virology a t the present time, the diversity of existing data permit certain generalizations of this type, but as noted earlier the tendency to generalize our experience is often premature. There are many evidences that the viruses are not a homogeneous group and it is probably unwise so to treat them. The tendency to reason by analogy is in many instances unwarranted in theory and dangerous in practice; this conclusion is related to the optimistic proposition that there is far more to be discovered than is already known.

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115. 116. 117. 118. 119. 120. 121.

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136. Puck, T. T., and Lee, H. H. (1954). J . Exptl. Med. 99, 481. 137. Puck, T. T., and Tolmach, L. J. (1954). Arch. Biochem. and Biophys. 61, 229. 138. Putnam, F. W. (1953). Advances in Prolein Chem. 8 , 175. 139. Reichmann, M. E., Rice, S. E., Thomas, C. A., and Doty, P. (1954). J. A m . Chem. SOC.76, 3047. 140. Rich, A,, and Watson, J. (1954). Nature 173, 995. 141. Ris, H. (1917). Discussion to paper of S. S. Cohen. Cold Spring Harbor Symposia Quant. Biol. 12, 49. 142. Ross, A. F . (1941). Phytopathology 31, 394. 143. Sigik, B., Puck, T. T., and Levine, S. (1954). J. Exptl. Med. 99,251. 144. Schachman, H. K., and Lauffer, M. A. (1949). J . Am. Chem. SOC.71,536. 145. Schlesinger, R. W. (1953). Cold Spring Harbor Symposia Quant. Biol. 18, 55. 146. Schwerdt, C. E. (1954). Paper presented a t meeting of Am. Assoc. Advancement of Sci., Berkeley, Calif. 147. Scott, T . F. M., Burgoon, C. F., Coriell, L. L., and Blank, H. (1953). J. Iirimunol. 71, 385. 148. Sharp, D . G . (1953). Advances i n Virus Research 1, 277. 149. Sinsheimer, R. L. (1954). Science 120, 551. 150. Simmons, N . (1954). Paper presented a t meeting of Am. Assoc. Advancement of Sci., Berkeley, Calif. 151. Smadel, J. E., and Hoagland, C. 1,. (1942). Bacteriol. Revs. 6 , 79. 152. Smith, J. D., and Stoker, M. G. P. (1951). Brit. J . Exptl. Pathol. 32,433. 153. Smith, K. M. (1952). Biol. Revs. 27, 347. 154. Smith, K. M. (1954). Proc Roy. SOC.B142, 196. 155. Smith, K. M., and Xeros, N. (1954a). Parasitology 44, 71. 156. Smith, I(. M., and Xeros, N. (1954b). Parasitology 44, 400. 157. Smith, L. (1954). Bacteriol. Revs. 18, 106. 158. Sprince, H., and Schoenbach, E. B. (1942). Proc. SOC.Exptl. Biol. Med. 49, 415. 159. Stanier, R. Y. (1954). i n “Cellular Metabolism and Infections” (E. Racker, ed.), p. 3. Academic Press, New York. 160 Stanley, W. M. (1939). J . Biol. Chem. 129, 405. 161, Stanley, W. M. (1940). J. Biol. Chem. 136, 437. 162. Stanley, W. M., and Knight, C. A. (1941). Cold Spring Harbor Symposia Quant. Biol. 9, 255. 163. Stent, G. S., and Fuerst, C. R . Personal communication. 164. Tamrii, I . , and Horsfall, F. L . , J r . , (1952). J. Exptl. Med. 96, 71. 165. Taylor, A. R., I3earcl, D., Sharp, D. G., and Beard, J. W . (1942). J.Infectious Diseases 71, 110. 166. Tavlor, A. R., Sharp, D. G., Beard, D., and Beard, J. W . (1943). J. Infectious Disenses 72, 31. 167. Taylor, A. R. (1945). J. Biol. Chem. 163, 675. 168. Theorell, H. (1951). i n “The Enzymes” (J. B. Sumner and K. Myrback, eds.), Vol. 2, Part 1, 11. 335. Academic Press, New York. 169. Volkin, E. (1954a). Federation Proc. 13, 315. 170. Volkin, I<. (1954b). J . A m . Chem. SOC.76, 5892. 171. Wang, T., and Commoner, B. (1954). Science 170, 1001. 172. Wasser, H. B. (1952). J . Bacteriol. 64, 787. 173. Watson, J. D., arid Crick, F. H. C. (1953). Nature 171, 737. 174. Weed, 1., L., and Cohen, S. S. (1951). J. Biol. Chem. 192, 693.

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175. Weidel, W. (1951). Z. Nalurforsch. 6b, 251. 176. Weil, M. L., Warren, J . , Breese, S. S., Russ, S. B., and Jeffries, J . (1952). J . Bacteriol. 63, 99. 177. Williams, R. C. (1953). Cold Spring Harbor Symposia Quant. Biol. 18, 185. 178. Wilmer, H. A . (1944). Proc. SOC.E z p t l . Biol. k f e d . 66, 206. 179. Woods, M. W., and Dubuy, H . G. (1943). Phytopathology 33, 637. 180. Wyatt, G. R . (1952). J . Gen. Physiol. 36, 201. 181. Wyatt, G. R . , and Cohen, S. S. (1952a). Nature 170, 846. 182. Wyatt, G. R . , and Cohen, S. S. (195213). Nature 170, 1072. 183. Wyatt, G. R., and Cohen, S. 5. (1953). Biochem. J . 66, 774. 184. Yanofsky, C. (1954). J . Bacteriol. 68. 577. 185. Zahler, S. A., and Moulder, J . W. (1953). J . Infectious Diseases 93, 159.

The Chemotherapy of Viruses R. E. F. MATTHEWS

AND

J . D. SMITH

Virus Research U n i t , f t g r i c u h r a l Research Council, Molten0 Institute, Cambridge, England

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Methods of Testing Compounds for Virus Inhibition., . . . . . . . . . . . . . . . . . . . A. Plant Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Methods of Assessing Inhibitory Effects.. . . . . . . . . . . . . . . . . . . . . . . . . . 2. Methods of Applying Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Choice of Virus and Hos .................................. 4. Mode of Action of Com ............................. B. Animal Viruses. . . . . . . . . . . . . . ................................. 1 . Screening Tests.. . . . . . . ................................. .......................... 2. Mode of Action of Compounds. . . . . . C. Bacteriophages, . . . . . . . . . . . . . . . . . . . . . . . .......................... 1 . Plaque Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. 2. Latent Period and Burst Size, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Intracellular Phage Development. . . . . . . . . 4. The Production of Immature Non-infective .......... 111. Structure and Multiplication of Viruses in Relation to Chemotherapy.. . . . A. Composition of Viruses., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Functions of Protein and Nucleic Acid in Virus Multiplication. . . . . . . . . 1 . Bacteriophages.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Plant Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Animal Viruses.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Structure of Nucleic Acids.. . . . . . . . . . . . . . . . 1 . The Components of Nucleic Acids.. . . . . . . . . . . 2. The Repeating Unit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The Linkage between Nucleotides. . . . . . . . . . . . 4. The Branching of the Chains., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. The Size of Nucleic Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. The Proportion of Bases in the Nucleic Acids. . . . . . . . . . . . . . . . . . . . . . . 7. Nucleic Acids Structure in Relation to the Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . The Lyosogenic Host Relationship. . . . . . E. Persistence of Virus Infections.. . . . . . . . . ............ IV. Effects of Purine and Pyrimidine Analogues .................................. A. Plant Viruses.. . . . . . . . . . . . . . 1 . Purine Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ies. . . . . . . . . . . . . . . . . . . . . . . . .

51 52 52 52 54 56 56 57 57 58 59 60 61 61 61 61 62 62 64

66 66 68

73 74 74

..................... ..................... 2. Pyrimidine Analogues.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49

87

.

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50

C . Animal Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Purine Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Pyrimidine Analogues., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Methods for Demonstrating Incorporation of Analogues into Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................ 1. 8-Azaguanine in Itibonucleic Acids . . . . . 2 . 5-Halogenated Uracils in Deoxyribonucl ................ E . Evidence that Incorporated Analogues Inhibit Growth through Incorporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Infectivity of Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Correlation between Inhibition and Incorporation. . . . . . . . . . . . . . . . . 3 . Delay in Inhibition of Bacterial Growth . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . In Vitro Evidence for the Absencc of Competitive Inhibition . . . . . . . F . Factors Affecting Incorporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Structural Features of the Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Host Metabolism ............................ ............................ G . The Design of New 1. Ring Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Substituted Purines and Pyrimidines . . . . . . . . . . . . . . . . . . . . . 3 . The Sugar Component . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Compounds Related t o a Base Known to be Incorporated . . . . . . . . . . . H Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Virus Inhibition b y Other Types of Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . A . Amino Acids, Amino Acid Analogues, and Related Compounds . . . . . . . . 1. Bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Animal Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Plant Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Vitamin Analogues and Related Compounds . . . . . . . . . . . . . . . . . . . . . . . . . 1. Bacteriophages . ............................................. 2 . Animal Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Plant Viruses., . ............................................ C . Metal Ions and Ch Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Plant Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................................... 3 . Animal Viruses . . . . 4 . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Dyestuffs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Animal Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Plant Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Bacterial Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Acridine Derivatives., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................ ........................................... 2 . Animal Viruses 3 . Plant Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Antibiotics . ............................................. 1. Animal v ........................................ 2 . Plant Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Plant Growth Regulators and Related Substances . . . . . . . . . . . . . . . . . . . . 1. Plant Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . .................. 2 Animal Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

.

89 89 92 92 93 94 95 95 96 96 96 98 98 101 102

105 106 106 108 108 108 110 113 114 114 114 115 118 119 119 121 123 123 124 124 125 125 126 126 127

128 130 131 131 132

CHEMOTHERAPY OF VIRUSES

H . Miscellaneous Compounds with Some Inhibitory Activity for Animal Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Sodium Monofluoroacetate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2,4-Dinitrophenol... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Thiosemicarbazones.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . l’henoxythiouracils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Diamidines., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. a-Haloacylamides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 . Polymerized Benzoid Sulfonic Acids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . Substances Affecting the Lysogenic Bacteriophage-Host Relationship. . 1 . The Lysogenization of Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Loss of Lysogenicity.. . . . . . . . . ..... VI. Incorporation Phenomena in Relation to Antimetabolite Action. . . . . . . . . . ......... VII. General Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51 132 132 133 133 134 135 135 136 136 136 137 137 140 142

I. INTRODUCTION If we exclude the lymphogranuloma-psittacosis group of large animal viruses which are now classed with bacteria, there is no well-established case of the effective use of chemotherapy in practice for any virus disease. Although spectacular results have been achieved in the chemotherapy of of diseases caused by cellular parasites, many workers have despaired of obtaining effective chemical means of controlling diseases due to viruses. The difficulties involved are concerned with the way viruses multiply. Cellular pathogens have a metabolism of their own which may differ in many respects from that of their hosts. There are thus many possible ways in which compounds could interfere with bacterial growth, for example, without affecting the host. Viruses, however, are much more intimately dependent on the host for their reproduction. Thus any compound that interferes with virus development is very likely to affect the host as well. Different types of compounds are known which interfere either with processes involved in the initiation of infection by viruses, or with their subsequent multiplication. There is a wide variety of natural and synthetic products which inactivate viruses in vitro and which, when applied to the host at or before the time of inoculation, reduce the number of virus particles which become established. We will be dealing here with only a few substances of this type. The term “chemotherapy” has been defined in various ways. We shall use the word rather loosely to include the effects of compounds which delay or inhibit to some extent virus multiplication or disease development. In fact most of the compounds that have some activity against plant and animal viruses are inhibitory only when applied before or at an early stage in the infection. Their action would be better described as prophylactic.

52

R. E. F. MATTHEWS AND J. D. SMITH

There have been two kinds of approach to the problem of virus chemotherapy. In some laboratories large numbers of compounds, selected almost randomly, are put through some screening procedure for virus inhibitory activity. Other workers choose particular compounds for testing because they have already been shown to have growth inhibitory properties in other systems. Neither type of work has yet produced compounds of practical use in disease control. Exponents of the first approach point out the fact that nearly all new types of chemotherapeutic agent effective against cellular pathogens have been discovered more or less by chance. Supporters of the second method tend to emphasize the theoretical importance of the results they obtain. In writing this account of virus chemotherapy we have accepted the editors’ invitation to present a point of view. There is a growing body of evidence suggesting that the genetic properties of viruses may reside largely in their nucleic acids, and a number of recent observations show that virus multiplication can be delayed by compounds which interfere with nucleic acid metabolism. As a class, analogues of the purine and pyrimidine bases may inhibit growth by a variety of mechanisms. Some are very probably “competitive inhibitors” in the strict sense, interfering either with the incorporat,ionof bases into nucleic acids or with the formation of the bases from percursors. Other analogues are incorporated into nucleic acids and appear to exert their growth-inhibitory effects through such incorporation. We have devoted most consideration to compounds of this latter type, their mode of action, and to the possibility of using them for effective control of viruses. 11. METHODS OF TESTING COMPOUNDS FOR VIRUSINHIBITION The methods that have been used to find inhibitory compounds and to examine their mode of action are discussed briefly in this section.

A . Plant Viruses 1. Methods of Assessing Inhibitory EJ’ects a. Measurement of Amount of Virus Produced. Tobacco mosaic virus has been most commonly used for this type of test because there are a variety of micromethods available for its estimation. To show that any reduction in yield of virus is due to inhibition of virus development rather than to reduction in the initial number of successful entry points, treatments should be commenced about 24 hr. after inoculation. We have calculated that not more than 10% and more probably about 0.1 % of the cells of tobacco leaf can be infected directly by inoculation with a highly infectious virus preparation. Thus subsequent increase in the amount of virus will reflect both the rate of multiplication of the virus and

53

CHEMOTHERAPY OF VIRUSES

the increase in numbers of cells in which the virus is multiplying. At present we cannot dissociate the effect of a compound on virus multiplication from its effects on rate of movement of the virus from cell to cell. b. Efect on the Development cf Systemic Disease. The majority of plant viruses cannot be accurately assayed. For these the activity of a compound can only be assessed by its effect on disease development. When

m a, c 0

1

I

0.01 M

.

0.003Mm

I

I

0 .-

E

c

u)

0.001 M

Days after inoculation

FIG.1 . Effect of leaf spraying treatments with various concentrations of 8-azaguanine on the systemic development of lucerne mosaic virus in tobacco. Six plants inoculated in each group.

treated plants develop no systemic symptoms, the possibility that the compound is merely masking the disease must be tested by subinoculation to indicator plants. Both the time taken for systemic disease to appear and the number of plants systemically infected a t the end of the experimental period can be used as a measure of the activity of a compound. Figure 1 illustrates results of this type. c. Reduction in Number of Local Lesions. For this type of test it is usual to employ host-virus combinations such as tobacco mosaic virus in Nicotiuna glutinosa where easily countable necrotic local lesions are produced. A reduction in the number of local lesions may be due either to interference

54

R. E. F. MATTHEWS A N D J. D. SMITH

with virus establishment or multiplication, reduction in susceptibility of the host, or to masking of the normal local lesion type. d. Virus Tumors. Growth measurements made on virus tumors in t,issue culture (e.g. Nickell, 1951) may have little relevance to the effect of a compound on the tumor-inducing virus. 2. Methods of Applying Compounds. Several methods have been used. These are: (1) Tissue cultures on agar or other medium (Kutsky and Rawlins, 1950; Norris, 1953). (2) Detached leaves or leaf discs floated on a solution containing the compound (Commoner and Mercer, 1951; Schlegel and Rawlins, 1954). (3) Compounds introduced into the stem through a glass fiber or a cotton wick (Beale and Jones, 1951). (4)Leaves dipped in a solution of the compound while attached to the plant (Yarwood, 1954). (5) Impregnation of leaves on whole plants, the leaves being dipped in solutions under reduced pressure (Kirkpatrick and Linder, 1954). (6) Soaking budwood or other excised vegetative organs in a solution of the compound (Stoddard, 1947). (7) Spraying a solution of the compound on the leaves of intact plants (Matthews, 1953a). (8) Watering the soil around the plant with a solution of the compound (Matthews, 1953a; Holmes, 1954). The floating leaf or leaf disc method has been most widely used. This technique has the advantage that the leaf tissue can be kept in contact with the known concentration of the compound for the duration of the experiment. Apart from this it has little to recommend it. Effects of a compound on the plant tissue cannot be adequately assessed. For example Commoner and Mercer (1951) found that solutions of thiouracil at about 4 x 10-6 M had no detectable effect on tobacco leaf discs. However, when sprayed on the plant, this compound is a highly effective inhibitor of plant growth at virus inhibitory concentrations (Matthews, 1953a; Bawden and Kassanis, 1954). It is known that marked metabolic changes occur in a short time following removal of a leaf from the plant. For example, in most species the protein content of the leaf tissue falls while the amount of soluble nitrogen-containing compounds rises (Ronner, 1950). Such changes may well affect the inhibitory activity of a compound towards the virus, causing either an increase or decrease in effectiveness compared with tests on intact plants. It is probable that a wide variety of compounds may have a slight depressing effect on virus multiplication when tested by the leaf disc method. For example Schneider (1954) tested 32 purine analogues on tobacco mosaic

CHEMOTHERAPY OF VIRUSES

55

virus by this method. The numbers of compounds causing a given reduction in yield of tobacco mosaic virus are plotted in Fig. 2A. The numbers of compounds are distributed with a peak at the group causing 10-20% reduction. Schneider considered that compounds recorded as - 18 % were having no effect while those causing -20% were effective. This is

Per cent change in amount

of virus

6 ,

Per cent change in amount of virus

FIG.2. Effect of compounds on amount of tobacco mosaic virus in leaf discs floated on solutions. A: 32 purine analogues (from the d a t a of Schneider, 1954. Amounts of inhibition a t 10 mg./100 ml., or the nearest level t o this). B: 22 pyrimidines and related compounds. (From Schlegel and Rawlins, 1954. Amounts of inhibition a t 0.1% or the nearest level t o this.)

an arbitrary division. Apart from the three compounds causing marked reduction (&azaadenine, 2-azaadenine, and 2,6-diaminopurine) it is difficult t o decide which cornpounds in this group were having a specific effect on virus development. The resuhs of Schlegel and Rawlins (1954) for 22 pyrimidines and related compounds are plotted similarly in Fig. 2B. In their experiments two compounds (diazouracil and thiouracil) fall outside a distribution with a mean of about 10% reduction.

56

R. E. F. MATTHEWS AND J. D. SMITH

With animal viruses, tissue culture techniques may have some advantage in the initial tests with new compounds, since tests with animals are expensive, and toxic effects must be avoided as much as possible. These considerations do not apply to plants, and thus many of the intermediate stages in testing can be avoided. There are two methods of applying compounds to plants that would be of general practical use: leaf sprays and watering on the soil. These methods appear to be the most suitable for screening tests. They are simple to carry out. Any successful results can be tested on a field scale by the same methods. A realistic assessment of damage to the host can be made and there is a minimum of interference with the normal plant processes. If small plants are used, the leaf-spraying method probably requires no more material than the leaf disc method, since with the latter the solution must be replaced every day or two. When whole plants are used, they may be grown as long as is necessary to assess the effect of a compound on virus development. These methods are not limited to the viruses which can be readily assayed in small quantities but can be applied to any host-virus combination using any desired method of virus inoculation. When whole plants are tested and disease development used as an estimate of inhibitory effects, natural variability in the material is readily seen. This variability tends to be overlooked when precise measurements are made on amounts of virus in carefully selected leaf discs. In attempting to free infected plants or parts of plants from virus by chemical treatment, actively growing material containing as little virus as possible should be used. For example with potatoes, tips of young sprouts would appear to be the most suitable material (Norris, 1953). 3. Choice of Virus and Host. For experiments designed to elucidate the mechanism of action of active compounds the obvious choice is a virus such as tobacco mosaic or turnip yellow mosaic which can be isolated in quantity and about which much is already known. However tests should not be confined to a single host-virus system. The results obtained with 8-azaguanine and 2-thiouracil show that compounds of this type at least can have a marked specificity and inhibitory effects may be dependent both on the virus tested and the host plant used. Where chemotherapy is being attempted for a specific virus of economic importance screening tests should be made, where possible, using the host in which control of the virus is desired. 4. Mode of Action of Compounds. For both animal and bacterial viruses methods are now available for studying a single cycle of virus multiplication. Until such methods have been developed for plant viruses, the information that can be obtained about the way in which compounds inhibit virus development will be limited. Growth curves showing the delay in

CHEMOTHERAPY OF VIRUSES

57

virus production in leaf tissue are of little value. The inhibition by one compound may be annulled by another, and this phenomenon can sometimes be used to indicate the way in which a compound acts. The effect of single applications of a compound a t various times, before and aft,er inoculation, on numbers of local lesions produced may show whether the compound interferes with processes connected with virus establishment. With inhibitory compounds that are structurally related to components of the virus, the possibility that they are incorporated into the virus can be examined for certain viruses and the effect of such incorporation on the biological and other properties of the virus can be studied.

B. Animal Viruses In testing compounds for inhibitory activity against animal viruses, most investigators have used one or more of the following kinds of test system: (1) Viruses grown in various types of tissue culture, compounds being introduced into the medium. (2) Viruses grown in the embryonated hen’s egg, compounds being injected either into the allantoic fluid, or the yolk sac. (3) Virus infections in small laboratory animals, usually mice; compounds being administered parenterally, in the diet, or inhaled as smokes or dusts. Various strains of influenza A and B, vaccinia, and strains of poliomyelitis viruses have been most widely used as test viruses. 1 . Screening Tests. When a compound has been found to possess some virus inhibitory activity, it is usually examined along with related substances in a variety of test systems and against a number of viruses. The object of a screening test is to discover among a large number of more or less randomly selected compounds new types of structure possessing inhibitory properties. For such a screening test a standard procedure involving a minimum of time and cost is required, so that as many compounds as possible can be tested. On the other hand it is important that a potentially active type of compound should not be overlooked. Any screening procedure will be a compromise between these two opposed requirements. Large scale screening programs have been carried out using only a tissue culture system for the initial tests; in some cases bacterial viruses have been used as a test system in work directed towards finding compounds active against animal viruses. Compounds are tested at some arbitrary concentration and, if toxic, may be retested at lower concentrations. Those showing some activity in tissue culture are then tested in animals, usually mice, perhaps with the embryonated egg as an intermediate step. Most of the compounds found to have some activity in tissue cultures or eggs are then found to have slight or negligible effects in mice. On the other hand, it is possible that compounds could be active in animals but in-

58

R. E. F. MATTHEWS A N D J. D . SMITH

effective in tissue culture or chick embryos. For example, certain 5-phenoxythiouracils, although only moderately active inhibitors of vaccinia virus in tissue culture, have a significant protective effect on the virus in mice (Thompson, el al., 1951b). Many compounds may be much more toxic for rapidly growing tissues or chick embryos than for an adult mammal, and thus might be tested in tissue cultures a t concentrations below those having antiviral activity. Furt#hermorea compound might be metabolized to an inactive material in tissue cultures more rapidly than in the adult animal. Some compounds have quite marked inhibitory properties for certain viruses but not others, using similar testing methods. In animal tests the method of introducing the compound may markedly influence activity. For example Fleisher (1949) found that the dye, J a m s green, injected intravenously gave mice some protection against the PR8 strain of influenza virus but had no effect given subcutaneously or intraperitoneally. The route of inoculation of the virus and the dosage given introduce further variables. Many compounds are known which have some protective effect in animals against small doses of virus, but none when larger doses are given. Likewise a compound may have some effect, for example, against a neurotropic virus inoculated intramuscularly but none when the virus is introduced directly to nervous t,issue. In preliminary screening tests it would seem desirable to use as low a dose of virus and as “mild” a route of inoculation as will regularly give a high proportion of disease in control animals. I n screening tests inhibitory activity is often assessed on amount of virus produced after a given time or on rate of virus production. It is possible that a compound could have an effect on disease development in an animal without having any marked effect on rate or amount of virus production. The extent to which consideration can be given to these variables in a routine screening procedure for large numbers of compounds will depend on factors of time and cost. 2. Mode of Action of Compounds. The mechanism of action of compounds has been studied in viruses growing in embryonated eggs and in a variety of tissue culture systems. Certain of these appear to offer particular advantages. The method in which pieces of intact chorioallantoic membrane are suspended in a culture medium has been increasingly used with influenza viruses. For example, Tamm el al. (1953a) and Tamm and Tyrrell (1954) have studied this method in relation to tests with Lee influenza virus. Fairly wide variation in the ability to support influenza virus multiplication occurs in membranes from different eggs. By planned randomization of pieces of membrane, groups of a t least 6 pieces being used, these workers

CHEMOTHERAPY OF VIRUSES

59

were able to obtain predictable growth of virus. They found that Lee influenza virus was absorbed by and multiplied in both the chorionic and allantoic layers of the membrane to about the same extent. On the other hand, Fulton and Isaacs (1953) found with PR8 influenza A that chorioiiic cells appeared to support no more than a siugle cycle of multiplication, no virus beiug released into the medium. In this method a tissue containing cells of relatively uniform age, isolated from other components of the egg, is suspended in a defined medium. The amount of virus in the inoculum can be related approximately to the number of cells in the culture. Perhaps more important for future work is the use of strains of mammalian tissue cells which can support growth of viruses in tissue culture. For example a strain of mouse fibroblasts (Strain L, Earle) can support the multiplication of a range of viruses (Syverton and Scherer, 1953). A stable strain of human epithelial cancer cells, HeLa (Scherer et al., 1953), can be maintained indefinitely in tissue culture. From sheets of such tissue grown on glass, cells free in suspension can be obtained by the use of trypsin. These cells support the growth of all the poliomyelitis viruses. A number of assay methods have been used t o measure the effects of compounds on virus production in animals or tissue cultures. In the multiplication of a virus (e.g. influenza) the production of virus-specific material may be measured in three ways: the development of antigen, hemagglutinating material, and infectivity for the chick embryo or mouse. These three methods of estimation may not necessarily give identical results. For example the presence of incomplete virus particles in an influenza virus preparation will decrease the ratio injectivity/hemaggZutinatingtiter. From a practical point of view infectivity measurement may be more important, and a precise method of measuring numbers of infective particles has been developed by Dulbecco (Dulbecco, 1952; Dulbecco and Vogt, 1953), in which the number of plaques on a continuous sheet of cells in tissue culture is counted. This is closely analogous to the plaque count method of assaying bacteriophages. The use of cultures containing an accurately determined number of animal cells together with the plaque method for assay of virus infectivity will be of great value in examining the mode of action of virus inhibiting substances. Using these techniques all the cells in a given suspension may be infected simultaneously, and a single cycle of multiplication isolated and studied.

C . Bacteriophages The use of bacteriophages in screening tests for compounds active against other viruses is of very doubtful value. On the other hand the bacteriophages do offer a system which is of great advantage in studying

60

R. E. F. MATTHEWS AND J. D. SMITH

the mode of action of a virus-inhibitory substance. The only comparable system is the plaque method of counting animal viruses on monolayer tissue cultures noted in the preceding section. The technical advantages of the bacteriophage-bacterium system are: (a) the possibility of accurate estimation of infective virus particles, (b) the comparatively short, time of the complete developmental cycle, (c) the ease with which all the cells in a bacterial population may be simultaneously infected with one or more virus particles, and (d) the suitability of bacteria for biochemical investigations. It is becoming increasingly evident that widely differing viruses show similarities in their mechanisms of multiplication. Examples of this are the existence of genetic recombinations between viruses both in bacteriophages (Delbruck and Bailey, 1946; Hershey and Rotman, 1949) and in influenza virus (Burnet and Lind, 1951a, b), and the discovery of “incomplete” noninfectious particles lacking nucleic acid both in plant viruses (Markham and K. M. Smith, 1949; Takahashi and Ishii, 1953) and in bacteriophages (de Mars et aE., 1952). Thus conclusions as to the mode of action of a virus-inhibiting compound derived from studies of bacteriophage may at least to some extent be relevant, when considering the action of the substance against other viruses. Many workers have tested the ability of compounds to inhibit bacteriophage multiplication without making use of the comparstively simple methods available to determine the point a t which these act in the bacteriophage growth cycle. For example, little information is given by the fact that, when los bacteriophage particles are added to lo7 bacteria in the presence of a given compound, the final yield of virus is less than that of controls without the inhibitor. Much of the work on substances inhibiting bacteriophage development has been carried out using the 7 phages active against B. coli “B” which are numbered T1-7 (Delbruck, 1946). (1) Plague Count. The techniques generally used in the manipulation of bacteriophages have been very comprehensively reviewed by Adams (1950). The fundamental technique on which all bacteriophage work depends is the measurement of numbers of infective virus particles by the plaque count method. A suitable dilution of the phage preparation is mixed with an excess of cells of a sensitive bacterial strain in weak liquid agar and the whole poured over the surface of an agar plate. Each infective phage particle by multiplication gives rise to a clear area (plaque) in the continuous surface of bacterial growth. Under suitable conditions the number of plaques is equal to the number of infective units placed on the plate. 2. Latent Period and Bur& Size. On the addition of a lytic phage to a,

CHEMOTHERAPY OF VIRUSES

61

sensitive bacterium there is a short period during which free phage disappears, being adsorbed to the bacteria, and a period of intracellular multiplication without liberation of phage, the latent period, terminated by the lysis of the bacteria and release of phage. The number of phage particles released per infected bacterium is generally called the burst size. The production of phage from a lysogenic bacterium following induction by a change in the external environment also involves a latent period followed by lysis of the bacteria. An inhibitory substance may affect the stability of the phage in vitro, its rate of adsorption, the latent period (it may prevent lysis altogether), or the burst size. The first two possibilities must be investigated separately; the latter two may be studied by the one step growth technique of Ellis and Delbruck (1939). 3. Intracellular Phage Development. The development of mature virus particles may be followed by breaking open the bacteria and assaying the phage thus liberated. In certain cases the appearance of phage-specific protein may be followed serologically. With the T-even coli phages the the formation of phage-specific deoxyribonucleic acid may be estimated by using the fact that the deoxyribonucleic acids of these phages are unique in containing the pyrimidine, 5-(hydroxymethy1)cytosine. 4. The Production of Immature or Noninfective Bacteriophage. With some inhibitory compounds a reduction in the burst size of infective particles is accompanied by the liberation of noninfective mature bacteriophage particles (in the case of 5-bromouracil), or of noninfective immature particles related to bacteriophage (in the case of proflavine). This possibility can be detected by examining the lysates in the electron microscope and by comparing the number of visible phage particles in purified preparations seen under the electron microscope with the number of infective units determined from plaque counts.

111. STRUCTURE AND MULTIPLICATION OF VIRUSESI N RELATION TO CHEMOTHERAPY A . Composition of Viruses An empirical definition of a virus has developed which involves essentially two properties, size and pathogenicity. Bawderi (1950) has defined a virus as “an obligately parasitic pathogen with dimensions of less than 200 mp.” Wit.h most viruses little is known of their nature other than that they would comply with this definition. However, those viruses which have been isolated and studied by physical and chemical means show quite a number of similarities. These common features probably reflect a minimum requirement of structure for genetic continuity. All the purified viruses contain protein and nucleic acid and at least in

62

R. E. F. MATTHEWS AND J. D. SMITH

plant and bacterial viruses these are the only structural components (although the small amounts of metal ions found in these viruses may also be an integral part of their structure). Some viruses contain only ribonucleic acid (RNA), others only deoxyribonucleic acid (DNA) (Table 1). Certain of the animal viruses have been reported to contain both types of nucleic acid. Components other than nucleic acid and protein, such as polysaccharides and lipids, have been found in preparations of many animal viruses. However, the analytical data on animal viruses must be regarded as equivocal owing to uncertainty as to the purity of the virus preparations and in some cases the analytical methods employed. TABLE 1 APPROXIMATE NUCLEIC ACIDCONTENT OF SOMEVIRUSES Type of virus Plant viruses Tobacco mosaic Potato X Turnip yellow mosaic Tobacco necrosis Tomato bushy stunt Bacteriophages T2 Animal viruses Influenza Insect polyhedral viruses

Nucleic acid content (yodry weight) 6% RNA 6% RNA 35% RNA 1420% RNA 14% RNA

45% DNA 1% RNA about 15% DNA

B . The Functions of Prolein and Nucleic Acid in Virus Multiplication Work on the bacteriophages has recently led to the conclusion that the virus protein and nucleic acid have distinct functions in the multiplication cycle. The protein forms the structure containing wit)hin it the nucleic acid. The protein is probably involved in adsorption to the host cell, whereas the riucleic acid comprises at least the major portion, if not the whole, of the genetic part of the virus. There is indirect evidence, summarized below, that these conclusions are also applicable to plant and animal viruses. 1 . Bacteriophages. Without presenting the experimental evidence we shall outline the sequence of events in the multiplication of a lytic bact,eriophage (the coli phage T2) on a sensitive bacterium (Hershey, 1952; Hershey and Chase, 1952; for a review see Putnam, 1953). The bacteriophage T2 consists of a head, hexagonal in cross-section, to which is attached a tail. The head consists of a protein ‘Lmembrane”within which is held the DNA.

CHEMOTHERAPY O F VIRUSES

63

The outside of the tail also consists of protein. As the result of collisions the phage particle becomes attached to the bacterium by its tail. This initial attachment (adsorption) is reversible. It is followed by irreversible steps in which the phage nucleic acid is injected into the bacterium through the tail. [The release of the nucleic acid from T4 phage can in fact be brought about specifically by the addition of a lipocarbohydrate preparation from the host bacterium (Jesaitis and Goebel, 1953).] The protein shell, consisting of the outside of the head and the tail, remains on the outside of the bacterium and takes no further part in the phage multiplication. It can in fact then be removed without affecting multiplication. Thus the virus DNA alone enters the bacterium and is responsible for organizing virus multiplication within the cell (although the possibility that a small amount of nonsulfur-containing protein may enter simultaneously has not been excluded in the experiments). Since the infecting virus particle has broken down, it cannot be recovered from the cell. Virus-specific DNA and protein are produced in the cell and after 9 min. (in broth) these begin to be organized into mature virus particles which may be liberated artificially by breaking open the cell. The number of new phage particles increases at a linear rate until about 21 min., the end of the latent period, when the cell bursts and all the newly formed virus particles are set free. By bursting open infected bacteria towards the end of the latent period two types of immature phage particle have been found. One (the doughnut) consists of a round structure similar in size to the phage head but containing only protein and virtually no nucleic acid, and the other consists of similar bodies with attached tails, practically devoid of nucleic acid. Although these types of particle occur together a t certain times the order of appearance appears to be: “doughnuts,” particles with tails but no DNA, and finally complete phage particles. 6. Plant Viruses. There is no such detailed knowledge concerning plant viruses, but with one a t least there is clear evidence that the nucleic acid is essential for infectivity. In plants infected with turnip yellow mosaic virus two kinds of virus particle are present. One type is a nucleoprotein containing about 35% RNA. This type is infectious. The other is an apparently identical protein containing no nucleic acid. This is not infectious (Markham and K. M. Smith, 1949). Markham (1951) suggested that the nucleic acid in the nucleoprotein must be inside a protein shell, a view supported by the X-ray data of Schmidt and his colleagues (1954). The relationship between the protein and nucleoprotein particles has not been clearly established, although experiments by Jeener (1954) suggest that the protein particles are precursors of the fully formed virus nucleoprotein. Plants in which

64

R. E. F. MATTHEWS AND J. D. SMITH

the virus was actively multiplying were placed for short periods in an . two types of particles were then isoatmosphere containing C L 4 0 ~The lated from the plant. More C14 was detected in the protein particles than in the virus nucleoprotein. Thus the protein particles may have some analogy with the nucleic acid-free ‘(doughnuts”found during the development of some bacteriophages. From plants infected with tobacco mosaic virus a protein fraction of smaller molecular weight than the characteristic virus rods can be isolated. This fraction reacts with antiserum to tobacco mosaic virus, does not contain nucleic acid and is noninfectious (Takahashi and Ishii, 1953; Jeener and Lemoine, 1953). There are several observations showing that the protein part of tobacco mosaic virus can be artificially modified without affecting the infectivity or genetic properties of the virus. For example, Miller and Stanley (1941) found that 70 % of the free amino groups of the virus could be acetylated without loss of infectivity. Harris and Knight (1952) by means of carboxypeptidase removed all the terminal threonine residues from tobacco mosaic virus without changing its infectivity. In both these cases normal virus was produced from infection by the altered particles. However, the protein part of viruses is certainly specific. Knight (1949) has shown that differences in the amino acid composition of strains of tobacco mosaic virus can be roughly correlated with degree of relationship based on biological properties; even closely related strains of the virus may have detectably different amino acid composition. Wang and Commoner (1954) isolated a fraction of tobacco mosaic virus (TMV) from infected tobacco leaves. This material (designated IS), although possessing biological properties indistinguishable from the ordinary virus, was found to differ in chemical composition and physical properties. They suggest as the most likely explanation for their results that I8 represents an “intermediate or alternative product of the specific biosynthesis of T.M.V.” They further conclude that the biological specificity of the of the virus may not be rigidly dependent upon the particular chemical structure ascribed to ordinary tobacco mosaic virus. However, the presence of small amounts of adsorbed host protein in Wang and Commoner’s I8 preparations could account for the difference in amino acid composition, the different isoelectric point, the precipitation in amorphous instead of paracrystalline form, and the apparent combination of a given weight of I8 with somewhat less TMV antibody than normal virus. 3. Animal Viruses. With these there is little experimental evidence covering the respective roles of the virus nucleic acid and protein. By infecting eggs with different sized inocula of influenza virus, preparations of the virus of widely: differing infectivity are obtained; heavy inocula pro-

65

CHEMOTHERAPY OF VIRUSES

duce preparations of low infectivity. Ada and Perry (1955) have shown that the infectivity of such preparations is correlated with their RNA content, increased infectivity being associated with a higher content of RNA.

C. The Structure of Nucleic Acids We are giving most attention in this review to the possibilities of inhibiting virus development with analogues which are incorporated into nucleic acids. The potentialities of this approach can best be dealt with after a consideration of t,he structure of nucleic acids. The structure and biological function of the nucleic acids have been recently reviewed by Markham and Smith (1954). 1 . The Components of Nucleic Acids. The nucleic acids are large polymerized molecules built up from purine and pyrimidine bases, a sugar, and phosphate. I n RNA the sugar is D-ribose and in DNA it is 2-deoxy-~ribose (both occurring in the nucleic acid as the furanose form). Four bases are so far known to occur in normal RNAs-the purines, adenine and guanine, and the pyrimidines, cytosine and uracil. NHz

0

C

C

II

I

H

.N.

Adenine

Guanine

0

II

I

C

/ \

C

N-

CH

I //\/

CH

C

0

N H

Cytosine

HN

II

/ \

CH

II

I

NC\ N /CH

0

H

Uracil

DNAs contain adenine, guanine, cytosine, and thymine (5-methyluracil) but do not contain uracil. Those from higher animals and plants also contain a fifth base, 5-methylcytosine, whereas in t>heDNAs from E . cola bacteriophages T2, T4, and T6 cytosine is absent and is replaced by

66

R. E . F. MAWHEWS AND J. D. SMITH

5-(hydroxymethy1)cytosine. In the T-even bacteriophage DNAs the hydroxymethylcytosine residues are esterified to a glucose molecule through the 5-hydroxymethyl group. NHz

0

c

/ \

HN

I

C

II

RC\

0

NH,

I

I

II

N/cH H

Thymine

N

/c\

C/CHs

II I CH HC\ /

0

N H

5-Methylcytosine

N

/c\

NC\

CHzOH

II

I

0

C

/

N /CH H

5-(Hydroxymethyl)cytosine

2. The Repeating Unit. The purine and pyrimidine bases, sugar, and phosphate can be considered as the basic structural units in nucleic acids. However, more complex units can be isolated by hydrolysis. These are the purine and pyrimidine glycosides (nucleosides), the sugar phosphates, and the nucleotides. Each nucleotide consists of a sugar joined by a p-glycosidic link to a purine or pyrimidine, with one of the sugar hydroxyl groups esterified with a phosphate. In the nucleotides of both DNAs and RNAs the glycosidic link is through Ng of the purines or NI of the pyrimidines. The nucleotides are the repeating unit along the nucleic acid chain. In this respect they bear the same relationship to the nucleic acids as the amino acids do to the proteins. 3. The Linkage Between Nucleotides. Chemical and enzymatic degradation of DNAs has shown that the majority if not all of the nucleotides are joined by phosphodiester links between the (OH) on Cs' of the sugar of one nucleotide to that on Ca' on the neighboring nucleotide (Fig. 3A). Likewise, the evidence from chemical and enzymatic degradation of RNAs has shown that at least most of the nucleotides are joined by phosphodiester links between the 3' position on one nucleotide and the 5' position of its neighbor. 4. Branching of the Chains. For both DNA and RNA there is no chemical evidence to support the idea that the chain may be branched, for example through triply esterified phosphate groups, although conclusive evidence that there is no such branching is difficult to obtain. We shall assume that both DNA and RNA are made up of unbranched chains of the type shown in Fig. 3B. 6. The Size of Nucleic Acids. Physical measurements on the best preparations of DNA from various animal and bacterial sources show that the molecular weight of the units in solution is extremely high being between

CHEMOTHERAPY OF VIRUSES

67

5 X lo6 and 10’. Physical data show that the molecule is very elongated and behaves as a fairly rigid rod. Under the electron microscope dried DNA films appear to be made up of long threads about 15-20 A in diameter. RNA chains appear to be much smaller. Several different types of chain

0

I

HO-P=O /

A

1 Sugar-base

Phosphate/ Phosphate/ Phosphate/

/

Sugar-base Sugar-base Sugar-base

B FIG.3. A : The internucleotide linkage in nucleic acids. In RNA the H on C1 is replaced by OH. B: Diagrammatic representation of part of a single nucleic acid chain.

can be distinguished by the identity of the terminal nucleotide residues and the arrangement of their terminal phosphate residues (Markham and Smith, 195213; Markham et al., 1954). From measurements on the proportion of such end groups, and assuming an unbranched structure, an estimate may be made of the mean chain length in an RNA preparation. In turnip yellow mosaic virus and tobacco mosaic virus this is about 50 nucleotide residues. Such methods, however, only give the length of the chain which is held together by covalent links and will not detect aggregates of short

68

R. E. F. MATTHEWS AND J . D. SMITH

chains held together by hydrogen bonding, such as have been postulated for DNA by Dekker and Schachman (1954). The high molecular weights found for some preparations of tobacco mosaic virus RNA suggest t,hat such aggregation may occur. There is spectrophotometric evidence for structural factors in the RNA which are destroyed by heating. These could be hydrogen bonds. 6 . The Proportions of the Bases in Nucleic Acids. I n all DNAs that have been examined adequately there is a 1 :1 molar ratio between adenine and thymine and between guanine and cytosine (or cytosine plus 5-methylcytosine, or 5-(hydroxymethy1)cytosine). I n RNAs no such uniformity exists although Elson and Chargaff (1954) have claimed that the proportions of purines and pyrimidines in animal ribonucleic acids obey rules similar in principle to those found for DNAs. They claimed that in a nu'mber of RNAs the ratio, adenine/uracil, guanine/cytosine, purineslpyrimidines, all approximately equaled 1, whereas in all the RNAs they examined the ratio (guanine uraciZ)/(adenine cytosine) was approximately 1 (i.e. the proportions of bases in the RNAs were such that the number of 6-keto groups equaled the number of 6-amino groups. These results, however, are based on selected analyses and there is no doubt that no such regularity in the proportions of the bases holds for the four plant virus nucleic acids which have been accurately analyzed. It will require much more accurate analysis of plant and animal RNAs before it can be concluded that such a lack of regularity is a distinctive feature of the plant virus RNAs. 7. Nucleic Acid Structure in Relation to the Reduplication of Genetic Material. If DNA (and in some viruses RNA) is the carrier of the genetical material of the cell or virus, then the structure must contain the required information and must be able to organize its own reduplication a t cell division or during virus multiplication. Furthermore, the information must be available in a form which can be used in the making of other cell components. The phosphate-sugar backbone of all DNAs and of all RNAs appears to be the same. It thus seems likely that the sequence of pyrimidine and purine bases is involved in some way in the transmission of the genetic information. A structure for DNA has been proposed on the basis of X-ray data which suggests a mechanism by which the chains could be duplicated. The proposed structure allows an unlimited variation in nucleotide sequence on individual polynucleotide chains and furthermore indicates explanations of certain genetical phenomena on the basis of chemical structures. a. The Watson-Crick Structure for D N A . The structure proposed by Watson and Crick (1953a, b, c; Crick and Watson, 1954) is based on the X-ray data of Wilkins and his colleagues (Wilkins et al., 1953; Franklin

+

+

CHEMOTHERAPY OF VIRUSES

69

and Gosling, 1953). It is also based on two general features of DNAs: (1) in intact DNA certain of the dissociable groups on the purines and pyrimidines (the NH2and C =0 groups) are involved in hydrogen bonding, (2) in practically all DNAs the molar ratios of adeninelthymine and of guaninelcytosine (or cytosine 5-methylcytosine or hydroxymethylcytosine) are equal to 1. The structure (illustrated in Fig. 4) comprises a pair of polynucleotide chains arranged as two regular right-handed helices coiled round the same axis and each displaced from the other by about W of the pitch of the helix. The phosphate groups are on the outside, the purine and pyrimidine bases facing inwards. The two helices are held together by hydrogen bonds between pairs of bases. The two bases in each pair lie in the same plane perpendicular to the axis of the helices. Thus the two backbone chains are joined by a series of flat “plated’ consisting of a purine on one chain hydrogen bonded to a pyrimidine from the other. The pairing is specific. As the phosphate-sugar backbones are regularly arranged in space around the helix axis, for stereochemical reasons only a purine plus a pyrimidine can form a pair. Allowing for the bases being in their most probable tautomeric forms (i. e. with the C=O groups at positions 6 and 2), only two types of pairing by hydrogen bonds are possible with this structure. These are adenine and thymine, and guanine and cytosine (or 5-methylcytosine or 5-(hydroxymethy1)cytosine). In each case one hydrogen bond is formed between t’he 6-amino group on one base and the 6-keto group on the other, and a second hydrogen bond is formed between the N atoms on position 1 (see Fig. 5 ) . This type of pairing accounts directly for the experimentally found equivalence of adenine and thymine and of guanine and cytosine in DNAs. The sequence of atoms in the phosphate-sugar backbones of the two chains runs in opposite directions (i. e. they are related by a diad perpendicular to the helix axis). There is a nucleotide on each chain every 3.4A in the direction of the helix axis and 1 turn is made every 10 nucleotides or 34 A. The distance of a phosphorus atom from the axis is about 10 A giving a molecular diameter of about 20 A. b. The Reduplication of D N A . The sequence of purines and pyrimidines on any one chain is completely unrestricted but that of its partner in the double helix is complementary. For example a sequence such as A.C.T.A. G. (where A represents adenine; G, guanine; C, cytosine; and T, thymine), is, by reason of the specific pairing, reflected in the partner chain as T.G.A.T.C. The st.ruct,ure thus provides a method of replication of the nucleotide sequence in any one chain through intermediary formation of an exactly

+

FIG.4. (Courtesy Scientijic American.) 70

CHEMOTHERAPY OF VIRUSES

71

complementary chain. If two chains of a double helix separate, and opposite to each is formed a complementary chain, then the result will be two double helices identical with the parent one. On separation of the two chains the purine and pyrimidine residues on each are exposed and free nucleotides (or polynucleotide precursors) may attach themselves by hydrogen bonds. Due to the geometry of the structure, only when the correct nucleotides have paired with the nucleotide residues on one of the separated chains is it possible by phosphodiester bond formation to form H Adenine

H

__-._---

Guanine

_---. H

(-)------

_--

FIG.5. The two pairs of bases in the Watson-Crick model. The dotted lines represent the hydrogen bonds holding each pair together.

the backbone of the complementary chain and so complete the double helical structure. One of the weaknesses in this replication theory is the failure of the structure to distinguish between cytosine and 5-methylcytosine. Another obvious difficulty lies in the means of separation of the two chains. They cannot come apart by movement perpendicular to the helix axis and complete separation by untwisting would involve about 150 turns per lo6 FIG.4. A model of the Watson-Crick structure of deoxyribonucleic acid showing the paired chains wound as a helix round the fibre axis. The phosphate-sugar backbones are represented by black wire, and the flat plates represent the pairs of bases joined by hydrogen bonds. The upper photograph is a top view showing clearly that the bases are on the inside of the structure while the phosphates are on the outside.

72

R. E. F. MATTHEW8 AND J. D. SMITH

molecular weight and the simultaneous breaking of about 3000 hydrogen bonds. Possibly replication of each chain is more likely to begin from one end with simultaneous untwisting of the original paired helices. Alternatively replication might begin at the ends of the chain and separation of the original paired chains take place through a series of breaks and rejoins in the phosphate-sugar “backbone” (Delbruck, 1954). In these methods of replication only small parts of the molecule are involved at any given time. There is physical and chemical evidence suggesting that the DNA molecule may be built up of a number of smaller units held together by hydrogen bonds (Dekker and Schachman, 1954). To reconcile this with the WatsonCrick structure Dekker and Schachman have suggested that the phosphatesugar “backbones” of the polynucleotide chains are not continuous but are interrupted on an average after every 30-50 nucleotides and that the breaks in the two chains are staggered relative to each other so that the smaller units are in fact held together by the hydrogen bonding. c. Genetic Implications of the Watson-Crick Structure. Watson and Crick have also suggested a theory of mutation based on tautomeric shifts in the structure of the bases during replication. For example if the 6-keto groups of a thymine residue (I) on a chain in the process of replication were to transitorily change to the enol form (11) (e.g. as the result of adsorption of energy during ultraviolet or X-irradiation) then the thymine would no longer be capable of pairing with adenine but could instead pair with guanine which could replace adenine in the new complementary chain. In a subsequent replication the guanine residue would pair with cytosine so that in the new double helix a thymine-adenine pair would be replaced by a guanine-cytosine pair. 0

OH II

2\

N

C

C

CH

II

I

/ \ /

0

N H

(11)

The way in which the information present in the DNA molecule may be transmitted to other structures in the cell to give rise for example to specific amino acid sequences in proteins or nucleotide sequences in ribonucleic acids is a matter for speculation. The Watson-Crick DNA model contains

CHEMOTHERAPY OF VIRUSES

73

a helical groove into which might be fitted an RNA or polypeptide chain, and the structure is sufficiently open to allow interaction between groups on such chains with specific groups on the purines and pyrimidines. d. The Structure of R N A . Largely because of the difficulties in obtaining sharp X-ray diffraction pictures of RNA no structure has yet, been proposed. Those pictures which have been obtained have suggested certain similarities with DNA (Rich and Watson, 1954) but are of too poor resolution to permit definite conclusions.

D. The Lysogenic Virus-Host Relationship Many bacteria can carry one or more bacteriophages in an intracellular form through an indefinite number of generations. Such bacteria have the property that, either “spontaneously” or as the result of an artificial treatment called induction, intracellular phage development will occur leading to lysis of the bacterium and liberation of bacteriophage particles active on related sensitive strains of bacteria. Unless lysogenic bacteria are actually in the process of developing mature virus particles following such induction, artificial disruption of the cells does not liberate any infective virus particles. The lysogenic bacterium thus carries the bacteriophage in a heritable form differing from the mature virus particle. This has been called by Lwoff “prophage” (Lwoff and Gutmann, 1950). I n one case it has been shown t,hat the prophage is linked to genetic characters carried in the bacterial chromosome. A parallel phenomenon has not yet been observed with animal or plant viruses but this is possibly due to the difficulty of its detection. Also it is not easy with animal and plant viruses to distinguish between a lysogenic phenomenon and a “latent” infection due to the persistence of mature virus particles in low concentrations. It may be that viruses such as the latent virus of the sugar beet (K. M. Smith, 1952) and that causing herpes simplex do exist in transmissible forms differing from the mature virus. If so, the elimination of such virus infections by chemotherapy may present an extremely difficult problem.

E. Persistence of Virus Infections Unlike the situation with most animal viruses, a plant, once infected usually never becomes free of infective virus even though disease symptoms may become milder with time. For example a tobacco plant, after infection for some time with tobacco mosaic virus, contains large numbers of crystalline inclusion bodies which consist almost entirely of virus. It is most unlikely that any chemical treatment would inactivate the virus in such inclusion bodies without being lethal for the host plant.

74

R. E. F. MATTHEWS AND J. D. SMITH

IV. EFFECTS OF PURINE AND PYRIMIDINE ANALOGUES ON VIRUSES A . Plant Viruses 1 . Purine Analogues a. 8-Azapurines and Related Compounds. i. Eflects on disease development. The guanine analogue 8-azaguanine, when sprayed on the leaves at concentrations around 0.005-0.01M in solution in 0.1 % NaHC03 , causes a marked delay or complete inhibition of systemic development with certain plant viruses (Matthews, 1953a). Among the viruses tested, the compound is most effective with lucerne mosaic virus in Nicotiana glulinosa and tobacco and cucumber mosaic virus in cucumbers and Nicotiana glutinosa. 0

II

C

8-Asaguanine

When young plants are mechanically inoculated with these viruses, they may be given complete protection against systemic development of the virus, provided the compound is sprayed on the leaves before inoculation, or up to about two days after inoculation. We have never observed the compound to prevent further systemic spread and development of a virus once systemic movement has begun. With cucumber mosaic viruses the compound when watered on the soil around the plants had no inhibitory effect on disease development when the virus was introduced by mechanical inoculation. Watering the compound on the soil prevented systemic spread in a proportion of plants when they were infected by means of aphids. Treatment of tobacco plants with 8-azaguanine causes a few days’ delay in the systemic development of tobacco mosaic virus, but has never prevented eventual systemic development. With turnip yellow mosaic virus the compound has no effect if fairly large Chinese cabbage plants (9-12 inches high) are used. However, with small plants (leaves 3 4 inches long) the compound has a delaying effect as with tobacco mosaic virus. With the following viruses the compound has no significant effect: henbane mosaic virus, potato viruses X and Y in tobacco, tomato spotted wilt virus in tomato, tropaeolum virus I1 in tobacco and pea mosaic virus in peas. 8-azaguanine causes a slight stunting and distortion of young leaves of

CHEMOTHERAPY OF VIRUSES

75

plants at concentrations around 0.01 M . The damaging effect varies to some extent with age and condition of growth of the plants. Very young, and rapidly growing plants, are more susceptible to damage. It is possible however to get complete inhibition of systemic development with lucerne mosaic and cucumber mosaic viruses without any detectable effects on plant growth. The adenine analogue, 8-azaadenine, has some inhibitory effect on lucerne mosaic, cucumber mosaic, and tobacco mosaic viruses but it also causes marked plant damage. The relative effectiveness of the compound as a virus inhibitor is therefore difficult to assess. Treated plants become stunted with yellowing, distortion, and necrosis of the leaves. At concentrations producing no plant damage the compound is ineffective as a virus inhibitor. 8-azahypoxanthine has no effect on the systemic development of any virus we have tested. However with lucerne mosaic virus, in leaves where only necrotic local lesions are produced, 8-azahypoxanthine is more effective than 8-azaguanine in reducing the number of local lesions. 8-Azahypoxanthine has not been observed to cause any plant damage. 8-Azaxanthine, 8-azaisoguanine, and dimethylamino-8-azaganine (8-azaguanine bearing two methyl groups substituted in the amino group) appear to have-no effect on virus development or on plant growth. We have tested a number of substituted triazoles. Among these only 4-(5)-amino-lH-l,2,3,triazoled-(4)-carboxyamide showed any activity. This compound was less effective than 8-azaguanine but caused a slight delay in the systemic development of cucumber mosaic virus in Nicotiana glutinosa and tobacco mosaic virus in tobacco. It has no effect on henbane mosaic virus in tobacco.

HzN

H

4-(5)-Amino-lH-l, 2,3,triazole-5-(4)-carboxyamide

5-hydroxy-1H-1 ,2,3-triazole, 4-(5)-hydroxy-lH-l , 2 , 3 -triazole-5-(4)carboxylic acid, and 4-(5)-amino-1H-l,2,3-triazole-5-(4)-carboxylic acid had no effect on these three viruses. ii. Effect on virus Production. When tobacco plants inoculated with tobacco mosaic virus are sprayed with 8-azaguanine about 24 hr. after in-

76

R. E. F. MATTHEWS AND J. D. SMITH

oculation (and every 2 or 3 days thereafter), the virus content of the plants sampled between about 3-15 days is less than in control plants. This delay in the production of virus has been demonstrated by infectivity measurements, serological estimations, extraction and estimation of the virus and by particle counts on electron micrographs of plant sap. The only compounds which have been found to delay virus production are those which cause a delay in systemic development of the virus (8-azaguanine, 8-azaadenine, and 4- (5)-amino-lH-l ,2,3-triazole-5-(4)-carboxyamide) . With turnip yellow mosaic virus in Chinese cabbage, the yield of virus is reduced by 8-azaguanine treatment if very young plants are used, but has no effect on larger plants. Even with tobacco mosaic virus, for a given time after inoculation and treatment with the compound the reduction in yield is quite variable. The reduction appears to be greatest in young, rapidly growing plants. The amount of virus in treated plants does not remain permanently less than in control plants with either of these viruses. After a variable time (about 2-3 weeks) the virus concentration reaches the concentration in control plants. With lucerne mosaic virus, some multiplication takes place in inoculated leaves (as judged by infectivity tests) even though no typical local symptoms may be produced, but virus production is much less. Where systemic development is prevented, the virus content of the whole plant must be a very small fraction of that in untreated plants. iii. Mechanism of action of 8-uzuguunine. (1). Reversal of inhibition by normal purines, The virus inhibitory activity of 8-azaguanine can be annulled if plants are sprayed with several naturally occuring purines or their ribosides and ribonucleotides. This effect can be demonstrated either by observations on the development of systemic disease, or by estimates of virus production. The activity of 8-azaguanine can be partly or completely annulled by treatments with the following: adenine, guanine, hypoxanthine, and the corresponding ribosides, and the adenylic acids (2’, 3’, and 5’ phosphates). We have not tested guanylic and inosinic acids. We have evidence suggesting that some of these compounds when supplied alone may stimulate virus production. The following compounds do not affect the activity of 8-azaguanine: xanthine, uric acid, theobromine, theophylline, caffeine, uracil, and thymine. (2). Incorporation of 8-azaguanine into virus nucleic acids. Nucleic acid was prepared from tobacco mosaic virus isolated by the ammonium sulfate precipitation method from control and 8-azaguanine-treated plants, On analysis by the paper chromatographic method of Wyatt (1951) the virus nucleic acid from treated plants was found to contain about 3 % less guanine than that from untreated plants. This deficiency was accounted for

77

CHEMOTHERAPY OF VIRUSES

entirely (within the limits of experimental error) by an increase in the adenine figure (Table 2). In the iso-propanol-hydrochloric acid solvent used, 8-azaguanine would run with and be estimated as adenine. The demonstration that 8-azaguanine was in fact incorporated into the nucleic acid as 8-azaguanylic acid is described in Section IV4. Using the size and density of the fluorescent sport of 8-azaguanylic acid obtained from a given amount of nucleotides as a rough estimate of the amount of incorporation, it was found that in tobacco plants infected for 14 days there was much less incorporation in the virus isolated from systemically infected leaves than in the virus from the inoculated leaves. This suggested that the 8-azaguanine is not randomly distributed among the virus particles, but that the early-formed virus contains more than the TABLE 2 MOLAR RATIOS OF BASES I N NUCLEIC ACIDO F TOBACCO MOSAIC VIRUSFROM CONTROL A N D 8-AZAQUANINE-TREATEDPLANTS Molar ratios (means of 7 determinations) Base

Control

8-Asaguanine treated

Difference control treated

Guanine Adenine Cytosine Uracil

0.979 1.103 0.817 1.101

0.943 1.134 0.815 1.110

-0.036 +0.031 -0.po2 -0.009

-

Difference required for significance at 0.01 P 0.024 0.031 0.021 0.058

virus formed later. Attempts to separate virus preparations into 8azaguanine-rich and 8-azaguanine-poor fractions have so far failed (Matthews, 1954). We have examined the virus nucleic acid from plants treated with the other 8-azapurines and the triazoles. No 8-azaguanylic acid could be detected in virus from plants treated with 8-azahypoxanthine1 8-azaxanthine, 8-azaisoguanine, dimethylamino-8-azaguanine,or 4-(5)-amino1H-l,2,3-triazole-5-(4)-carboxylic acid. However virus from plants treated with either 8-azaadenine or 4-(5)-amino-lH-1,2,3-triazole-5-(4)carboxyamide contained 8-azaguanylic acid in small amounts. Thus incorporation into the nucleic acid was found only in those cases where a compound caused a delay in virus production. 8-azaguanylic acid can be detected in the nucleic acid of turnip yellow mosaic virus when young plants are used. We have not yet tested any of the other compounds with this virus. (3). The infectivity of virus containing 8-azaguanine. It is possible to

78

R. E. F. MATTHEWS AND J. D. SMITH

compare the infectivity of two virus preparations by estimating the total amount of virus material present by some physical or chemical means and comparing these estimates with those obtained by infectivity measurements on the same preparations. In material from 14-day-old tobacco mosaic virus infections no difference in infectivity between virus from control and 8-azaguanine-treated plants could be detected. However material from 3-day-old infections from treated plants consistently gave a lower ratio of infectivity to total virus than did control preparations when total virus was measured by three different methods (Matthews, 1954). The data suggested that roughly half the virus particles were rendered nonTABLE 3 INFECTIVITY

OF

TURNIP YELLOW MOSAIC VIRUS CONTAININQ 8-AZAQUANINE Mean number of local lesions per half leaf

Expt. No. (1)

Material used for inoculum Purified virus preparations Purified virus preparations

Method of equalizing amount of virus material in inoculum

Control

8-Azaguanine containing

Optical density at 260 mp

37.5

13.6

14.6

6.5

37.7

22.3

59.3

34.6

Nucleic acid content determined by serological chromatographicmethod (3a) Unclarified plant Virus nucleic acid content of sap determined by Sap serological chromatographic method (36) Purified virus Phosphorus content preparations (2)

infectious. However there are several possible alternative explanations for this result. Firstly the 8-azaguanine preparations might have contained a contaminant not present in the control preparations. Such an explanation is unlikely since the contaminant would have to contain nucleic acid, precipitate with TMV antiserum, and be stable to freezing, slow thawing of the plant extract, heating to 55OC., and salt precipitation. Secondly, normal immature noninfectious forms of the virus might exist and these might form a larger proportion of the 8-azaguanine-containing preparations since less multiplication had taken place. Such immature forms would have to have the properties noted above for a contaminant. Thirdly, although freshly expressed plant sap diluted in water was used for the infectivity tests there might have been slight differences in the degree of aggregation of the rod-shaped particles in virus from control and 8-azaguanine-treated plants. Such changes could markedly affect the results

79

CHEMOTHERAPY OF VIRUSES

of infectivity tests. We have recently carried out similar experiments with turnip yellow mosaic virus. This virus has spherical particles. Thus the problem of degree of aggregation does not occur, so infectivity tests can be made on purified virus preparations. The experiments summarized in Table 3 show that, comparing equal amounts of virus nucleic acid, 8-azaguanine-containing virus is less infectious than control material. The occurrence of virus nucleoprotein and protein particles in plants infected with turnip yellow mosaic was described on p. 63. 8-Azaguanine had no detectable effect on the proportions of these two types of particles in plant,s in which the virus was multiplying rapidly (Matthews, 1955b). PRODUCTION O F

“PURPLE”

TABLE 4 AND “BLUE” METABOLITES IN

~~

Metabolite produced Compounds used to treat germinated seeds 8-Azaguaniue Dimeth~~laniino-8-azaguanine 8-Azaadeninc 8-Azahypoxanthine 8-Azaxanthine 8-Azaisoguanine 5-Hydrosy-IH-l,2,3-triazole 5-(4) -Hydroxy-IH-l,2,3-triazole-4-(5)-carboxylic acid 5- (-1) -Aniino-lH-l,2,3-triazole-4-(5)-carboxylic acid 5-(-1)-Amino-IH-l,2,3-triaeole-4-(5)-carboxyamidc

PEAS

~

“Purple”

“Blue”

++ ++

+ ++ ++ ++ ++

-

Inhibitory activity for plant viruses

-

.-

+

(4). Metabolism of 8-azapurines in plant tissues. The metabolism of 8azaguanine i i i plant tissues has been studied mainly in young germinated peas (Matthews, 1955a). Young germinated peas were placed in the dark in dishcs cwntaining a solution of the compound and harvested after 3-6 days. The washed material was frozen, minced, aiid extracted with hot 50 % ethanol and the extract concentrated. The metabolites were isolated by paper chromatography, advantage being taken of the marked fluorescence in ultraviolet light of many of the compounds. Most of the 8-azaguaiiine absorbed by young peas is fairly rapidly deaminated to 8-azaxanthine. In addition two other highly fluorescent compounds are producd in trace amounts. One of these gives a bright purple fluores(wiw, while thc other appears bright blue under ultraviolet light. The “purple” and “blue” metabolites have not yet been identified. There

80

R. E. F. MAlTHEWS AND J. D. SMITH

is some evidence to suggest that they may be 4,5-substituted triazoles. Their production in peas from the various 8-azapurines may be of interest in relation to the antivirus activity of the latter (Table 4). Of the six 8-azapurines tested only the two with inhibitory activity for plant viruses produce the purple metabolite. Thus it is possible that the “purple” compound may be involved in the inhibitory action of 8-azaguanine and 8-azaadenine. The relationships so far established may be summarized as follows: iA~8-az “purple” metabolite

ITkV

t

~

8-azaguanylio acid in RNA

5- (4)-amino-lH-l,2,3triazole-4-(5)-carboxyamide in TYV

in TMV

8-azaguanine in-

,

aadenine

and p e a

I

L8-azaxanthine

1.

inpeas,

“blue” metabolite

b. 2-Azapurines. 2-Azaadenine markedly decreased the multiplication of tobacco mosaic virus when tested by the leaf disc method (Schneider, 1954). NH,

I

C

2-Azaadenine

In Schneider’s test 2-azaadenine was more effective than 8-azaguanine. When tested by the leaf-spraying method this compound was inactive against tobacco mosaic virus. It was also inactive against several other plant viruses (Matthews, 1953a). c. Indene Derivatives. The 1,2,4 ,g-tetraazaindene analogue of guanine had no effect on tobacco mosaic virus (Matthews, 1954) or on cucumber mosaic virus and henbane mosaic virus.

CHEMOTHERAPY OF VIRUSES

81

0

II c

/’\ dl\ NH

HN6

5-Amino-7-hydroxy-l ,2 4,6-tetraazaindene

d. Thiopurines. We found thioguanine and 6-thiopurine to be inactive when tested by leaf spraying against tobacco mosaic virus, cucumber mosaic virus, and henbane mosaic virus. S

II

C

6-Thioguanine

Thioguanine, 2-thioxanthine, and 2-methylthioadenine had no marked effect on tobacco mosaic virus multiplication tested by the leaf disc method (Schneider, 1954). e. Oxadiazolo-pyrimidines. The guanine analogue 5-amino-7-hydroxy~3,1,2)oxadiazolo(5,4-d)pyrimidinehad no effect on the development of several plant viruses (Matthews, 1953a). 0

II

N 5-Amino-7-hydroxy(3,12)oxadiazolo(B 4-d)pyrimidine

f. Other Substituted Purines. Schneider (1954) tested a variety of methylated and halogenated purines against tobacco mosaic virus by the leaf disc method. None of these had any marked effect on virus development.

82

R. E. F. MATTHEWS A N D J. D. SMITH

However, he confirmed the observation of Mercer et al., (1953) that 2,6-diaminopurine had a strong inhibitory effect on virus production. He found that this inhibition could be annulled by adenine, adenosine, adenylic acid, or guanine. Schneider noted that 2,6-diaminopurine caused no observable damage in tobacco leaf discs. However, when sprayed on growing plants, this compound is a very strong inhibitor of plant growth (Matthews, 1953a). Its delaying effects on systemic virus development could not be dissociated from its effect on the host. 2. Pyrimidine Analogues a. Thiopyrimidines. i. Thiouracil. Commoner and Mercer (1951, 1952) showed that, in tobacco leaf discs floated on solutions containing low concentrations of 2-thiouracil, the production of tobacco mosaic virus was greatly reduced. 0

II

C

HN

I s=c

/ \

CH

II

CH

\ / NH

2-Thiouracil

Matthews (1953a) found that when sprayed on plants this compound caused yellowing and marked inhibition of growth in the young leaves. A slight delay occurred in the production of systemic symptoms by lucerne mosaic and cucumber mosaic viruses, a delay that might well be explained a t least in part by the difficulty of observing symptoms on the yellowed leaves. The effect on the systemic development of these viruses is much less than that produced by 8-azaguanine. Thiouracil has no effect on the systemic development of henbane mosaic virus. However, with tobacco mosaic virus, thiouracil is a more effective inhibitor of virus multiplication than 8-azaguanine (Mercer el al. 1953) and is also more effective in delaying systemic development of the virus. Bawden and Kassanis (1 954) found that in addition to inhibiting tobacco mosaic virus, thiouracil reduced the multiplication of potato viruses X and Y and henbane mosaic in tobacco leaves floated on solutions containing the compound. With Rothamsted tobacco necrosis virus the compound was effective in tobacco but not in the French bean. They found the compound had no effect on broad bean mottle virus in the broad bean. With tobacco mosaic virus Bawden and Kassariis (1954) found that thiouracil has its greatest inhibitory effect under conditions which would,

CHEMOTHERAPY OF VIRUSES

8s

without the treatment, lead to the production of most virus. Mercer et al. (1953) showed that the inhibitory activity of thiouracil could be reversed by uracil but not by cytosine or thymine. Jeener and Rosseels (1953) cultured inoculated tobacco leaves on a solution containing 2-thiouracil-S36. They then isolated the virus from leaves in which the yield of virus was reduced by about 50% and found activity in the nucleic acid fraction but not the virus protein. The virus nucleic arid was hydrolyzed in 1 N HCl a t 100°C. for 30 min. and subjected to chromatography in iso-propanol-hydrochloric acid. They found that all the S36moved with an R, about 6 % greater than that of uridylic acid, a position that might be expected for thiouridylic acid. From the radioactivity measured, they calculated that about 20% of the uracil was replaced by thiouracil. It should be possible t o detect this amount of incorporation by ordinary chromatographic procedures. We have attempted to do this without success (Matthews, 1954). Inoculated plants were sprayed with thiouracil. Yields of virus from treated plants were less than 50% of the controls, sometimes much less. The virus was isolated, nucleic acid prepared, hydrolyzed, and subjected to chromatography by the methods used by Jeerier and Rosseels. We then eluted the uridylic acid band together with the area of paper ahead of it. The concentrated eluate was then subjected to paper electrophoresis a t pH 9, when thiouridylic acid might be experted to run ahead of uridylic arid, since thiouracil has a considerably greater mobility than uracil a t this pH. We also attempted to isolate thiouridylic acid by chromatography of the eluate in the ammonium sulfate-water solvent, where uracil and thiouracil have widely differing mobilities. In no case could we detect the presence of thiouridylic acid. With the quantities of material examined the printing process used to locate the spots should have been able to detect thiouridylic acid if only 2 4 % of the uridylic acid was replaced by this compound. It seems unlikely that the activity obtained by Jeener and Rosseels was due to contaminating thiouracil. However, the position expected in the iso-propanollhydrochloric acid solvent for thiouridylic acid is also the position in which sulfate would appear. In our experiments thiouracil was applied to the plants every 2-3 days. This difference in method of application could possibly account for our failure to detect incorporation. The relative infectivity of tobacco mosaic virus from thiouracil-treated tobacco leaves has not been adequately investigated. Commoner and Mercer (1952) considered that the treatments had no effect on infectivity of the virus produced. However their results showed only that the reduction in amount of virus found after treating for a given time with varying concentrations of thiouracil, followed the same trend when measured by

84

R. E. F. MA’ITHEWS AND J. D. SMITH

amount of virus isolated or by infectivity of the leaf extracts. In fact, their data could be used to suggest that virus from thiouracil-treated leaves was less infectious. Bawden and Kassanis (1954) detected no difference in the infectivity of virus from treated and control plants, although they state that their methods would not have detected “minor” changes. Jeener (1954) inoculated half leaves of tobacco with equal concentrations of tobacco mosaic virus from control plants and from plants treated with thiouracil. He then estimated the amounts of virus after 2-8 days and found that less was produced in half leaves inoculated with virus from thiouraciltreated leaves. His results suggest that the virus was less infectious. However, he does not state on what basis the two inocula were equalized. If concentration was estimated by a method which included virus protein, his results could mean that in virus from thiouracil-treated leaves there was more of the noninfective virus protein, and less nucleoprotein. Further work on the effects of thiouracil on tobacco mosaic virus should include firstly, an attempt to isolate and characterize thiouridylic acid from the virus RNA; and secondly, infectivity measurements (using local lesion counts on half leaves of Nicotiana glutinosa) on virus preparations from control and thiouracil leaves, equalized with respect to am0unt.s of virus nucleic acid and treated in a manner calculated to minimize aggregation of the virus. ii. Other thiopyrimidines. Mercer et al. (1953) found using the tobacco leaf disc method that 2-thiothymine and 2-thiocytosine reduced the multiplication of tobacco mosaic virus, and that the inhibition could be annulled by uracil but not by cytosine or thymine. In tests by the leaf-spraying method we have not found 2-thiothymine or 2-thiocytosine to have any detectable effect on the systemic development of tobacco mosaic virus or cucumber mosaic virus, nor do these two compounds have any marked effect on plant growth. We have found that 2,4-dithiouracil has a delaying effect on tobacco mosaic virus production when sprayed on tobacco plants at concentrations around 0.01 M in solution in warm water. However the effect is much less than that produced by 2-thiouracil. If applied in water the effects of the compound on plant growth are slight. Applied at the same concentration in solution in 0.1% NaHC03, however, the material is extremely toxic, killing young plants in 2-3 days. The following compounds had no effect on tobacco mosaic virus in tobacco and cucumber mosaic virus in cucumber when tested by the leaf spraying method: 4-methyl-2-thiouraci1, 4-propyl-2-thiouraci1, 2-thioorotic and 2-mercapto-4-methylaminoacid, 4,5-diamino-2-mercaptopyrimidine, pyrimidine. b. V a r i m l y Substituted Pyrimidines. Schlegel and Rawlins (1954) tested

CHEMOTHERAPY OF VIRUSES

85

a variety of substituted pyrimidines against tobacco mosaic virus by the leaf disc method. None of these had any marked effect except 2-thiouracil and diazouracil (Fig. 2B). We have found the following pyrimidines to be inactive against tobacco mosaic virus, cucumber mosaic virus, and henbane virus tested by leaf spraying: 5-bromouracil, 5-chlorouracil,4-aminod-iodopyrimidine, 5-nitrouracil, and a variety of hydroxy- and amino-substituted pyrimidines other than those found in nucleic acids. c. Substituted Triazines. The following triazines had no marked effect on the development of tobacco mosaic virus in tobacco and cucumber mosaic virus in N . glutinosa tested by leaf spraying: 2-methyl-4,6-dihydroxy3,5-triazine; 2-benzyl-4,6-dihy1,3,5-triazine; 2-phenyl-4,6-dihydroxy-l, 5-triazine. droxy-l,3,5-triazine; and 2-a-phenylpropyl-4,6-dihydroxy-113, OH

I

A

N1

6N

II

I

HjC-C2

'C \a/

\

.NOH 2-Methyl-4,6-dihydroxy-l,3 5-triszine

These compounds had little effect on the host plant except the 2-phenyl compound which caused a sudden and complete cessation in chlorophyll formation in the very young leaves only, without any noticeable effects on leaf growth. d. Substituted Pyridines. The uracil analogue 2 ,4-dihydroxypyridine had no effect on tobacco mosaic virus, cucumber mosaic virus, or henbane mosaic virus, tested by leaf spraying. OH

I

A

HC6

II

HC'

aCH

I

2C-OH

\I/

N 2,4-Dihydroxypyridine

B . Bacteriophages 1. Purine Analogues a. 8-Azaguanine. The presence of 8-azaguanine during growth of T2

86

R. E . F. MATTHEWS AND J. D. SMITH

through several lytic cycles on B. coli “B” has been found to have no effect on the final phage titer (Wooley et al. 1952b; Bourke et al., 1952). We have also found the addition of 0.01 M 8-azaguanine before and during phage growth to have no significant effect on the burst size of T2r or T2r+ on B. coli “B” in a synthetic medium, and no 8-azaguanine could be detected in the DNA from T2 grown under these conditions. Although 8-azaguanine inhibits the growth of B. coli “B” and is incorporated into the bacterial ribonucleic acid, replacing guanine residues, no incorporation into the bacterial DNA can be detected (Lasnitzki et al., 1954). There is no definite evidence that ribonucleic acid is concerned in T2 multiplication and the failure of 8-azaguanine to inhibit T2 development is presumably simply due to the fact that in B. coli “B” it is not incorporated into DNA. However, in Bs.cereus, whose growth is more strongly inhibited by 8-azaguanine than the growth of B. coli, we have been able to demonstrate the incorporation of small amounts of 8-azaguanine (about 1 % of the nucleic acid guanine) into the bacterial DNA. Under these conditions about 25 % of the guanine residues in the Bs.cereus RNA has been replaced by 8-azaguanine. It is consequently possible that 8-azaguanine may be found to be incorporated int.0 phages active in bacteria other than B . coli. b. 8-Azahypoxanthine. I n B. coli “B,” 8-azahypoxanthine inhibits growth and is converted to 8-azaguanine which is incorporated into the bacterial ribonucleic acid. No 8-azaguanine or 8-azaadenine (or 8-azahypoxanthine) could be detected in T2r+ grown on B. coli “B” in the presence of 8-azahypoxanthine. c. 8-Azaadenine. Like 8-azahypoxanthine, 8-azaadenine inhibits growth of B. coli “B” and is converted to 8-azaguanine which is incorporated into the bacterial RNA. We have some evidence that 8-azaadenine itself may also be incorporated into the RNA. 8-Azaadenine has no effect on the lysis of B. coli “B” by T213 and the bacteriophage produced has the same specific infectivity (phage titer)/(Dnao)as control T2r+. d. 2-Azaadenine. 2-Azaadenine strongly inhibits the growth of B . coli “B” apparently without being incorporated into the bacterial RNA. The growth inhibition is reversed by adenine. We have found that T2r+ grown on B . coli “B” in the presence of 50 pg./ml. 2-azaadenine and isolated by the method of Herriott and Barlow (1952) has a specific infectivity (phage titer)/ ( 0 2 6 0 ) about 0.5 times that of oormal T2r+, or of T2r+ grown in the presence of 2-azaadenine adenine. The ultraviolet absorption spectra of the preparations are the same. We have not yet been able to detect any incorporation of 2-azaadenine into the nucleic acid of these abnormal T2r+ preparations and the possibility of a direct inactivation of the bacteriophage by 2-azaadenine has not yet been excluded. e. Other purine analogues. Wooley et al. (195213) found that the following

+

CHEMOTHERAPY OF VIRUSES

87

substances reduced the yield of T2 grown on B. coli “B” in “multicycle” growth experiments: 5-hydroxy-7-methyl-tetrazolo(A)pyrimidine;2-p-hydroxyphenyl-5-amino-7-hydroxy-v-triazolo(d)pyrimidine and 2-p-carboxyphenyld ,7-diamino-v-triazolo(d)pyrimidine. 2. Pyrimidine Analogues a. 5-Halogenated pyrimidines. i. Incorporation into bacterial DNAs. The pyrimidines, 5-bromo-, S-iOdO-, and 5-chlorouracil may be regarded as structural analogues of thymine in which the methyl group has been replaced by a halogen atom. 0

1I /\

HN

0

1I

II I CH YC\ /

0

N H

Thymine

/”‘

/c\

C

I YC\

1I

C

HN

0

/CH N H

5-Bromouracil

5-Bromouracil inhibits the growth of Lactobacillus casei (Hitchings et al., 1950a) and Streptococcus faecalis R (Weygand et al., 1952) under conditions where growth is dependent on externally supplied thymine. I n both cases the inhibition is competitively reversed by thymine. Weygand and his co-workers found that, after growth of S.faecalis R in a medium containing 5-bromouracil labeled with the radioactive isotope B1$2, the labeled 5bromouracil was incorporated into the bacterial nucleic acids. They did not demonstrate which type of nucleic acid contained the analogue. The growth of B. coli may be made thymine dependent by culturing in a synthetic medium containing sulfanilamide (1 mg./ml.), vitamin-free casein hydrolyzate (1 mg./ml.), xanthine (25 pg./ml.) and thymine (Winkler and de Haan, 1948). Dunn and Smith (1954) found that in this medium 5-bromouracil and 5-iodouracil inhibit growth of B. coli “B,” the degree of inhibition being increased by increasing the ratio (halogenated pyrimidine)/ thymine. Under these conditions large amounts of 5-bromouracil or 5-iodouracil are incorporated into the bacterial DNAs, quantitatively replacing some of the thymine residues. No 5-bromouracil is incorporated into the RNAs. 5-Bromouracil (Zamenhof and Griboff, 1954), 5-iodouracil, and 5-chlorouracil (Dunn and Smith, 1954) also inhibit the growth of thymine-requiring mutants of B. coli. The inhibition is reversed by thymine and all three analogues are incorporated into the bacterial DNAs replacing thymine. For a given ratio (5-halogenated uracil)/(thymine) during growth, the ef-

R. E. F. MATTHEWS AND J. D. BMITH

88

fectiveness of the analogues in replacing thymine in the DNA increases in the order 5-iodouracil, 5-bromouracil, and 5-chlorouracil (Table 5). ii. Incorporation into bacteriophage nucleic acids. The bacteriophages T2r and T5 can be grown on B. coli “B” in the supplemented sulfanilamidecontaining medium (see above). The latent period is about 90 min. Lysis with T2r+ under these conditions is not complete (see Section V. B.l). Under these conditions Dunn and Smith (1954) showed that large amounts of 5-bromouracil and 5-iodouracil could be incorporated into the bacteriophage DNAs. The bacteria were grown in the medium containing 8 X 1t6 M thymine and infected with 3 phages per bacterium a t a density of 1.5 X lo9 bacteria per milliliter. 2 X lea M 5-bromouracil or 5-iodouracil was previously added at a bacterial density of 108 per milliliter. Lysis occurred at TABLE 5 LEVELSOF INCORPORATION OF THREE5-HALOOENATED URACIL5 THYMINE-REQUIRINQ STRAINOF B . coli

Concentrations of thymine and 5-halogenated uracil in medium Thymine 2 4 2

x x x

10-6 M 10-6

M

10-6M

5-Halogenated uracil 10-8 M 5-chlorouracil 2 X lo-* M 5-bromouracii 2 X lo-* M 5-iodouracil

INTO A

Percent thymine residues Molar ratio replaced by 5-halogenated 5-halogenated uracil uracil in thymine bacterial DNA 50

70

60

42

100

57

approximately the =me time as controls without added analogue. The amounts of 5-bromouracil and 5-iodouracil formed in T2r preparations are given in Table 6. Proof that the halogenated pyrimidines are in fact incorporated into the DNAs of these systems and methods for their quantitative estimation in nucleic acids are described in section IV. D. I n all the deoxyribonucleic acids containing hdogenated pyrimidines the relative proportions of adenine, guanine, and cytosine [or hydroxymethylcytosine in T2r] are unchanged, but the amount of thymine is decreased so that the relative proportion of thymine halogenated pyrimidine to the total bases is equal to that of thymine in the corresponding normal nucleic acid (Table 6). From this we conclude that the 5-halogenateduracils only take up positions in the nucleic acid which are normally occupied by thymine residues. This is exactly parallel to the relation between 8-aeaguanine and guanine in plant virus RNAs. When B . coli “B” is grown in a glucose-ammonium sulfate medium with-

+

89

CHEMOTHERAPY OF VIRUSES

out sulfanilamide, 5-bromouracil does not inhibit growth except at high concentrations (0.01 M ) . This inhibition is not reversed by thymine and no 5-bromouracil is incorporated into the bacterial DNAs. However, when T2r+ is grown on B. coli ICB”in the synthetic medium in the presence of 0.01 M 5-bromouracil the bacteriophage progeny contains 5-bromouracil in 5-bromouracil = 0.28). in its DNA (ratio 5-bromouracil thymine iii. Properties of bacteriophage containing halogenated pyrimidines. “Bromouracil”, “Iodouracil”, and normal bacteriophage preparations behave similarly during their purification procedures (centrifugation and

+

~

TABLE 6 MOLARPROPORTIONS OF THYMINE A N D HALOGENATED URACILIN DNAs FROM B . coli AND T2r GROWN I N THE PRESENCE O F 5-CHLOROURACIL, 5-BROMOURACIL, A N D 5-IODOURACIL Moles/100 moles estimated bases Deoxyribonucleic acid

B . coli “B” B . coli “B” “5-bromouracil” B . coli ‘IB” “5-iodouracil” B . coli 15TB . coli 15- “5-chlorouracil” T2r T2r “5-bromouracil” T2r “5-iodouracil”

5-chloro- 5-bromoThymine uracil uracil 27.2 16.0 19.4 22.4 5.9 30.5 6.4 15.4

4.5

24.1 -

+

Thymine 5-iOdO- halogenated uracil uracil

14.5

27.2 24.7 23.9 22.4 19.5 30.5 30.5 29.9

* The nucleic acids were hydrolyzed in perchloric acid, and in the case of T2r, Wyatt’s (1953) data were used to allow for the 5-(hydroxymethy1)cytosine and precipitation of T2r at pH 4). Under the electron microscope “bromouracil” and “iodouracil” preparations show phage particles indistinguishable from those of normal preparations. The ultraviolet absorption spectra of “bromouracil”, “iodouracil”, and normal phage do not differ detectably. The majority of the particles in ‘Lbromouracil”and “iodouracil” preparations are, however, noninfectious when plated on B . coli “B.” As these only contain bacteriophage particles of normal shape and size and have the same ultraviolet absorption spectra as normal preparations the number of infective units per phage particle (specific infectivity) can be compared by the ratio (plaque count)/optical density at 260 mp. Measurement of this showed that in various “bromouracil” and “iodouracil” preparations 50-98 % of the particles were noninfective. For example a preparation of

90

R. E. F. MA’ITHEWS AND J. D. SMITH

T2r in which 79 % of the thymine residues in the phage nucleic acid were replaced by 5-bromouracil contained 70 % noninfective particles. The fact that the abnormal preparations do contain active particles raises the important question as to whether all the phage particles containing 5-bromouracil are incapable of initiating phage growth or whether inactivation by bromouracil incorporation depends on either (1) a certain number of thymine residues being replaced, or (2) the replacement of thymine residues occupying particular positions in the nucleic acid. The data available at present do not allow a clear distinction between these possibilities. Let us consider the T2r preparation described above, where 79% of the thymine residues were replaced by 5-bromouracil and 70% of the virus particles were noninfectious. Allowing for the error in both the plaque counts and the estimations of 5-bromouracil, the results could be interpreted as showing that about 75 % of the particles contained 5-bromouracil and no thymine and were noninfectious and the remaining 25 % were infectious and contained no bromouracil. The 5-bromouracil was added before infection and at the t,ime of phage addition about 20% of the thymine residues in the bacterial DNA were replaced by bromouracil so that the foregoing interpretation would appear impossible. However in this experiment the burst size was small so that a selective transfer of host thymine (compared with host bromouracil) to the first virus progeny could result in a very unequal distribution of bromouracil in the progeny. Probably the most direct method of demonstrating that some of the phage particles containing bromouracil were infectious would be to show the transfer of bromouracil from the infectingvirus particle to the phage progeny. b. Other Pyrimidines. Wooley and his colleagues (195213) have found that 4hydroxy-2,5 ,6-triaminopyrimidine and 2,4-diamino-6-hydroxy-5(p-carboxyamidopheny1)-pyrimidinereduced the yield from multicycle growth of T2 on B . coli “B.” The growth rate of the bacteria was somewhat reduced.

C. Animal Viruses 1. Purine Analogues a. 8-Azapurines. 8-Azaguanine had no effect on vaccinia virus in tissue culture (Thompson, 1947; Thompson et al., 1950), Theiler’s GD VII virus in minced mouse brain cultures (Rafelson et al., 1950), Russian spring and summer encephalitis virus (Friend, 1951), PR8 influenza and mumps virus in chicken embryo tissue culture (Cushing and Morgan, 1952), or influenza viruses A and B in embryonated eggs (Takemoto et al., 1954). 8-Azahypoxanthine and 8-azaxanthine were also tested with some of these viruses and found to be ineffective. 2 , 6 ,8-trichlorob. Halogenated Purines. 2,6-Dichloro-8-hydroxypurine,

CHEMOTHERAPY OF VIRUSES

91

purine, and 2 ,6-dichloro-7-methylpurine were inhibitory for vaccinia virus in tissue culture. The last named compound was about as effective as 2,6diaminopurine but gave no protection in mice (Thompson et al., 1950). The activity of these compounds was not annulled by normal purines. c. 2,6-Diaminopurine. 2,6-Diaminopurine was the most inhibitory purine compound tested by Thompson et al. (1950) against vaccinia virus in minced embryonic tissue culture. The inhibitory activity was annulled by adenine, yeast nucleic acid, adenylic plus guanylic acids, coenzyme I, and hypoxant,hine, but not by xanthine or uracil. 2,6-Diaminopurine had no in vitro effect on vaccinia virus. Mice inoculated intracerebrally with a neurotropic strain of the virus were not protected by treatment with the compound. 2,6-Diaminopurine was inhibitory for Russian spring and summer encephalitis virus in minced embryonic chicken or mouse tissue culture and in two types of tumor tissue (Friend, 1951). At low2M both tumor and virus growth stopped. At M only the virus was affected while at M the compound had no effects. There was marked inhibition if the compound was added up to 24 hr. after inoculation with virus. The inhibition was reversed by adenine but not by guanine. PR8 influenza and mumps viruses grown in chicken embryonic tissue culture were unaffected by 2,6-diaminopurine (Cushing and Morgan, 1952). The compound inhibited development of MEF-1 Lansing poliomyelitis in tissue culture, the inhibition being partially reversible by adenine or guanine (Brown, 1952). 2,6-Diaminopurine had no effect on infection by poliomyelitis virus grown in human epithelial cancer cell tissue cultures, except a t concentrations that were markedly inhibitory to cell respiration (Gifford et al., 1954). Moore and Friend (1951) showed that 2 ,6-diaminopurine given intraperitoneally was effective in increasing the percentage of mice surviving an intraperitoneal inoculation with RSSE virus, if the compound was given within 24 hr. before or after virus. The compound was ineffective against large doses of virus. 2,6-Diaminopurine was effective if given intraperitoneally with virus given subcutaneously, but not with virus given intracerebrally. The compound had no effect on mice infected intraperitoneally with West Nile encephalitis, Ilheus encephalitis, or Louping Ill, or on mice infected intracerebrally with Lansing poliomyelitis virus or Bunyamwera virus. The cytoplasmic Kappa factor in Paramecium contains DNA and in its properties has some analogy with viruses. Williamson et al. (1952) tested about 70 purines and pyrimidines for their ability to interfere with the killer action of a strain of Paramecium aurelia. 2 ,&Diaminopurine was unique in being able to free animals of killer activity. 8-haguanine, %azaadenine,

92

R. E. F. MATTHEWS AND J. D. SMITH

5-bromouracil, and 5-aminouracil were among the compounds with no effect. After 24 hr. of incubation with the compound under test, animals were allowed to undergo 8-10 fissions in fresh medium without the compound before being tested for killer activity. The action of 2,6-diaminopurine was annulled by adenine, adenosine, and adenylic acid. This interesting specific action of 2,6-diaminopurine warrants further investigation. 2. Pyrimidine Analogues. 5-Bromouracil, 5-nitrouracil, dithiothymine, and isobarbituric acid gave slight but reproducible inhibition of vaccinia virus in chick embryonic tissue culture (Thompson et al., 1949a). Certain amides of 5-aminouracil (e.g. 5-p-nitrobenzamidouracil) showed similar activity. 5-Chlorouridine inhibited propagation of Theiler’s GD VII virus and uptake of labeled phosphate by RNA fractions in minced one-day-old mouse brain. Uridine partially reversed these effects (Rafelson et al., 1951). 6-Aminouracil, 2-chloro-4-dirnethylaminopyrimidinel 2,4-diamino-5-nitroso-6-hydroxypyrirnidinel5,6-diaminouracill 6-imino-5-nitrosohydroxyuracil, and 2 ,6-diamino-4-propoxy-s-triazine had no effect on influenza viruses A and B in embryonated eggs (Takemoto et al., 1954). Wooley et al. (1952a) list a number of heavily substituted pyrimidines which were ineffective against St. Louis encephalitis or PR8 influenza in mice.

D . Methods for Demonstrating the Incorporation of Analogues into Nucleic Acids In this section we shall briefly describe methods which may be generally applied to demonstrate the incorporation of unnatural bases into nucleic acids. The isolation of the base from nucleic acid hydrolyzates is not alone adequate proof of its incorporation. For example we have often detected free azaguanine as a contaminant in nucleic acid preparations from bacteria and plant viruses grown in the presence of 8-asaguanine. In those cases where the nucleic acid normally has a constant composition (DNAs and plant virus RNAs) purine and pyrimidine analyses showing that the analogue quantitatively replaces part of the natural base (Tables 2 and 6) strongly suggest but do not prove that the analogue has been incorporated. Proof can be obtained by the isolation and characterization of the appropriate nucleoside or nucleotide of the analogue. The isomeric ribonucleoside 2’ and 3’ phosphates are obtained from RNA by hydrolysis in 1 N KOH a t 20°C. (assuming the nucleotide of the analogue is stable under these conditions). The 5’ deoxyribonucleotides may be obtained from DNA by enzymic hydrolysis. The nucleotides are best separtated by a combination of paper chromatography and paper electrophoresis. These methods are illustrated below for

93

CHEMOTHERAPY OF VIRUSES

8-azaguanine in RNA (Matthews, 1953b, 1954) and the 5-halogenated uracils in DNA (Dunn and Smith, 1954). 1. 8-Azaguanine in Ribonucleic Acids. When alkaline hydrolyzates of tobacco mosaic virus nucleic acid from 8-azaguanine-treated plants were subjected to paper electrophoresis a t pH 9 in borate buffer, a band which fluoresced in ultraviolet light under alkaline conditions was found running in a positive direction slightly ahead of uridylic acid, the fastest moving of the naturally occurring nucleotides a t this pH. This band, which was absent in control preparations, moved about 1.4 times as fast as 8-azaguanine-approximately the mobility to be expected for 8-azaguanylic acid. Material isolated in this way was freed of traces of uridylic acid and nucleoside diphosphates by chromatography in iso-propanol-ammonia TABLE 7 APPROXIMATE R, VALUESFOR GUANINE,AZAGUANINE AND CORRESPONDING NUCLEOTIDES

Compound

iso-propanolammonia (RI)

Guanine Guanylic acid

0.33 0.08

8-Azaguanine 8-Azaguanylic acid

0.27 0.05

Ammonium sulfate-water* (R,) Nucleoside 3’ phosphate Nucleoside 2’ phosphate Nucleoside 3’ phosphate Nucleoside 2’ phosphate

0.16 0.33 0.42 0.17 0.33 0.40

* From Markham and Smith (1952a). (Markham and Smith, 1952a). The material had the ultraviolet absorption spectrum expected for 8-azaguanylic acid and analyses showed the presence of 8-azaguanine and phosphorus in a ratio of approximately 1:l. The chromatographic behavior was that expected for 8-azaguanylic acid (Table 7). In particular, the fluorescent material divided into two spots in the ammonium sulfatewater solvent, running in the positons expected for the 8-azaguanosine 2’ and 3’ phosphates. The following simplified procedure can be used to demonstrate the presence of 8-azaguanylic acid in alkaline hydrolyzates of ribonucleic arid, and t o estimate the amount present relative to guanylic acid. The material is is first subjected to chromatography in the iso-propanol-ammonia solvent, when adenylic, rytidylic and uridylic acids move ahead of guanylic acid. A strip of paper containing the guanylic acid spot is then cut from the chromatogram, and subjected to electrophoresis a t about pH 1.7 in phosphate

94

R. E. F. MATTHEWS AND J. D. SMITH

buffer, when 8-aeaguanylic acid moves ahead of guanylic acid towards the positive electrode. The two spots can then be cut out, eluted, and the amounts present estimated spectrophotometrically. The amounts of 8-azaguanylic acid we have found in plant virus nucleic acids are too small to be estimated at all accurately by this method. The analytical figures in Table 2 suggest that about 3 % of the guanine was replaced by 8-aeaguanine in these preparations. 2. 6-Halogenated Uracils in Deoxyribonucleic Acids. 5-Bromouracil and 5-chlorouracil are liberated with the natural bases from DNAs on hydrolysis with 72% w/w HClO, a t 100°C. for 1 hr. (Marshak and Vogel, 1950). They are separated from the other bases by two-dimensional paper chroRt VALUES

OF

TABLE 8 &HALOGENATED URACIL5 Solvents i s 0 -propanol-

water-HC1*

(Rf)

Pyrimidine ~

Thymine 5-Chlorouracil 5-Bromouracil 5-Iodouracil Uracil

* From Wyatt

n-butanolwater-NHst

(Rf) ~

0.79 0.70 0.75 0.75 0.69

0.50

0.16 0.19 0.26 0.21

(1951).

t From Markham and J. D. Smith (1949). matography in iso-propanol-water-hydrochloric acid (Wyatt, 1951) and n-butanol-water-ammonia. (Markham and J. D. Smith, 1949). In the first solvent 5-bromouracil and 5-chlorouracilrun approximately in the position of thymine and ahead of the other three bases, while the second solvent separates thymine from 5-bromouracil and 5-chlorouracil (Table 8 gives the Rf values of these substances). Only that part of the chromatogram rontaining the thymine and 5-halogenated uracil need be run in the second solvent so avoiding “trailing” of the guanine spot due to its insolubility in ammonia. The 5-bromouracil and 5-chlorouracil are identified by paper chromatography, electrophoretic mobility, and ultraviolet spectroscopy (Table 8). When iodouracil-containing DNA is hydrolyzed in perchloric acid (or when iodouracil itself is heated with perchloric acid in the presence of DNA) it is quantitatively converted to uracil and may be estimated as such. The above method is the most convenient for estimating the 5-halo-

CHEMOTHERAPY OF VIRUSES

95

genated uracil content of a DNA and its purine and pyrimidine composition. In the case of T-even phage DNA, 5-(hydroxymethy1)cytosine is destroyed during this hydrolysis and a separate hydrolysis in formic acid (Wyatt, 1953) is necessary for its estimation. Deoxyribonuclease followed by rattlesnake venom diesterase hydrolyzes DNA to the 5’ nucleotides. By this means the 5’ nucleotides of the halogenated uracils may be isolated from the appropriate nucleic acid. Their separation from the other nucleotides depends on the fact that at pH 9 the 5-halogenated uracils bear a negative charge presumably due to the dissociation of the 2- or 6-hydroxyl groups and that on paper electrophoresis in 0.05 M borate buffer, pH 9, the nucleotides of 5-bromo-, chloro-, and iodouracil migrate towards the anode more rapidly than the natural nucleotides. The substances so isolated have the expected ultraviolet absorption spectra and chromatographic behavior of 5-bromodeoxyuridylic, 5-chlorodeoxyuridylic, and 5-iododeoxyuridylic acids, respectively. On hydrolysis with perchloric acid they give phosphate and the halogenated uracil (with the exception of 5-iododeoxyuridylic acid which gives uracil and phosphate). The molar proporions of phosphate and 5-bromouracil from 5-bromodeoxyuridylic acid are 1 :l. On treatment with smake venom 5’ nucleotidase the 5-bromodeoxyuridylic acid is converted to a substance with the expected properties of the nucleoside, 5-bromodeoxyuridine.

E. Evidence that Incorporated Analgues Inhibit Growth Through Incorporation In a given system a compound may cause growth inhibition through a variety of mechanisms. However, there is a number of lines of evidence suggesting that, with the purine and pyrimidine analogues which are incorporated into nucleic acids, growth inhibition is mainly due to the formation of nucleic acids which are not capable of functioning normally. This evidence is summarized below. 1. Infectivity of Viruses. Preparations of tobacco mosaic and turnip yellow mosaic viruses containing 8-azaguanine and bacteriophage T2 containing 5-bromouracil are less infectious than control preparations, compared on a nucleic acid basis. This is the most direct evidence that nucleic acid containing these analogues cannot function normally. However there is as yet no proof that the analogues interfere with the actual reduplication of the virus nucleic acids. For example the reduced infectivity of the virus preparations could be due to interference by the analogues with the release of nucleic acid from the virus. Likewise the possibility that incorporation of an analogue into the nucleic acid results in a change of vital importance in the protein part of the virus cannot be ruled out. 8-Azaguanine-containing and normal tobacco mosaic virus could not be distinguished ser-

96

R. E. F. MATTHEWS AND J. D. SMITH

ologically, but such tests would be far too insensitive t o detect small changes in protein structure. 2. Correlation between Inhibition and Incorporation. With 8-azapurines and related compounds, growth inhibition was observed only when the analogue was found to be incorporated into the nucleic acid (Matthews and Smith, 1954) (Table 9). 3. Delay in Inhibition of Bacterial Growth. The type of bacterial growth inhibition observed with 8-aaaguanine and the 5-halogenated pyrimidines does not support the idea that these act through competitive inhibition EFFECTSOF 8-AZAPURINES

AND

TABLE 9 RELATEDCOMPOUNDS MOSAIC VIRUS

ON

B . coli

TMV

Compound 8-Amguanine 8-Azaadenine 8-Azahypoxanthine 8-Azaxanthine 8-Azaisoguanine 4-(5)-Amino-1H-112,3-triazole-6-(4)-carboxyamide 4-(5)-Amino-iH-l,2,3-triazole-6-(4)-carboxylic acid 5-Hydroxy-1H-112,3-triazole

AND

TOBACCO

B . coli

Incorporation Incorporation as 8-azaInhibition as 8-azaof developguanine Inhibition guanine into RNA ment into RNA of growth

-

-

-

-

+

+

+

+

-

-

-

-

-

-

not tested

with natural purines or pyrimidines. Inhibition does not usually begin until 1-2 generation times after the addition of the analogue, and thereafter increases progressively. Reversal of inhibition by the corresponding natural purine or pyrimidine is only complete when this is added before, or simultaneously with the analogue. A similar delay was noted by Kidder and Dewey (1949) in the reversal by guanylic acid of growth inhibition of Tetrahymena by 8-azaguanine. The time lag in the onset of growth inhibition presumably represents the time required for the accumulation of a certain percentage of ineffective RNA in the cells. In Fig. 6 are shown growth curves of B. coli in the presence of 8-azahypoxanthine (which is incorporated into the bacterial RNA as 8-aaaguanylic acid). 4. Evidence fur Absence of Competitive Inhibition. Although an ana-

97

CHEMOTHERAPY OF VIRUSES

logue may act through the formation of abnormal nucleic acid, part of the inhibition during virus multiplication in the presence of the analogue may also be due to competitive inhibition by the latter or its derivatives (e. g. nucleotides etc.) of the enzymes concerned in the metabolism of natural purines and pyrimidines. The relative importance of these two types of inhibition is not easily measured, but there are several kinds of experimental evidence suggesting that 8-azaguanine and the 5-halogenated pyrimidines act solely by their incorporation into nucleic acid. Carlo and Mandel (1 953) found 8-azaguanine had no significant effect on the incorporation of C'*-labeled guanine or 4-amino-5-imidazolecarboxyamide into the nucleic acids of liver or sarcoma 537 of mice. Under their Control

._21 L

I n c

D QJ

-

0

I

1

1

I

50

100

150

200

Minutes

FIG.6. Growth curves of B . coli in a synthetic medium in the presence of 0.005 M and 0.01 M 8-azahypoxanthine. 8-Azahypoxanthine was added at zero time.

conditions 8-azaguanine itself would be incorporated iuto the ribonucleic acids and would inhibit growth of the sarcoma. The rates at which purine and pyrimidine analogues are converted to nucleosides and nucleotides have been studied to some extent using cell-free enzyme preparations. Friedkin and Roberts (1954) measured the rates of format,ion of a number of pyrimidine deoxyribosides catalyzed by a horse liver enzyme system according to the reaction: Pyrimidine

+ 2-deoxyribose-1-phosphate

--t

pyrimidine deoxyriboside

+ inorganic phosphate

Some of these are given in Table 10. Friedkin (1952) showed that 8-azaguanine, 2-thiouracil and 5-aminouracil will participate in a similar reaction with ribose-1-phosphate to give the ribonucleosides. This is also catalyzed by an enzyme from horse liver.

98

R. E. F. MATTHEWS AND J. D. SMITH

F . Factors Agecting Incorporation The effectiveness of an analogue in inhibiting growth through replacement of a normal component in the nucleic acid will depend firstly on the structural features of the analogue and secondly on the metabolism of the host. 1 . Structural Features of the Analogue. In theory we can distinguish four possible types of analogues in relation to their effects in a given system: (1) Analogues too different from the normal base to be incorporated, and therefore inert. (2) Analogues sufficiently like the normal base to be incorporated into TABLE 10 RATESO F FORMATION OF SOMEPYRIMIDINE DEOXYRIBONUCLEOSIDES* Pyrimidine

p M inorganic P released/0.25 p M pyrimidine (after 30 min.)

Thymine Uracil 5-Chlorouracil 5-Bromouracil 5-Iodouracil 5-Aminouracil 2-Thiouracil Orotic acid 5-Methylcytosine Control (no pvrimidine)

0.22 0.22 0.06 0.11 0.14 0.21 0.12 0.01 0.01 0.01

* From Friedkin

and Roberts (1954).

the nucleic acid, but different enough to render the nucleic acid nonfunctional. (3) Analogues so similar to the normal base that they are incorporated into nucleic acids which function normally. (4)Analogues which are incorporated and allow the nucleic acid to function in a modified way. We cannot make firm predictions as to the possibility of an analogue being incorporated into nucleic acids. Still less can we say how the analogue, when incorporated, will affect the functioning of the nucleic acid; in fact it is possible that by studying such effects we may be able to assess the relative importance of various features of nucleic acid structure. However the influence of metabolic factors is always likely to make the interpretation of such results difficult. With RNA for which no detailed structure has been proposed we can only rely on a structural comparison of possible analogues with the known nat-

CHEMOTHERAPY OF VIRUSES

99

urally occurring bases. For example we would consider it unlikely that a thymine analogue such as 5-bromouracil would be incorporated into RNA. In the case of DNA the Watson-Crick model defines certain restrictions on the types of purines and pyrimidines which may enter into the nucleic acid structure. Positions 1 and 6 in the purine and pyrimidine rings must have the configurations necessary to form the hydrogen bonds linking the pairs of bases in the two chains of the double helix. It would, however, be unwise to exclude analogues because they will not fit into this model. This has become apparent from the recent discovery that a “thymineless” strain of B. coli contains small amounts of 6-methylaminopurine in its DNA and that under certain conditions of growth these amounts may be increased tenfold (Dunn and Smith, 1955). Although 6-methylamino-purine is an analogue of adenine, quantitative analysis of the abnormal DNA shows that the 6-methylaminopurine apparently replaces not adenine but thymine residues. It is impossible t o fit 6-methylaminopurine into such positions in the Watson-Crick model. H

\

N

I

C

N-

H

6-methy laminopurine

Presumably an abnormal type of DNA is built up, and cells which contain an increased amount of 6-methylaminopurine in their DNA dhow morphological changes and are inhibited in their growth and eventually die. The recent discovery (Wyatt and Cohen, 1953; Sinsheimer, 1954; Volkin, 1954) that the DNA of T-even bacteriophages contain the base 5-(hydroxymethy1)cytosine esterified with glucose a t the 5-hydroxymethyl position further illustrates the difficulties in making predictions regarding the incorporation of analogues. (Actually in the Watson and Crick model the 5-hydroxymethyl position occurs in an open part of the structure and there appears to be no difficulty in fitting the relatively large glucose residue.) There is one structural feature which may always be necessary for incorporation. This is the ability to form a glycosidic link at the correct position in the ring. It is unlikely that any purine or pyrimidine which is unable to form an N-glycosidic link in the 9 and 3 positions, respectively,

100

R. E. F. MATTHEWS AND J. D. SMITH

can be incorporated into nucleic acids. Examples are guanine analogues in which the nitrogen of position 9 is replaced by carbon (p. 81) or oxygen (p. 81). Another condition for the formation of an N-glycosidic link is that a free hydrogen should be available on the nitrogen. Thus, whereas guanine can form a glycosidic link on Ne , 7-methylguanine cannot (unless of course addition takes place across the double bond between atoms 8 and 9). 0

HzN

N 7 -Methy lguanine

Modifications of the natural purines and pyrimidines can be of two types; those in which a substituent group has been added or altered, and those where one or more ring atoms have been changed. Of the analogues known to be incorporated the 5-halogenated pyrimidines belong to the first type and 8-azaguanine to the second. As would be expected from their relation to thymine, the 5-halogenated uracils have only been found to be incorporated into DNAs. On the other hand 8-azaguanine appears to be incorporated in substantial amounts only into RNAs. However recently we have obtained evidence for the presence of small amounts of 8-azaguaninein the DNA of Bs.cereus (Section IV. B). The replacement of the methyl group by chlorine, bromine or iodine and replacement of the CH group of position 8 in guanine by N alters both the size and shape of parts of the molecule and the electronic configuration of the compound as a whole. Both these changes are presumably of importance in deciding whether a compound will enter into the nucleic acid structure and the extent to which it will alter nucleic acid function. Their relative importance is difficult to assess. Under comparable conditions the degree of incorporation of the 5-halogenated uracils increases in the order 5-chloro-, 5-bromo-, and 5-iodouracil although from the data of Pauling (1939) one would consider that the bromine atom was closest in size to the methyl group. On the other hand the discovery of glucosehydroxymethylcytosine in T-even phage DNA9 suggests that the size of the group in position 5 of DNA pyrimidines is not a limiting factor from structural considerations. Probably the electron attracting character of the halogen atom is the major factor determining the changes

CHEMOTHERAPY OF VIRUSES

101

in properties of DNA-containing 5-halogenated uracils. The pK values of the OH groups of both 8-azaguanine and the 5-halogenated uracils (probably that on the 6‘ position) are about 2 units lower than those of the corresponding normal compounds, and the amino group of 8-azaguanine is less basic than that of guanine. A detailed physico-chemical comparison of these compounds and their nucleosides and nucleotides might provide valuable information. 2. Host Metabolism. A compound potentially capable from the structural point of view of replacing a normal base in a nucleic acid will only do so if the metabolism of the system tested can deal appropriately with the analogue. There are a number of ways in which host metabolism can influence its incorporation. The analogue must not be altered or degraded to an inactive material. The compound must be synthesized into the type of molecule involved in the final condensation to form the nucleic acid. This is presumably some type of nucleotide or small polynucleotide. The polynucleotide “precursor” containing the analogue must be formed at a a rate of the same order of magnitude as that determining the formation of the natural “precursor.” Thus incorporation of the analogue may be prevented or restricted either because the normal base itself is not used for nucleic acid synthesis or because the analogue cannot be taken to the polynucleotide “precursor” as easily as the normal compound. We will now consider some examples. a. Route of Incorporation of Bases into Nucleic Acids. Bacterium coli will not normally use externally supplied thymine or thymidine in its DNA synthesis, and 5-bromouracil, when added to the medium, is not incorporated into the bacterial DNA. The thymine “polynucleotide presursor” is presumably normally synthesized by a route not involving thymine or or thymidine. However, when B. coli is grown in a medium containing sulfanilamide and supplemented with certain amino acids and purines, the the rate of synthesis of the thymine “polynucleotide precursor” by the normal route is greatly reduced so that the cells become dependent on externally supplied thymine for their growth. Under these conditions 5-bromouracil inhibits growth and is incorporated into bacterial and bacteriophage DNAs. b. Rate of Synthesis of Nucleic Acid. Although when grown under conditions where it is nonthymine requiring, B . coli does not incorporate 5-bromouracil into its DNA, T2 bacteriophage grown on such bacteria in the presence of 10-* M 5-bromouracil have about 30 % of their thymine residues replaced by bromouracil. This is probably a consequence of the greatly enhanced DNA synthesis of cells after infection (the rate is about 4 times that of uninfected cells) so that the cell in effect behaves as though it were thymine deficient. This case is of interest because it provides an example

102

R. E. F. MATTHEWS AND J. D. BMITH

of a clear-cut differentiation between incorporation into host and virua nucleic acids. c. Conversion of Analogues to Their Nucleosides and Nucleotides. The results obtained by Zamenhof and Griboff (1954) with a thymine-requiring strain of B. coli suggest that the degree of inhibition by 5-bromouracil is limited by its rate of conversion to the nucleoside. The bacteria, which incorporate 5-bromouracil into their DNA, are more strongly inhibited by 5-bromouracil deoxyriboside than by 5-bromouracil itself. Conversely thymidine is much more effective than thymine in antagonizing inhibition by 5-bromouracil. This example emphasizes the importance of testing the nucleosides and nucleotides of analogues for their virus chemotherapeutic activity. d. Conversion of the Analogue to an Inactive Material. In plant tissues 8-azaguanine is fairly rapidly deaminated to 8-azaxanthine, a compound which has no effect on any plant virus tested. This inactivation undoubtedly limits the effectiveness of the compound. Animal tissues likewise convert 8-azaguanine to 8-azaxanthine. The fact that 8-azaguanine has not been found to be inhibitory for any animal virus may well be due in part to this inactivation. It may sometimes be possible to limit such inactivation by administration of a compound inhibitory for the inactivating enzyme. For example, Dietrich and Shapiro (1953a) found that flavotin (a riboflavine analogue) enhanced the carcinostatic activity of 8-azaguanine for a tumor in mice. In viuo adminstration of flavotin inhibited tumor xanthine oxidase. Guanase is susceptible to product inhibition and they suggested that flavotin had its effect by causing xanthine to accumulate, thus inhibiting the conversion of 8-azaguanine to 8-azaxanthine by guanase. Dietrich and Shapiro (1953b) found that a guanase in mouse liver extracts was inhibited by various pteridines including xanthopterin. However we have found, with tobacco mosaic virus in tobacco, that the application of xanthopterin with 8-azaguanine did not increase virus inhibition. Shapiro et al. (1952) showed that 6-formylpteridine enhanced the carcinostatic activity of 8-azaguanine. In vitro the compound inhibited the deamination of 8-azaguanine by tumor extracts.

G . The Design of New Analogues Bearing in mind the factors affecting incorporation already observed in Section IV. F, we shall consider what purine and pyrimidine analogues may be of possible chemotherapeutic value. The number of compounds which can be considered as purine or pyrimidine analoguesis verylarge. Numerous 5,8membered bicyclic ring structures which could be considered as analogues of purine or pyrimidine have been synthesized (Patterson and Capell,

CHEMOTHERAPY OF VIRUSEB

103

1940). Few of these have so far been prepared with subtituents correspondingto those for any of the nucleic acid bases. Of such compounds that have been prepared few have been tested for incorporation into nucleic acids or for virus-inhibiting properties. The effects of some of these compounds on the growth of certain microorganisms have been studied, and this information may give some guide to their possible action as analogues of nucleic acid bases. We list below some of the compounds with modified ring systems and appropriate substituent groups which have already been synthesized, and some purines and pyrimidines which have been found to affect the growth of certain bacteria. 1. Ring Modifiations a. Purine Analogues. i. Deazapurines. The adenine analogues l-deazaadenine (Kogl et al., 1948) and 3-deazaadenine (Salemink and van der Want, 1949) have been synthesized.

H 1-Deazaadenine

H H 3-Deazaadenine

ii. Benzimidazoles. The benzimidazoles may be considered as purine analogues in which NO and NI have been replaced by carbon atoms. 5,6-Dimethylbenzimidazole occurs naturally as part of the structure of vitamin B12. Other substances with the functions of vitamin BIZ(pseudovitamin BI2s)occur in certain microorganisms where the 5,6-dimethylbenzimidazole is replaced by adenine (Dion et al., 1952), or 2-methyladenine (Brown et al., 1955). Thus in at least one substance of biological importance the benzimidazole and purine nuclei can replace each other. Van der Want (1948) has prepared the adenine analogue 4-(7)-aminobenzimidazole and the corresponding guanine analogue 6-amino-4-hydroxybenzimidazole has been prepared by Gillespie et al. (1954). NHz

I

H

H

4-(7)-Aminobenzimidazole

iii. Benztriazoles.

Gillespie et al. (1954) have also described the synthe-

104

R. E. F. MATTHEWS AND J. D. SMITH

sis of a series of benztriazoles containing a hydroxy or methoxy group at position 4 and a nitro or amino group a t position 6. An example is 4-hydroxy-6-aminobenztriazolewhich may be regarded as a guanine analogue. OH

H2N- H H 4-Hydroxy-6-aminobenz triazole

b. Pyrimidine Analogues. i. sym-Triazines. The sym-t,riazines are analogues of pyrimidines where C6is replaced by a nitrogen. The uracil analogue, dihydroxy-s-triazine has been described (Brandenberger, 1954).

K

N

N

0H' 2,6-Dihydroxy-s-triazine

ii. 1 ,d,d-Triazines. The 1,2,4triaeines are also analogues of pyrimidines. Prusoff et al. (1954) have prepared 3,5-dihydroxy-6-methyl1,2 ,Qtriazine (4-azathymine) which corresponds to thymine.

0. H 3,5-Dihydroxy-6-methyl-1,2,4-triazine

This substance inhibits the growth of Lactobacillus casei and the inhibition is antagonized by thymine (Elion et al., 1954). In the presence of mathymine, an enzyme in Streptococcus faecalis transfers deoxyribose from thymidine to form a compound with the expected properties of aeathymine deoxyriboside (Prusoff, 1954). 8. Substituted Purines and Pyrimidines a. Antagonists of Thymine. Certain 5-substituted uracils other than the 5-halogenated uracils inhibit the growth of Laclobacillus casei and the inhibition may be competitively reversed by thymine. These include 5-amino-

CHEMOTHERAPY OF VIRUSES

105

uracil, 5-nitrouracil (Hitchings et al., 1950a), and a-thiothymine( Strandskov and Wyss, 1945). 5-Nitrouracil is most inhibiting when growth is in a medium cont,aining folic acid and no thymine. 5-Ethyluracil similarly inhibits growth of a thymine-requiring B. coli (Zamenhof and Griboff, 1954). 5-Aminouracil and 2-thiothymine also inhibit growth of a thymine-requiring strain of B. coli and this inhibition is antagonized by thymine. Dunn and Smith (1955) could not detect any incorporation of these analogues into the B. coli DNA, but instead the small proportion of 6-methylarninopurine normally present in the DNA of this organism was greatly increased after growth in the presence of either of the two inhibitors. 5-Methyl-4-hydroxypyrimidine,although differing considerably from thymine in its substituent groups, will replace thymine in the growth of L. casei (Hitchings et al., 1950b). b. Antagonists of Uracil. The action of several substituted pyrimidines which inhibit the growth of L. casei is antagonized by uracil but not by thymine. These include 5-hydroxyuracil (Hitchings et al., 1950a), 2-thiouracil, and 2-thio-4-n-propyluracil (Wolff and Karlin, 1951). c. Other Pyrimidines. Although there is as yet little evidence concerning their mode of action the phenoxythiouracils (Section V. H.4) and the ahaloacylamides (Section V. H.6), active against certain animal viruses, are pyrimidine analogues with a large substituent in the 5 position. The diamidines which are active against some animal viruses (Section V. H.5) could condense with ketonic esters or P-diketones to form 2-substituted pyrimidines. d. Substituted Purines. Hitchings and his colleagues have found that a large number of substituted purines will inhibit the growth of L. casei. These include 2-thioadenine, 2-aminopurine, 2-amino-6-methylpurine, 2,6-diaminopurine, substituted 2,6-diaminopurines and 6-mercaptopurine (Elion et al., 1950b, 1951, 1953). On the other hand, l-methylguanine, l-methylxanthine, and 6-methylaminopurine will partially replace t.he adenine requirement of L. casei (Elion and Hitchings, 1950a). The relation of this latter finding to the fact that 6-methylaminopurine may under certain circumstances replace thymine residues in a strain of B. coli (Dunn and Smith, 1955) is not a t present clear. Evidently elucidation of the role of this substance in nucleic metabolism may be of considerable importance in the design of new analogues likely to be incorporated into nucleic acids. 3. The Sugar Component. The possibilities of introducing variants of the normal sugar components into nucleic acids appear to be limited as most changes in the sugar configuration will make the construction of the nucleic acid “backbone” impossible. All RNAs that have been examined

106

R. E. F. MAWHEWS AND J. D. SMITH

are hydrolyzed by alkali or ribonuclease, the 2’ ,3’ cyclic phosphates being intermediates in the formation of nucleoside2’ and 3‘ phosphates (Markham and Smith, 1952a; Brown and Todd, 1952). If the 2’,3’ cyclic phosphate is an essential intermediate in RNA synthesis then it seems unlikely that any sugar could replace D-ribose. Should this not be so, then analogues of D-ribose with modifications at CZmight be incorporated. In DNA nucleotides with modifications at the 2’ position of 2-deoxy-~-ribosemight provide substances which could replace normal nucleotides in DNA formation. 4. Compounds Related to a Base Known to be Incorporated. Once it has been established that a particular analogue with certain changes in the ring structure or in substituent groups can be incorporated into nucleic acids, it may be possible to vary the form in which the analogue is supplied to the organism. Firstly, the nucleosides and nucleotides should be prepared and tested for the reasons noted earlier. Secondly, it may be possible with some analogues to prepare compounds corresponding to precursors of the normal bases. For example, 4-(5)-amino-lH-l, 2,3 triazole-5(4)-carboxyamideis the triazole analogue of 4-(5)-arninoimidazoled(4)-carboxyamide which in some systems can act as a precursor of purines. The triazole analogue was less effective than 8-azaguanine in the systems so far tested. However the riboside and ribonucleotides of the triazole compound have not yet been examined.

H, Discussion The results obtained so far with 8-aeaguanine and the 5-halogenated uracils suggest that in due course a variety of analogues may be found which can be incorporated into nucleic acids rendering them biologically nonfunctional. One of the major problems in controlling virus infections by this means is that such analogues are likely to be incorporated not only into the virus nucleic acids but also those of the host. Thus the problem is to obtain substantial inhibition of virus multiplication without disorganizing the functioning of the host nucleic acids. There are several ways in which, at least from a theoretical standpoint, this condition may be realized. One of them depends on the relative rates of synthesis of virus and host nucleic acid and may be illustrated by considering the case of deoxyribonucleic acids. DNA is present in all cells, excepting those animal cells where the nucleus has undergone disorganization, and (with the exception of the DNA-avidin complex in egg white) is restricted t o the cell nucleus or equivalent structure in the case of bacteria. One of the striking facts about DNA is its relative stability in the cell. Among nondividing cells of the same organism the composition and amount of the DNA per haploid set of chromosomes is constant. This is not true for dividing cells, where doubling of the amount of DNA takes place at some time before cell division.

CHEMOTHERAPY OF VIRUSES

107

The rate of replacement of DNA phosphorus in “non-dividing” cells is very low and may possibly be attributed to a small amount of cell division in the tissue studied. In most mature animal and plant tissues little cell division occurs and during multiplication of a DNA-containing virus in such tissues there would presumably be rapid synthesis of virus DNA and little or no synthesis of host DNA. Under these conditions a specific analogue of a DNA component might be incorporated into the DNA of the virus t o a far greater extent than into that of the host. RNA is present in all cells, excepting certain animal spermatozoa, both in the cytoplasm and (in smaller amount) in the nucleus. I n contrast to DNA the amount and composition of RNA in different cells of the same organism is not constant and in most cells phosphorus and bases are incorporated a t appreciable rates into RNA in nondividing cells and in the absence of net RNA synthesis. Here the situation during the multiplication of a RNA-containing virus in the presence of an analogue of an RNA component is different from that considered above for a DNA-containing virus. It is unlikely that an analogue will be incorporated into the virusRNA and not into the host-RNA. On the other hand, taking the generally accepted view that a t least most of the genetic information in a cell resides in its DNA and not in its RNA, the formation of nonfunctional RNA in a host cell need not be disastrous for its survival. There is evidence, both from the relative rates of RNA phosphorus turnover in nucleus and cytoplasm (Jeener and Szaforz, 1950; Barnam and Huseby, 1950), and from the RNA metabolism of enucleated cells (Linet and Brachet, 1951) that RNA synthesis takes place in or is controlled by the nucleus. It is an attractive and plausible hypothesis that the DNA provides the patterns for the RNA molecules in the cell (excluding any which might be dependent on cytoplasmic heritable factors) and can bring about their synthesis without the intervention of pre-existent RNA. If it is further postulated that only the virus RNA is able to bring about its own synthesis, then a host cell containing biologically nonfunctional RNA would have the possibility of recovery, whereas virus particles containing nonfunctional RNA would be incapable of further multiplication. Certain of the purines and pyrimidines, as well as being components of nucleic acids, are found in other important compounds such as coenzymes. Toxicity for the host could well arise from the incorporation of unnatural bases into molecules of this type. As examples, 8-azaadenine and thiouracil might be built up into compounds corresponding to vitaaminBIZ and uridine diphosphate, respectively. When the virus nucleic acid contains a base not present in the host nucleic acids, it may be possible to inhibit virus development without affecting host nucleic acid metabolism. The only case so far known of a

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R. E. F. MATTHEWS AND J. D. BMITH

nucleic acid base peculiar to viruses is the occurrenceof 5-(hydroxymethy1)cytosine in the DNA of the T-even phages. However, it.should be recalled that of some 200 known plant viruses in only about 6 has the nucleic acid composition been determined whereas of the animal viruses none has been adequately analyzed from the point of view of nucleic acid composition. Furthermore the recent isolation of 6-methylaminopurine in small amounts from apparently normal B . coli DNA emphasizes the possibility that, even with those nucleic acids that have already been studied, new bases may be found in small amounts on more careful analysis. In many types of therapeutic drug the size, shape, and nature of substituent groups often play an important part in determining how the drug is distributed in and retained by different tissues. Such factors are often of great importance in drug action. With the analogues of purine and pyrimidine bases so far known to be incorporated into nucleic acids, the possibilities of varying substituent groups in order to alter the distribution or increase the concentration of the compound attained in the tissues appear to be very limited. If however other bases are found which, like 5-(hydroxymethy1)cytosine-glucose, have large substituents, then there might be wider scope for alterations of this type.

V. VIRUSINHIBITION BY OTHERTYPES OF COMPOUNDS It would not be possible to enumerate the large numbers of compounds that have been tested, particularly against animal viruses. The great majority of compounds have proved quite inert, and among those with activity there are only a few types of compounds having any marked inhibitory effects. With most groups there is little information to indicate how the inhibitory effects are produced. The results obtained by different workers are often difficult to compare since a variety of testing methods have been employed. In the following sections we survey the information available for certain groups of compounds. No attempt has been made to record every observation A . Amino Acids, Amino Acid analogues, and Related Compounds 1 . Bacteriophage. Several amino acid analogues are known to inhibit the multiplication of bacteriophages. However, all of these have an inhibitory effect upon growth of uninfected bacteria and none brings about the “cure’, of an infected cell. a. 6-Methyltqptophan. The tryptophan analogue, 5-methyltryptophan inhibits the growth of B . coli “B” but does not kill the cells, nor does it inactivate free bacteriophage T2 and T4.

CHEMOTHERAPY OF VIRUSES

HaC

109

CHI. CH(NH3) C OOH

5-Methyltryptophan

The addition of 5 X le4 M 5-methyltryptophan to B . coli cells infected with T2 or T4 up to midway through the latent period totally prevents lysis and virus liberation (Cohen and Anderson, 1946; Cohen and Fowler, 1947). It also stops the synthesis of DNA by the infected cell. The inhibition of virus liberation and DNA synthesis is reversed by tryptophan. If 5methyltryptophan is added later than half way through the latent period, the bacteria lyse but the amount of virus liberated is less than that of controls. Presumably under these conditions mature virus particles are present before addition of the inhibitor. The time at which the inhibited bacteria lyse after reversal by tryptophan is independent of the period during which 5-methyltryptophan alonehas been present. The presence of 5-methyltryptophan thus completely blocks virus development. As this concentration of 5-methyltryptophan stops bacterial growth, there is no differentiation in the acion of 5-methyltryptophan on the development of uninfected host cells or of bacteriophage. If however infected cells are left in 5-methyltryptophan for a period greater than 50-70 min. (the latent period is about 23 min.) an increasing proportion (up to 85%) of the infected cells irreversibly lose their ability to liberate bacteriophage on dilution in tryptophan-containing medium. These cells cannot multiply or produce bacteriophage. In this way it is possible to reduce the number of infected cells in a mixed population of infected and uninfected bacteria. b . Methionine Sulfoxide. CHa

I so I CH, I CHs I

CH(NH3)C OOH Methionine sulfoxide

OH

I

co

I I

CH, CH,

I

CH(NH2)COOH Glutamic acid

Methionine sulfoxide acts in several systems as an antimetabolite of glutamic acid. Its action on B. coli “B” infected with T2r+ is very similar

R. E. F. MA’ITHEWS AND J. D. SMITH

110

to that of 5-methyltryptophan (Fowler and Cohen, 1948). At 5 X M, DL-methionine sulfoxide inhibits lysis and phage liberation and this effect is reversed by glutamic acid. It differs from 5-methyltryptophan in that it does not completely block intracellular virus development. After a longer treatment with methionine sulfoxide a proportion of the infected cells irreversibly lose their ability to liberate bacteriophage. c. Aminomethanesulfonic Acid. Aminomethanesulfonic acid, which can be considered as a glycine analogue, inhibits the multiplication of the bacteriophage P1 on B. coli “B” (Spizizen, 1943). Its mode of action and its effect upon uninfected bacteria have not been studied. HzN

- CHt - COOH Glycine

-

HIN CH1 - SOsH Aminomethanesulfonic acid

d. Amino Acids and Adsorption of Bacteriophage. Certain mutants of T4 bacteriophage are unable to adsorb to the host cell (B. coli “B”) in the absence of some amino acid cofactors, the most important being L-tryptophan (Anderson, 1945). Delbruck (1948) showed that the action of L-tryptophan as an adsorption cofactor is antagonized by indole and to a lesser extent by skatole. e. Natural Amino Acids. Fowler and Cohen (1948) found that L-leucine and L-serine inhibited the multiplication of T2r+ in B . coli “B.” The inhibition by leucine was overcome by DL-valine, DL-isoleucine, and DL-norleucine. The amino acids, L-cystine and L-cysteine, caused a loss of infected bacteria (i. e. these were no longer able to produce phage and did not survive.) 1. Animal Viruses a. Natural Amino Acids. Eaton et al. (1951b) found that the basic amino acids, arginine, lysine, and ornithine, at concentrations of 1-10 mg./ml. retarded the growth of influenza (PR8 and Lee viruses) and mumps virus in in tissue cultures of minced chorioallantoic membrane. L-Lysine was about twice as active as DL-lysine. Amino acids with 2-amino groups appeared to be most effective. Arginine retarded virus development without altering tissue respiration, glucose utilization, or potentiality for growth. Arginine did not inactivate the virus in witro. Addition of egg yolk annulled the inhibitory activity of arginine, but RNA, adenylic acid, methionine, and creatine did not. Rafelson et al. (1950) using Theiler’s GD VII virus in minced mouse brain culture found that lysine, and, to a lesser extent histidine and tryptophan, inhibited virus growth. These compounds also inhibited the uptake of labeled inorganic phosphate into lipid- and protein-bound fractions in the tissue culture. The virus inhibition by lysine was partially overcome by methionine. Pearson el al. (1952), using the same test system, found that

111

CHEMOTHERAPY OF VIRUSES

most of the naturally occurring amino acids had some inhibitory effect at 3 mg./ml. At 1 mg./ml. only lysine, serine, and ornithine were inhibitory. L-Lysine was the most inhibitory. L-Aminoadipic and ketoadipic acids, compounds known to be metabolites of lysine, were as inhibitory as lysine. D-Aminoadipic acid was ineffective. The inhibition by lysine was partially annulled by leucine and tyrosine as well as methionine. b. fld-ThienyZaZanine. Thompson and Wilkin (1948) found that 0-2thienylalanine inhibited the growth of vaccinia virus in chick embryonic tissue culture. The inhibition was annulled by phenylalanine but not by methionine.

Phenylalanine

8-2-Thienylalanine

Rafelson et aZ. (1950) found that ~~-2-thienylalanine inhibited growth of Theiler’s GD VII virus in minced mouse brain, but had little effect on uptake of labeled inorganic phosphate into lipid- and protein-bound fractions. Pearson et al. (1952) found that the three analogues, ~~-@-2-thienylalanine, ~~-p-2-furylalanine, and DL-1-napthylalanine were inhibitory for Theiler’s virus at concentrations of 0.1 mg./ml. The 0 , m, and p-fluorophenylalanines were also inhibitory. p-2-Thienylalanine was active against the MEF 1 strain of Lansing type poliomyelitis virus in tissue culture, the inhibition being annulled by DLphenylalanine (Brown, 1952). N-dichloroacetyl-a-(p-nitropheny1)-glycine was also active with this system but the inhibition was not annulled by phenylalanine. j3-2-Thienylalanine did not inhibit poliomyelitis virus grown in tissue cultures of human epithelial cancer cells except at concentrations that were markedly inhibitory to host cell respiration (Gifford et aZ., 1954). The compound also had no significant effect on PR8 influenza or mumps viruses in chicken embryonic tissue culture (Cushing and Morgan, 1952) or on influenza (PRS or Lee virus) in embryonated eggs (Takemoto el aZ., 1954). c. DL-Methoxinine. Thompson (1947) found that DL-methoxinine, an analogue of methionine, was inhibitory for vaccinia virus in tissue culture. NHz

I

CH~-S-CHz-CHz-CH-COOH Methionine

NHz CHs-0-CHZ-CHz-CH-C Methoxinine

I

0 OH

Ackermann (1951a) tested methoxinine against PR8 influenza virus growing in intact chorioallantoic membrane tissue culture. The compound was

112

R. E. F. MATTHEWS AND J. D. SMITH

an effectiveinhibitor at concentrations of 0.025 M . The inhibition could be annulled by L-methionine but not by D-methionhe, betaine, choline, cysteine, or creatine. Virus-inhibitory concentrations of the compound had no effect on respiration of the membrane. Ackermann and Maassab (1954b) found that methoxinine was inhibitory for influenza in tissue culture if added up to 2-3 hr. after inoculation. This was about the duration of the period before virus release began. These authors contrast this effect with that produced by a-amino-p-methoxyphenylmethanesulfonic acid, which was found to act only at the initial stage of infection, and in delaying release of virus from the tissue at a latter stage. d. DL-Ethionine. DL-Ethionine did not inhibit poliomyelitis virus in tissue cultures of human epithelial cancer cells (Gifford et al., 1954). NHn

I

CH,-CHr-S-CH*-CHa-CH-COOH Ethionine

Brown and Ackermann (1951) tested the effect of DL-ethionine on the Lansing strain of poliomyelitis virus grown in minced brain tissue from human embryos. The compound inhibited the growth of the virus and the inhibitory effect could be annulled by methionine. The compound had no in vitro action on the virus and did not irreversibly affect the host tissues. Ackermann (1951) found DL-ethionine was also active against influenza virus in membrane tissue culture, the effect being annulled by DL-methionine. DL-Ethionine was inhibitory for the MEF 1 Lansing type poliomyelitis virus, the inhibition being partly annulled by methionine, but not by betaine or homocysteine. However, the compound had no effect on influenza (PRS or Lee virus) in embryonated eggs (Takemoto, et al., 1954). e. SuZjonic Acids. Thompson (1947) found that a-aminomethanesulfonic acid (resembling glycine), a-aminobutanesulfonic acid (resembling valine), and a-aminophenylmethanesulfonic acid (resembling phenylalanine) were inhibitory for vaccinia virus in tissue cultures. Ackermann (1952) tested the effect on PR8 influenza virus in tissue culture of three substituted sulfonic acids related structurally to tyrosine and phenylalanine. a-Aminophenylmethanesulfonic acid, a-amino-P-phenylethanesulfonic acid, and a-amino-p-methoxyphenylmethanesulfonicacid were all markedly inhibitory in tissue culture at 5 X 10-4 M . In embryonated eggs 8 mg. of each compound inhibited influenza virus while t8heL D ~ for o the embryos was about 30 pg. Under certain conditions the a-amino-pmethoxyphenylmethanesulfonic acid gave some protection to mice against WS influenza. The compounds were not virucidal and did not affect respiration in tissue

CHEMOTHERAPY OF VIRUSES

113

cultures. The inhibitory activity was not annulled by phenylalanine or tyrosine. Breakdown products were apparently not responsible for the inhibition since ammonium ions, bisulfite ions and p-methoxybenzaldehyde did not affect virus propagation. cr-Amino-/3-phenylethanesulfonic acid was inhibitory for M E F 1 Lansing poliomyelitis virus in tissue culture (Brown, 1952). Ackermann and Maassab (1954a) used minced chorioallantoic membrane in tissue culture to study the effect of p-methoxyphenylmethanesulfonic acid on the PR8 strain of influenza virus. Some early stage in virus development is affected by this compound. If this early stage (about the first 30 min.) was allowed to proceed in the absence of inhibitor, later addition did not prevent virus reduplication, but release of virus from the tissue was impaired. f. 6-Methyltryptophan. Rasmussen et al. (1951) showed that the effect of tryptophan deficiency in prolonging the incubation period and reducing the incidence of paralysis in mice infected with Lansing poliomyelitis virus was enhanced by feeding 6-methyltryptophan in the diet. With 3 % 6-methyltryptophan in a diet with 9 % casein growth was retarded but the mice otherwise appeared in good condition. Brown (1952) found 6-methyltryptophan ineffective against M E F 1 Lansing poliomyelitis virus in tissue culture. g. Methionine Sulfoximine. At 150 mg./kg. body weight methionine sulfoximine had a slight delaying effect on the early stages of growth of Lansing poliomyelitis virus in mice. 0

1I II

C HI-S-C

HzCHz-C H-C

I

00H

NH NHz Methionine sulfoximine

3. Plant Viruses. Richkov (1951) tested a number of amino acids and related compounds against tobacco mosaic virus, leaves being dipped in solutions of the compound. Norleucine arid taurine were the most effective inhibitors of the necrotic reaction in Nicotiana glutinosa. He stated that glutamic acid, threonine, lysine, and cysteine were particularly powerful inhibitors of both the necrotic reaction and the multiplication of the virus in tobacco leaves. Schlegel and Rawlins (1954) tested several amino acids and amino acid analogues against tobacco mosaic virus by the leaf disc method. L-ISOleucine appeared to be inhibitory whereas D-isoleucine was not. DL-Benzoylalanine had no effect whereas ethionine caused some reduction in virus multiplication. Matthews and Proctor (unpublished data) found the following amino acid analogues sprayed on leaves at about 0.01 M had no

114

R. E. F. MATTHEWS AND J. D. SMITH

significant effect, on disease development with tobacco mosaic virus in tobacco, cucumber mosaic virus in cucumber, or lucerne mosaic virus in tobacco: 3,4-dihydroxyphenylalanine,@-2-thienylalanine, allothreonine, alanine ethyl ester hydrochloride, benzoylalanine, acetylglycine, ethionine, acetylmethionine, 6-benzoyllysine, acetyltryptophan. 4. Discussion. The action of amino acid analogues on virus multiplication is specific in so far as the inhibition is generally reversed by the appropriate natural amino acids. The mode of action of the amino acid analogues is not clear a t present. They may simply affect virus growth by the competitive inhibition of a reaction involving the natural amino acids or they may be taken further along the path of protein synthesis possibly giving rise to virus protein containing the analogue. In that case their mode of inhibition would in certain respects resemble that of those purine and pyrimidine analogues which are incorporated into nucleic acids. The inhibition of virus growth by natural amino acids is perhaps more difficult to understand. In many cases the inhibition is reversed by other amino acids bearing no obvious relationship to the inhibitor. Thus their action may not be due to direct interference with the enzymes concerned in protein synthesis.

B. Vitamin Analogues and Related Compounds 1 . Bacteriophages a. Sulfonamides. The growth inhibition of B . coli by sulfanilamide may may be partially reversed by the addition of methionine, xanthine, valine, and thymine and is due to failure of the inhibited bacteria to synthesize folic acid or a related substance concerned in the synthesis of these metabolites (Winkler and de Haan, 1948). Rutten, et al. (1950) tested the ability of B . coli “B” grown in the presence of sulfanilamide, methionine, xanthine, valine, and thymine to support the multiplication of the T1-7 series of bacteriophages. Sulfanilamide does not affect the free bacteriophages nor their rate of adsorption onto host bacteria. Bacteria grown in sulfanilamide concentrations up to 2000 mg./l. and infected with T1, T3, and T7 lysed and produced phage, but when grown in concentrations of sulfanilamide above 500 mg./l., bacteria infected with T2, T4, and T 6 failed to lyse and grew a t the same rate as uninfected bacteria. (The actual level of sulfanilamide required to suppress the multiplication of these phages depends on the number of generations through which the bacteria have been grown in the sulfanilamide medium. Dunn and Smith (1954) found that under their conditions in a similar medium containing 10oO mg./l. sulfanilamide T2r infected B . coli “B’, would lyse with phage production although lysis with T2r+ was incomplete.) The concentrations of sulfanilamide required to inhibit multiplication of the T-even phages corresponded to that where the bacteria

CHEMOTHERAPY OF VIRUSES

115

begin to show a requirement for valine and thymine, and Rutter and coworkers concluded that the inhibition might be concerned with the metabolism of one or both of these substances. The fact that only the T-even phages were inhibited by sulfanilamide remained unexplained until Wyatt and Cohen (1953) found that the deoxyribonucleic acids of T2, T4, and T6 were unique in containing 5-(hydroxymethy1)cytosine instead of cytosine. 5-(Hydroxymethy1)cytosine has not been found in any other deoxyribonucleic acids including those from B . coZi, and T5 and T7. It would therefore seem probable, as Cohen (1953) has suggested, that the sulfonamide inhibition of T2, T4, and T6 multiplication is due to the prevention of formation of 5-(hydroxymethyl)cytosine, which, like the introduction of a methyl group into the pyrimidine ring to form thymine, might conceivably be folic acid dependent. Cohen attempted to reverse the inhibition of T4 multiplication in the sulfanilamide medium by adding 5-(hydroxymethy1)cytosine and its deoxyriboside, but without success. However, the inactivity of hydroxymethylcytosine and its nucleoside may perhaps now be explained by the recent discovery (Sinsheimer, 1954; Volkin, 1954) that in the T-even deoxyribonucleic acids this base only occurs as a glucoside linked through the hydroxymethyl group. The effect of hydroxymethylcytosine-glucose in this system has not yet been tested. If this explanation of the action of sulfanilamide on the multiplication of T-even bacteriophages were correct it would provide the first example of inhibition of virus development based on a known chemical difference between host and virus. b. Deoxypyridoxine. Wooley and Murphy (1949) have shown that deoxypyridoxine (2,4-dimethyl-3-hydroxy-5-hydroxymethylpyridine) reduces the yield of T2r+ growing on B . coli at concentrations which do not affect bacterial growth. The inhibition is reversed by pyridoxine, certain fatty acids, pyruvic acid, and glucose-6-phosphate. It is not reversed by glucose. 2. Animal Viruses. There are numerous records of the effects of various vitamin analogues on animal viruses. None of the observations summarized in Table 11appear to have led to any further developments. However work on benzimidazole compounds provides one of the more interesting recent developments in animal virus chemotherapy. Benzimidazole is found as part of the vitamin Blz molecule.

H

Beneimidazole

TABLE 11 EFFECTS OF SOMEVITAMIN ANALOWES A N D RELATEDCOMPOUNDS O N ANIMAL VIRUSES Analogue

Natural compound

Pyridine-3-sulfonic acid

Nicotinic acid

Pyridine-3-sulfonic acid

Nicotinic acid

Pyridine-l-sulfonic acid m-Dethiobiotin

Nicotinic acid Biotin

D-Norbiotin

Biotin

7-(3,4-ureylene cyclohexyl) butyric acid DL-Homobiotin

Biotin

2-Methylnaphthoquinone (synthetic vitamin K)

Vitamin K

Pantoyltaurine Oxythiamine

Pantothenic acid Thiamine

Deoxypyridoxine

Pyridoxal

Deoxypyridoxine

Pyridoxal

Triethylcholine

Choline

Biotin

4- (Imidazolidone2)-caproic acid 2,4-Diamino-5,6dimethylpyrimidine 4-Aminofolic acid

Folic acid Folic acid

Virus and test system Influenza A in chicken tissue culture Influenza A in embryonated eggs Vaccinia in tissue culture Vaccinia in tissue culture MEF-1 poliomyelitis in tissue culture Vaccinia in tissue culture MEF-1 poliomyelitis in tissue culture Vaccinia in tissue culture

Vaccinia in tissue culture PR8 influenza and mumps in chicken tissue culture PR8 influenza and mumps in chicken tissue culture MEF-1 poliomyelitis in tissue culture MEF-1 poliomyelitis MEF-1 poliomyelitis in tissue culture Vaccinia in chick embryo tissue culture Vaccinia in chick embryo tissue culture 116

Effect

References

Nil

Cushing and Morgan (1952) Takemoto, et al. (1954)

Nil Nil Nil Nil Stimulated virus production Inhibitory

Thompson (1947) Thompson (1947) Brown (1952) Thompson (1947) Brown (1952)

Inhibitory Thompson but less so (1947) than benzoquinone or hydroquinone Nil Thompson (1947) Inhibitory Cushing and (mumps Morgan more sen(1952) sitive) Inhibitory Cushing and Morgan (1952) Nil

Brown (1952)

Nil

Brown (1952)

Nil

Brown (1952)

Nil

Thompson, e t al. (1949a)

Nil

Thompson, et al. (1949a)

117

CHEMOTHERAPY OF VIRUSES

This compound has been shown to have some inhibitory effect on a number of viruses, e.g. vaccinia virus in tissue culture (Thompson, 1947; Thompson, el al., 1950); MEF-1 poliomyelitis virus in tissue culture (Brown, 1952); and Theiler's GD VII virus in tissue culture (Rafelson et al., 1950). Brown et al. (1953) tested the effect of henzimidazole on experimental poliomyelitis in mice and monkeys. Subcutaneous administration of 250 mg./kg. body weight prolonged the incubation period with intracerebrally inoculated Lansing type 2 poliomyelitis and reduced mortality produced by minimal infective amounts of the virus. The MEF-1 strain. was not affected in adult mice and in suckling mice the compound appeared t o enhance infection. I n monkeys the incubation period was slightly prolonged with type 1 Mahoney strain but mortality was not reduced. Tamm, et al. (195313) examined the effect of various alkyl substitutions in benzimidazole on the activity against influenza B (Lee strain) in membrane culture. They found that alkyl substitution in one or both rings markedly affected the virus inhibitory power. The more active compounds were 2 , 4 , 5 6,7-pentamethylbenzimidazole,5 6-diethylbenzimidazole, and 2-ethyl-5-methylbenzimidazole. Tamm et al. (1954) found that certain chloro-substituted benzimidazoles (for example the 5,6-dichloro-compound) were 2 to 3 times as active as the corresponding alkyl-substituted compounds in suppressing influenza virus growing on chorioallantoic membrane cultured in tubes. Because the 5,6-dimethylbenzimidazolemoiety of vitamin Blz and the adenine and guanine bases in nucleic acids are linked to pentoses, Tamm and his co-workers then examined a series of N-glycosides of variously substituted benzimidazoles. )

)

' I

HCOH

HIOH HC

I

'i

' I

HCOH HIOH

1

HC

I

CHiOH CHzOH 5,8-Dimethyl-l-~-~-ribofuranosyl5,6-Dichloro-l-~-~-ribofuranobenzimidazole (a-ribazole); sylbenzimidazole (DRB) part of vitamin B12

118

R. E. F. MATTHEWS AND J. D. SMITH

The riboside of the 5,6-dichloro-compound (DRB) was 92 times as active as the reference compound benzimidazole. Compounds containing other sugars were much less active. For example, when the ribofuranoside was changed to the 6-membered ribopyranoside, the inhibitory activity was lowered to ?+jthat of DRB, whereas the arabinopyranosyl compound was ?&as active. DRB was 6 times more active than the 5- or 6-monochlorosubstituted ribofuranosylbenzimidazole. Methyl substitution at position 2 caused a considerable reduction in activity, an opposite effect to that noted for alkyl-substituted benzimidazole. 5,6-dimethylbenzimidazolehas inhibitory activity. This activity is lost on conversion to a-ribaxole. Virus inhibition by DRB was not blocked by vitamin Biz, DNA, a-ribazole or a mixture of adenosine, guanosine, adenylic acid, and guanylic acid. DRB has no detectable effect in vitro on the infectivity or hemagglutinating power of the Lee strain of the virus, nor on its ability to adsorb on to the membrane. When membranes were treated with DRB and then washed several hours before infection with virus, some inhibitory activity remained. DRB had no effect on respiration of the membranes at concentrations 6 times the virus inhibitory level. The compound had a slight effect on membrane proliferation at virus inhibitory concentrations. Besides the Lee strain DRB also inhibited the MB strain of influenza B and the PR8 and FM1 strains of influenza A. DRB was inhibitory for the Lee virus in chicken embryos and mice at concentrations causing no observed host toxicity except transient drowsiness and roughing of the fur in mice. More recently Tamm (1954) has shown that the 4,5,6-(or 5,6,7)trichloro-l-p-D-ribofuranosylbenzimidazole (TRB) is 8 times as active as DRB on a molar basis, and 760 times as active as benzimidazole in the inhibition of Lee influenza virus in chorioallantoic membrane tissue culture. The activity of TRB was not annulled by a mixture of adenosine, vitamin Blz , folic acid, and coenzyme I. Both DRB and TRB had a marked inhibitory effect on mumps virus in embryonated eggs. 3. Plant Viruses. Very few vitamin-like compounds have been tested against plant viruses. Richkov et al. (1946) found that thiamine caused no in vitro inactivation of tobacco mosaic virus, but reduced the amount produced in leaves. Weintraub et al. (1952) found thiamine had a variable effect in reducing the number of tobacco mosaic virus lesions in Nicotiana glutinosa leaves floated on solutions of the compound. We have found the following, when sprayed on the plants, to be ineffective against tobacco mosaic, cucumber mosaic, and lucerne mosaic viruses: benzimidazole, 3-acetylpyridine, 6-hydroxynicotinic acid, and ascorbic acid. Schlegel and Rawlins (1954) using leaf discs found the following compounds had no effect on multiplication of tobacco mosaic virus: nicotinamide, nicotinic acid, isonicotinic acid hydrazide, pyridine-3-sulfonic acid, riboflavin, and

CHEMOTHERAPY O F VIRUSES

119

thiamine. Schneider (1954) tested benzimidazole against tobacco mosaic virus in leaf discs. The figures for reduction in amount of virus fell within the range of compounds having a slight inhibitory effect (Fig. 2).

C. Metal Ions and Chelating Agents The multiplication of some bacterial viruses is dependent on the concentration of certain metal ions. Virus multiplication may thus be controlled by removal of the necessary cations either directly, through displacement by other cations, or by chelating agents such as citrate. I n other cases organic acids and other substances known to bind metal ions are found t o inhibit virus multiplication although in these systems no specific cation requirement has been demonstrated. It is thus convenient to discuss metal ions, organic acids, and chelating agents together as their mode of action in inhibiting virus multiplication may be related. 1. Bacteriophage. Specific cations are known to be essential a t certain steps in the multiplication of some bacteriophages. They may be required either for the adsorption of phage onto sensitive bacteria or for multiplication steps after phage attachment has taken place. a. Cation Requirement for Adsorption. The rates of adsorption of some bacteriophages to their hosts are markedly affected by the concentration of cations in the medium. In many cases this is not a specific effect of any individual cation. At low cation concentrations the phages T1 and T 2 are not appreciably adsorbed onto B. coli (‘B’’ but on increasing the concentration of uni- or bivalent cations the adsorption rate approaches the theoretical maximum where 100% of the collisions between phage and bacterium lead to adsorption (Puck et al., 1951; Garen and Puck, 1951). At higher cation concentrations the adsorption rate is decreased. The cation requirements for adsorption are characteristic of individual phages (Puck, 1953) and are concerned with the initial reversible attachment between phage and bacterium. b. Cation Requirement after Adsorption. Several phages require specific cations for multiplication stages after adsorption. These include a typhoid bacteriophage (Fildes et al., 1951) and T5 (Adams, 1949) which require Ca++ ions, and T1 which will multiply in the presence of CaH, Mg+, S+, Mn* or Ba++ ions (Adams, 1949; Fildes et al., 1951). With these phages the specific ion is not necessary for adsorption or stability of the free phage (although with T 5 a low concentration of any divalent ion, e.g. Mg++, is necessary to prevent inactivation of the free phage). i. Calcium. The T5-B. coli “B” system has been studied in most detail. B. coli “B” does not itself require detectable amounts of calcium for its normal growth, but, in the absence of calcium, T5-infected bacteria fail to lyse. On addition of calcium such starved bacteria lyse, liberating phage

120

R. E. F. MA’ITHEWS AND J. D. SMITH

after a period approximately equal to the normal latent period counted from the addition of the calcium. A prolonged starvation of calcium leads to an irreversible loss of the ability to produce phage. These bacteria do not however survive, as even in the absence of calcium, adsorption of T5 leads to the death of the bacterium (in the sense that it can no longer multiply). Luria and Steiner (1954) have shown that in the absence of calcium T5 fails to transfer its nucleic acid into the host bacterium. T5 labeled with Ps2was adsorbed onto B. coli “B” in the absence of calcium. If no calcium was added 80 % of the specifically adsorbed P32could be removed from the bacteria by shearing forces in a Waring blendor. This treatment shears off the head and part of the tail of the attached phage (Hershey and Chase, 1952). Ten minutes after the addition of 5 X M Ca++ a similar treatment only removed 10 % of the adsorbed Pa2and most of the bacteria were subsequently capable of lysing and producing phage. Thus in order that lysis and phage production may occur T5-infected bacteria require calcium only for a few minutes after adsorption, although Luria and Steiner found that for a full yield of phage either calcium or magnesium must be present throughout the latent period. As T5 cannot form a lysogenic relation with its hosts the effect of calcium on lysogenic systems cannot be studied with this phage. This has however been possible with the lysogenic strain of B. coli (Lisbonne) which carried the phage H+ (Beumer and Beumer-Jochmans, 1953). B . coli (Lisbonne) will conserve its lysogenicity for phage H+ when multiplying in the absence of calcium although calcium is necessary for the multiplication of H+ on the related sensitive nonlysogenic strain of the bacterium and also for the maximum production of H+ from the lysogenic strain (Lisbonne) by spontaneous induction. Thus in this system calcium is required for the multiplication of phage but not of “prophage.” In all the cases where calcium is required a t some stage in phage development virus multiplication may be controlled in a bacterial population by the addition of citrate, oxalate, or other calcium-binding substances. In no instance, however, can an infected bacterium be “cured.” ii. Manganese. Huybers (1952) has shown that the lysogenic strain of Bs. megatherium 899 (1+) which carried the phage (1) requires manganese for phage development after induction by ultraviolet irradiation. The bacterium itself requires manganese for growth but at much lower concentrations than those needed for phage development. Addition of Ca++, Co*, or Zn* ions to the bacteria after induction by ultraviolet irradiation prevents lysis and development of bacteriophage although bacterial growth is still possible at a reduced rate. The inhibition of phage development by these ions is reversed on the addition of manganese and appears to be due

CHEMOTHERAPY OF VIRUSES

121

to the displacement of manganese from some site concerned in phage development. Although phage will not develop in the absence of manganese, there is no irreversible loss of lysogenicity. iii. Phenyl mercury borate. The organic mercury complex merfene (a complex between phenyl mercury borate, CeH6--Hg-O-B(OH)2 , and phenyl mercury hydroxide, C.&-Hg-OH) is toxic to bacteria and destroys uninfected B. coli “B” and B. coli “B” infected with T2 a t about the same rate (Latarjet and Morenne, 1954a). However, with the lysogenic strain of B. coli KI 2 which carries the phage A, merfene has a considerably greater inhibitory effect on phage development than on bacterial division (Latarjet and Morenne, 1954b). If phage development is induced in B . coli K12 by ultraviolet or X-irradiation and the bacteria are immediately diluted into medium containing merfene (8 X 10-lo to 6 X 1WBw/w) and plated after 45 min. incubation a t 37”C., the number of infectious centers (phage-producing bacteria) relative to the controls without merfene is decreased while the relative number of bacterial colonies (bacteria able to divide but not able to produce phage) is increased. After exposure to merfene concentrations greater than 6 X 10-9 w/w, none of the bacteria will produce phage but the number of surviving bacteria is greatly reduced. The effect of merfene on phage production in B . coli K12 is antagonized by cystine, cysteine, and thioglycollic acid. 2. Plant Viruses a. Some Egects of Organic Acids and Metal Ions on Infection by a Tobacco Necrosis Virus. For several plant viruses there is a daily variation in the susceptibility of the host plant (Matthews, 1953~). Maximum susceptibility is attained during daylight, the minimum being towards the end of the dark period. In an attempt to eliminate this variation bean plants were grown under continuous artificial illumination for several days before inoculation with a tobacco necrosis virus. Under these conditions plants produced almost no local lesions. No major differences in the free amino acids or sugars could be detected by paper chromatography between plants grown under normal glasshouse conditions and in continuous light. However, in the plants grown under continuous light, the organic acid fraction showed a striking increase of acids with Rj values corresponding to citric and succinic acids (Matthews and Proctor, unpublished data). It was then found that these, and other organic acids, when sprayed on the plants, could greatly reduce the number of local lesions produced by tobacco necrosis virus. Of a number of acids tested di- and tricarboxylic acids were effective but most monocarboxylic acids had little effect. Some acids were toxic for the plant, but the reduction in number of local lesions was not correlated with toxic effects or with the pH of the solutions used. Citric

122

R. E. F. MATTHEWS AND J. D. SMITH

and succinic acids were particularly effective without causing any marked plant damage. These acids appear to interfere with the process of virus establishment rather than multiplication. For example, in one experiment bean plants were given a single spray with water or with 0.02 N succinic acid at various times before and after inoculation. The ratios of numbers of lesions (acid treated)/(water treated) plants at times close t o inoculation were as follows: 15 min. before inoculation, 0.14; immediately before inoculation, 0.28; immediately after inoculation, 0.50; 15 min. after inoculation, 0.56. In plants sprayed more than about 2 hr. after inoculation, acid treatment had no effect. This period corresponds quite well with the CO2-sensitive period for this type of virus found by Kalmus and Kassanis (1944). They found that susceptibility of bean plants to a tobacco necrosis virus was greatly reduced where plants were placed in an atmosphere containing 3 0 4 0 % C02. This treatment was effective up to about 4 hr. after inoculation. We have found that the effect of succinic acid can be partially or completely annulled by spraying the plants with solutions of certain metal nitrates. The reduction in numbers of lesions produced by t,he organic acids and the extent to which the metal ions annul the effect has been quite variable from experiment to experiment. We have not yet established the major causes for the variation. However, certain points have been established. Caesium, calcium, magnesium, barium, aluminum, and, in some experiments, sodium ions can annul the effect of succinic acid. Potassium and a number of other ions appear to be ineffective. Under some conditions magnesium nitrate sprays significantly increase the number of successful virus entry points, as is illustrated in Table 12. These results suggest that, as with certain bacterial viruses, metal ions may play some part in the process of infection by a tobacco necrosis virus. The organic acids may act by sequestering metal ions in the leaf. It may be interesting from the point of view of plant virus control that a class of compounds which must be universally present in susceptible leaves can, in increased concentrations, inhibit virus establishment. The variation in the magnitude of the effects we have obtained may turn out to be due to variation in the natural state of the plants with respect to organic acids and metal ions over the experimental period. b. The Efect of Zinc Ions 072 Plant Viruses. Stoddard (1947) stated that zinc sulfate was an effective chemotherapeutic against the X disease of peach. Rumley and Thomas (1951) treated 12 carnation mosaic infected carnation cuttings with zinc chloride (0.025%) and 12 with calcium chloride (0.2 %). About half the resultant plants appeared to be free of virus. Weintraub et al. (1952) found that zinc sulfate or zinc chloride at concentrations around 1/2000 markedly reduced the number of local lesions pro-

123

CHEMOTHERAPY OF VIRUSES

duced by tobacco mosaic virus in Nicotiana glutinosa leaves floated on solutions of the compound. The treatment was effective when applied 6 hr. after inoculation. Zinc sulfate had no detectable in vitro effect on the infectivity of tobacco mosaic virus. Yarwood (1954) showed that when tobacco mosaic virus-inoculated bean leaves were dipped in 0.001-0.03 % zinc sulfate or 0.1-1.0 % calcium chloride for 10 min. a t 10 min. after inoculation the number of local lesions was greatly increased compared with water controls. However, using the virus, host, and method of application described by Weintraub et al. (1952), Yarwood confirmed that zinc treatments reduced the number of local lesions. The reason for this difference in the effects in bean and in N . TABLE 12 EFFECTOF SUCCINIC ACIDA N D MAGNESIUM NITRATE O N NUMBER OF LOCAL LESIONS PRODUCED BY A TOBACCO NECROSIS VIRUSI N BEANS

Treatment Control 0.01 N succinic acid 0.001 M magnesium nitrate 0.01 M magnesium nitrate 0.01 N succinic acid 0.001 M magnesium nitrate 0.01 N succinic acid 0.01 111magnesium nitrate

+ +

Mean number of local lesions per leaf

Significance of difference between control and treatment

19 8 27

-

13

0.001 P 0.01 P 0.001 P 0.05 P

24

No significance

36

glutinosa is not clear. Yarwood found that a variety of other.meta1 salts increased the number of lesions produced by tobacco mosaic virus in beans. We found that zinc nitrate sprayed on the plants before inoculation caused a very marked reduction in the number of local lesions produced by a tobacco necrosis virus in beans. None of about 12 other metal nitrates had a comparable effect. 3. Animal Viruses a. Organic Acids. Sodium succinate, citrate, pyruvate, and other intermediate products of carbohydrate metabolism had no significant effect on the multiplication of vaccinia virus in tissue culture (Thompson, 1947). b. Metal Ions. Schmidt and Rasmussen (1952) showed that the growth of influenza in embryonated eggs could be inhibited by cobaltous ion and that the inhibition could be annulled by histidine, cysteine, or sodium thioglycolate. In mice the maximal tolerated dose of cobalt was of no value against influenza virus. 4. Discussion. It seems probable that most, if not all, viruses will even-

124

R. E. F. MA'ITHEWS AND J. D. SMITH

tually be found to require metal ions at some stage during infection or multiplication. At present, the possibilities of chemotherapy by interference with such requirements appear to be limited, particularly for animal viruses. However, the virus inhibitory effect of sodium monofluoroacetate (Section V. H.l) might possibly occur through the metalchelating activity of the citrate which accumulates in certain tissues of treated animals. In plants, variation in susceptibility at different times of day, in leaves of different ages, and between different varieties of a species might sometimes be connected with the balance between organic acids and metal ions in the leaf. Further work should be done to determine whether a substantial decrease in plant susceptibility could be maintained either by the application of organic acids, other chelating agents, or compounds which cause organic acids to accumulate, or by breeding for varieties with a naturally high level of appropriate organic acids. D . Dyestugs 1 . Animal Viruses. Hoyle (1949) tested a number of basic dyes of the triphenylmethane group for inhibitory activity on influenza virus grown in the chorioallantoic membrane of eggs given a large dose of the virus. He found that certain of these dyes, particularly dahlia violet and crystal violet, could retard and reduce the intracellular growth of influenza A virus. With dahlia violet, a t the toxic dose for the embryo tissue, he found a reduction in the amount of virus. At the concentrations tested all the acid dyes were nontoxic for the embryo and had no effect on the virus. Hoyle considered that, since basic dyes combine with RNA in vitro, they might act i n vivo by interference with the metabolism of RNA. Fleisher (1949) tested a variety of azoquinone-imide and xanthene dyes. Nile blue A, cresyl violet, and Janus green B weremost effective in in vitro neutralization of the PR8 influenza virus. I n in vivo protection tests with mice only Janus green B and safranine-pyrazolon-sulfonamide had any effect. This effect was evident only if the chemical was given in doses close to the L D ~ ofor the chemical. Janus green B had some effect if introduced up to 48 hr. after inoculation. Both agents were more effective against the PR8 than the WS virus. Janus green B was only effective in mice when given intravenously. No effect was observed following subcutaneous or intraperitoneal injection. Within the limited number of compounds tested for in vivo activity only safranine structures were active. An azo coupling and the absence of a straight carbon chain or naphthol compound coupled to the dye appeared to be necessary for activity.

xo

CHEMOTHERAPY OF VIRUSES

125

A number of dyes may interfere with the development of foot and mouth disease in guinea pigs (Ciaccio et al., 1954). The effects varied from a complete suppression of infection to a delay in primary and/or secondary lesions. The effect varied with dye, size of virus inoculum, and the treatment schedule. Only malachite green and brilliant green were effective when injected 3 hr. after virus. These workers considered that some direct action between dye and virus is involved, since the compounds had direct neutralizing activity in vitro and since greatest effects were obtained by simultaneous injection of dye and virus. Congo red inhibited PR8 and Lee influenza viruses in the embryonated egg but had no effect in mice (Takemoto et al., 1964). Trypan red reduced mortality in herpes febrilis in mice (Hurst et d., 1952b). 1. Plant Viruses. Takahashi (1948), using a detached leaf technique, found that malachite green decreased the amount of tobacco mosaic virus produced in Nicotiana glutinosa leaf tissue. GCCH a) 2

II

D

-

C II

-

0

(CHa)z

Malachite green

The growth of virus tumors from Rumex acetosa L. was found by Nickel1 (1951) to be inhibited by malachite green, methylene blue, and crystal violet a t concentrations above 0.1 p.p.m. - Norris (1953) cultured shoots from virus X-infected potato tubers on agar containing malachite green. Of 16 shoot tips cultured for 3 weeks on malachite green (3 p.p.m.), 1 was subsequently found to be free of virus. Norris attributed the freedom of this shoot from virus to the treatment. Although the numbers involved in the experiment hardly warrant this assumption, it is well-known that potato varieties chronically infected with virus X rarely produce virus-free shoots spontaneously. 3. Bacteriophages. Various dyestuffs have been reported to be inhibitory for bacteriophage growth but their mode of action has not been studied and in most cases their effect on bacterial growth not adequately investigated. These include malachite green, homofuchsin, safranine 0 (Fitzgerald and Babbitt, 1946)) and Janus green (Bourke et al., 1952).

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R. E. F. MATTHEWS AND J. D. SMITH

E . Acridine Derivatives 1. Bacteriophages. Proflavine (2,6-diaminoacridine) has an interesting action on the multiplication of certain of the T series of B . coli bacteriophages resulting in the liberation of incomplete virus particles. Proflavine does not appreciably inactivate free bacteriophage. The addition of 2 4 pg./ml. proflavine to B. coli “B” infected with T2, T5, or T6 bacteriophages prevents the production of infective phage particles although lysis occurs after the normal latent period. Complete suppression of bacteriophage production occurs only when the proflavine is added before the formation of the first mature virus particles inside the bacterium (Foster, 1948). Addition of proflavine after the formation of intracellular virus particles prevents their increase in number (de Mars et al., 1952). The effect of proflavine can be reversed by its removal, or dilution, or washing the cells at any time up to just before the end of the latent period. Evidently proflavine inhibits a late stage in phage multiplication. On examination under the electron microscope, lysates of proflavinetreated B. coli infected with T2 are found to contain round tailless particles instead of the normal-tailed bacteriophages (de Mars et al., 1952; Levinthal and Fisher, 1953). The round particles are hollow and so usually appear flattened in their centers and are often referred to as “doughnuts.” The number of doughnuts formed per burst is approximately equal to the normal yield of bacteriophage particles. They have the properties of bacteriophage “heads” which have lost all or most of their nucleic acid. They fix complement with bacteriophage antiserum, but will not block bacteriophage neutralizing antibody which is now known to react with the tail portion of the particle. A “doughnut” contains more than 75% of the normal phage content of sulfur, but less than 15% of its phosphorus. The “doughnuts” are hollow with an outline about the size of bacterioof the mass of bacteriophages. They do phage heads and have about not adsorb to sensitive bacteria. Particles apparently identical with “doughnuts” are found in normal T2-infected bacteria when these are prematurely lysed towards the end of the latent period (de Mars et al., 1952; Levinthal and Fisher, 1953). During the course of development these disappear and are replaced by mature virus particles. Proflavine does not affect the synthesis of DNA by the infected cell (Levinthal and Fisher, 1953) and would appear to act either by arresting phage development before the nucleic acid has entered the incomplete protein shell, or at a stage when the DNA is not bound within the protein head and the tail portion of the phage has not yet been formed. This action is possibly a consequence of the known ability of acridines to com-

127

CHEMOTHERAPY OF VIRUSES

bine with nucleic acids. Although proflavine may prevent the liberation of active phage, it does not allow the survival of the infected cell. 2. Animal Viruses. Thompson (I 947) found that atebrin, proflavine, and 9-aminoacridine were inhibitory for vaccinia virus in tissue cultures. Nitroakridin 3582, rutenol, and 2-nitro-5-aminoacridine, in doses that were just subtoxic, retarded growth of small inocula of influenza in embryonated eggs but had no effect in mice (Rasmussen and Stokes, 1951). There workers considered that the compounds had their effect through making the chorioallantoic membrane atrophic. Eaton et al. (1951a) tested 3-nitro-6,7 -dimethoxy-9- (2-phenyl-4-diethylamino-butylamino)-acridine, and 3-nitro-6,7-dimethoxy-9-(2-hydroxy-3-diethylamino-propylamino)-acridine, and a related chloroacridine against mumps and the PR8 and Lee influenza viruses. Allantoic infections of mumps were slightly inhibited by both chloro- and nitroacridines, but only the nitro-compounds had any effect on influenza Lee strain. They considered, chiefly from this specificity, that these compounds were not having their effect through virucidal action or through damage to host cells. Briody and Stannard (1951) found that proflavine injected a few hours before or after inoculation with virus inhibited the multiplication of type B influenza virus in embryonated eggs but did not influence type A strains. The compound also inhibited vaccinia virus if given up to 2 hr. after inoculation. Proflavine was not virucidal a t the concentrations used. It had an effect on influenza B a t to W Othe concentration that was highly toxic for the chick embryo. Mumps and Newcastle disease virus were unaffected by concentrations of proflavine that were active against vaccinia. Proflavine was inhibitory for MEF-1 Lansing poliomyelitis in tissue culture (Brown, 1952). CHa I NH-CH(CHz)sN

Proflavine (2,g-diaminoacridine)

/CH,CH3

Mepacrine (atebrin)

Takemoto et al. (1954) record proflavine as having no effect on influenza virus A and B in embryonated eggs. a. Mepacrine. Among many substances tested for activity against Eastern equine encephalomyelitis and louping ill in mice, mepacrine was by far the most active (Hurst et al., 1952a). Mepacrine protected mice

128

R. E. F. MA’ITHEWS AND J. D. SMITH

against these viruses inoculated intramuscularly provided the compound was administered before involvement of the central nervous system was established. The compound was effective against viruses of varying virulence and against large inocula. Other acridines, even fairly closely related to mepacrine, had no comparable activity. All compounds with some activity were taken up by cells of the reticulo-endothelial system which is probably the chief site of multiplication outside the nervous system of the viruses studied. Mepacrine markedly inhibited the growth of the equine encephalomyelitis virus and greatly reduced the virus titer in the blood. The compound caused no in vitro inactivation of the virus. Mepacrine had no effect on louping ill in sheep, the natural host of the virus. Sheep tolerated lower doses of the compound on a body weight basis. Hurst et al. (1952b) tested mepacrine against a range of further viruses in mice. Some beneficial effect was noted with the following viruses, provided treatments were begun before or soon after infection: Western equine encephalomyelitis, Rift Valley fever, herpes febrilis, lymphocytic choriomeningitis, and St. Louis encephalitis. The compound had no effect against 16 other virus infections in mice, rabbits, or chickens. 3. Plant Viruses. When tobacco leaves were infiltrated with 0.05% trypaflavin solution there was a marked depression of tobacco mosaic virus multiplication (Richkov and Smirnova 1948). An 0.001 % solution depressed the necrotic reaction of Nicotiana glutinosa to tobacco mosaic virus. Trypaflavin precipitates tobacco mosaic virus from solution, but the virus was still infectious after dialysis. Acriflavine and 5-aminoacridine had no marked effect on tobacco mosaic virus multiplication tested by the leaf disc method (Schlegel and Rawlins, 1954).

F . Antibiotics 1 . Animal Viruses. There appears to be only one example of successful treatment of an infection that may be caused by a small animal virus using the antibiotics that are effective against the psittacosis-lymphogranuloma group. Andrewes and Niven (1950a, b) found that mice suffering from gray lung disease could be clinically cured by aureomycin and terramycin. Even chronic lung lesions were susceptible to treatment. The virus appeared to be eliminated. A number of other antibiotics were ineffective. However there is considerable doubt as to whether the agent of the gray lung disease is a typical virus (Andrewes and Niven, 1950a, 1953). Several newly isolated antibiotics have been found to have slight effects on some animal viruses. “Erlichin,” a preparation from culture filtrates of a nonstreptothricin-producing strain of Streptomyces lavandulae was

CHEMOTHERAPY OF VIRUSES

129

inhibitory for influenza A and B viruses in vitro (Groupe et al., 1951). The preparation had an effect against A but not B virus in embryonated eggs. “Erlichin” was ineffective against several bacteria, fungi, pox viruses, and bacterial viruses. A filtrate from Penicillium stoloniferum (8450) had antivirus activity in mice (Powell and Culbertson, 1953). Intraperitoneal doses of 8450 could protect mice against 100-1000 LD60 of MM or Semliki Forest virus inoculated subcutaneously. Oral administration of 8450 was ineffective, and the preparation had little effect when the virus was introduced intracerebrally. 8450 protected roller tubes of monkey testicular cultures against all 3 types of poliomyelitis virus as was also found by Hull and Lavelle (1953) and had some effect against a type 2 strain in mice. Cochran el. al. (1954) found that 100 ml. doses of crude filtrate of 8450 reduced morbidit,y and increased the incubation period in monkeys subcutaneously infected with poliomyelitis type 1 Mahoney strain. Netropsin, an antibiotic from Streptomyces netropsis, caused a substantial increase in the percentage of mice surviving intracerebral inoculation with neurotropic vaccinia virus (Schabel et al., 1953). One half the maximum tolerated dose of antibiotic was given intraperitoneally twice daily for 5 days beginning 30-60 min. after inoculation. The preparation had no effect on infections with the following viruses in mice: feline pneumonitis, influenza A, Western equine encephalomyelitis, and Lansing poliomyelitis. Shope (19534 described experiments with material produced by Penicillium funiculosum which had therapeutic activity against swine influenza virus in mice. The culture slowly lost its ability to produce material active against this virus. Subsequently it was found that the culture produced a substance designated “Helenine” which was effective against Columbia SK virus and Semliki Forest virus infections in mice (Shope, 195313). Groupe et al. (1954) described a product (“xerosin”) from Achromobacter xerosis n. sp. which had some suppressive effect on influenza in mice. Parenteral administration of “xerosin” reduced mortality following intracerebral but not intravenous doses of influenza A virus. The material transiently suppressed the development of pneumonia following infection with large inocula of influenza B virus. 1. Plant Viruses. As might be expected, most of the antibiotics developed for use against bacterial diseases have no inhibitory effects against plant viruses. Manil (1947a, b) found that penicillin, streptomycin, tyrothricin, actinomycin, and actinomycetin tested by several methods had no effect on tobacco mosaic virus. Penicillin, streptomycin, aureomycin, chloromyceh, and terramycin had no effect on tobacco mosaic and potato yellow dwarf viruses when introduced into the plants or mixed with inocula (Beale and Jones, 1951). Leben and Fulton (1952) using a screening test

130

R. E. F. MA’ITHEWS AND J. D. SMITH

in which inoculated half leaves were placed on agar in the dark, found that streptothricin and terramycin inhibited the development of local lesions by tobacco mosaic and tobacco ringspot viruses in cowpea leaves. The fact that Leben and Fulton found terramycin to have some effect on tobacco mosaic virus, while Beale and Jones did not, could be due to differences in the method of testing or to the fact that different host plants were used. Kutsky (1952) with tobacco stem tissue cultured in vitro found that terramycin, streptomycin, and subtilin had no effect on the amounts of tobacco mosaic virus produced. Schlegel and Rawlins (1954) using the tobacco leaf disc method found that an antibiotic from Nocardia formica reduced the amounts of tobacco mosaic virus produced. The following antibiotics sprayed on the leaves were ineffective (at concentrations not severely toxic to the host) against tobacco mosaic virus in tobacco, cucumber mosaic virus in cucumber, and lucerne mosaic virus in tobacco (Matthews and Proctor, unpublished data) : aureomycin, terramycin, chloromycetin, streptomycin, bacitracin, and clavicin. Kirkpatrick and Linder (1954), using a leaf infiltration method for applying compounds to whole plants, found that chloromycetin reduced the multiplication of tobacco mosaic virus in tomato plants and of a fruit tree virus in cucumbers. 3. Bacteriophages. The effect of most antibiotics on bacteriophage multiplication closely parallels that on bacterial growth, and penicillin actually accelerates the lysis of phage-infected bacteria (Himmelweit, 1945; Nicolle and Faguet, 1947; Elford, 1948). a. Chloromycetin. Chloromycetin at sufficiently high concentrations inhibits the synthesis of bacterial proteins but not that of RNA or DNA. It has no effect on T1 bacteriophage particles nor does it prevent their adsorption to B. coli “B” but, if added to B. coli at bacteriostatic concentrations shortly after infection with TI, lysis is prevented and no intracellular phage formation occurs (Bozeman et al., 1954). When added later lysis is not suppressed but further phage formation is prevented. Its action is reversible up till almost midway through the latent period. Sub-bacteriostatic concentrations of chloromycetin allow lysis and reduce the virus yield in about the same proportions as they reduce bacterial growth. Similar effects of chloromycetin on Staphylococcus and B. coli phage growth have been described by Edlinger (1951). b. Aureomycin. Aureomycin has no effect on free T3 bacteriophages but bacteriostatic concentrations added at the time of infection will prevent lysis and intracellular phage production of T3 in B. coli “B” (Altenbern, 1953). Under these conditions after about 50 min. in aureomycin the infected bacterium irreversibly loses its ability to produce phage. As infection with T3 kills B. coli “B” cells, the bacterium thus can neither multiply

CHEMOTHERAPY OF VIRUSES

131

nor produce phage. The effect of aureomycin on the viability of uninfected cells a t these concentrations is small. c. Other Antibiotics. Streptomycin a t bacteriostatic concentrations delays lysis by Staphylococcus bacteriophages (Faguet and Edlinger, 1949; Edlinger and Faguet, 1950) and terramycin a t sub-bacteriostatic concentrations retard or inhibit lysis of Staphylococcus by bacteriophages (Edlinger and Faguet, 1951). Asheshov and his colleagues (1949) have described several unidentified substances from Aspergillus species which inhibit the growth of several Staphylococcus, Streptococcus, and B . coli bacteriophages. These substances have no effect on the free phages or their adsorption, and their mode of action is unknown.

G . Plant Growth Regulators and Related Substances 1. Plant Viruses. Limasset et al. (1948) found that 2-methyl-4-chlorophenoxyacetic acid had a temporary inhibitory effect on the development of potato viruses X and Y in tobacco plants, the effects being most marked when the compound was applied with the inoculum. Limasset and Cornuet (1949) suggested that the very low concentration of tobacco mosaic virus t,hey found in tobacco meristems might be due to the high concentration of auxin known to occur in this tissue. Locke (1948) found that 2,4-dichlorophenoxyaceticacid masked the symptoms of potato leaf roll. He also considered that the concentration of virus was reduced. Augier de Montgremier and Morel (1948) obtained results suggesting that less tobacco mosaic virus developed in tobacco tissues with a high content of naphthaleneacetic acid. CHZ. COOH

I

Naphthaleneacetic acid

Kutsky and Rawlins (1950) using tobacco stem tissue cultured on agar, found that naphthaleneacetic acid at lO-4% in the medium reduced the amounts of tobacco mosaic virus produced after 3 4 weeks to about 20-40 % of that in controls. Nickel1 (1950) found that concentrations between 0.001-1 p.p.m. indoleacetic acid, 2,4-dichlorophenoxyacetic acid and naphthoxyacetic acid stimulated growth of virus tumors from Rumex acetosa L. At concentrations of I p.p.m. and above, these compounds inhibited tumor growth. 2,3,5-Triiodobenzoic acid inhibited growth a t all concentrations tested. Kutsky (1952) using the same methods as Kutsky and Rawlins (1950)

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R. E. F. MATTHEWS AND J. D. SMITH

found that indolebutyric acid was effective in reducing the amount of tobacco mosaic virus produced. Phenylacetic acid, phenylpropionic acid , and phenylvaleric acid were ineffective a t concentrations near the maximum that the plant tissue could tolerate and produce normal growth. Nichols (1952) sprayed tobacco plants with naphthaleneacetic acid and indolebutyric acid. He found that daily sprays a t 100 mg./l. retarded development of symptoms and decreased severity of tobacco mosaic symptoms. No concentration of these substances was found which would prevent all mosaic symptoms without plant damage. 2. Animal Viruses. Wooley et al. (1952a) tested a large number of compounds for activity against several viruses in mice. The following 4 compounds gave rise t o longer incubation periods and to fewer deaths with poliomyelitis virus: 2 ,4-dichlorophenoxyacetic acid; 3 ,4-dichlorophenoxyacetic acid ; 2,4-dichlorophenoxypropionicacid; 2,5-dichlorophenoxyacetic acid. None of these compounds had any effect on St. Louis encephalitis or influenza infections. Takemoto et al. (1954) list a number of substituted phenoxyacetic acids which were ineffective against influenza in the embryonated egg.

H . Miscellaneous Compounds with Some Inhibitory Activity for Animal Viruses I . Sodium Monofluoroacetate. Sodium fluoroacetate inactivates the enzyme system necessary for the oxidation of citric acid. By intraperitoneal injection of sublethal doses of fluoroacetate, which increased the concentration of citrate in the mouse lung, Ackermann (1951b) demonstrated the blocking of the citric acid cycle in that organ. The injection of similar concentrations of the compound gave a marked inhibition of the development of influenza virus A in the lung. The compound had no effect in vitro on the infectivity or hemagglutination properties of the virus. Similar results were found for the Lansing strain of poliomyelitis in mice (Ainslie, 1952). The early phase of growth of this virus in mouse brain and spinal cord was suppressed or delayed by the intraperitoneal injection of fluoroacetate at 6 mg./kg. body weight (but not a t 3 mg./kg.) 1 hr. before intracerebral inoculation with virus. There was a delay in the onset of illness. However, Mogabgab and Horsfall(l952) found that sodium fluoroacetate gave only a slight delay in mult,iplication of PR8 influenza virus in mouse lung ( 3 4 mg./kg.) or of PR8 and Lee viruses in the allantoic sac (at 2 mg./kg.). These quantities of the compound caused a 10-20 % mortality in mice and 100% mortality in the chick. Sodium monofluoroacetate at 0.006 mg./ml. had no effect on MEF-1

CHEMOTHERAPY OF VIRUSES

133

Lansing poliomyelitis virus in tissue culture (Brown, 1952). The compound, when injected intraperitoneally, delayed virus production and slightly prolonged the survival time of mice inoculated intracerebrally with Eastern equine encephalomyelitis virus (Watanabe et al., 1952). Francis et al., (1954) treated monkeys with 5 mg./kg. body weight doses of sodium monofluoroacetate 1 or 3 days after subcutaneous inoculation with type 1 poliomyelitis virus. This treatment gave a significant reduction in the number of animals developing paralysis but had no effect on the average incubation period. These authors considered that fluoroacetate was having its effect in some non-neural tissue. They could demonstrate no accumulation of citrate in the brain following treatment. 2.1,4-Dinitrophenol. Thompson (1947) showed that 2,4-dinitrophenol (DNP) was inhibitory for the growth of vaccinia virus in tissue culture. D N P inhibits multiplication of PR8 influenza virus in flask cultures of chorioallantoic membrane (Ackermann and Johnson, 1953). There appeared to be no permanent alteration in the ability of the tissues to support virus synthesis. At the concentrations used the compound had no in vitro action on the virus. I n minced preparations of chorioallantoic membrane D N P caused a marked stimulation of ATPase. I n intact tissues there was a correlation between virus inhibition and stimulation of respiration and the release of phosphorus. 3. Thiosemicarbazones. Hamre et al., (1951) tested a number of thiosemicarbazones for activity against various viruses in intranasally infected mice. Benzaldehyde thiosemicarbazone and some p-substituted analogues were most effective.

S Benealdehyde thiosemicarbazone

These compounds were only effective in mice if fed in the diet for 2 days before inoculation and 10 days after infection a t levels near the maximum tolerated by the animals. Thompson, et al. (1951a) found that benzaldehyde thiosemicarbazone prevented the multiplication of vaccinia virus in chick embryonic tissue cultures a t a concentration of 1 pg./ml. in the medium. When fed in the diet the compound gave a marked protection to mice inoculated intracerebrally with vaccinia virus. The compound had no effect on vaccinia virus i n vitro. Derivatives with substitutions in the para position of the benzene nucleus or in the 4 position of the thiosemicarbazone portion of the molecule had reduced activity. Minton et al. (1953) found that mice infected intracerebrally with a strain

134

R. E. F. MATTHEWS AND J. D. SMITH

of vaccinia virus could be given protection by the administration of isatin thiosemicarbazone and 5-nitrothenaldehyde thiosemicarbazone. The compound could be administered either intraperitoneally or in the diet. Protection of the mice did not appear to be associated with inhibition of virus proliferation in the brain. Thompson et al. (1953) found that benzaldehyde thiosemicarbazone was a strong inhibitor for vaccinia virus in mouse and chick embryonic tissues. They considered that the following properties of the thiosemicarbazone molecule are associated with a high degree of antivaccinial activity: (1) The presence of the =N-NH-CS-NH* group, as such. (2) The presence of a cyclic component. Heterocyclic thiosemicarbazones may be active (e.g. the pyridine, quinoline, isatin, or thiophene compounds). 4. Phenoxythiouracils. Thompson et al., (1951b) studied the effect of a variety of phenoxythiouracil compounds on the development of several viruses. 5-(2,4-Dichlorophenoxy)-4-hydroxy-2-mercaptopyrimidine [ (Dichlorophenoxy)thiouracil] was the most effective compound. OH

S (Dichlorophenoxy) thiouracil

Although only moderately active inhibitors of vaccinia virus in chick embryonic tissue culture, (dich1orophenoxy)thiouraciland certain related compounds had a significant protective effect in mice on the development of the 1 HD strain of vaccinia virus. Virus was inoculated intracerebrally and compounds were administered intraperitoneally or in the food. The protective effect could be demonstrated only when treatment was begun soon after infection. Treated animals appeared to have a lower virus titer in the brain. There was no evidence that these compounds were virucidal. Modifications of the pyrimidine nucleus lead to a loss or considerable decrease in activity. Some variation in the benzene nucleus is permissible for retention of activity. In general, the presence of an electron-attracting group (chloro or bromo) in the para position of the benzene nucleus appeared to enhance activity whereas electron donor groups (alkyl, alkoxyl) in the same position reduced activity. Phenoxythiouracils do not inhibit growth of Bacillus subtilis, Salmonella typhosa, or tubercle bacillus. They gave no protection in mice against influenza A, herpes, St. Louis encepha-

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CHEMOTHERAPY O F VIRUSES

litis, MM, or Rift Valley fever viruses. 2-Thiouracil had no effect on vaccinia infections in mice. 6. Diamidines. McClelland and van Rooyen (1949) found that certain aromatic amidines, particularly hexamidine, had some inhibitory action against influenza virus in embryonated eggs, the effect being dependent on route of inoculation. Hexamidine showed some action on the virus in vitro but no protective effect in mice. Mumps virus was inhibited in tissue cultures by 0.5-5.0 pg./ml. of pentamidine or stilbamidine added a t the same time or before inoculation. For similar conditions influenza viruses A and B were inhibited by 5-20 pg./ml. of pentamidine and 20 pg./ml. of stilbamidine (Eaton et al., 1951~). NH

NH

\\C

/

- e - ( C H z )

6-0 a

NHz

C

[

NHz Pentamidine

NH ~

/

c

~

-

c

-

H

=

c -H

~

YNH c \

NH2

NHz Stilbamidine

The amount of pentamidine necessary for inhibition increased with increasing dose of virus. Pentamidine affected growth and oxygen consumption of fibroblasts a t concentrations down to about 5 pg./ml. Eaton et al., (1952) found that pentamidine had a definite virucidal action in influenza virus B in vitro a t concentrations achieved in experiments in the allantoic sac. Stilbamidine and propamidine did not show this effect. The compounds had no effect on influenza in mice. 6. a-Haloacylamides. Several chloro- and bromoacylamides inhibit the multiplication of vaccinia virus in chick embryonic tissue culture. 5-chloracetamidouracil was inhibitory while the corresponding 5-fluoro-compound was not. This suggested that an alkylation reaction may be involved in the activity (Thompson et al., 194913). OH

I

5-Choracetamidouraci1

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R. E. F. MA'ITHEWS AND J. D. SMITH

5-Chloroacetamidouracil was no more toxic in mice than related substances of much lower antiviral activity. '7. Polymerized Benzoid Suljonic Acids. Of 182 compounds tested against PR8 and Lee influenza viruses in the embryonated egg, by Takemot0 et al. (1954), the most effective inhibitor consisted of polymerized sodium salts of substituted benzoid sulfonic acids. At the concentrations used the material did not inactivate the virus in vitro. It was ineffective in mice.

I . Substances Agecting the Lysogenic Bacteriophage-Host Relationship 1. The Lysogenizatim of Bacteria. A temperate bacteriophage is defined as one which is able to enter into a lysogenic relationship with one of its hosts (Jacob et al., 1952). After infection with a temperate phage the phage-bacterium system may develop in one of three ways: (1) the bacterium may lyse and produce phage; (2) the bacterium may become lysogenic; (3) the genetic material of the phage may disappear either with or without survival of the infected bacterium. Lwoff et al. (1954) have described conditions where the response of Salmonella typhi-murium to a temperate phage may be modified by treatment with certain substances. This system is of considerable interest as it provides a model where the fate and subsequent mode of development of the infecting particle may be altered by chemical means. The importance of findings from this type of system in the problem of the establishment of virus infection in other types of cells depends largely on whether or not the lysogenic type of virus-host relationship is a unique property of bacteriophages. There are indications but no direct evidence that similar situations may occur with some animal and plant viruses. Cells of Salmonella typhi-murium infected with phage A either lyse and produce bacteriophage or become lysogenic. The relative proportions of these two types of response depends on the multiplicity of infection. In a synthetic medium after infection with a multiplicity of 1 phage per bacterium, 5 % of the bacteria become lysogenic, while at a multiplicity of 40 this increases to 90%. At a given multiplicity of infection a number of of substances, when added to the synthetic medium before or a t the time of infection, reduce the proportion of lysogenizations. These are citric, fumaric, succinic, malic, lactic, pyruvic, and malonic acids, chloromycetin, cyanide, 5-methyltryptophan, and also inhibitors such as 2,4-dinitrophenol and azide when given at concentrations allowing bacterial growth nt a reduced rate. None of these substances has an appreciable effect on the viability of free phage or its adsorption to the bacterium. Exposure to certain doses of ultraviolet light or a temperature of 42°C. also has a similar effect.

CHEMOTHERAPY O F VIRUSES

137

From a study of the effects of 5-methyltryptophan, citrate, and heating applied a t various times after infection, it appears that the fate of the invading phage genetic material (that is, whether it will produce phage and lyse the bacterium, or become “prophage” giving a lysogenic bacterium) is irreversibly decided between 6 and 9 min. after infection. The latent period is 32 min. The inhibitors 2,4-dinitrophenol and azide when applied at concentrations which are bacteriostatic have an interesting effect on bacteria infected with the phage Ac and which is quite different from that described above. (At such concentrations these substances have no effect on phage viability or adsorption.) The phage Ac is a mutant of phage A differing in the fact that it gives a much smaller proportion of lysogenization. This proportion is independent of the multiplicity of infection. 2,4-Dinitrophenol or azide was added to bacteria simultaneously infected with phage Ac a t a multiplicity of 20. Increasing the length of exposure to the inhibitor u p to 30 min. increased the proportion of surviving bacteria from 0.1 to loo%, while the number of bacteria capable of lysing and yielding phage correspondingly decreased. The bacteria which survived were not lysogenic and were sensitive to the phage Ac (if given later in the absence of inhibitor). In this case the genetic material of the bacteriophage is apparently permanently lost. 2. Loss of Lysogenicity. A number of lysogenic virulent strains of Corynebacterium diphtheriae carry bacteriophages which are able to transfer the property of virulence and toxigenicity to related avirulent strains. When the bacteriophage liberated by the virulent strain is added to a culture of the avirulent strain, most of the cells are lysed but a proportion become lysogenic and virulent. Certain virulent strains carry simultaneously two or more different bacteriophages but only one type is able to transfer virulence to avirulent strains. Hewitt (1954) has found that, after culture in the presence of certain antibiotics (notably streptomycin) or certain other growth-inhibiting substances (for example arsenite, and copper and cobalt salts), resistant cultures of such strains could be obtained which have lost one of the viruses they originally carried, and in some cases the virulence-transferring properties of these cultures have been modified. In these cases it is not clear whether the streptomycin (or other substances) acts directly on bacteria carrying the virus causing a loss of lysogenicity (or “curing”), or whether it merely selects bacteria which are not carrying the virus. VI. INCORPORATION PHENOMENA IN RELATION TO ANTIMETABOLITE ACTION In most examples of competitive inhibition by analogues of essential metabolites the inhibitor is considered to combine with the enzyme which

138

R. E. F. MATTHEWS AND J. D. SMITH

uses the metabolite, reducing the amount of enzyme available for the normal process and thus inhibiting growth. The inhibitor and metabolite can each combine reversibly with the same site on the enzyme surface. On this hypothesis the inhibitor is usually considered t o act only in the form in which it is supplied, or in some slightly modified or degraded form. However, a variety of examples are now known, or suggested, in which the inhibitor is metabolized along the same path as the natural metabolite until a t some stage an inhibitory or nonfunctional product is formed. These examples vary from a simple phosphorylation to the incorporation of purine and pyrimidine analogues into nucleic acids already described. Umbreit and Waddell (1949) studied the effect of deoxypyridoxine on tyrosine decarboxylase from Streptococcus faecalis. Deoxypyridoxine had no effect on the conversion of pyridoxal to pyridoxal phosphate in the presence of ATP. Deoxypyridoxine phosphate interfered strongly with the combination of pyridoxal phosphate and enzyme. They suggest that deoxypyridoxine has its inhibitory effect in vivo as deoxypyridoxine phosphate. Stekol and Weiss (1950) administered t o rats ethionine CI4 labeled in the methylene carbon of the ethyl group. They were able t o isolate radioactive choline and creatinine from the tissues. The activity of the choline was in the trimethylamine portion of the molecule. They suggested that the radioactive ethyl group had replaced a methyl group in this port.ion of the molecule and that ethionine may have its growth inhibitory action at least in part through the in vivo synthesis of ethyl analogues of compounds which participate in “transmethylation” reactions. Fluoroacetate is synthesized in vivo into a tricarboxylic acid (probably fluorocitrate (Peters et al., 1953)). This compound inhibits competitively the conversion of citrate to isocitrate by aconitase. I n preparations of mitochondria the inhibition is apparently irreversible presumably due to structural factors (Peters, 1952). Hughes (1954) studied the inhibition of cozymase synthesis by 5-fluoronicotinic acid in several bacterial species. The suggested route of cozymase synthesis is: (2) nicotinamide riboside (3) nicotinic acid (1) nicotinamide +

-

-

-

nicotinamide ribonucleotide (4) cozymase Hughes found that fluoronicotinic acid inhibited the synthesis of cozymase from nicotinic acid, nicotinamide, nicotinamide riboside, and to a lesser extent nicotinamide ribonucleotide. The inhibition was competitive when inhibitor and metabolite were added simultaneously but noncompetitive when the inhibitor was added before the metabolite. Prior incubation of

139

CHEMOTHERAPY OF VIRUSES

cells with inhibittor gave an about tenfold increase in the inhibition of cozymase synthesis from nicotinic acid. During the incubation 5-fluoronicotinic acid was taken up and bound by the cells. Although compounds containing 5-fluoronicotinic acid have not yet been identified, Hughes considered that the compound is metabolized along the same route as nicotinic acid and a t some stage or stages the fluoro-compounds act as potent inhibitors of cozymase synthesis. Diphosphopyridine nucleotidases from certain sources (e.g. pig’s brain) catalyze an exchange reaction in which the nicotinamide part of cozymase is replaced by isonicotinic acid hydrazide (INH) (Zatman et al., 1953). NH2 I

NH

I

c=o

Nicotinamide

Isonicotinic acid hydrazide

Certain DNPases, such as that from beef spleen, are inhibited by INH. The cozymase analogue containing I N H is a t least twice as potent an inhibitor as I N H for beef spleen DPNase. These observations suggest that possibility that a metabolite analogue which is inactive or only slightly inhibitory in one form might be a potent inhibitor if supplied to the organism at another stage in the synthetic sequence. For example in the following sequence: A

enzyme b )

c

enzyme

enzymed

Product

A’, an analogue of A , might be ineffective as an inhibitor because it is too different from A to be metabolized to B’ (the corresponding analogue of B ) by enzyme a. However, if the A’ is supplied to the organism in the compound B’, then this might be inhibitory for enzyme b, or might be taken by enzyme b to an inhibitory compound C’. Alternatively, having been taken over the step from A to B , A’ might then be metabolized all the way to replace A in the final product which is then rendered nonfunctional. Thus whether an analogue is inert, inhibitory, or able t o replace the metabolite in normal growth for a particular organism may well depend on the form in which it is supplied. This can be illustrated from the work of Tamm and his collaborators on influenza virus inhibition (Section V. B.2). 5,6-Dimethylbenzimidazole is inhibitory, but when this compound is supplied as

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the riboside there is no inhibition. Conversely, 5,6-dichlorobenzimidazole is much more inhibitory when supplied as the riboside. It is thus becoming apparent that quantitative data on growth inhibition by antimetabolites and on the annulling of inhibition by the normal metabolite may be difficult to interpret unless the fate of the antimetabolite in the system studied is known. Mathematical treatments of data on growth inhibition have been developed from the kinetics of enzyme inhibition in vitro. Such treatments will not adequately account for the results obtained when an inhibitor is incorporated into more complex molecules following one or perhaps several of the synthetic routes in which the normal metabolite takes part. Here inhibition by a number of different mechanisms may occur simultaneously.

VlI. GENERAL DISCUSSION With plant virus diseases there are two situations in which chemical control would be most desirable. Stocks of many economically important plants which are reproduced vegetatively are heavily or completely virusinfected. Chemical treatment to free individual plants of the viruses would allow nucleus stocks of virus-free plants to be built up. For this type of treatment severe but temporary host damage would not be important. There are several reports of such chemotherapeutic cures but they require confirmation. At the present time heat treatment (Kassanis, 1954) appears to offer a more hopeful solution for some infections of this type. There are numerous crops grown on a large scale for which a chemical means of protection against virus infection would be of great value. For example sugar beets are regularly infected with the serious aphid-transmitted yellows disease. A spray treatment which delayed disease development for even a few weeks would be useful. It is perhaps with crops of this type that control by chemical means may be first successfully applied. The situation is different with economically important trees and other perennial crops which suffer from serious virus infection, such as the swollen shoot disease of cocoa and the quick decline of citrus. With these plants, which are grown for many years and which are normally subject to continued reinfection by insects, the problem of obtaining effective protection by chemical means may be much more difficult. Although control of the major virus diseases may not be obtained for some time, chemical treatments might, in special circumstances, find practical application in the fairly near future. For example Holmes (1954) found that systemic necrosis and death of lines of tobacco hypersensitive to tobacco mosaic virus could be prevented by watering the plants with 5 mg. of thiouracil (0.01% in water) daily for 4-12 days. Systemic disease

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could be prevented if applications of the compound were begun after the local reaction had developed. Thus applications of thiouracil might be used in breeding work to enable the necrotic local reaction to be detected, and then to allow the plant to survive and set seed. With many human and animal viruses the ideal agent may be one which, administered after diagnosis of infection, would satisfactorily modify the TABLE 13 STAGESI N VIRUSDEVELOPMENT WHICH VARIOUS SUBSTANCES ARE KNOWNOR PRESUMED TO INHIBIT Mechanism of inhibition

Inhibiting substance

1. Specijk inactivation of free

Phage receptor substance from host cell wall Indole Changes in cation concentrations Citrate (or deprivation of calcium ions) 2,4-Dinitrophenol

virus particles before attachment t o host cell 2. Prevention of attachment of virus to host cell receptors 3. Prevention of release of nucleic acid into the host cell 4. Loss of intracellular virus genetic material before multiplication begins 5 . Inhibition of production of virus components 6. Interference with the assembly of complete virus particles from virus components 7. Inhibition of release of virus from the infected cell

8. Incorporation into the virus nucleic acid with consequent formation of non-infective virus particles

Virus T2 bacteriophage T2 bacteriophage Various T phages T5 bacteriophage Salmonella typhi-murium bacteriophage

Some amino acid and vitamin analogues Proflavine

Bacteriophages and animal viruses T2 bacteriophage

p-Methoxyphenylmethanesulphonic acid 8-Azaguanine

PR8 influenza virus

5-Halogenated uracils

Bacteriophages

Plant viruses

course of the disease without affecting the build-up of natural immunity. There is little indication that such control will be available for any virus in the near future. On the other hand, the development of compounds giving adequate protection against certain viruses, when administered before infection, has perhaps been more nearly attained. For many of the substances showing some virus inhibitory activity, there are large numbers of related compounds already available (e.g. compounds of the plant-growth-regulator type, the acridines and dyestuffs). Only in a few instances do the potentialities of such groups appear to have

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been assessed adequately from the practical point of view (e.g. the work of Hurst and his collaborators on the effects of mepacrine and related compounds on animal viruses). Tamm and his co-workers have increased the activity of benzimidaxole many hundredfold against influenza virus in tissue culture by systematically exploring the effect of various substitut,ions in the molecule. There are few examples such as this in the literature where the observation that a certain compound has inhibitory activity for a virus is followed by synthetic work directed specifically towards increasing the activity against the particular virus. In the past, the information available about virus multiplication has been of little value as a guide in developing inhibitory compounds. However, in certain systems some of the processes involved are now known in considerable detail, and certain of these appear to be general features of virus multiplication. It is thus becoming possible to study virus inhibitory substances in relation to their action at definite steps in virus development. We conclude with a summary illustrating the various stages in virus development a t which compounds are known or presumed to have their inhibitory effects (Table 13). References Ackermann, W. W. (1951a). J. Exptl. Med. 99, 337. Ackermann, W. W. (1951b). J. Exptl. Med. 93, 635. Ackermann, W. W. (1952). Proc. SOC.Exptl. Biol. Med. 80, 362. Ackermann, W. W., and Maassab, H. F. (1954a). J . Exptl. Med. 99, 105. Ackermann, W. W., and Maassab, H. F. (1954b). J . Expt2. Med. 100, 329. Ackermann, W. W., and Johnson, R. B. (1953). J . Exptl. Med. 97, 315. Ada, G. L., and Perry, B. T. (1955). Nature 176, 209. Adams, M. H. (1949). J . Zmmunol. 62, 505. Adams, M. H. (1950). i n “Methods in Medical Research” (J. H. Comroe, Jr. ed.), Vol. 2. Yearbook Publishers, Chicago. Ainslie, J. D. (1952). J. Ezptl. Med. 96, 9. Altenbern, R. A. (1953). J. Bacteriol. 66,288. Anderson, T. F. (1945). J . Cellular Comp. Physiol. 26, 17. Andrewes, C. H., and Niven, J. S. F. (1950a). Brit. J. Exptl. Pathol. 31,767. Andrewes, C. H., and Niven, J. S. F. (1950b). Brit. J. Exptl. Pathol. 31, 773. Andrewes, C. H., and Niven, J. S. F. (1953). J . Pathol. Bacteriol. 66, 565. Angier de Montgremier, H., and Morel, G. (1948). Compt. rend. 227, 688. Asheshov, I. N., Strelitz, F., and Hall, E. A. (1949). Brit. J . Exptl. Pathol. SO, 175. Barnam, C. P., and Huseby, R. A. (1950). Arch. Biochem. 29, 7. Bawden, F. C. (1950). “Plant Viruses and Virus Diseases,” 3rd ed., 327 pp. Chronica Botanica, Waltham, Mass, Bawden, F. C., and Kassanis, B. (1954). J . Gen. Microbiol. 10, 160. Beale, H. P., and Jonea, C. R. (1951). Contribs. Boyce Thompson Znst. 16 (8), 395. Beumer, J., and Beumer-Jochmans, M. P. (1953). Ann. inst. Pasleur 84, 328. Bonner, J. (1950). “Plant Biochemistry.” Academic Press, New York.

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Tumor Viruses' J. W. BEARD, D. G. SHARP,

AND

EDWARD A. ECKERT'

Department of Surgery, Duke University School of Medicine, Durham, North Carolina

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Rabbit Papillomatosis and Avian Leukosis ......................

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B. Virus of Erythromyeloblastosis. . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Chemical Constitution.. . . . . . . . . . . . . . . . . . . . . . . . . . 3. Enumeration of Virus Particles. . . . . . . . . . . . . . . . . . 4. Enzyme Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6. Antigenic Constitution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Erythromyeloblastosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Rabbit Papillomatosis ....................... C. Chicken Tumor I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Host Response to Other Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..............................................................

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188 191 193

I. INTRODUCTION The implication of viruses as specific carcinogens has now been firmly established. For many years the only known host of viruses inducing neoplasia was the chicken, and there was but small inclination to interpret the phenomenon in the bird as bearing significantly on conditions occurring in the mammal. A new era of interest in viruses as potential carcinogens 1 Preparation of this paper was supported by a research grant to Duke University from the National Cancer Institute of the National Institutes of Health, U. S. Public Health Service; by a grant from the American Cancer Society, on recommendation of the Committee on Growth; by a grant from the Damon Runyon Memorial Fund for Cancer Research, Inc.; and by the Dorothy Beard Research Fund. 2 Scholar in Cancer Research, American Cancer Society, Inc.

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was introduced by the work of Rous and his co-workers in the demonstration of the tumor attributes of the virus-induced rabbit papilloma (12, 140, 141), and observation of the progression of the growth to outright carcinoma (108, 137-139, 142, 167). Full appreciation of the import of these studies of a mammalian virus tumor might have been retarded had it not been for the timely recognition of the viral etiology (15-17) of mouse mammary carcinoma. Here was a growth conceded to be exemplary of mammalian cancer and intensively studied (128) for many years as a typical tumor. It is of compelling significance that the discovery of the agent was the result not of a search for an etiological virus but of recognition of the need for demonstration of an extrachromosomal factor in the origin of the tumor which could not be accounted for on the basis of the known influences of the genetic constitution and the hormonal status of the host (3, 55, 95, 162). Corollary to the findings with the rabbit papilloma and the mouse mammary carcinoma, there is no longer a basis for excluding the virus tumors from the field of classical oncology or for evasion of the responsibility for exhaustive investigation of the bearing of viruses on the occurrence of cancer in general. Among the many problems of the virus tumors, there is none more critical than the nature and properties of the essential causative agents. It is in this field, however, that progress has lagged behind the advances made in the study of other aspects of virus tumor phenomena. Nevertheless, it is encouraging that substantial beginnings have been made in this direction. At least two remarkably different tumor viruses, the agent of the rabbit papilloma and that of avian erythromyeloblastica leukosis, have been obtained in preparations of purity and amount sufficient for quantitative characterization. The experimental data provide a foundation not only with respect to knowledge of the properties of these agents but for quantitative clarification of many questions of known host-virus relations not susceptible to final resolution except by study of the isolated viruses. The status of the problem of the virus of mouse mammary carcinoma has been reviewed recently by Dmochowski (55) and that of the chicken sarcoma by Claude and Murphy (49), Foulds (77), Duran-Reynals (56), and Harris (94). It is the purpose of this paper to discuss the physico-chemical find8 Because of the earlier uncertainty of the etiological relationships between erythroleukosis and myeloblastosis (see following discussion), the general term “erythromyeloblastosis” has been employed here, and in the individual reports cited, to designate the disease under study. As the result of further evidence gained in the study of another form of avian leukemia, erythroleukosis, it has been concluded as stated by Burmester (58), that the condition designated here as erythromyeloblastic leukosis is actually an essentially pure strain of myeloblastosis. Since there is now little doubt that this is the case, the properties of the virus’ described may be regarded (58,66) as those of the virus of myeloblastosis.

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ings derived through study of the purified papilloma and erythromyeloblastic leukosis viruses arid their application to the interpretation of certain biological phenomena associated with the growths which they cause. There will be considered, also, some of the implications of these findings with respect to virus diseases in general. 11. RABBITPAPPILLOMATOSIS AND AVIANLEUKOSIS

A . Papillomatosis The characters of the rabbit papilloma and of the cancers deriving from it, as well as the biological phenomena associated with the induction of the malignant process, have been adequately summarized by Rous (137-139), Kidd (log), and Syverton (167). In brief, the epidermal papilloma, produced in various wild or domestic rabbits by experimental virus tmnsmission, arises as the result of infection of the cells of the basal layer. These proliferate rapidly and, a t first, in an orderly fashion. With enlargement and spreading at the base, the young cells are thrown into folds and papillae which, together with increasing amounts of the older elements dying and keratinizing without desquamation, come to form the warty mass characteristic of the disease. In the beginning, the growths exhibit every evidence of a benign process. With time, however, the cells become unruly and the growth less contained. Extension begins into the connective tissue layers, resulting in cysts and cell strands, until all appearance of orderliness is lost. Although the papillomatous growths through these stages exhibit, by definition, the attributes characteristic of tumors (12, 140,141), there is little evidence of true malignancy. In some rabbits, both domestic and cottontail, the warts continue to grow, irregularity of histological arrangement increases until after several months, in the natural course of events, the growths have progressed to outright squamous cell carcinoma of classical behavior, eroding other tissues, metastasizing, and finally killing the host. In all of this, the process has been a progressive transition from a benign, orderly character through subtly increasing disorder to unquestionable malignancy. Although the transformation of papilloma to carcinoma may be influenced experimentally (110,136,143), it is characteristic that malignancy in rabbit papillomatosis is not the immediate effect of infection with the virus. The ultimate relation of the virus to the cancer derived through its influence constitutes one of the more interesting mysteries of the virus tumor phenomena. Rarely is the virus demonstrable (147, 163) in extracts of domestic rabbit warts, and the infectious agent has never been recovered from the cancers of this host (164). The infectious activity of extracts of cottontail rabbit warts varies greatly from one donor host to another, but may be apparently high in some cases of naturally occurring growths. Yet,

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except under conditions of dubious significance (167), the agent is not demonstrable by infectivity in the cancers of cottontail rabbits. That the antigency of the virus continues for long periods in the carcinomatous growths transplanted to other hosts is readily demonstrable (164) by immunological tests of the host bearing the transplants. Even this evidence of the agent may ultimately disappear (109). It is notable, and probably significant with respect to the nature of tumor viruses, that throughout the papillomatous and carcinomatous processes, until the final stages mentioned, the infectious agent expresses the usual viral influences in the induction in the immediate host, as well as that bearing transplants, of typical antiviral immune bodies (32, 105-107) which fix complement and precipitate and neutralize the agent. In this behavior, the papilloma virus is entirely analogous, antigenically, to the generality of other agents which do not induce tumors.

B. Avian Leukosis Relatively little attention has been given in recent years to the highly malignant forms of avian leukosis, which were the first of the neoplastic conditions recognized (70, 71) to be of viral etiology. There has been much interest in the leukosis complex as a serious economic hazard in the poultry industry, and much of the more recent work has been concerned with problems of transmission, host resistance, and prevention. The results of the early studies have been reviewed by Ellermann (70), Furth (79), and, exhaustively, by Olson (131). An informative and well-illustrated chapter on avian leukosis was written by Jungherr (101) in the textbook by Biester and Schwarte, and some of the special problems of the complex have been summarized by Burmester (40). Since much of the present discussion is concerned with one form of leukosis, it is desirable to emphasize again certain features of the so-called complex. The literature on this subject is entirely confusing, for which a variable nomenclature is partly responsible. Many of the uncertainties are referable to lack of definition of experimental conditions and broad generalizations from inadequate observations. In 1941 a tentative pathological classification was suggested by a committee (102) which provided a uniform nomenclature for the terminology and analysis of data from various laboratories. The implications of this classification are stated in Jungherr’s definition (101) “ that the diseases of the avian leukosis complex are such which are primarily characterized by autonomous proliferation of essential blood-forming cells, and are as a rule, due to oncogenic viruses.” It was emphasized that this classification was based entirely on a pathological definition without any etiological implication. Thus, other virus-induced neoplasms, such as the fibrosarcomas as exemplified by the Rous tumor

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(chicken sarcoma I) which some investigators (72) have regarded as related to leukosis, are not included under the general term of the avian leukosis complex. The histogenesis of the avian sarcomas has long been in question. Many have followed the views of Carrel (47), Carrel and Ebeling (48), and others (76) that the cell of origin is the monocyte or tissue histiocyte. There has been no general concurrence (77, 117) with this hypothesis, and recently Sanford et al. (145) have concluded, with good evidence, that the host cell of the Rous sarcoma virus is the fibroblast. Consequently, until further, more definitive information is available, there is no tangible basis for regarding avian sarcomas as derivatives of any elements of the bloodforming organs or as intimately related to the recognized forms of leukosis. As the simplest concept in the practical approach t o the many problems of leukosis, the complex can be divided into two categories of pathological conditions, lymphomatosis and erythromyeloblastic leukosis. The former is manifested (101), in general, as a series of neoplastic diseases involving cells of lymphocytic origin varying, pathologically, principally in the location of the primitive cells. With few exceptions (40), these occur in the tissues and do not appear in the circulation. Localization of the cells in association with nerve trunks results in neurolymphomatosis (range paralysis). When the cells are grouped in the iris, grey eye and blindness follow. Most frequently there is infiltration of the viscera, liver, spleen, lungs, and other organs, giving rise to visceral lymphomatosis. Isolated tumors of typical lymphosarcomatous characters are seen. Osteopetrosis (marblebone or big leg) occurs naturally, only sporadically, although it is often associated with cases of visceral lymphomatosis. For this reason it has been classified with lymphomatosis. However, the neoplastic changes involve primarily the cellular elements of the periosteum, and thus pathologically this condition is more closely related to the fibrosarcomas than to any disease of the “blood-forming cells.” These are the forms of leukosis of greatest economic importance. The second category is comprised of erythroblastosis and myeloblastosis or a combination, erythromyeloblastosis, of the two forms. Both conditions are intensely malignant leukemias characterized by the occurrence in the circulating blood of very large numbers, up to 2.5 million per mm.3 of primitive cells derived from the progenitors of the erythrocytes or those of the myelocytes. The characters and possible origin of the cells were discussed by Furth (79). Erythromyeloblastic leukosis is of infrequent occurrence in nature. I t has been demonstrated conclusively that both lymphomatosis and erythromyeloblastic leukosis are of viral etiology and, in contrast with rabbit papillomatosis, malignancy is the first evidence of infection. In the instance of visceral lymphomatosis, virus transmission has been confirmed by Burmester and Cottral (41) in the serial passage of virus in cell-

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free filtrates of preparations of the R. P. L. 12 tumor. Whereas visceral lymphomatosis has been regularly transmitted with viral preparations, it has not always been possible t o transmit this disease without the occurrence of neurolymphomatosis and osteopetrosis. The viral etiology of erythromyeloblmtic leukosis has been repeatedly confirmed (59, 70, 81) since the initial observations of Ellermann (70). Major questions of the nature of the leukosis complex are those of the etiological specificities of the various entities. Are all forms of lymphomatosis caused by the same virus or variants of it? Are erythroblastosis and myeloblastosis different anatomical manifestations of infection by a single agent or are there two viruses or variants of one? Is one virus or variant of it responsible for both lymphomatosis and erythromyeloblastosis? Studies on pathology, histogenesis, and transmission have not yielded the solution nor have the results of the immunological investigations thus far made. Contrary to the findings in papillomatosis, the occurrence of lymphomatosis or erythromyeloblastic leukosis is not associated with the consistent formation in the host of significant neutralizing antibodies. Neutralizing antibodies for the agents of some forms of leukosis have been produced in rabbits (66, 80, 103, 104) with the respective viral concentrates but have not been applied to the systematic elucidation of questions of etiological specificity. Progress has been further impeded by the lack of the quantitative methods of virus bioassay necessary for detection and measurement of the activity of neutralizing antiserums. Some of these obstacles have been overcome in recent findings with the purified erythromyeloblastic leukosis virus described later (65, 66). With this material, strong neutralizing and precipitating antibodies have been produced in the natural host and findings of this sort hold much promise for future investigations. Aside from the possible future developments, it would appear at the moment that lymphomatosis in its various anatomical forms differs decisively from the conditions of erythromyeloblastosis. This is seen in the cell-type involved and in the distribution of the respective neoplastic cells in the body. Lymphomatosis is a contagious disease (42) occurring frequently under natural conditions, as contrasted with either erythroblastosis or myeloblastosis, both of which are seen very infrequently and have none of the attributes of contagious diseases. In the laboratory, experimental transmission by filtrates (virus) of diseased tissue is specific for lymphomatosis or for erythromyeloblastic leukosis. One form of ery thromyeloblastic leukosis (58) has been carried by means of virus apparently unchanged for more than five years. Some forms of lymphomatosis, notably the R. P. L. 12 strain (43), have likewise been transmitted repeatedly and specifically by filtrates. The induction of lymphomatosis by virus is characterized by long survival periods (41), averaging 137 days in 189 birds

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inoculated intraperitoneally at 1-3 days of age. Both erythroblastosis and myeloblastosis (59) can be transmitted routinely with short latent periods, 10 t o 100 days, by filtrates of the plasmas from diseased birds. Although these facts tend to emphasize differences between the phenomena of lymphomatosis and erythromyeloblastosis, there are data suggesting relationships. As noted before, erythromyeloblastic leukosis is rarely observed under natural conditions but has been derived repeatedly by passage of cellular material from cases of naturally occurring lymphomatosis. The disease studied a t Duke (58), the B. A. I. A strain, followed cell passage from rieurolymphomatosis (92) and the R. P. L. 3 strain was likewise obtained in the same way from cases of neurolymphomatosis (40). There has been no evidence, however, that the conversiop could be effected by virus alone. There is no information for decisions with respect to the etiological specificities of erythroblastosis and myeloblastosis. It is evident that, even when the leukosis complex is reduced to only two principal categories of malignancy, the issue of etiological classification remains thus far almost hopelessly uncertain. Much of the literature is more of a deterrent than an aid in the clarification of the problem. All of these virus-induced conditions are neoplastic and transmissible by either cells or by agent free of cells, but transmission by cells is a phenomenon of transplantation which is entirely different from induction of the disease by virus alone. Results obtained with cells cannot be interpreted as bearing directly on the properties of the agent. A considerable part of the literature is ineffective because of the failure to discriminate between tumor transplantation and virus transmission. Clearly the leukosis complex constitutes an extraordinarily attractive material for the investigation of the most fundamental problems of the virus etiology of tumors. The host is readily available, and the diseases are easily passed either by transplantation or virus infection. The processes are particularly attractive as potential sources of isolated viral agents needed for many critical experiments. The posriibilities for the direct approach are well-exemplified by the simplicity of the isolation of the virus of erythromyeloblastic leukosis. There seems little doubt that other representative agents of the complex could be obtained by systematic exploration of the field a s suggested by the isolation of a particulate component (153) in one form of lymphom a tosis.

111. PURIFICATION

A . Papilloma Virus Purification of the papilloma virus presents but few problems, and the agent has been obtained in homogeneous preparations by investigators in several laboratories (11, 114, 146). The simplicity of isolation of the virus

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and the homogeneity of the concentrates are due to its high concentration in the naturally occurring warts of cottontail rabbits and to the nature of the material from which it must he separated. Virus is present throughout the amorphous mass of denatured constituents of keratinized papilloma cells which constitute the major portion of the warty growths. Most of it can be recovered by a single extraction of the ground or homogenized (169) material with saline solution. After clarification of the extracts by low speed centrifugation or celite filtration, the virus is sedimented by spinning for an hour at 40,000 X g. Large volumes of extract can be handled by initial sedimentation in the Sharples centrifuge (169). There is sedimented a t the same time small amounts of colorless or pigmented extraneous material which remains in flakes and small masses, when the virus is redispersed by pipetting. Much of this is removed by low-speed centrifugation. Repetition of this sequence of packing and redispersion 3 to 5 times usually results in preparations that give single, sharp boundaries on sedimentation in the analytical ultracentrifuge and on migration in the Tiselius apparatus and exhibit little evidence of extraneous material in electron micrographs (Fig. 1). No virus has been obtained in measurable quantities from the warts of domestic rabbits; that is to say, no opalescence was visible in concentrates carried through 3 or more centrifugal cycles; no nitrogen could be determined; nor were sedimenting boundaries seen in ultracentrifuge diagrams. It is significant that these experiments were made before the electron microscope became available, and further study may well reveal physical evidence of the agent in domestic rabbit warts. Calculations based on the known infectivity of the virus (29, 129) show that as many as lo8 particles could be present in 1 g. of domestic rabbit warts without causing a lesion on the inoculation of 0.1 ml. of a 10% suspension of the warty tissue. The yield (11) of virus from cottontail rabbit warts varies greatly from amounts barely measurable, 0.008 mg., to as much as 1.0 mg. per gram of papillomas. The impression is frequently given that all warts from cottontail rabbits contain much virus. This is not the case; some growths arising under natural conditions and many of those induced experimentally in these animals are almost entirely inactive, and no virus can be obtained from them by centrifugation. It would be expected that the virus yielded in the purification process would represent but a fraction of that actually present in the growths. Even so, calculations from the known size and density of this virus indicate the presence of no less than about 10*2.6 particles per gram of warts which yield 0.5 mg. of virus per gram.

B . Virus of Erythromyeloblaslosis The agents of several forms of leukosis have been concentrated by centrifugation (39,80,85, 103, 104, 111, 166), and various immunological

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studies have been made. Stern and IGrschbaum (186) observed a sedimentation rate of 582 s for material obtained from bone marrow of birds

FIG.1. Rabbit papilloma virus (159). 23,OOOX. FIG.2. Virus of erythromyeloblastic leukosis in dilute plasma. 23,OOOX. FIG.3. Virus of erythromyeloblastic leukosis in a purified preparation. 23,OOOX.

with erythroleukosis. There would appear t o be no douht that the agents responded in each case to the sedimeiitatioii fields employed, hut there is no basis for judging the characters of the concentrates. The problem of purifying the virus of erythromyelotilastic leukosis differs greatly from that of papilloma virus isolation. The iiifrctious agent

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has often been demonstrated in the blood plasma (64,70,81,92) of diseased chicks, although the physical amounts have been unsuspected. Investigation showed that the plasma from such chicks contained also a particulate component (8, 152), subsequently identified as the virus, reaching occasionally the high levels of 10l2 particles per milliliter of plasma (127) and visibly clouding the fluid. This number of particles corresponds to about 1.5 mg. of hydrated virus per milliliter of plasma calculated on the basis of 140 nip diameter (of hydrated particles) and density 1.059 (151). Electron micrographs of plasma, cleared of cells by two centrifugations a t 2300 X y for 15 min. filtered with celite, and passed through Selas filters, revealed an essentially homogeneous population of particles of variable size as illustrated in Fig. 2. Most of these particles and a proportional part of the infectious unit (67) were sedimented in gravitational fields of 20,000 X y applied for 43 min. (152). Further treatment consisted in repetition of low, 2300 X y for 15 min., and high-speed spinning. Most of the plasma protein was discarded in the first cycle and all serological evidence (00) of it in the second, as described in a later section. No material of this sort has been obtained from normal chicken plasma. A serious impediment to the investigation of the concentrated material has been the property of the particles to cohere in small clumps in the second and subsequent cycles, a behavior unlike that of the papilloma virus, which was readily redispersible after repeated sedimentation. This property of the leukosis virus has introduced special problems of characterization and investigation, since much of the work must be done with virus sedimented a single time. For this reason, it has been necessary to resort to virus particle counts instead of nitrogen or other chemical determinations for estimation of virus mass in correlation studies.

IV. PHYSICAL AND CHEMICAL PROPERTIES A . Papilloma Virus 1. Physical Properties. This agent (Fig. 1) is an essentially spherical entity of small variation in individual particle size and shape. In unshadowed preparations (I 57), the periphery of the particle image in electron micrographs is indistinct, and the central portion shows a rounded region of relatively high electron-absorbing power which is regarded as the region corresponding to the nuclear apparatus. The diameter of the images, under these coiiditioiis of dehydration and exposure to the heat of the electron henin, is about 44 mp, a value small in contrast with 65.0 mp calculated for the hydrated particle (158). The particles are of relatively rigid structure. They flatten but little, compared with the larger viruses, on drying from saline solution as seen iii shadowed preparations (Fig. 1) (159). The general appearance of the papilloma virus and its behavior

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during the procedures for preparations of screens for electron micrography do not differ greatly from the analogous behavior of other small viruses. There is essentially no evidence in purified concentrates of material extraneous t o the characteristic virus particles. Virus from human warts is indistinguishable morphologically (123) from the agent of the rabbit growths. Sedimentation diagrams (13, 129, 146, 158) of good preparations of the papilloma virus reveal single, sharp boundaries indicative of great uniformity in size and shape (corroborative of the characters of the images seen in electron micrographs) and uniformity in particle density. Because of these properties, sedimentation data have been of much value in establishing the homogeneity of purified preparations and in further analysis of the physical characters of the agent. Sedimentation boundaries are usually single, but one or two secondary boundaries are sometimes seen (13, 146). Examination of preparations of different batches of warts have revealed variations (129) in the sedimentation constant of the virus from 266 to 287 s. This was ascribed, a t first, to natural variations in the size, shape, or density of virus particles from different sources. Schachman (146) observed values ranging from 269 to 200 s but presented evidence that variation in sedimentation rate was related to viscosity effects due to a n impurity corresponding, perhaps, to one of the sedimentation boundaries which he described, that of 170 to 190 s. Schachman’s interpretation may be a more likely explanation than that virus particles vary from one batch to another. The nature of the 3rd component, 390 s, has not been disclosed. The virus boundary, 266 to 287 s, was very stable (13) in pH regions of 2.9 t o 9.9. Above and below these hydrogen ion concentrations. there were observed other boundaries corresponding to degradation products of the virus particles. Estimations of the hydrated density of the virus have been made (158) by sedimentation in solutions of bovine serum albumin in which, because of of the large size of the albumin molecules, there would he expected little, if any, osmotic effect on the virus particles. A value of 1.133 was obtained in this way. The partial specific volume of the agent determined by pycnonietric measurement was 0.761, and the reciprocal or dry density was 1.31. The water content of the particle calculated from these measurements was 58 % by volume. Migration of the papilloma virus on electrophoresis (146, 156) proceeds with a single sharp boundary on both sides of the isoelectric point, which is a t pH 5.0 (13). Schachman (146) has observed a secondary component in some of his preparations, possibly the material represented by the sedimentation rates of 170 to 190 s noted above, most of which could be removed by precipitation a t the isoelectric point and redispersion of the virus a t p H 3.5. The single boundary seen in electrophoresis, together with that

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observed in sedimentation diagrams, constitutes excellent evidence of the monodispersity of the purified papilloma virus. The agent of papillomatosis is very stable. It has been recovered in considerable amounts, 0.108 to 1.0 mg. per gram of warts, from growths stored in glycerine solutions for 4-5 years (11,169). The infectious properties, measured approximately, are stable (13) through the range of about pH 4.2 to 7.2. 2. Chemical Constitution. The chemical constitution of the papilloma virus is not remarkably different from that of most other animal viruses, varying chiefly in the small amount of fat associated with it. Probably the best estimate of lipid content (170) is about 1.5 % whereas other viruses (9) range to levels as high as about 50%. It cannot be said that the virus certainly contains any fat, since the little found may have been a contaminant. A similar situation exists with bacteriophages which contain little lipid material. Nucleic acid is present to the extent of about 8.7%, and all was of the deoxypentose type (170). There was no evidence of carbohydrate in excess of that which might have been contained in the nucleic acid. The amino acid content of the agent was studied by Knight (114).

B. Virus of Erythrom yeloblastosis 1. Physical Properties. One of the more interesting features of viral properties and behavior is the extreme of differences between the agents. This is seen first in the variety of pathological states which range from specific lethal and necrotizing effects of virus on the motor cells in poliomyelitis, for example, to the stimulation of host cells to rapid multiplication in the chicken sarcomas and in leukosis. Despite the small sizes of the agents, wide differences are seen in chemical constitution, such as a range of 5 t o 45 % nucleic acid, 1.5 to more than 50% fat; only one virus, that causing influenza, is known to contain conjugated carbohydrate (113,168); and only one was recognized (45, 96), until recently, to bear an intrinsic component with enzymatic activity. I t seems no less than fitting, and possibly significant, that a virus producing disease a t one limit of the pathological spectrum, malignancy in erythromyeloblastic leukosis, should possess properties a t one extreme of physical (149), chemical, and biological behavior. The virus of avian erythromyeloblastic leukosis, Figs. 2 and 3, is a particulate material of spheriodal shape and of variable size with an average diameter of about 120 mp in the dried state (152). Unusual features of the physical constitution of the virus were a t once evident in the applicat,ion of the techniques of electron micrography. When the particles, concentra,ted and washed by sedimentation from plasma, are dried on the collodion membranes from saline solution by the usual procedures in preparation for electron micrography, there are observed particles of the most bizarre shape

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and size (8, 152). The shapes vary from spheroids t o filaments and tadpolelike images. Removal of salt by washing with water leaves behind indistinct images poorly defined but containing within them various small regions of high electron-absorbing power. Drying the particles from water suspension yields little that can be seen. In order to portray the particles in their natural form unaffected by the stringent osmotic and disruptive forces exerted in the presence of drying and crystallizing salt, it was necessary t o devise a special technique applicable to the virus. This was accomplished (152) by drying the preparation of particles on the surface of agar, which takes up both salt and water, and fixation of the particles with osmic acid for 5 or more minutes. When the fixed particles were taken off the agar surface in a collodion film, the characteristic, almost spherical particles were routinely demonstrable in either dilute plasma (Fig. 2) or concentrated preparations (Fig. 3). Even with this treatment, however, the particle surfaces are not smooth but seem irregularly sunken and distorted. At the periphery of many particles, there is seen a fan-shaped protuberance as if the membrane had sagged and spread on the agar surface before fixation was effected. It is a common experience that, although the essential characters of the particles are not greatly altered in the purification process, the best electron micrographs are obtained withdilute plasmas4 of high particle content. Repeated sedimentation does not improve the homogeneity of the particle population over that seen in dilute plasma, and irregularity in shape of individual particles increases. There is good reason to suspect damage of the virus particles in this process for this has been found t o occur in the similar procedures for purifying such agents as the bacteriophage and the papilloma virus. I n the latter case sedimentation boundaries of the virus lose sharpness after repeated packing of the particles. It is likely that the virus of erythromyeloblastic leukosis is unusually fragile and easily torn and fragmented in the purification procedure. This would account for the appearance of more small amorphous particles in resedimented concentrates than were present in the starting plasmas, which should have been eliminated in the process of centrifugal fractionation. The characters of the internal structure and of the enclosing membrane are well-illustrated in particles partially fixed and very lightly shadowed with metal (152). The limiting membrane is clearly shown surrounding a body substance, within the central portion of which there is located material of high electron-absorbing power. This occurs not as a single mass but as a group of moieties lying close together. Corroboration of the multiplicity of the nuclear entities was seen in the salt-dried, pleomorphic particles in which several areas appeared scattered within the stretched and elongated structures. The occurrence of pleomorphism identical with 4

Agar sedimentation technique (150).

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that seen with the erythromyeloblastic leukosis virus was apparent (6, 51, 69) with the agent of Newcastle disease; the relation of the phenomenon to salt effects was suspected by Bang (6), although he was unable entirely to clarify the matter. Excellent micrographs of spheroidal particles of Newcastle disease virus can be obtained (152) with formalin-treated preparations, but the agent needs further study with the agar technique. The findings have established the basis for judgment that the virus particles of erythromyeloblastic leukosis, and probably those of Newcastle disease, are gel-like, nonrigid structures of high water content and elastic or easily deformable surrounding membranes. This has been substantiated in part by the results of studies (151) on the density and water content of the erythromyeloblastic leukosis virus. Sedimentation diagrams revealed (151) a somewhat diffuse but always single boundary. The extent of boundary spread was approximately that expected of a population of particles varying in size as seen in electron micrographs. The size distribution is about the same as that of purified influenza virus (160). Because of the high concentration of the particles in plasma, good sedimentation diagrams were obtained with the particles as they occurred naturally in this medium. The sedimentation constant of concentrated virus sedimenting in saline solution was about 693 s. Sedimentation of the particles in bovine serum albumin gave a value of 1.059 for hydrated density. This value is extraordinarily small in comparison with analogous findings of 1.104, 1.104, and 1.100 for influenza viruses A and B and the swine influenza virus, respectively, (161) and 1.133 for the papilloma virus (158). Dry density estimated by sedimenting the virus in DnO was 1.29. The water content of the erythromyeloblastic leukosis virus is consequently very high, almost 80 % by volume. Although numerous studies have been made for measurement of electrophoretic mobility of the virus, as will be discussed in the appropriate place, data have not yet been obtained suitable for quantitative analysis of the electrophoretic patterns of the visible migrating boundary. For this, high concentration of the particles is necessary. Visible boundaries have been observed (66), however, with preparations of material sedimented a single time and containing 1OI2 particles per milliliter, corresponding to about 1.5 mg. of hydrated virus per milliliter. In this concentration the particles scatter light in the Tiselius apparatus, and the preparations have a tendency to gel because of residual fibrinogen or, possibly, because of intrinsic properties of the virus still undefined. Nevertheless, it has been determined that the particles migrate with a sharp single boundary in the pH region of 7.0 to 8.5. The boundaries were of such character that it would have been possible to detect a secondary population of particles constituting

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25% or more of the total population. The presence of residual plasma protein, known to be in the preparations, was detected in one sample as the albumin boundary and readily separated from the particulate material by difference in migration rate. That the boundary in question represented the migrating particles has been established beyond reasonable doubt. 2. Chemical Constitution. Only preliminary analyses have been made (126) of the chemical constitution of the virus. The material employed was derived from large volumes of plasma before the variation in particle content was realized and before a means was available for selecting only those plasmas containing significant numbers of the particles. As a consequence, the concentrates contained relatively large proportions of plasma constituents other than virus. Material obtained in this way was of variable constitution. It appeared, however, that the agent is high in fat content, perhaps 30 % or more, low in nucleic acid, and contains much phosphorus associated with phospholipids. Significant analyses can be made only with material from plasma of high virus content to minimize the influence of possible extraneous material. 3. Enumeration of Virus Particles. The procedures (150, 152) for counting virus particles in the work with the virus of erythromyeloblastic leukosis deserve special emphasis. Not only have they been indispensable in this case, but they are applicable to the study of other agents procurable in small amounts and in unpurified preparations. In erythromyeloblastic leukosis, the host is small; the optimum age of the chicks for infection is 3 days, and the virus reaches a high concentration in the plasma while the chicks are 3 weeks or less in age. Thus, the amounts of plasma available are usually about 1 to 3 ml. Clearly, chemical estimations of virus in plasma or in small amounts of purified material are out of the question. With the counting technique, however, volumes corresponding to 1 t o 2 X of plasma are sufficient for significant accuracy of particle enumeration. These volumes are diluted to 1 ml., placed in a rectangular centrifuge cell of 1 cma2bottom surface (150) and sedimented on agar. Thus all particulate material responding t o the centrifugal field sedimenting the characteristic particles comes to rest on the surface and is fixed with osmic acid and taken off with collodion. Where extraneous material is present, it is seen along with the virus particles, and a far better opportunity for recognition of it is afforded in this way than with the usual methods involving washing of the film t o remove salts before electron micrography. This technique is not advocated as a substitute for such methods as spraying (119). It can be used, however, in the presence of relatively high concentration of soluble proteins; with preparations not fractionated by centrifugation; and with preparations of particle content as low as lo7 per milliliter. A unique ad-

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vantage of the procedure is its applicability to the study of the precipitiri reaction in plasma or concentrates as employed in recent investigations (65, 66). 4. Enzyme Activity. A finding of much interest in the study of this agent has been the observation that the plasmas from chicks with erythromyeloblastosis exhibit (125) the capacity to dephosphorylate adenosine triphosphate and, as recently determined, inosine triphosphate (88), whereas plasmas from normal birds are essentially devoid of adenosinetriphosphatase activity, as previously noted (78). Thus the plasmas possessed three attributes specific to the disease : particles, virus infectivity, and the enzyme activity. There appeared to be little doubt that the particles represented the virus. Preliminary experiments (125) indicated that enzyme activity was associated, a t least in part, with a material which was sedimented and concentrated in the purification process in the significant fractions containing the virus particles. This experience suggested a probable intimate relationship between the characteristic particles seen in electron micrographs, the enzyme and the infectious unit. Experiments were undertaken to determine the nature of the relation. In order to obtain the most information about the nature of the material with enzyme activity, the studies were made with plasma. Direct counts of the particles were made by sedimentation on agar (150, 152) and enumeration in electron micrographs. Enzyme activity was measured by electrometric titration of the acid liberated by dephosphorylation, and infectious capacity was estimated (59) by titration in 3-day-old chicks employing the latent period procedure. Investigations of the quantitative relations between particle, enzyme, and infectious unit were made in a study (67, 127, 155) of the relationship (1) as it developed in the plasma during onset of the disease; (2) on electrophoresis of plasma; and (3) on ultracentrifugation of the plasma. The results showed a very close correlation between number of particles and enzyme activity in plasmas of widely varying content of these two attributes. A good, but more variable correlation, was observed with virus infectivity. The best results were seen with plasmas of highest enzyme and particle content. Sedimentation experiments were made by centrifugation of plasma in special centrifuge cells (148, 154) and sampling the supernatant fluid after various periods of spinning, I n these studies, the respective sedimentation rates of the three properties determined from the data were remarkably similar, 645 s, 597 s, and 700 s for particles, enzyme activity, ahd infectious unit, respectively (151). The sedimentation constant of the particles measured directly was 693 s. Inasmuch as these differences include all variations in the technique of counting the particles, measurement of enzyme, and titration of infectivity, as well as unknown factors such as differences in plasma viscosity

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and difficulties in sampling the column of fluid in the ultracentrifuge cell, it must be concluded that the three attributes display essentially identical sedimentation properties. Electrophoretic fractionation (67, 155) yielded entirely analogous results with respect to the mobilities of the particles, the enzyme activity, and the infectious unit. In studies made through the pH range of 6.5 to 8.5, the mobilities of the particles and enzyme were identical and were similar, as later observed (66), t o the mobility of the visible migrating boundary. The results of infectivity measurements (67) were not so decisive, but considering the small number of experiments feasible and the variation in titration results to be expected, it was evident that the mobility of virus activity was not significantly different from the mobility of the particles and that of the enzyme. The experiments revealed that the particles, the enzyme, and infectivity appeared with quantitative simultaneity in the plasma with onset of the disease and were inseparable by sedimentation or electrophoresis. It is notable that, while the physical findings alone cannot be interpreted (10) as unequivocal evidence that the enzyme moiety is an intrinsic constituent of the virus, the results do eliminate, with high probability, the existence of more than one significant population of particles. The enzyme must be associated inseparably with the virus and is either a constituent of the agent or adsorbed to it. All doubt of the relation was eliminated (65, 66) in the experiments recounted in another part of this paper by the quantitative precipitation of enzyme activity along with the virus particles by means of the serum of chicks hyperimmunized with the virus. Further consideration indicates that the adsorption, if it occurred, must have taken place inside the cell from which the virus was derived. It seems unlikely that, if the enzyme were a low molecular weight material appearing in the plasma separately from the particle, it should always have been adsorbed quantitatively. If adsorption did occur in the cell, it must have been specific and in quantitative proportion; thus, no matter what the direction of approach, the conclusion must be that the enzyme is a specific component of the virus. The relation found with the agent of erythromyeloblastic leukosis strongly suggests reconsideration of earlier indications (97, 120, 121) of enzymes associated with other viruses. Methods are now available for quantitative examination of the problem. It seems very well-established that the influenza virus does exert enzymatic activity (45, 63). It is possible that a more detailed investigation might reveal significant relations with other agents. An examination of the behavior of the adenosinetriphosphatase of the virus reveals (87) the properties typical of an enzyme. It is activated somewhat by Na+ and K+, acting separately or together and in a similar

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way by Ca* and MgH, separately or in mixtures of the two. A very strong activating influence is seen, however, with either or both Ca++ and M g H in the presence of either or both monovalent ions. The concentrations of the ions optimum for the effects in a mixture of all were Na+ and K+, 0.05 M ; and Ca* and M e , 0.04 M . The activating influence of the ions can be extinguished by chelation of the divalent ions. There is a strong dependence of the activity on hydrogen ion concentration with a sharp single optimum a t pH 7.16. Dephosphorylation involves hydrolysis only of the terminal phosphate of adenosine triphosphate, there being no effect on adenosine diphosphate either by hydrolysis or dismutation. Adenylic acid, which was not a reaction product and which was investigated separately, was not affected. The inosinetriphosphatase activity (88) of the virus parallels that exerted in the dephosphorylation of adenosine triphosphate. Activation of the enzyme occurs under the same conditions; the effects of Ca* and Mg++ in enhancing the reaction are exerted in the presence of optimum concentrations of Na+ or K+ or both. The pH region of optimal activation is likewise a t the same point, about 7.2. The enzymatic activity of the virus is a property not only of fundamental significance but is also of inestimable practical value in the continued investigations of this virus. Estimates of virus amount can be made simply and accurately within the limits of possible variation of individual virus particle activity from one preparation to another and with precision in repeated measurements on the same preparation. By this means, virus can be followed through a multiplicity of procedures in which the enzyme, a stable material, is not inactivated. A sphere of greatest practical usefulness thus far has been in the selection of chicks as the source of virus for various studies. It has been noted before that the virus content of the plasmas from chicks even in advanced stages of the disease varies through at least a thousandfold, although probably greater, range. A micro method (86, 124) for accurate estimation of enzyme activity and employing only 3-X volumes of plasma permits the rapid and repeated screening of hundreds of small diseased chicks to provide the agent in useful quantities. This use of the dephosphorylating activity of the erythromyeloblastic leukosis virus is comparable with that of the hemagglutinative properties of the influenza viruses with the advantages of greater accuracy and facility of application. 6. Stability. The constituent of the virus of erythromyeloblastosis responsible for its infectious properties is relatively unstable, necessitating a constant source of fresh material for practical routine investigation. Fortunately, however, the portion of the particle exerting enzyme activity is much more resistant to change. Both properties can be destroyed without significant alteration of the morphology of the particles. Investigation of

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thermal inactivation (62) showed that essentially all infectivity disappeared within 10 min. at 56°C; about 99 % was lost a t 51°C. for 80 min.; and almost 90% at 46°C. in 160 min. In this region the rate of inactivation was an essentially linear function of the temperature. A t 37°C. more that 90 % of the infectivity was lost in 20 hr., and only 1% remained after 3 weeks a t either 3°C. or - 15°C. The stability of the enzyme was far greater; little change occurred in 160 min. a t 46"C., and no diminution in activity was detected a t 37°C. for 20 hr. or a t 3°C. or -15OC. after 3 weeks. Calculations from the data in the range of 46 to 56°C. gave the value 89,000 cal. per mol. for the Arrhenius constant for destruction of the infectious property which is in the range of analogous values with other viruses (115). The corresponding value for destruction of enzymatic activity was 98,000 cal. per mol. The pH region (61) optimum for stability could not be established accurately but was approximately the same, about 6 to 9, for both infectivity and enzyme activity, although the rate of inactivation of the latter was far less than that for infectivity. Under all of the conditions of the studies, there was no electron micrographic evidence of change in the particles. For purposes of continuity it should be noted here that the infectious activity of the virus can be neutralized (65, 66) completely by specific immune serum induced with the agent in rabbits and in chickens. Such serums, however, have no effect to diminish enzyme activity. Serums from chickens hyperimmunized with the virus likewise precipitate (65, 66) the specific particles. Enzyme activity is associated in quantitative proportions with the particles remaining after precipitation or with those removed from suspension by action of the serum. This constitutes a phenomenon of much practical value in the study of the immunological properties of the virus. 6. Antigenic Constitution. Immunological studies (66) have revealed the presence in the particle concentrates of material with the capacity to induce, in the rabbit, immune bodies specific to the virus, to normal chick tissue, and to the Forssman antigen. There have been reports of such findings with concentrated preparations of several viruses. Amies (1) and Amies and Carr (2) found chick antigen in preparations of avian sarcoma virus. Kabat and Furth (80, 103, 104) made similar observations with the concentrates of the agents of two forms of leukosis, sarcoma 13 and erythroleukosis. Knight (112) reported the presence of normal chick antigen in preparations of purified influenza virus derived from chick embryos and mouse tissue antigen when the agent had been obtained from mouse lung. Cohen (50) likewise found chick and, in addition, Forssman antigen in influenza virus concentrates. Interpretations of the results have differed. Amies (l),Amies and Carr (2), and Knight (112) regarded the normal tis-

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sue antigens as components of the respective viruses, whereas Kabat and Furth (103, 104) and Cohen (50) considered them as contaminants extraneous to the virus. Though the implications of results such as these are of obvious importance in their bearing on the properties and nature of viruses, little has been done to establish the necessary quantitative correlations. The respective findings, especially those with the chicken tumor virus, are frequently cited (90, 91, 94) as significant. It is plain, nevertheless, that the interpretations are entirely without basis a t the present time. In no instance, except that of Knight’s (112) work with influenza virus in which “highly” purified preparations were employed, has any evidence been advanced of the physical constitution of the virus preparations. Although the contentions about chicken tumor viruses may eventually be substantiated, the evidence to support them is still to be obtained. In the work with the virus of erythromyeloblastic leukosis (66), there were observed effective cross reactions between virus concentrates and normal chick tissue with the respective immune bodies induced in rabbits, guinea pigs, and mice. That is to say, virus Concentratesfixed complement in the presence of antinormal chick tissue immune bodies, and, conversely, the complement fixation reaction was obtained with chick tissue in the presence of the viral antibodies. Forssman antigen was demonstrable in the viral concentrates with the use of antibodies to guinea pig tissue induced in the rabbit. Absorption of antiviral immune serums with normal chick tissue removed essentially all capacity to fix complement with the viral concentrates. The Forssman antibody could be removed from antiviral immune serum by absorption with sheep cells. That the antiviral immune serums contained antibodies specific t o the agent was evidenced by the fact that the infectious capacity of the virus was effectively neutralized with the serums. The specificity and immunological activity of the virus in the concentrates and in plasma was further established by the induction of strong neutralizing and precipitating antibodies (65) in chicks injected with both formolized and fully infectious agent. Despite the obvious interpretation of normal chick and Forssman antigens as contaminants of the virus Preparations, there were certain indications suggesting a more than incidental relationship of the materials to the virus particles. The plasma was by no means a complex source of the virus in the purification procedure; indeed, it would be expected that the albumins and globulins would be washed away easily in the fractionation process. Plasmas from normal chicks fractionated in the manner employed to purify the virus yielded but little that could be seen, and this gave only a trace of complement fixation. I n addition, electron micrographs of filtered plasma revealed, as shown in Fig. 2, little particulate material other than the virus. Of still greater significance was the observation that the

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chick tissue antigen was not lessened beyond the second sedimentation of the virus and that plasma from normal chicks exhibits only traces of Forssman antigen. I t was for these reasons that investigations were made to determine whether chick and Forssman antigens could be correlated quantitatively with the particles themselves. In these experiments, the particles were counted as before (150, 152) and compared with the activity of chick tissue and Forssman antigens, as measured with antinormal chick tissue and antiForssman (guinea pig kidney tissue) immune bodies from the rabbit. It has already been mentioned (65, 66) that enzyme activity is unaffected by immune serum which completely neutralizes virus activity. Consequently, enzyme activity could be used as another means for correlation with virus particles, and parallel estimations were made of this property wherever possible. The relationship of these various attributes was studied on (1)ultracentrifugal fractionation of the plasma constituents; (2) comparative sedimentation; (3) electrophoresis; (4)tryptic digestion; and (5) precipitation with specific antiserum from chicks hyperimmunized with viral concentrates and the virus in plasma. I n all of the studies, except those involving centrifugal fractionation, the virus tested for immunological reactions consisted of concentrates of the agent sedimented a single time; experiments with the precipitin reaction were made also with the virus in plasma. The plasma from normal chicks, as well as that from the diseased birds, gave the complement fixation reaction with antinormal chick-tissue antibodies. With both materials the reaction was characterized by high titers, as measured by the point of 50% lysis of sheep red cells in the system employed, and, in contrast, by incomplete fixation by any amount of plasma greater than that giving the endpoint. The reaction involving sedimented virus differed greatly, exhibiting clear-cut complete fixation diminishing sharply with dilution to an endpoint titer at 1:77, for example, in comparison with 1 :512 obtained with the plasma from which the virus was derived. Thus the reaction with virus antigen differed, both qualitatively and quantitatively, from that with plasma. The findings with studies on Forssman antigen were quite unlike those with chick tissue; normal plasma contained scarcely detectable amounts of this material, and i t was present only in those plasmas containing virus. I n the experiments on ultracentrifugal fractionation through three cycles of sedimentation, the virus particles and enzyme activity were recovered in proportional quantities. There was no loss of complement-fixing capacity between the second and third cycles of sedimentation or afterward, and the Forssman antigen content of the third cycle concentrates was essentially the same as that of the original plasma. These experiments indicated that both chick tissue antigen (except that associated with the

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plasma proteins) and Forssman antigen were concentrated essentially quantitatively with the virus particles. The relations were even more clearly shown, in comparative sedimentation, by sampling suspensions of concentrates spun in the centrifuge for varying periods. Here, the rates of sedimentation of particles and Forssman antigen were the same; the apparent sedimentation rate of the chick tissue antigen was slightly greater, but the difference lost significance after taking into account the residual complement-king capacity of the plasma proteins remaining in the preparat,ion of particles sedimented once. The experiments with electrophoresis gave analogous results; in the pH regions of 7.0 and 8.5, there was a close correlation, in the same experiments, of the mobilities of the particles, enzyme activity, complement-fixing capacity with chick tissue immune serum, and Forssman antigenicity. The preparations of virus concentrates employed for the experiments with electrophoresis were of relatively high concentration. As a consequence the boundary of the migrating particulate material was visible and its mobility measurable. The boundary was single and sharp and moved at a rate indistinguishable from the rates measured by estimates of particle number, enzyme activity, chick tissue antigen, and Forssman antigen made directly on electrophoretic fractions of the concentrates. Digestion of the virus concentrates with crystalline trypsin resulted in the proportional destruction of the chick and Forssman antigens, the enzyme characteristic of the particles, and the infectious capacity of the virus. There was, however, no electron micrographic evidence of diminution in particle number. This lack of apparent change in the physical characters of the virus particles, despite possible major constitutional damage, has a parallel in the results of the treatment of vaccinal elementary bodies with pepsin (53) which alters the structure of the bodies without change in their form. The results of precipitation of the virus particles with specific viral immune serums from hyperimmunized chicks were followed by electron microscopy. Treatment of plasmas from diseased birds or preparations of concentrates with the chicken immune serum caused quantitatively proportional precipitation (65, SS) of the particles and the enzyme activity to dephosphorylate adenosine triphosphate. Both chick tissue and Forssman antigens were found in the precipitates of the concentrates in close proportion to the enzyme activity of the precipitated material. Precipitates of the particles from plasma removed essentially all material which could be seen in the electron micrographs. These experiments demonstrate, without equivocation, the absolute relationship of the enzyme to the virus particles and provide still another criterion of the intimate association of chick tissue and Forssman antigen with the virus particles. The foregoing experiments demonstrated the presence of chick tissue and

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Forssman antigens in concentrates of the virus as measured by complement fixation and by the Forssman reactions with Forssman antibodies induced in the rabbit with guinea pig tissue. These antigens could not be separated from the virus particles or from the enzyme activity by the physical methods of sedimentation and electrophoresis. The findings could be interpreted as indicating either the presence of a population of antigenic particles different from those of the virus, of which there was no evidence, or that the antigenic behavior was a property of the virus particles themselves. I n the latter case, it was conceivable that the antigens might be intrinsic constitutional elements of the virus. As it turned out, the problem was relatively simple of solution, and it could be demonstrated that the antigens were, indeed, vital constituent parts of the particles. This was accomplished with the use of the specific neutralization and precipitin reactions with immune serum from rabbits and chickens. It was noted before that the serum from rabbits immunized with viral concentrates neutralized the infectious property of the agent in concentrates or in plasma. An equal and qualitatively similar neutralization of the agent was effected with serums from the rabbit immunized with normal chick tissue or normal chick serum. Absorption of antiviral immune serum from the rabbit with normal chick tissues, removed not only a large part of the complement fixing capacity but, likewise, a proportional amount of the neutralizing activity. The virus was strongly neutralized by Forssman antibodies from rabbits immunized with guinea pig tissue, and these neutralizing antibodies were removed quantitatively by absorption of the immune serum with sheep red blood cells. Treatment of virus in plasma with antiviral immune serum from the chicken neutralized the virus, and, in proper proportions of serum and virus, precipitated the agent quantitatively. In the precipitin reaction it was observed that, with precipitation of the virus particles, there was proportional precipitation of the Forssman antigen. These measurements were made by comparing the number of particles with the Forssman antigenicity remaining in supernatant plasmas after precipitation of the particles with graded amounts of immune serum. Analogous studies with virus concentrates and chicken immune serum revealed the precipitation of the normal chick tissue antigen along with the particles estimated by enzymatic acrivity. From these results it could be concluded, with assurance, that the chick tissue and Forssman antigens were integral elements, not only of the virus particle as a whole, but of that portion of the particle exerting the activity of the agent to transmit the disease. This relationship, so definitely evident, effectively excluded the possibility that nonspecific adsorption of the antigens on the particles was a contributing factor of the phenomenon.

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With correlation of the findings with both rabbit and chicken immune serums, there may be postulated at least three viral antigens constituting the infectious entity of the agent; (1) one indistinguishable from normal chick host tissue; (2) Forssman antigen behaving independently of the host, tissue antigen; and (3) a third material antigenic in the chicken, which would not be expected to react immunologically to homologous chick tissue or to Forssman antigen which is present in the chicken. In its physical attributes the virus of avian erythromyeloblastic leukosis does not differ fundamentally from other viruses thus far examined. The agent is a particulate body separable as discrete autonomous entities which maintain, apart from the cell, their full potentialities to transmit specific disease. There has been no evidence of need for the postulation of any extra-particulate factor for the initiation of the infectious process; every demonstrable element, including the chick tissue and Forssman antigen, appears as fully a part of the particle as any other. The mechanism whereby the host tissue antigens become incorporated in the particle is, of course, entirely obscure, but there is no reason to suppose that it is different from that governing the formation of any other component of the particles or different, fundamentally, from that determining the assembly of the constituents of other virus particles. This kinship of the virus to the substance of the host, so clearly evident, implies a relationship to the host of basic significance with respect to the nature and origin of the agent. The data provide a substantial experimental basis for the concept that the agent originated initially from the host cell, maintaining through the years a part of its host identity and developing, with time, the properties of autonomous existence and the newer structure unique to the agent. Where the properties of this virus fit into the pattern of host-virus relationship suspected for the viruses of other avian neoplasms and the influenza virus is not clear. The interpretations with avian tumor viruses are vulnerable, principally, in the lack of clarity of the findings; and those with the influenza virus lose force in the absence of evidence that the host tissues are not adsorbed on the agent. It would be incautious to generalize from the virus of erythromyeloblastic leukosis to other filterable agents; nevertheless, the evidence in this case emphasizes the the need for further investigations in this field. Still more important implications are those directing attention to yet undisclosed viruses that may be related to the causation of some human tumors. It has not been possible yet to demonstrate such agents, and a possible structural relationship of virus to host cell might explain, in part, the difficulties thus far experienced. One point should receive emphasis: there has been no more evidence that the virus of erythromyeloblastic leukosis arises de novo from the cells of healthy chickens than is available with respect to the analogous phenome-

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lion with other viruses. Within the limits of the knowledge thus far gained, this virus has behaved as an agent extrinsic to the host cell, and the occurrence of the disease is fully dependent on the introduction, anew, of the agent into the host. AND HOSTRESPONSE V. VIRUS INFECTIVITY

A recent advance in the study of the tumor viruses has been the development of methods and procedures for the bioassay of infectious capacity which have greatly extended the possibilities for work in this field. I n the early studies, these agents did not appear to behave on titration as other viruses. The results were more erratic and variable, so much so, that extraordinary efforts have been made to discover the principles of host-virus relations permissive of accuracy compatible with quantitative estimates of activity. Procedures are now available for the titration of three tumor viruses, those causing rabbit papillomatosis (29-31) , avian erythromyeloblastic leukosis (57, 59), and the chicken sarcoma I (25, 26, 28, 35) with an accuracy comparable with that attained with the vaccinia and influenza viruses (27). Both the development of methods and the interpretation of the results have been dependent on correlation of biological activity with virus mass estimated by physical or chemical measurements. In order to discuss the findings, i t is necessary first to consider the basis for the developments. Two types of response, described succinctly by Bryan arid Shimkin (37), are available for study, namely, the quantal response, which is manifested in incidence of effect in relation to dose, and the latent period, which is the time required for the formation of a detectable lesion by a given dose. The methods for the titration of viruses in general have been built principally about the quantal response, and a considerable body of thought has evolved in the understanding and interpretation of the observed results. Because of the prevalance of application, the present concepts depending on this type of response will be considered first. The application of analytical methods to the study of dose-response with animal viruses began with the work of Parker in 1938. With vaccinia (132) and myxoma (133) viruses, Parker found that a plot of the per cent frequency of positive inoculations with successive dilutions of virus against log dose yielded a n S-shaped curve. It was observed that the sigmoid distribution resembled more or less closely that Poisson probability curve which is a consequence of the chance presence or absence of one or more particles in suspension. On the basis of this appearance, grounded on earlier work (89,93) on the enumeration of bacteria, Parker concluded that whether the inoculation was positive or negative was dependent only on the chance presence or absence of a virus particle in the inoculum. From this evidence

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alone, it was concluded further that a single vaccinia or myxoma virus particle, i.e. one elementary body, was the infectious unit of the disease and that the dilution value at the point of 50 % positive inoculations on his curves was, per se, a measure of the number of virus particles in suspension (0.69 particles per unit inoculum at the 50% point, IDGO according to Poisson’s bionomial theorem). This view was modified subsequently by Parker el al. (134) to include considerations of the probability of an infectious virus particle reaching a susceptible host cell. The concept that the pattern of response is significant as an indicator of the number of particles in the tested preparations is no longer in vogue. Instead, theories of the applicability to the problem of viruses of the Poisson laws of probability have been amended but still constitute the basis for the analyses of titration results with many of the agents (46, 94, 98, 118). The present view has been well-expressed by Lauffer and Price (1 16) as the result of their study of titration results with plant viruses. It was their opinion that “the only tenable theory at present available for explaining the character of the quantitative response of hosts to various doses of viruses is that the probability of infection is related to the probability of finding the minimum requisite number of infectious units in an element of volume which comes into intimate contact with a susceptible focus in the host.” Thus, it was believed that the Poisson distribution, although devoid of meaning as a measure of the number of virus particles in any inoculum, still was acceptable as the best dose-response relation to be expected in the use of the quantal response in virus titration. Experience has shown that the Poisson distribution is inapplicable as a standard quantal dose-response relation in the titration of the tumor viruses. This was first evident and discussed (31) in relation to studies on the papilloma virus, the results of which stimulated the search for hypotheses more compatible with the experimental data. These have been found in recourse to concepts which have evolved in the study of biologically active materials other than viruses. The application of the biomathematical procedures derived in the study of the effects of these materials, principally various drugs, to the elucidation of host-virus relations has been largely due to the work of Bryan, first in the study of the papilloma virus (31) and more recently with the virus of chicken sarcoma I (25, 26, 28,35). The influence of this investigator has been a substantial aid in the formulation, likewise, of the interpretations reached in the study of the virus of avian erythromyeloblastic leukosis. The contribution in this field has been the recognition and practice of the application to the study of viruses, as well aa to carcinogenic hydrocarbons (37,38) of the concepts and the methods of biomathematical analysis derived from the investigations in the bioassay of drugs in the animal host.

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The essence of the well-established concepts of host response to drugs is recognition (14, 18-20, 54, 82, 172) of the influence of host factors, i. e. simply, the variation6 in host resistance or susceptibility or capacity to resist effect in the determination of the charactersa of the response of the population. The quantal response (18-20, 82) is elicited, as noted before, by the administration of graded doses of the material under study to representative individuals or groups of hosts of an appropriate population. If the dose range has been properly chosen, the smallest doses will produce no result, while all of the largest doses will be effective. I n the range of doses between 0 and 100% effective, the number of positive inoculations will increase with increase in dose. When the number of positive inoculalations is plotted in per cent incidence against the logarithm of dose in quantity or dilution, there is generally obtained a sigmoid or S-shaped curve. Curves of this sort are characteristic of the quantal response and have been observed with diverse drugs (22, 44, 75); with viruses (74, 116, 132, 134) as mentioned before; and with carcinogenic hydrocarbons (37,38). The idea that the characters of this S-shaped dose-response curve are dependent on host behavior was based initially on the findings of Trevan (172) with digitalis and Behrens (14) with k-strophanthin. From the data with these materials, it was concluded that the S-shaped curve was actually an integrated frequency curve characterized by variations in the resistance or susceptibility of the individuals of the host population. Thus it was agreed that, for a given material, the characters of the curve, its shape and steepness, were functions of these host factors which, in most cases, were normally distributed. The S-shaped curve could be transformed into a linear relationship by conversion of frequency in per cent to normal equivalent deviation units (82) or probits (18-20) (see Bryan (27) for reference to methodology). The results obtained in these studies on drugs have provided an abundance of data (22, 44, 75) available for the developmemt, with suitable modification, of the biomathematical relations needed for titration of the tumor viruses and for interpretation of the findings. With this basis it has been learned that the character of dose-response with the tumor viruses is determined principally by the influence of the host. It has been learned, further, that the complexity of host influence is such as to eliminate with present knowledge the usefulness of the quantal response as an instrument 6 This term signifies, simply, variation as such without connotations with respect to the mechanisms responsible for it. 8 Character as employed here, bears no implications relative to the nature of the lesion or change occurring. The term is statistical or mathematical, referring only to the form of response as evident, for example, in the distribution of incidence of positive takes in relation to dose; whether the distribution is curved or linear, normal or skewed; or some other function of dose.

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for accurate bioassay of the tumor viruses. Constructively, however, analyses made with this type of response have revealed the principles providing the basis of methods of titration employing latent period relations which are already in use with several nontumor viruses (83,84, 122). The findings with the tumor agents have, in all probability as will be seen, a significant bearing on the knowledge and interpretation of dose-response relations with viruses in general. BlOO.

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FIQ.4. The relation of incidence to graded doses of the vaccinia (V), rabbit papilloma (P), myxoma (M), fowl sarcoma (FS) and erythromyeloblastic leukosis viruses and to those of methylcholanthrene (MCA). The units of the abscissa are intervals of log,, dilution, and the direction of increasing doses is from left to right (57).

A . Erythromyeloblastasis Host response to the virus of erythromyeloblastic leukosis has been investigated by both quanta1 (57, 73) and latent period responses (57, 59). For the experiments described in detail here, graded doses of virus were prepared at 5-to-10-fold dilution intervals. Test chicks were arranged in dose groups varying from 30 to 120 3-day-old birds, and each chick was inoculated intravenously with a single dose in 0.1 ml. volume. After an interval of several days, blood smears for diagnosis of onset of disease were obtained (130) daily for a time and at 2-day or longer intervals, as indicated, thereafter. The number of dose groups were from 3 to 6, involving total numbers of 90 to 180 chicks of some strains of birds, White Leghorns of Line 15 described later, or as many as 360 to 720 birds of other strains, such as New Hampshires. In the initial studies employing New Hampshire chicks (57, 68), t,here was observed a very high resistance to infection. This was broadly distributed, the incidence varying in one experiment, for example, from 69.8 7% with the largest dose to 20.9 with the dose 3.5 decimal dilutions lower. When incidence in per cent was plotted against log dose, the response was

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not obviously sigmoid but essentially linear, Fig. 4. Although there has never been any evidence of a systematic trend toward curvature, even in

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experiments with highly susceptible chicks, the possibility cannot be ignored that the apparently linear relation was really the manifestation of a very shallow sigmoid curve. When per cent incidence for different doses in the same experiment was converted to probits (18-20, 82) and plotted against log dose, there was obtained a linear relationship (see Fig. 5 ) which, on the basis of the concept

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that the response was determined by host factors, was interpreted to indicate a normal distribution of resistance and susceptibility in the chick population. The regressions in different experiments were characterized by shallow slope and by considerable variation in slope. Analyses of the latent period relations were made with the same data. Various tests showed that the most useful transformation of the latent period response was to be found in the reciprocal in days in its relation to log dose. In this case there was a linear regression of the means of the reciprocals of the latent periods for the different dose groups on log dose, which revealed only an occasional point deviating beyond the chi square level of 5% in tests of heterogeneity of distribution of points about the regression lines. As in the case of the quantal response analyses, the slopes of the regression lines were very shallow compared with variation within dose groups, and considerable variation was observed in the slopes. The data revealed further an extremely broad spread of latent periods within the dose group. For example, when the mean latent period response to a given dose was 31 days, the latent periods for individual chickens varied through the range of 19 to 54 or more days. At this stage in the work it could be concluded that the population of chicks under study were, as a group, highly resistant to infection with the virus (as shown by the number of virus particles required a t the IDso, an example of which is given later) and that the distribution of individual resistance or susceptibility was very broadly distributed with respect both to quantal and latent period responses. Either relationship could be used (57) for the purpose of bioassay but with the restrictions of low accuracy and the need for large numbers of test chicks. Furthermore, as will be seen, these quantal and latent period response data are both spurious as representative of the response of the host in the natural state. The unsatisfactory status of the data as the basis for bioassay led to further studies in a relatively highly susceptible, inbred line of White Leghorn chicks [Line 15, developed a t the Regional Poultry Research Laboratory, East Lansing, Michigan (173, 174)]. The results of the investigation with these birds have greatly clarified the nature of the response to this agent. This waa accomplished by analysis of incidence in relation to latent period within individual dose groups. When the frequency in per cent positive inoculations was plotted against the latent period in days, a skewed curve was the result. However, a plot of frequency in per cent positive against log latent period gave a curve in which the experimental data followed a normal distribution through a part of the course and then deviated sharply from it. This is illustrated in Fig. 6A in which, for the lower curve toward the left (dose 1O-I ml. of virus-containing plasma), the per cent positive inoculations was plotted in the ordinate against unit log

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latent period. The same treatment for the data of two smaller doses, and 10-a.8ml. of plasma, gave the other two curves which are of the same shape as the first but which shift along the log latent period axis in the direction of increased latent period as the dosage is decreased. It was observed, as shown, that the data for the respective doses followed a normal

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FIQ.6A. Schematic curves representing (continuous lines) the distribution of latent periods observed with three doses, lo-*.', and 10-8.8 ml. of blood plasma, containing the virus of erythromyeloblastic leukosis. The heights of the respective points of each curve represent the per cent of the chickens in the dose group becoming positive per unit interval of log time indicated in the abscissa. Beginning at the left, these data follow the curve of a normal distribution of latent period responses t o the points of deviation indicated by the vertical line. The deviate distribution thereafter is shown by the terminal portion of the continuous line. FIG.6B. Time-frequency distributions of chick host responses t o 6 graded doses, 10-1, 10-1.7, 10-2.4, lo-*..', 10-8.8, and 10-4.6 ml. of blood plasma, containing the virus of erythromyeloblastic leukosis. The initial limbs of the respective truncated distributions were drawn as normal distributions with the standard deviation, 0.058, characteristic for the chickens employed (59), and the second limbs were drawn with lines fitted by sight. The distributions indicated by the initial limbs correspond t o those of the curves of Fig. 6A t o the points of deviation, and the distributions of the second limbs t o those indicated by the terminal portions of the continuous line of Fig. 6A.

distribution over a portion of the curve and then deviated sharply from it as indicated by the vertical line in each case. Thereafter, the distribution was greatly different as shown by the terminal, almost horizontal tails of the continuous line. The vertical line is employed only to indicate the discontinuity in the distribution of latent period. These results show that the point of deviation of the experimental data from the theoretical curve was related to dose and consequently to latent period. These characters are shown more simply for a similar experiment in Fig. 6B. Here per cent

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positive inoculations like those used for the construction of the curves of Fig. 6A are accumulated and converted, from standard tables (75), to probits which transform the relationship from the curve of Fig. 6A to the linear relation with log latent period of Fig. 6B. By this means there was obtained a series of truncated linear relationships (21, 99), one for each of 6 successive 5-fold dilutions of virus. The first limb of each distribution was linear in the region in which the frequency in per cent positive followed the normal distribution in Fig. 6A. The points of deviation from the normal distribution in each dose group are shown in the breaks in Fig. 6B, indicated by the vertical lines for the three doses of Fig. 6A, after which the experimental data seemed to follow other approximately linear distributions corresponding to the terminal portion of the continous lines of Fig. 6A. The interpretation of the phenomenon of truncation seemed clear, namely, that the first limb represented a normal distribution of response based on a normal distribution of natural host resistance or susceptibility of the chicks in the individual dose groups. The deviation was regarded as due to a change in the state of the host developing during the period of observation. This could be explained as the result of the development of some form of acquired resistance related to the experience of the host with the virus. The second limb, therefore, was the expression of the influence of the sum of natural and acquired resistance exerted during the latter period of observation. When the dose of virus was very large and the latent period was shortened, as occurred occasionally in the inbred White Leghorn chicks, there was insufficient time for development of acquired resistance, and the first limb prevailed (59). When such truncated dose-response curves were analyzed for many individual experiments, the following characters were observed as illustrated in part in Fig. 6B: (1) the slopes of the initial limbs of the distribution were highly uniform and characteristic for a given strain of chickens; (2) the points of truncation with respect to incidence and latent period were related to dose but showed large variation; (3) the slopes of the second limbs were highly variable; and (4) there was radical and unsystematic variation in total per cent positive in relation to dose. These findings provided the explanation of the previously observed variations seen in both quanta1 and latent period responses described earlier in this section. It was evident that any analyses made with total incidence or total latent period must be based on the responses of chicks which had undergone, not only considerable but variable, change in their status of resistance or ability to respond during the period of observation. Both total incidence and total latent period observed in the titration of this virus, therefore, are clearly functions of the combined influences of natural and acquired resistance. From this it seems obvious that any measure of host response expected

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to reflect only the influences of the natural state of resistance and, therefore, the influences which are least complex and variable, must be limited to those data contributing to the first limb of the truncation. Use of the quantal response for this purpose is eliminated immediately because of the considerable variation in per cent positive both a t the point of truncation, which could not be defined precisely, and at the end of response where the effects of change in resistance and susceptibility were greatest. There is left the latent period response which can be employed in the following way as described by Bliss (21) and Ipsen (99). It was observed, as noted, that the slopes of the initial limbs of the regressions of accumulated incidence in probits on log latent period were independent of dose and characteristic of a given strain of chicks and could be regarded as constant. If lines of such slope are drawn through the data of the first limbs and extended, if necessary, t o intercept the level of the median log time at probit 5, Fig. 6B, median log latent periods are obtained for the various dose groups. Variation in the median log latent period then will include only those natural variations in the responses of the different dose groups. When this was done, there was observed (59) a true linear relation between the median log latent periods of the various dose groups and log dose. Whereas the earlier analyses involving all of the data had revealed (57) wide variations in the slopes of the regression of log latent period on log dose, in this case the variation was small, 0.092 to 0.117, permitting use of a standard slope, 0.104, for analysis of small numbers of data (estimation*of virus potency from the data with 1, 2, or 3 dose groups). The fit of the individual points to the regression line was excellent. The variation expected of estimates of virus potency was indicated by the value (Gaddum precision index; see Bryan (27)) X = 0.6, as contrasted with values of X = 0.5 for the influenza (in chick embryos) and vaccinia viruses. I n the relations of total quantal and total latent period responses described (57) earlier, the average respective values for X were 3.8 and 1.02. Studies by means of these latent period procedures of chicks of various strains and sources revealed (68) a behavior qualitatively alike in all. Natural resistance among the individuals of the different populations was normally distributed but varied greatly in the spread, as represented by the standard deviation, s, of log latent period within the individual dose group, as for example, s = 0.228 for New Hampshire chicks as contrasted with s = 0.058 for White Leghorns of Line 15. Certain other strains of White Leghorns showed even greater spreads. The different chicks likewise varied greatly in median population resistance as measured by differences in median effective dose and in great differences in the capacity and time required for development of acquired resistance. This median resistance of the highly susceptible inbred White Leghorn chicks to the virus of

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erythromyeloblastic leukosis was remarkably similar to that (30, 129) of rabbits to the papilloma virus. In the experiment of Fig. 6B, the number of virus particles required to infect 50 % of the chicks injected was 3.9 X lo7 as compared with the number of papilloma virus particles, of the order of 107, needed (30,129) to cause warts in 50 % of the rabbits inoculated. The spread of individual resistance in leukosis was shown in the same experiment; 2.2 X 1010 particles were needed to infect 95 % of the chicks while 5 % of them were susceptible to as few as lo6 particles, a variation of at least 10,000-fold1compared with an analogous spread of approximately 500-fold in the susceptibility of individual rabbits to the papilloma virus. Age (60) was a very potent factor in the influence of host response. Chicks, 10, 17, and 24 days old at the time of inoculation, were 5.4, 9.0, and 39.2 times as resistant as 3-day-old chicks. Route of inoculation was likewise (60) a large influence. This was manifested in differences in apparent resistance of 3-day-old chicks in which the multiples of unit resistance compared with response to virus inoculated intravenously were 1.8, 15.2, 17.5, and 66.6 for virus given into the medullary canal of the tibia, into muscle, into the peritoneal cavity, and into the subcutaneous tissue, respectively.

B. Rabbit Papillmnatosis Most of the studies of the response of rabbits to the papilloma virus have been made with purified materials of which the mass of dose was known (29, 30). Experiments were usually made with serial 2-fold dilutions. Each dose, in 0.1 ml. volume, was rubbed into l-inch squares of abraded skin of each rabbit of the test group. In fairly large rabbits, as many as 24 such spaces could be conveniently arranged and, consequently, that many different doses or 12 doses in duplicate could be administered in each animal. This technique was quite different from the use of an individual for every dose of the virus of erythromyeloblastic leukosis and chicken sarcoma I, a factor likely of influence on the character of dose-response. The quanta1 response (29-31) showed that the observed incidence in per cent positive plotted against log dose was described by an approximately S-shaped curve (Fig. 4). Per cent incidence in relation to log dose was converted to probits yielding the points shown in Fig. 7A. Tests were made to determine whether these experimental data with the papilloma virus represented a Poisson distribution as postulated by Lauffer and Price (116). It is seen that the data were a fairly good fit to the curve for one or more particles as illustrated in Fig. 7A; as a matter of fact, the fit was no worse than for some of Parker's data (see Fig. 5 ) with vaccinia and myxoma viruses (32). There were two disturbing features, however; (1) it was known that was a number of the order of lo7 which the median effective dose, IDSO, which seemed to eliminate any factors associated with particle distribution

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in the inoculums; and (2) there w m a consistent deviation, Fig. 7A, of points from the curve in the region of the highest doses. It was evident, also, that the points of Fig. 7A were not linearly arrayed so that the data

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FIG.7. A: The points are the percentage responses, expressed as probits, of rabbits t o graded doses of papilloma virus observed at the termination of the experiment on the 34th day after inoculation. These points and the points of the closed circles of C and D are the same as those indicated by the open triangles of B. The curve represents the theoretical Poisson distribution fitted t o the observed papilloma data a t the point of ID60 . B: Per cent positive inoculations in probits observed in the rabbits a t successive intervals after inoculation (31). C: Linear regression (broken line) extrapolated from the response represented by the first limb of the truncated distribution (5 points of largest doses) to indicate the responses (open circles) which might have been observed in the absence of effects of acquired resistance. D: Poisson curve drawn through the calculated IDso of the hypothetical'response shown in C.

actually observed in the experiment did not indicate that the responses of the rabbits were dependent on a normal distribution of host factors. Further studies were made in an effort to determine the factors responsible for the observed relationships. If it were assumed that the characters of rabbit response were really dependent on normal variations in resistance or susceptibility, the relation of frequency of positive inoculations in probits (the points of Fig. 7A) should have been linear (18-20, 82) with log dose. Since this was not the case, it seemed reasonable to suppose that

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the deviation might have been due to alteration in the capacity of the rabbit to respond during the course of the experiment. This was the more attractive, since the latent period was long and, because the largest doses were quickly effective, there might be ample cause and time for the expression of a new state of resistance related to the experience of the rabbit with the virus and with the warts appearing early. This was investigated by plotting, in probits, the frequency distribution of positive inoculations observed at different periods after inoculation against log dose. It was seen, Fig. 7B, that the distribution at the 20th day after inoculation was actually linear. At 22 days there occurred a break or truncation which persisted to the 28th day. At this time there occurred a second break. The distribution a t 34 days, which is identical with the closed circle points of Figs. 7A, C, and D, revealed a well-defined double truncated curve consisting of an initial, essentially linear limb; a middle section indicating a sharp deviation of response to lower incidence; and a third limb which was essentially linear and of a slope very close to that of the first limb. These results were interpreted as indicating that the first limb of the relation, that corresponding to the frequency of warts developing early, was probably linear and dependent on a normal distribution of natural resistance and susceptibility of the individual rabbits. The break at the second limb suggested onset of acquired resistance. The third portion of the curve, representing the data derived from warts appearing late, was likewise practically linear and was regarded as the response characterized by an essentially normal distribution of summed natural and acquired resistance. The nature of the acquired resistance was not determined, but there was a definite relation (31) between the time of truncation of the curve and that at which retrogression of warts occurred in some animals. The manner of papilloma virus inoculation should be emphasized. Each rabbit received a succession of doses in contrast with the chicks, each of which was injected with a single amount of virus. It seems likely that acquired immunity related to the large doses of papilloma virus might influence the infectious or growth processes associated with the smallest doses, an effect not occurring in the chicks in which acquired resistance was related only to the one dose. The results suggest that the only segment of the quanta1 response curve related directly to the natural state of resistance was that determined by the data of the first warts to appear (the 5 points corresponding to the largest doses as shown in the 34-day curve of Fig. 7B). These are the responses which seemed to be the deviate ones in relation-to the Poisson curve. Although it would not be sound statistically because of the small number of data available, nevertheless, it would be interesting to project a linear relation through these points. When this is done (Fig. 7C) it is

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seen that the line, which represents the linear relation that might have occurred if the resistance of the rabbits had not changed, becomes widely separated from the observed data in the region of the smaller doses. These, then, were the truly deviant points, although they lay closest t o the Poisson curve. The distance vertically from the observed relations (closed circles) to the calculated line may then be an indication of the number of lesions suppressed a t each respective dose level by the development of acquired resistance, while the horizontal distance provides an estimate, in terms of virus potency, of the level of acquired resistance above that of natural resistance. calculation of the IDsoof the animals of natural resistance from the hypothetical relationship gives a value of about g. of virus in contrast with the IDSo of 10-*.3g. actually observed in the animals under the conditions of natural plus acquired resistance. The calculated linear relationship resembles closely, in the broad spread of animal response and high median population resistance, the analogous regression line observed (see Fig. 5) with the leukosis virus, being of relatively shallow slope lying, in value, between the slopes of the lines of vaccinia in rabbits and leukosis in chicks. It is possible now to construct the Poisson curve, as in Fig. 7D, across the hypothetical true relationship between incidence and log dose as it might have occurred had acquired resistance not intervened. It is evident that this hypothetical “true” relation bears no more resemblance to the Poisson distribution than the relation seen (Fig. 5 ) with the virus of erythromyeloblastic leukosis. It was quite clear in the beginning that the quantal response did not provide data suitable for quantitative studies of host response of rabbits to the papilloma virus. Variations in total incidence (29-31) were very large, the reasons for which are evident from the preceding discussion. For example, duplicate titrations of virus on the two sides of 12 animals gave endpoints differing by 87%. Analyses of the data with the same virus preparation in a comparison of 4 rabbits of high resistance with 4 susceptible animals showed a difference of 1011%. It was for this reason that a procedure based on latent period was sought. I n a significantly large series of experiments (29) there was observed a linear relation between log dose and the latent period expressed in days. With the data in the same rabbits mentioned above, the difference in response on the two sides of the animals was only 7.2 % by the latent period procedure, and that, observed with the same two groups of rabbits was only 67% instead of 1011% observed with the quanta1 response. By correlating the quantal response observed in a large number of rabbjts with latent period measurement (30), it was possible to express the results of the latent period procedure in terms of the quantal response unit, the IDso. The amount of virus, 10-8.3g.,corresponding to the observed IDSO gave a latent period of 26.45 days. It should be empha-

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sized that the latent period data employed in the bioassay of the papilloma virus represent the total response related both to natural and acquired resistance. Although this relation is of practical usefulness for virus bioassay, it might be possible to devise a better procedure involving only those responses related to natural resistance.

C. Chicken Tumor I Systematic studies on procedures for bioassay of the chicken sarcoma I virus have been made by Bryan and his associates (25, 26, 28, 33-35, 36) who have provided the data for the present discussion. Quanta1 response data are unsatisfactory for titration of this agent because of excessive variations. For this reason, recourse is had to the latent period response. The most recent experimental design of the technique (28, 35) consists in the administration of a single dose of virus to each chicken in dose groups of 20 to 60 3-to-5-week-old birds. The data of particular interest for this paper were obtained with 50 birds per dose group in the titration of a sample [CT581 (28, 35)] of frozen chicken sarcoma virus. The treatment of the data here was the same as that for the results obtained with the virus of erythromyeloblastic leukosis illustrated in Fig. 6B. When accumulated per cent positive inoculations within the individual dose groups were converted to probits and plotted against log latent period [the reciprocal of the latent period was employed by Bryan (28)], there was obtained the series of distributions shown in Fig. 8. It is seen that the distributions observed with four of the doses were truncated in a manner identical with the analogous phenomenon with the virus of erythromyeloblaatic leukosis (Fig. 6B). The distribution with the highest dose is essentially linear as is that for the smallest dose. There seems no reason to doubt that truncation with the chicken sarcoma I virus was based, as postulated for that of erythromyeloblastic leukosis, on change in the state of the birds due to the development of acquired resistance during the experiment. The linear distribution seen with the largest dose indicated that the tumors appeared before development of acquired resistance, whereas the relation occurring with the smallest dose had the same slope as the second limbs of the truncated responses and, in all probability, was due throughout its course to both natural and acquired resistance. Similar relations were seen with the small doses of erythromyeloblastic leukosis virus as indicated in Fig. 6B. The character of this dose-response to the sarcoma virus is essentially identical with that to the virus of erythromyeloblastosis,and analyses based on total quanta1 or total latent period response must be variable and must reflect a variable and complex state of host factors changing within the experimental period. This can be circumvented for the sarcoma virus, for purposes of bioassay, by use of only those data observed before exertion of

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of the influence of acquired resistance when the latent period is highly correlated with dose. Thus, it is found (28, 35) that the reciprocals of the mean latent periods for the various dose groups, under these conditions, are a linear function of log dose, indicative of a normal distribution of host susceptibility and resistance to the virus. The quantitative aspects of the

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FIQ.8. Time-frequency distributions of chick host response t o graded doses, from left to right, of chicken sarcoma I virus (0.2 ml. per chick of dilutions of 10-8, 10-4,10-6, 10-8, and 10-7 of an extract of frozen chicken sarcoma containing 1 gram-equivalent of tumor per milliliter). The lines were drawn as the best fit t o the observed data. regression can be illustrated by the precision index (27), X = 0.6 (35,28), computed from the variations of the data of all samples about a common regression line determined in repeated titrations of a single sample of virus. Analyses of the quantal response of chickens to the sarcoma virus show that the relation of frequency of positive inoculations in per cent to log dose is S-shaped (see Fig. 4),and in some of the experiments of Bryan (28) the sigmoid curve constructed with the observed total quantal response data was not greatly different from the Poisson distribution. It is evident from the characters of the data of Fig. 8 that the resemblance is not significant.

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D. Host Response to Other Viruses In the foregoing discussion there have been reviewed experiments establishing the basis for the development of procedures suitable for the bioassay, with relatively high accuracy (271, of the infectious activity of three tumor viruses. The results have shown that dose-response with these agents is influenced primarily by host factors concerned with resistance and susceptibility. Of greatest significance for the development of the procedures was the observation that the state of host influence was not constant during the periods of the individual studies but was subject to change in an irregular manner. Analyses of the data have resulted in the concept that the characters of dose-response during a portion of the period of observation are determined by a normal distribution of natural host resistance but are altered subsequently by progressively increasing resistance arising as the result of experience of the host with the virus. The total response, namely, that measured by all of the data available a t the end of the period in which the hosts continue to respond, is thus dependent on the summation of factors concerned with natural host resistance and that evolving as the result of variable acquired resistance. It has been an observation of long standing that dose response with the tumor viruses is highly variable and because of this, bioassay of the agents could not be accomplished with the accuracy needed for quantitative work. An explanation of these findings is apparent in the demonstration of change in host influence. When this was realized and taken into account by the utilization only of those data accumulated during the period in which change was not evident, procedures were immediately available for bioassay of these tumor viruses with an expected accuracy equivalent to that experienced with vaccinia virus in rabbits and influenza virus in chick embryos. The results have clearly indicated deficiencies in the total quantal response eliminating its applicability to the titration of tumor viruses because no means could be devised for accurate selection of those quantal responses which occurred during the period before change began. It was possible, however, t o employ frequency in relation to time as an indicator of the level of response related only to the state of natural resistance by means of which the basis was established for the application of latent period relations suitable for bioassay in relation to uncomplicated host response. It should be emphasized, for the reasons considered, that no form of final (i.e. at “infinite” time) quantal response yet employed, for example, that suggested by Fagraeus (73) for the erythromyeloblastic leukosis virus, can be expected to yield results of quantit,ative value. This is especially so in the case of methods based on the minimum infectious dose (M.I.D.) or the smallest dose producing effect, as advocated by Carr and Harris (46) for the agent of chicken tumor I. A glance at the curves of Fig. 8 reveals fully the fu-

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tility of attempting to obtain significant information under the chaotic conditions of change in this region of dose-response. It has been suggested that these results with the tumor viruses may contribute to a better understanding of the titration data with other agents which, in the main, are based on quantal response. The point to be examined is whether dose-response with other viruses is determined principally by host factors. In Fig. 5 there are shown data obtained with several agents. The points represent per cent positive inoculations converted to probits and plotted against log dose. In the charts a t the right, tests were made of the fit to the linear relation expected if dose-response was related principally to host factors normally distributed among the individuals of the respective populations. At the left the Poisson curve was drawn through the same points. It is seen a t the left that the fit of the data to the curves varies from excellent for the bacteriophage (74), good for influenza virus [data of preparation XI1 A, Table 3, (171)], to poor for vaccinia in rabbits and mice [Fig. 4, Table I11 and Fig. 2 (134)], on to impossible with the viruses of leukosis and rabbit papillomatosis, a s seen for the latter in Figs. 7A and D. As good a fit as any to the Poisson curve was seen with the results in the induction of tumors with methylcholanthrene [Table 2 (38)], a relation which could hardly be expected to depend on the chance distribution of the molecules of the chemical in the inoculums or even in the minute unit volumes of fluid which come into contact with individual cells.7 In contrast, the points in every instance fitted closely, in compatibility with the number of data available, the linear regressions drawn at the right. The principal lessons to be learned here are (1) contrary to the thesis of Lauffer and Price (116), the dose-responses with various viruses obviously may and do deviate in differing degrees from the Poisson distribution; and (2) the slopes of the dilution curves, expressed here as regressions of probits on log dose, are not constant but vary greatly from one agent to another. These results indicate the inapplicability of the Poisson curve as a standard dose-response curve. Furthermore, the fit of the data varies not only from one virus to another but with different samples or strains of the same agent in the same host species [see (31) for analyses of Parker’s four experiments with vaccinia (132)]. Lauffer and Price (116) cited data from plant virus titrations closely fitting the Poisson distribution but likewise mentioned results which did not conform. Consideration of representative findings (4, 5, 135, 144, 165), with the plant viruses reveals numerous aberrations 7 Some other explanation (e.g. that proposed by Iversen and Arley (100) involving a “hit theory” and infrequent successful collisions between hydrocarbon molecules and certain critical intracellular “giant molecules”) will have to be found, therefore, if quantal responses with carcinogenic hydrocarbons are observed to follow consistently a Poisson “one-or-more-event” distribution.

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of the data which must be corrected by appropriate constants to bring them into line with the Poisson distribution. It is evident, nevertheless, that the data do not always fit and that appropriate analysis reveals differences in the slopes of the dose-response to plant viruses in analogy with the results of animal titration. Methods of bioassay of plant viruses which take these factors into account were adapted by Spencer and Price (165) from procedures developed in the field of pharmacology by Bliss and Marks (23,24). For purposes of the practical bioassay of virus infectivity, the major point for consideration is the determination of a dose-response which is applicable to the problem at hand and reproducible under standard conditions. With the bacteriophage, the Poisson distribution seems to meet these criteria, but the conditions of test are unique in virology. A few phage particles in the presence of relatively great numbers of host bacteria in a fluid medium choose, as it were, only those hosts which are fully susceptible, and all others are automatically rejected. The data, however, are described as well by the linear relation referable to a normal distribution of host factors. Thus, while the response is related to chance distribution of phage particles in suspension, this is possible only because both host variation, as far as can be discriminated by the test, and the median resistance of the host population are essentially zero. Since, within the significant response range, a normal curve of standard deviation of s = 0.5 closely approximates the Poisson curve, either relation can be used to describe dose-repsonse for practical bioassay purposes. Nevertheless, the happenings with the bacteriophage cannot be used as a model to predict the course of events with other viruses unable to operate under the same conditions. With the other agents, such as those of Fig. 5 , host influence plays its role in varying degree to shift the distribution of response farther from the Poisson. In all cases, however, the responses lie close to the linear regression, the more so when host influences subject to interfering change are discovered and eliminated. It is probable that detailed investigation will reveal, especially with such agents as vaccinia, myxoma, fibroma, and other viruses, the distorting effects of change in host response during the experimental period. There is no knowledge in virology more firmly ingrained than that pertaining to host variations in resistance and susceptibility to infection, and few reports concerned with dose-response to viruses fail to invoke the influence of these factors in the interpretation of the results. With the hypothesis that the character of response is determined principally by variation in host factors, it may be relatively simple to correlate the findings with many or all viral agents. It has been seen that the results with an occasional agent, the papilloma virus for example, do not appear at once to conform, but application of the concept provides the guidance for search of those factors responsible for the aberration. Of broader significance are

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the indications that the characters of host response to viruses are no different in principle from those to other biological materials. This is not meant to imply that the factors which govern response to digitalis, for example, are precisely the same quantitatively and qualitatively, as those which determine response to the papilloma or any other virus; instead, it is the distribution of the summation of factors which determines the character of response. Much has been learned of the effects of genetic constitution, age, nutrition, hormone balance, and other factors on the capacity of the host t o respond to viruses. As it is evident that virus effects on host cells constitute a spectrum of disease conditions, it must be recognized that an analogous spectrum of host factors of variable quantitative and qualitative distribution must govern the character of host response. VI. CONCLUSION There have been reviewed in this chapter the results observed in the study of the physical, chemical, and biological properties of purified preparations of two tumor viruses which are associated with the induction of specific malignant conditions in hosts of two classes of vertebrates, the bird and the mammal. These agents differ remarkably in many respects of structure and behavior, but both are similar in consisting of discrete, characteristic particles of complete autonomy of physical existence outside the respective host cells. In these respects, the two agents do not differ in principle from other viruses studied in purified preparations. It was scarcely to be predicted that the agents, both responsible for or immediately associated with the induction of cancer, should be so different. The papilloma virus in the generality of its physico-chemical properties and its biological behavior can scarcely be distinguished from other viral agents. I n contrast the virus of erythromyeloblastic leukosis is outstanding in its deviation from the average physical structure; its pronounced expression of enzymatic activity; and its kinship in antigenic constitution with the host cell in which it originates. It was noted earlier that the virus of erythromyeloblastic leukosis differs in its action from the papilloma virus in the induction of malignancy simultaneously with the initiation of the infectious process, whereas malignancy associated with the latter is a function of progressive change dependent on time. Indeed, the occurrence of cancer in association with the papilloma virus is so remote from the initial impression of the agent on the cell that uncertainty exists (167) of its immediate relation to ultimate malignancy. The fundamental basis for the difference is a problem for the future, and speculation of more than the most superficial possibilities is unproductive, the more so since both agents, particularly the virus of erythromyeloblastic leukosis, are available for further study. It requires no great imagination to realize (10) the possible

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influence of the specific enzyme in the immediate utilization of the stores of energy waiting within the cell for the purposes of virus multiplication and diversion of the metabolic and genetic processes of the host cell. The presence of antigen in the virus identical with that of the host cells suggests the basis for the mutual compatibility of host and parasite, which permits the symbiotic relationship optimum for the continued existence of both host cell and virus. The precise mechanism responsible for the alteration of the genetic status of the normal cell to that of the uncontrolled, individual primitive cell is entirely obscure. There have been many discussions of the possible nature and origin of the tumor viruses (52, 90, 91). For once, it would seem, the simple experimental facts observed with the virus of erythromyeloblastic leukosis have very nearly paced, if not actually outdistanced, fancy. It has been surmised that viruses, (7) including those causing tumors (90,91),may represent evolutionary products not greatly remote from cell constituents. At the extreme of this concept is the idea that tumor agents analogous to viruses might arise de mvo under proper stimulus and cause cancer without the need for transfer of an extrinsic infectious principle. Historically, and practically too, in view of the facts establishing the specificity of viruses accumulated in the last half century, such postulates seem unattractive. Nevertheless, it would appear reasonably well-established that the intracellular processes associated with multiplication of the virus in erythromyeloblastic leukosis are so similar to some aapects of the analogous normal mechanisms of the cell that components indistinguishable immunologically from host constituents become incorporated in some manner within or about the virus particle. Many schemes might be suggested as to how this would be accomplished, but there exists at the moment no experimental basis for interpretation. It is in its expression of enzymatic activity, particularly one which might well be concerned with the vital transfer of energy, that the virus has surpassed the prognostications. The occurrence of the enzyme is of significance not only for future explorations with this agent but in pointing up the need for quantitative reexamination of other viruses, thought at one time or another to possess enzyme activity. The experiments establish more firmly another principle of primary significance. The fact that it has required almost fifty years for the characterization of an agent, the virus of erythromyeloblastic leukosis, present in as simple a medium as chicken blood plasma in concentrations such that the material visibly clouds the fluid, is ample evidence that the possible presence of virus in less obvious amounts in more complex sources has certainly not been eliminated by the efforts thus far made. It is evident, further, that the infrequent occurrence of the usual viral immune bodies in chickens with erythromyeloblastic leukosis dispels one more objection to

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the idea that other tumors, particularly those in man, might be of viral etiology simply because specific immune bodies have not been demonstrated in those conditions. It seems entirely possible that the lack of immune body formation may be related, as in the case of erythromyeloMastic leukosis, to virus mass too small for the induction of antibody response. Typical antibodies in high titer result when the immunizing dose is sufficiently large. It is not improbable, also, that the constitutional element of the virus exercising the antigenicity of chick tissue may be of such large proportion or so strategically arranged as t o block or diminish, except under artificial conditions, the immunological effects of other constituents of the agent differing constitutionally from chick tissue antigen. There is no reason to doubt that other cancer viruses might resemble in these respects the agent of erythromyeloblastic leukosis.

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139. Rous, P. (1943). i n “Virus Diseases.” The Messenger Lectures, Cornell Univ. Press, Ithaca. 140. Rous, P., and Beard, J. W. (1934a). J. Exptl. Med. 60,701. 141. Rous, P., and Beard, J. W. (1934b). J. Exptl. Med. 60, 741. 142. Rous, P., and Beard, J. W. (1935). J. Exptl. Med. 62, 523. 143. Rous, P., and Kidd, J. G. (1938). J. Exptl. Med. 67,399. 144. Samuel, G., and Bald, J. G. (1933). Ann. Appl. Biol. 20, 70. 145. Sanford, K. K., Likely, G. D., Bryan, W. R., and Earle, W. R. (1952). J. Nail. Cancer Znst. 12, 1317. 146. Schachman, H. K. (1951). J . Am. Chem. SOC.73, 4453. 147. Selbie, F. R., Robinson, R. H. M., and Shope, R. E. (1948). Brit. J. Cancer 2 , 375. 148. Sharp, D. G. (1950). Biochim. et Biophys. Acta 6, 149. 149. Sharp, D. G. (1953). Advances i n Virus Research 1, 277. 150. Sharp, D. G., and Beard, J. W. (1952). PTOC. Soc. Exptl. Biol. Med. 81. 75. 151. Sharp, D. G., and Beard, J. W. (1954). Biochim. et Biophys. Acta 14.12 152. Sharp, D. G., Eckert, E. A., Beard, D., and Beard, J. W. (1952). J. Bacteriol. 63, 151. 153. Sharp, D. G., Eckert, E. A., Burmester, B. R., and Beard, J. W. (1952). Proc. SOC.Exptl. Biol. Med. 79, 204. 154. Sharp, D. G., Lanni, F., Lanni, Y. T., and Beard, J. W. (1951). Arch. Biochem. 90,453. 155. Sharp, D. G., Mommaerts, E. B., Eckert, E. A., Beard, D., and Beard, J. W. (1954). J . Null. Cancer Znst. 14, 1027. 156. Sharp, D. G., Taylor, A. R., Beard, D., and Beard, J. W. (1942). J. Biol. Chem. 143, 193. 157. Sharp, D. G., Taylor, A. R., Beard, D., and Beard, J. W. (1942). PTOC.Soc. Exptl. Biol. Med. 60, 205. 158. Sharp, D. G., Taylor, A. R., and Beard, J. W. (1946). J. Biol. Chem. 169, 289. 159. Sharp, D. G., Taylor, A. R., Hook, A. E., and Beard, J. W. (1946). Proc. SOC. Ezptl. BioE. Med. 61, 259. 160. Sharp, D. G., Taylor, A. R., McLean, I. W., Jr., Beard, D., and Beard, J. W . (1944). J. Biol. Chem. 166, 585. 161. Sharp, D. G., Taylor, A. R., McLean, I. W., Jr., Beard, D., and Beard, J. W. (1945). J . Biol. Chem. 169, 29. 162. Shimkin, M. B. (1945). Publ. Am. Assoc. Advance. Sci. No. 22, 85. 163. Shope, R. E. (1935). Proc. SOC.Exptl. Biol. Med. 32, 830. 164. Smith, W. E., Kidd, J. G., and Rous, P. (1952). J. Exptl. Med. 96, 299. 165. Spencer, E. L., and Price, W. C. (1943). Am. J . Botany SO, 280. 166. Stern, K. G., and Kirschbaum, A. (1939). Science 89, 010. 167. Syverton, J. T. (1952). Ann. N . Y . Acad. Sci. 64, 1126. 168. Taylor, A. R. (1944). J. Biol. Chem. 163, 675. 169. Taylor, A. R. (1946). J. Biol. Chem. 163, 283. 170. Taylor, A. R., Beard, D., Sharp, D. G., and Beard, J. W. (1942). J. Infectious Diseases 71, 110. 171. Taylor, .4. R., Sharp, D. G., McLean, I. W., Jr., Beard, D., Beard, J. W., Dingle, J. H., and Feller, A. E. (1944). J. Immunol. 48, 361. 172. Trevan, J. W. (1927). Proc. Roy. Soc. B101, 483. 173. Waters, N. F. (1945). Poultry Sci. 24,259. 174. Waters, N. F. (1951). Poultry Sci. SO, 531.

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Morphology and Development of Insect Viruses KENNETH M. SMITH Virus Research Unit Agricultural Research Council, Molten0 Institute, Cambridge, England

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Polyhedral Viruses: IUuclenr T y p e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Lepidoptera.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Hymenoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Diptera.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. The Polyhedral Viruses: Cytoplasmic Type... . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Lepidoptera.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... IV. The Granuloses or Capsular Diseases VII. Development of Insect Viruses.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References.. . . .

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I. INTRODUCTION The early work on insect viruses was almost exclusively devoted to the study of the polyhedral diseases and this is understandable enough, since they were so readily recognized on the optical microscope by their characteristic intracellular inclusions. From the intensive study of insect viruses by several workers during the past decade, it is becoming increasingly clear that insects, like plants and the higher animals, are susceptible to a multiplicity of viruses, of which the polyhedral viruses are only a part. The development of the electron microscope has given us considerable knowledge of the morphology of the virus particles and their relationship to the various and interesting types of intracellular inclusions. Furthermore, the recent advances in the technique of cutting ultra-thin sections, which now allow sections to be cut of the virus particles themselves, are likely to yield interesting information on the internal arrangement of the ultimate virus particle. Before the advent of the electron microscope there existed much confusion of thought regarding the polyhedral diseases. The actual polyhedra were variously considered to be organisms, to contain the virus, and to be crystalline aggregates of the virus itself. It was shown by Komarek and Breindl (1924) with the optical microscope, and by Bergold (1947)with 199

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the electron microscope, that the second theory was the correct one and that the virus particles were contained within the polyhedral crystals. Weak alkalis dissolve the polyhedra, leaving the virus particles behind inside the membrane which enclosed the crystal. This procedure is, in effect, a microdissection technique but the pH has to be carefully adjusted, otherwise the virus particles, which are themselves susceptible to the effects of alkalis, would also be dissolved. This fact is shown by the difficulty experienced in dissolving the cytoplasmic polyhedra, which have no protective membrane, without also dissolving the virus particles. The polyhedral bodies from the different types of polyhedroses appear to be genuine crystals. They are not, however, nucleoprotein and the arrangement of the virus particles inside the crystal is not regular but haphazard according to the manner in which they are drawn into the crystal at the time of its formation. These facts differentiate the polyhedra sharply from the plant virus crystals which are nucleoprotein and are composed only of the virus particles themselves. With the knowledge of the insect virus diseases we have at present, which is admittedly scanty, we can put them in four arbitrary groups as follows, Group I. The polyhedral virus diseases. These are subdivided into (a) nuclear polyhedroses, and (b) cytoplasmic polyhedroses. Group 11. The granuloses or capsular virus diseases. Group 111. Viruses without intracellular inclusion. Group IV. A miscellaneous collection of apparent viruses which require further study.

11. THEPOLYHEDRAL VIRUSES:NUCLEAR TYPE Polyhedral viruses which appear to start their development in the cell nuclei have been described for many species of lepidopterous larvae (the claasical case, of course, being silkworm jaundice), one or two species of hymenoptera, and one species of diptera. I n the lepidoptera the polyhedra are found in the skin, tracheae, fat, and blood cells, but also, in certain species, in the imaginal wing and limb buds, nerve ganglia, and Malpighian tubules. Among the hymenoptera, in the sawflies, the polyhedra occur in the nuclei of the digestive cells of the mid-gut epithelium, and in the TipuZidue (diptera) they develop in the blood cells.

A . Lepidoptera The number of polyhedral viruses which attack the larvae of lepidoptera is very large. More than 50 of these viruses have been observed a t Cambridge alone, although not all were of the nuclear type. It might, perhaps, be more accurate to say that more than 50 species of lepidoptera have been infected or found infected with polyhedral viruses. Until these various

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viruses have been more carefully examined by serological and other means to determine possible relationship, it is not wise to say definitely that they are all separate and distinct. The polyhedra themselves vary greatly in size and shape, although it is probably true to say that the shape is usually constant for a given virus. Thus in the nuclear polyhedral diseases of the scarlet tiger moth larva, Panaxia dominula, the polyhedra all tend to be cubic in shape. Although they vary greatly in size in a single individual, the polyhedra in the same nucleus are all the same size. Analyses of the amino acid composition of some insect viruses and their polyhedral bodies have been made by Wellington (1954). She finds that the proteins characteristic of insect virus inclusion bodies are very similar to one another in amino acid composition. Although many significant differences can be observed between them, these proteins form a group with very similar properties. It seems probable that most, if not all, nuclear polyhedra are surrounded by a membrane which remains behind with the contained virus rods when the polyhedral body is dissolved in weak alkali (Fig. 1A). This membrane is of a similar composition t o that of the polyhedra themselves (Wyatt, 1950). The polyhedra from the different diseases of lepidopterous larvae vary greatly in their resistance to treatment with alkalis, the most resistant so far known being those from an Australian pasture caterpillar, Pterolocera amplicornis Walker, which require 60 min. exposure to 4 % sodium carbonate at a temperature of 56°C. to dissolve them completely (Day et al., 1953). There is very little homogeneity in the sizes of virus rods in the nuclear polyhedqoses of lepidopterous larvae. Thus the size of the virus rod varies even within one nucleus. This is particularly so in Lymantria monacha which shows greater variation than any other species in the length of the virus rods obtained from polyhedra by dissolving them in weak alkali. A particular polyhedral body may contain an almost complete range of virus rods from longer than average (about 290 mp) to less than half length (Xeros snd Smith 1955). It has a.lso been shown (Smith and Xeros 1954) that the so-called spherical developmental forms described by Bergold (1950, 1952, 1953) are not spherical forms at all but bundles of half-length rods enclosed within an inner capsule (Fig. 1A). These occur in Lymantria monacha, L . dispar, and Bombyx mom', and the proportion of half-length to normal-sized rods varies from one species to another and seems to be highest in L . monacha. Bundles of 3 or 4 or more virus rods in a capsule also occur in the polyhedra of various species; these are commonly observed in L. monacha and Abrmas grossulariata. In B . mori the rods are mostly single, but each rod is in a capsule.

FIG. 1 . A: Nuclear polyhedral disease of Lyrnantria nionacha: polyhedra treated with weak alkali; note part of dissolved crystal and membrane with contained virus; note also the liberated virus, single and half rods and bundles, in their c a ps ul ~ s . x 21,000. 13: Cytoplasmic polyhedra from Phlogophora nieticulosu treated with weak alkali; note the honeycomb structure with holes hut no virus particles. x 19,900. C : Section through cytoplasmic polyhedra from Phlogophora meficirlosu; note the spherical virus particles in a matrix of polyhedral protein; composite virus particles can be seen in the cytoplasm. X 14,910. From Smith and Xeros (1054~). 202

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B . Hymenoptera Nuclear polyhedroses of the hynieiioptera have so far only been observed in the larvae of sawflies and recorded from the following species, the European pine sawfly, Neodiprion sertifer (Geoff r.), and the European spruce sawfly, Diprion hercyniae (Htg.). What is probably the European pine sawfly has been observed infected with a polyhedral disease in Scotland by Morison and sent to the writer. In addition, the larvae of the black currant sawfly, Nematus olfaciens, is occasionally attacked by a polyhedral virus (Smith, 1954). Although the polyhedral viruses of sawfly larvae appear to be nuclear ill development, the polyhedra are fourid only in the nuclei of the digestive cells of the mid-gut epithelium. In Neodiprion sertijer the polyhedra average 1 p in diameter and are frequently almost spherical. The virus particles are rods measuriiig about 250 X 50 mp and appear to be enclosed in a capsule (Bird and Whaleri, 1953). These authors caomment on the presence of spherical particles together with the rods, but from their appearance such particles could also be half-length rods. In Diprion hercyniae, rod-shaped virus particles with dimensions of about 250 X 50 mp, and spherical particles, varying in size from less than 80 to more than 160 mp in diameter have been isolated froni purified polyhedra by Bird (1952). In a study on the electron microscope of thin sections of infected nuclei arid material extracted from infected cells, it appears that polyhedra arise as ultramicroscopic bodies about 160 mp in diameter, and contain spherical particles about 20 mp in diameter which increase in number and size as the polyhedra grow. Bird considers that multiplication and development of the virus particles takes place within the growing polyhedra as well as iu the free state within ail infected nucleus. This does not seem t o fit in with Bergold’s thesis (1950, 1953) that “virus particles of all developing stages are occluded and fixed rigidly by aggregation of the inclusion body proteins.” C . IXptera Only one authentic. nuclear polyhedrosis of a dipterous larva is knownThis affects the leatherjacket, the larva of the crane fly, Tipula palisdosa, and shows some uriusually interesting characteristics. I t was first recorded arid briefly described by Remiie (1923) who, however, described it as a disease of the fat body. The virus mas rediscovered by Smith and Xeros, (1954b), who have made a more intensive study of it. Although the disease is referred to as a polyhedrosis, the “polyhedra” are actually shaped like the segment of an orange and they form inside the blood cells. The virus causes a form of leukemia in that the number of blood cells is enormously increased and pack the blood cavities. The in-

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FIG.2. A: Section through a diseased blood cell of Tipula paludosa (Diptera) infected with a polyhedral disease; note the greatly enlarged nucleus containing virus rods and a t top center one of the crescent-shaped polyhedral crystals, (met,hrtcrylste not removed). X 11,880. €3: Section through tip of a polyhedral crystal from the same disease; note the many virus rods inside. (methacrylate not removed). X 15,840. C: Section through II diseased blood cell as in A , hut with the methacrylate removed and shadowed with gold-palladium; note the nucleus enlarged almost to the cell limits, the horseshoe-shaped central body, the periceiitral hody and the virus rods developing in the surrounding less dense area (from Xeros, and Smith 1955). X 7,000. D : Section through part of a polyhedral body showing virus rods arid their capsules (methacrylate removed and shadowed) (from Xeros, and Smith 1955). X 24,750.

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fected larva is much paler in color and, if the integument is punctured in a late stage of the disease, there is a rush of blood cells showing the presence of the characteristic crescent-shaped crystals in each one (Fig. 2A). These inclusion bodies do not appear till the disease is well advanced and then they begin to develop on the nuclear membrane in the blood cells. They have one surface exposed t o the nuclear sap and as they grow the other protrudes more and more into the cytoplasm. Sections through infected blood cells, photographed on the electron microscope (Fig. 2C), show the virus particles developing in the nucleus and then becoming incorporated into the growing polyhedra. The so-called polyhedra of this disease are like the polyhedra of lepidopterous larvae in so far as they contain virus bodies occluded in a presuniably noninfectious matrix. In other respects they are very different. They are Feulgen-positive throughout and stain only moderately well with bromophenol blue, even after 15 min. of mild acid hydrolysis, whereas the classical polyhedra do not stain with normal Feulgen and stain intensely with bromophenol blue after acid hydrolysis (Xeros and Smith, 1955). Suspensions of lepidopterous polyhedra dissolve rapidly in weak alkali, but those of Tipula hardly react at all. When treated with 1 N NaOH, however, the inclusion bodies from Tipula pull out into thin worm-like shapes and, when returned to distilled water, resume their normal appearance (Smith and Xeros, 195411). It, was, therefore, difficult to determine whether there were any virus bodies occluded in these peculiar crystals. However, when they were treated first with XaOH, then with sodium thioglycolate, and filially with NaOH again, most of the crystalline material dissolved, leaving behind a sponge-like matrix c.oiitaining apparent rod-like bodies. That these were actually the virus rods was confirmed by means of thin sections of untreated crystals in which the virus rods were clearly seen (Fig. 2B). These rods appear similar t o the virus rods desmibed in polyhedroses of lepidoptera, each being apparently inside a capsule. They measure about 160 nip in length (Fig. 2D).

111. THEPOLYHEDR.\L

VIRUSES:

CYTOPLASMIC TYPE

-4.Ikpidoptera It was first, shown hy Smith and Wyckoff (1950) that there existed an entirely cliff erent type of polyhedral body which contained spherical virus particles instead of rods. They were found in the larvae of two tiger moths, Arctia caja and A . villica, but they have since been found in maiiy other species of lepidopterous larvae and are just as, if not more, common than the nuclear polyhedra. It has further been shown that these larvae frequently suffer from hoth types of polyhedral virus a t the same time and this can be quickly demonstrated by a simple staining technique. In a

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smear prepared from a caterpillar infected with both types of polyhedroses and stained with Giemsa, the polyhedra containing the spherical virus pick up the stain readily and are thus sharply differentiated from the nonstaining type of polyhedra containing rod-shaped virus particles (Smith, et al., 1953). Another inportant discovery is that these polyhedra form in the cytoplasm of the cells of the mid-gut and not in the cell nuclei (Xeros, 1952; Smith and Xeros, 1953). The reaction of the cytoplasmic polyhedral crystal t,o treatment with weak alkali is again different from that of the classical nuclear polyhedra. Instead of dissolving completely, leaving behind a number of virus rods in an enclosed membrane, this new type of polyhedra dissolves only partially. There is left behind a kind of skeleton structure filled with small circular holes and there is 110 membrane (Fig. 1B). In some cases it has been observed that instead of leaving a honeycomb-like structure behind when treated with weak alkali, the cytoplasmic polyhedra resolve into what appears to be a heap of horseshoe-shaped plates. It is possible in these cases that the plates are so arranged one upon another so that circular spaces are formed into which the virus fits. In examination in the electron microscope of the cytoplasmic polyhedra from many different diseased caterpillars, and treated with different strengths of sodium carbonate, great difficulty was experienced in seeing the actual virus particles. It therefore seems as if this technique was unsuitable and that in most cases t,he polyhedral protein was comparatively resistant to the action of the alkali arid the virus particles were dissolved away. This would account for the numerous empty holes in the polyhedral shell. A different approach to the problem was therefore made and thin sections were cut of fixed larvae infected with the cytoplasmic polyhedra. A number of different species were examined in this way including: Bombys mori, Abraras gross uluriata, arid Phlogophora meticulosa, but only P. meticulosa is dealt with here. Thin sections of the gut revealed an interesting state of affairs. If the sections through the polyhedral crystals were thin enough, they could be seen to consist of a mass of spherical bodies approximately 00 mp in diameter, held together in a matrix of polyhedral protein (Fig. IC). IJnder higher magiiificatioii each of these spherical bodies could be seen to be composite, consisting of a number, usually four, of very small units each about 15 mp in diameter (Fig. 1C). The composite bodies were also to t)c found loose in the cytoplasm together with a certain iiumber of thc sriiall, single units. The size of the composite virus bodies from P. meticiilosu (about 00 m p ) is approximately the same as that of the spherical virus partirles from Arctia villica (Smith and Wyrkoff, 1950; Bergold, 1952) which is about 85 mp. The electron micrographs of the spherical virus bodies for -4. uillica suggest that these also may be composite. The q u r s -

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tion of the composite nature of virus particles is an interesting one and it is discussed in a later section. In our comparison of the two types of polyhedral diseases, nuclear and cytoplasmic, we find certain phenomena are characteristic of each type of disease. Thus, it appears that the nuclear type viruses are always rods and this is so even in such different species of insects as the larvae of lepidoptera and diptera. On the other hand, the viruses occluded in the cytoplasmic polyhedra have so far all been spheres. Furthermore, the nuclear polyhedra all seem to be enclosed in a membrane, but this is not the case with the cytoplasmic polyhedra. We thus have the rather interesting phenomenon of first, a rod-shaped virus correlated with a nuclear site of development, a particular type of crystal, and a characteristic disease. Secondly, the spherical virus is correlated with a cytoplasmic site of development, a quite different type of crystal from the foregoing, and again a characteristic disease. These correlations may not hold good as investigations proceed, but a t the moment they appear to do so. IV. THE GRANULOSES OR CAPSULAR DISEASES The granuloses attack many species of lepidopterous larvae but they have not as yet been recorded from any other species of insect. This type of virus disease was first recorded by Paillot (1926), rediscovered by Steinhaus (1947) and later by Bergold (1948). The inclusion bodies consist, as the name implies, not of polyhedral crystals but of huge numbers of very small granules or capsules. When subjected to the action of weak alkali the capsular material is dissolved revealing, as a rule, a single virus rod. The site of formation of the granules appears to differ in the granular diseases of different insects. In Cacoecia murinana (Hbn.) the development of the virus and granules is in the cytoplasm of most organs including blood cells (Bergold, 1948). In Sabulodes caberta Gn., the omnivorous looper, however, the granules appear to form in the nucleus and not in the cytoplasm (Hughes and Thompson, 1951). Similarly with a granulosis disease of Natada nararia, the nettle grub, a pest of the tea plant in Ceylon (Smith and Xeros, 1954a), the formation of the granules takes place in the nuclei of the hypodermis and fat body. Suspensions of granules from graiiulosis-infected N . nararia were mounted on grids, and after drying, were treated with sodium carbonate solutions of varying strengths and for varying times. The micrographs (Fig. 3A-C) show the course of dissolution of the granules which measure approximately 360 X 200 mp in size. First the outer capsule disintegrates and partially dissolves, leaving an expanded residue on the grid. Within this outer capsule a second inner capsule (about 290 X 45 mp) is revealed. As the action of the alkali proceeds this

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FIG.3. A: Granulosis disease of Natada nararia; granules untreated but shadowed (from Smith and Xeros, 1954~). X 19,200. R : Thesamegranulesafter treatment with weak alkuli; note the collapsed outer capsule, the inner capsule and two liberated virus roda (from Smith and Xeros, 1954~) . X 20,000. C: The virus rods after treatment with weak alkali, the contents of the rods are seen partially dissolved, sometimes from the centre and sometimes from the ends, leaving the intjniatc membraneesposed (from Smithand Xeros, 1954~) . X 20,000. D : An apparent virus from the pine looper caterpillar, Bupalus piniarius. X 19,200.

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inner capsule in turn dissolves to reveal the much thinner virus rod (about 290 X 45 mp). Furthermore, as the virus rod in turn is subjected to alkali disintegration, the intimate membrane enclosing the virus material itself is seen. In some rods the virus material is dissolved away from each end and only the middle part of the virus material remains. In others the virus material is dissolved out of the middle region of the rod, leaving two small remnants, one at each end. The only membrane, as distinct from capsules, actually observed is the intimate membrane surrounding the actual virus material. No membranes surrounding the inner or outer capsules were observed. In the descriptions published by different workers on the various granulosis viruses there is some confusion regarding what is virus rod and what is its capsule. Bergold (1948) and Steinhaus et al. (1949) published the first electron micrographs of granules treated with weak alkali. In these first descriptions the pictures were interpreted to mean that one virus particle was released from each capsule, leaving a rod-shaped cavity in the granule substance. Another interpretation, however, is also possible, namely that the alkali treatment caused the dissolution of an outer capsule, leaving two virus rods each within an inner capsule which together with the outer capsule had constituted a single granule. Figure 2D in Steinhaus, et al. (1949) and Fig. 164B in Steinhaus’s book (1949) definitely suggest that there are two rods per capsule in the granulosis of Peridroma margaritosa. The intermediate stages of dissolution are, however, largely lacking and it is not clear whether each rod is in a separate inner capsule or whether both (if there are two) rods are within one inner capsule. Bergold (1952, 1953) states that only occasionally there is a “double rod,” i.e., two rods, in one granule; but the majority of his published pictures of Cacoecia murinana granules, including those published in 1948 when he supposed that there was only one virus rod per granule, show two virus rods per granule. The possibility that the granules of Cacoecia mvrinana may have two virus rods per granule has also been pointed out by Tokuyasu (1953). Steinhaus and Thompson (1949) described a new granulosis of Junonia coenia in which there was without doubt only one virus rod per granule. They showed pictures of the virus still enclosed in “some of the granular material” but did not identify this material as an inner capsule. Thompson (1951) in reporting a granulosis of Pieris rapae gave no details of the inner morphology of the granules. Wasser and Steinhaus (1951) reported a granulosis of Argyrataenia velutinana stating that apparently one rod was enclosed in each capsule. In their illustration of partially dissolved granules, however, the distribution of the electron absorbing material suggests that two rods may be present in each capsule. Hughes and Thompson (1951) and Hughes (1952) showed there was one virus rod per granule in the granulosis of Sabulodes caberata. Tanada (1953) described with elec-

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tron micrographs a granulosis of P . rapae. The granules formed in this disease have only one virus rod per granule. Tanada states that there was a great variation in the width of the virus rods, from 40 mp t o 100 mp. It is clear from the published pictures, however, that this great variation is not entirely due t o variations in the width of the virus rods themselves, but that the virus rods are about 40 mp wide, and those bodies considered by Tanada to be very wide virus rods are actually rods still surrounded by their inner capsules and having a diameter of about 90 mp. The intact undissolved capsules are about 185 mp in diameter, and the flattened intimate membranes about 50 mp in diameter, that is, wider than the virus rods. I t is important that the virus rods should be freed of capsular material before measurement of their diameter. Whether they are free or not free of inner capsules, material ran be checked by observing the diameter of the collapsed intimate membranes of completely dissolved rods (Smith arid Xeros, 1954).

v. I'IRUSES

WITHOUT INTRACELLULAIt

INCLUSIONS

There are one or two virus diseases of insects in which no intracellular iiiclusions have been observed. These include sacbrood of honeybee larvae, the virus paralysis of adult bees, a virus disease of Cirphis unipuncta (Haworth), the cosmopolitan army worm, and a new virus affecting the larva of the dipterous insect, Tipula paludosa. It is only in the last two diseases that the viruses have been observed arid in which any description of their morphology is therefore possible. Larvae of Cirphis unipuncta infected in the late third instar soon appear swollen and somewhat darker than normal insects. The cuticula of the diseased larvae have a waxy appearance arid in some cases the middle part of the insect is slightly enlarged. There is no liquefaction and disintegration characteristic of the polyhedral diseases and no intracellular iiiclusions are visible under the optical microscope. Examination under the electron microsrope c;f clarified arid filtered suspensions prepared from the caterpillars revealed the presence of large numbers of spherical t o ovoid bodies measuring approximately 25 mp in diameter. These particles could not be observed in normal caterpillars (Wasser, 1952). Superficially the virus particles resemble preparations of some of the smaller near-spherical plant viruses. In the spring of 1954 a new virus was discovered in this laboratory in one or two leatherjackets, larvae of the cranefly, Tipula paludosa, which had been collected during a search for these larvae infected with the polyhedral disease previously described. A short account was later published by Xeros (1954). The virus causing this disease is an interesting one in many ways and there appear to be no iiitracellular inclusions. Infected larvae

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21 1

in a late stage of the disease are very much paler in color than the normal, but the first sign of the disease is seen in the fat body which becomes bright violet in color. The best method to diagnose the infection is to wash the larva in cold water, put it still wet in a clean glass tube, and examine under a bright light. The larva then adheres to the side of the tube arid the violet coloring of the fat body is apparent. If a larva in a late stage of the disease is punctured, an iridescent fluid is obtained which reserghles a purified preparation of one of the spherical plant viruses. A drop of this iridescent fluid, diluted but not otherwise treated, when photographed o k t h e electron microscope is seen to consist of huge numbers of small particles which are roughly hexagonal i n shape and which tend to arrange themselves in a regular manner. They are about 100 mp in diameter. Multiplication of the virus appears to start in the cytoplasm of the fat body which becomes greatly enlarged. Sections of infected fat body when photomphed on the electron microscope suggest that the whole material of the ?&n seems t o be converted into virus (Fig. 4A). The arrangement of the virus particles inside the fat body is very interesting; in addition t o the large number of isolated particles there occur numerous masses or agglomerations of virus particles which look superficially like some of the intracellular inclusions of other insect viruses. These masses, however, appear to be composed only of virus particles (Fig. 4B-D). The hexagonal virus particles, measuring about 100 mM in diameter, mentioned above, are not apparently the ultimate particle. When photographed a t a magnification of 40,000 times, numbers of very minute spheres can be seen inside the larger one. Moreover, thin sections of the virus particles themselves reveal their apparently composite nature (Fig. 5).

irI. APP.UWNTVIRUSES, IXSUFFICIENTLY STUDIED A number of apparent virus diseases which need further study are briefly discussed in this section. In 1924 Paillot described a peruliar disease of the caterpillar of the large white butterfly, Pieris brassicae. In affected larvae the blood is viscous and milky in appearawe, and the blood cells contain numerous refringent bodies which are characterist ic of the disease. Examination of the diseased blood cells under the optical mic+roscopeshows a diffused cytoplasmic mass togcther with the refractile bodies which appear to arise from the mitochondria of the cell. Thcre arc also present numerous granules measuring less than 0.1 p in diamcter, which Paillot considered to be the actual disease agent. Xo studies with the elertron microscope have been carried out on this disease, so that the actual virus has not been obsrrved. In 1953 Arvy described a disease of the caterpillars of Jfalacosoma neitstria which seems to hear sonic rcscmhlanc.c to the foregoing. Long baton-like inclu-

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KENNETH M. SMITE

FIQ.4. A: Section through part of the fatbody of a leatherjacket, TipuZa paludosa, infected with a new virus; note how the entire substance of the organ seems to be converted into virus; the large masses on the right of the photograph are virus aggregates. X 6,500. B: Section through one of the agglomerates of virus particles. X 19,350. C: Section through a small virus aggregate photographed at a higher magnification; note how the virus particles seem t o be joined together in colonies. x 24,750. D: Section through one of t,he virus aggregates shown in A, photographed at a higher magnification (Elections with methacrylate removed and shadowed with gold-palladium). X 6,637.

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sions apparently of a crystalline nature develop in the cytoplasm of the leucocytes. No elect,ron micrographs of the actual virus have as yet been published. An apparent virus disease of a dipterous larva, Camptochironomustentans,

FIG. 5. Sections through the virus particles in the same disease of Tipula paludosa a8 shown in Fig. 4 , but without removal of the methacrylate; note the darker

center in some of the particles and apparent membrane. X 62,400.

has been described by Weiser (1949). Cytoplasmic inclusion bodies develop in the fat body; Weiser shows photographs taken using the optical microscope with dark ground illumination in which the ovoid inclusion bodies appear t o be filled with minute granules which may be the virus itself. During a study made with the electron microscope of the blood of house crickets, GryZZuZus domesticus, Gregoire (1951) observed large masses of

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SMITH

virus-like bodies in apparently healthy insects. Their average size was approximately 223 X 57 mp and they consisted of a central, dense, rodshaped structure, surrounded by a relatively transparent area. If this is confirmed as a virus disease, it will be interesting as a case of diagnosis by finding the virus particles by means of the electron microscope i n the absence of any visible disease symptoms. In studying possible virus infections of the pine looper caterpillar, Bupalus piniarius, the writer observed with the electron microscope a number of curious spindle or boat-shaped bodies (Fig. 3D) measuring about 400 mp. These were isolated from one or two caterpillars which had died arid for which no causal agent or intracellular inclusion could be observed with the optical microscope. There is a suggestion in one or two of the electron micrographs that these spindle-shaped bodies are composite and are made up of numbers of long thin rods, but confirmation of this and further study must wait until fresh material can be obtained. Two diseases of the honeybee and its larva are usually accepted as due to viruses although these have not been certainly observed with the electron microscope. These are sacbrood disease of the larvae and paralysis of the adult bee (Burnside, 1945; Butler, 1943). Finally two possible virus diseases are mentioned, the blue disease of the larvae of the Japanese beetle, Popilla japmica Newm., (Dutky and Gooden, 1950) and a somewhat similar disease of the larvae of the eockchafer, Melolontha vulgaris F., (Wille and Martignoni, 1952).

VII. DEVELOPMENT OF INSECT VIRUSES Bergold (1950, 1953) states that the polyhedral viruses of insects are organisms with a complicated life cycle during which several polymorphic forms are produced. His method of studying the development of the viruses was to use as his starting material purified and dried polyhedral bodies. These were treated with alkali, and the virus contained within the crystals and thus liberated was studied with the electron microscope. Thc various “developmental forms” observed were then arranged in an arbitrary fashion as stages in the “developmental rycle.” Bergold justifies this met,hod by the following statement: “During the process of inclusion body formation, virus particles of all developing stages are occluded and fixed rigidly by aggregation and crystallization of the inclusion body protein.” But it has been shown (Smith and Xeros, 1954c) by sections through the infected nuclei of silkworms in early stages of the disease before the formation of the polyhedral bodies that the fully .formed virus rods, without capsules or what Bergold refers to as “developmental membranes,” are extruded from the central chromatic mass into the ring zone. Moreover,

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FIG.6. A : Section through diseased fatbody of larva of Tipula paludosa, without removal of methacrylate; note the virus aggregates and apparently regular spacing of the virus particles. X 18,OOO. B : Section through a diseased nucleus of the silkworm, infected with a nuclear polyhedrosis; note the virus rods emerging from the central chromatic mass and the peripheral chromatin. The emerging virus rods are longer than the free rods, and appear to be dividing. Methacrylate removed and shadowed with gold-palladium (from Smith and Xeros, 1954a). X 12,520. C : Similar t o B a t a later stage; the virus rods are now free in the nuclear ring zone but have no capsules. Methacrylate removed and shadowed with gold-palladium (from Smith and Xeros, 1954a). X 13,120.

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KENNETH M. SMITH

there is some evidence that the virus rods forming in the chromatic mass, or nuclear net, are longer than normal and may divide or break across (Fig. 6B). Again, sections of diseased blood cells of the dipterous larva, l‘ipzda paludosa, show the virus rods forming in the outer zone of nuclear material long before the “polyhedra” are formed. The sections also show numbers of fully formed virus rods being incorporated into the crystal (Fig. 2C, D). It is clear that thin sections of larvae a t different stages in the progress of disease, fixed with osniic acid, and without the removal of the metharrylate offer much greater opportunities for the study of virus development than collections of polyhedral crystals subjected to treatment with alkali. This is brought forcibly home in the study of the cytoplasmic polyhedra arid the spherical viruses associated with them. I n the case of the caterpillar of the angleshades moth, Phlogophora meticulosa, it was found impossible to see the virus in cytoplasmic polyhedra treated with many different strengths of alkali. In thin sections, however, fixed with osmic arid, it was possible not only t o see that the polyhedra were full of spherical virus particles but also that these particles were composite (Fig. 1C). Smith and Xeros (1953b, 1953~)have shown with both the optical and electron microscopes that the typical rod-shaped viruses of the nuclear polyhedroses develop about halfway through the course of the disease, partly on the peripheral chromatin, but mainly in the chromatic net or mass. After formation and liberation into the ring zone the rods acquire an inner capsule, which is dense to the electron beam, and are occluded in the crystallizing polyhedral protein that accumulates in the ring zone. Hughes (1953) who has carried out similar studies on the development of a nuclear polyhedral virus affecting the alfalfa caterpillar, Colias philodice, eurytheme, Bdvl., seems to agree with this. He says that there is some evidence that the virus particles occur primarily in the central portion of the nucleus at an early stage in the formation, probably indicating that their formation may be related in some way to the chromatin remains. In the nuclear polyhedrosis of Tipula paludosa, referred to above, the situation seems somewhat similar though more information is needed since me do not know the exact intranuclear site of formation of this virus. In mounts of whole blood cells, either fresh or fixed with osmic acid, and examined with the light microscope, the nucleus appears dense and homogeneous except for a small, highly refractile body, the “central body,” which is often horseshoe shaped. As the nucleus grows so does the central body and the remainder of the nuclear material becomes less and less dense. In sections examined with the electron microscope it is possible to identify the same structure as a very electron-dense, horseshoe-shaped body (Fig.

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2C). I n these sections the electron microscope shows that there is also a pericentral body surrounding the central body. This was not observable in studies with the light microscope of whole cells fixed in osmic acid. Around the mass of central and pericentral bodies there is a false ring zone filled with a less dense material. In this latter material can be seen the virus rods in their inner capsules. By the time the crescent-shaped polyhedra begin to form in the nuclear membrane the nucleus has greatly enlarged and become less dense (Xeros and Smith, 1955). It is interesting to compare the development of some of the insect viruses with that of certain viruses infecting higher animals. Studies have been made by Morgan et al. (1954) on the structure and development of the viruses of herpes simplex, vaccinia, and fowl pox. In some beautiful photographs of ultra-thin sections of these viruses i t is possible to see that the particles are enclosed in membranes in a similar manner to the insect viruses. In herpes simplex, the nuclei of infected cells contained small dense primary bodies (30-40 mp in diameter) as well as slightly larger and less dense particles (4G50 mp in diameter) surrounded by a single membrane (70-100 mp in diameter). In the cytoplasm most of the particles possessed a double outer membrane (120-130 mp in diameter). It is suggested that the initial site of virus development is restricted to the nucleus where primary bodies form and become enclosed by a single outer membrane. Upon release into the cytoplasm these particles seem t o acquire a second outer membrane and presumably represent the mature virus. This phenomenon of the development of membranes to enclose the virus particles is also extremely characteristic of the insect viruses. As we have seen in the case of the nuclear polyhedrosis of the silkworm, the virus rods are freed into the nuclear ring zone without their enclosing capsules, and these are developed later when the rods are occluded in the polyhedra. Similarly with the granulosis disease of the nettlegrub, the virus, surrounded by its intimate membrane, is enclosed within an inner capsule and further surrounded by an outer capsule. Superficially perhaps, the closest resemblance to the herpes simplex virus particles is shown by the sections of the recently discovered virus of the leatherjacket. In Fig. 5 , although the sections are much thicker than those of Morgan et al., there can be seen the suggestion of an outer membrane and a central darker body. The various types of virus particles, discussed in this paper, together with their enclosing membranes and capsules, are shown more or less diagrammatically in Fig. 7. The difference between the nuclear and cytoplasmic polyhedral viruses is emphasized.

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FIG.7. A: Virus rods from Lymantria mmacha; note bundles and single and half rods with and without capsules. B: Virus rods from Bombyx mori, the silk worm, single rods, with and without capsules, half rods and one bundle with capsule. C: Virus rods with capsules from a polyhedrosis of Tipula paludosa. D : Membrane from dissolved polyhedral crystal, containing short thick rods from the larva of Abraxas grossulariata, the currant moth. The rods apparently consist of bundles of thinner rods. E: Similar composite rods from a nuclear disease of a larva of Panazia dominula, the scarlet tiger moth. F: Composite spherical virus particles from a cytoplasmic polyhedrosis of Phlogophora meticu2osa. (This drawing is greatly enlarged compared with the other drawings which are roughly on the same scale.) G : Hexagonal virus particles from a new disease of Tipula paludosa. H : Sections of the virus particles shown in G ; note the apparent membrane and internal structure.

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ACKNOWLEDGMENTS Acknowledgments are due to Miss S. Vernon-Smith for taking the electron micrographs, and to Miss M. E. Short for making the drawings in the text figure. REFERENCES Arvy, Lucie. (1953). Rev. hematol. 8, 204-212. Bergold, G . H. (1947). 2.Naturjorsch. 2b, 122. Bergold, G . H. (1948). 2.Naturforsch. 3b, 338-342. Bergold, G. H . (1950). Can. J . Research 628, 5-11. Bergold, G . H . (1952). i n “The Nature of Virus Multiplication” (P. Fildes and W. E . Van Heyningen, eds.). Cambridge Univ. Press, New York. Bergold, G . H. (1953). Advances i n Virus Research 1, 91-139. Bird, F. T. (1952). Biochini. e t . Biophys. Acta 8, 360-368. Bird, F. T., and Whalen, M. M. (1953). Can. Entomolgist 86, 433437. Burnside, C. E . (1945). A m . Bee J . 86, 354-355. Butler, C. G. (1943). Bee World January, 11 pp. Day, M. F., Common, I . F. B., Fabrant, J. L . , and Potter, C. (1953). Australian J . Biol. Scz. 6, 574-579. Dutky, S. F., and Gooden, E. I,. (1950). Sac. A m . Bacteriologists PTOC.A17, 22-23. Gregoire, C. (1951). J. Gen. Microbiol. 6, 121-123. Hughes, K. M. (1952). J . Bacteriol. 64, 375-380. Hughes, K . M. (1953). Hilgardia 22,391406. Hughes, K. RI., and Thompson, C. G. (1951). J . Infectious Diseases 89, 173-179. Komarek, J., and Breindl, V. (1924). 2.angew. Entomol. 10, 99. Morgan,C., Ellison, S. A., Rose, H. M., and Moore, D . H. (1954). J . Exptl. ikfed. 100, 195-202, 301-310. Paillot, A . (1924). Compt. rend. 179, 1353-1356. Paillot, A. (1926). f‘ompt. rend. 182, 180-182. Rennie, J. (1923). Proc. Roy. Phys. SOC.Edinburgh 20, 265. Smith, K. M. (1954). Discovery 16, p. 455458. Smith, K . M., and Wyckoff, R . W. G . (1950). Nature 166,861. Smith, K. M., Wyckoff, R. W. G., and Xeros, N. (1953). Parasitology 42,287-289. Smith, K . M., and Xeros, N. (1953a). Parasitology 43, 178-185. Smith, K . M., and Xeros, N. (1953b). Nature 172, 670. Smith, K.M., and Xeros, N. (1954a). Parasitology 44, 400-406. Smith, K. M., and Xeros, N. (1954b). Nature 173. 866-867. Smith, K.M., and Xeros, N. (1954~). Parasitology 4 4 , 7 1 4 . Steinhaus, E . A. (1947). Science 106,323. Steinhaus, E. A. (1949). “Principles of Insect Pathology.” McGraw Hill, New York. Steinhaus, E . A., Hughes, K. M., and Wasser, H. B. (1949). J . Bacteriol. 67, 219224. Steinhaus, E. A,, and Thompson, C. G. (1949). Science 110, 276-278. Tanada, Y. (1953). Proc. Hawaii Entomol. S O C 16, . 235-260. Thompson, C. G . (1951). J . Econ. Entomol. 44, 255. Tokuyasu, K. (1953). Enzyrnologia 1 6 , 6 2 4 . Wasser, H . B. (1952). J . Bacteriol. 64, 787-792. Wasser, H. B., and Steinhaus, E. A. (1951). Virginia J . Sci. 2, [N.S.] 91-93.

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Weiser, J. (1949). Ann. paraaitol. humaine el comparde 14, 259-264. Wellington, Eunice F. (1954). Biochem. J . 67, 334-338. Wille, H., and Martignoni, M. E. (1952). Schweiz. 2.Allgem. Pathol. u . Bahleriol. 16, 470-474. Wyatt, G . R . (1950). Thesis (unpublished). Cambridge University, England. Xero8, N. (1952). Nature 170, 1073. Xeros, N. (1954). Nature 174, 562. Xeros, N., and Smith, K. M. (1955). PTOC.Intern. Conf. Electron Microscopy, London, 1954.

Multiplication of Plant Viruses in Insect Vectors KARL MARAMOROSCH The Rockefeller Institute f o r Medical Research, Aew York, Aew York

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , , . . . . . . , . , . . _ . . . . . . . . . . . . . . 11. Rice Stunt V i r u s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Aster Yellows Virus. . . . . . . . . . . . , ., . . ... , , .. , . . . . . . . . . . A . Heat-Induced Incubation Period. . . . . . . , , . . . . , . . , . . , . , , , . , , . . . . . . . . . B . Assay of Virus in Insect Vectors. . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . C. Correlation of Incubation Periods.. . . . . . . . , . , . . :. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . D. Dosage Effect. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Effect of Low Temperature. . . , . . . . . , . , , . , . . , . . , . , , . .. . .. .. .. .. F. Serial Passage. . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Measurement of Virus Concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Clover Club Leaf Virus.. . . . . . . . , . , . . . . . . , , , . . , , . . . . . . . . . . . . . . . . . . . . . . . V. Wound Tumor Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Corn Stunt Virus.. . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Curly Top Virus. , . , . . . . . . . . . , . . , , . . . . . . . , . . . . . .. . .. . .. . . . . . . .. .. VIII. Possible Multiplication in Nonvector Species of Leafhoppers.. . . . . . . . . . . IX. Possible Multiplication in Other Groups of Arthropod Vectors. . . . . . . . . X . Conclusions.. . . . , . . , . . . . . . . . . . . , , , . . . . . . , . _ ., , , . . . . . . . . . . . . . . . . . . . . . . . A. Significance of Plant Virus Multiplication in Insects. . . . . . . . . . . . . . . . References.. . , . . . . . , , . , . . , , . . . . . . . . . . . , . . . . . . . . . . . . , , , . . . . . . . . , . . . . . . . . . . . . . . ,

221 223 225 225 227 229 229 229 230 232 233 235 237 238 241 243 245 245 248

I. INTRODUCTION Virus diseases of plants are, for the most part, arthropod-borne. The word vector is commonly used t o indicate the invertebrate transmitter necessary for the successful passage of certain disease agents from the infected to the noninfected host. The term derives from the Latin vehere, to carry. Arthropod vectors are quite often, however, not merely carriers; they may serve as alternate hosts or constitute important reservoirs of certain disease agents. Most vectors of plant viruses are insects of the families Aphididae (aphids), Cicadellidae (leafhoppers) and Aleyrodidae (white flies). Only a few viruses are transmitted by insects of other families, like the Coccidae (mealybugs) and the Thysanoptera (thrips) . Transmission of plant viruses by Acarina (mites), arthropods other than insects, was recently established (Slykhuis, 1953a,b; Flock and Wallace, 1955). In order to ascertain that an arthropod is a vector of a given plant virus in nature, the infection must be reproducible experimentally by use of the 221

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arthropod under controlled conditions through feeding it on susceptible plants with adequate uninfected controls. In practice, this test alone is generally considered sufficient for the establishment of a virus-vector relationship. Animal virologists usually require additional criteria such as a close association of the arthropod with the infected host, regular visits to the healthy host under conditions suitable for transfer of the virus, and the presence of the virus in the arthropod host (Meyer, 1953). These additional requirements are thought necessary because animal viruses, unlike most insect-borne plant viruses, are manually transmissible. Rare visits of an insect not ordinarily associated with a given plant may be sufficient to cause infection and the production of disease signs may appear many years thereafter when no insects of the vector species are present in the area. In cases where there is no close, easily observed association between vector and plant the discovery of the vector becomes a very tedious and difficult task. This is clearly manifested in a number of virus diseases of stone-fruit trees. In the transmission of plant viruses two major types of virus-host relationship are usually recognized. Vectors of one type may acquire a virus in short feeding periods and transmit it almost immediately. In every known case such vectors soon lose the virus. Vectors of the second type may also acquire virus by feeding for short periods on infected plants, although generally longer acquisition feeding is required. I n contrast to the first type, however, these vectors are unable to transmit virus to healthy plants immediately. An incubation period of considerable length occurs between the time of acquisition of virus and the time of its transmission to a susceptible host. Only after an incubation period of many hours, days, or even weeks, do vectors of this type become infective. Plant viruses are retained by such vectors for considerable lengths of time, often for the remainder of their lives, without need of replenishment from infected plants. The virus-vector relationship in this case represents a highly specialized adaptation of the virus to the arthropod host. This obligatory relationship between the vector and the transmitted virus is indicated by a high degree of specificity; in the majority of cases one or a few closely related species are found to transmit a given virus. This relationship is often called biological and the transmission is then known as biological transmission, Problems of plant virus transmission by arthropod vectors have been critically reviewed (Storey, 1939; Bawden, 1950; Black, 1953a,b, 1954; Maramorosch, 1953b, 1954). Recently Day reviewed the literature pertaining to the mechanism of transmission of both plant and animal viruses (1955) and the problems of specificity of arthropod vectors (Day and Bennetts, 1954). Excellent evidence presented by a number of workers in the past two

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decades has established beyond reasonable doubt that several plant viruses reproduce in their leafhopper vectors. The present chapter will be concerned with the multiplication of these viruses in Cicadellid leafhoppers.

11. RICESTUNTVIRUS The first experimental evidence of plant virus reproduction in insect vectors came from the work of Fukushi in Japan in the years 1935 to 1939 (Fukushi, 1940). I n 1933 Fukushi demonstrated that the rice stunt virus passed through eggs of infective females of Nephotettix apicalis to the progeny. This was the first known case of plant virus “inheritance” in a n insect and experiments were designed to find whether the virus was merely carried over to the progeny or whether it actually multiplied in the animal host. It was found that the virus did not pass to the progeny from the male parent. To insure that the insects used in the tests obtained no virus from plants where eggs were deposited, Fukushi removed each individual nymph while i t was in the act of emerging from the egg; thus the nymphs had no opportunity to feed on plants that may have contracted the disease in consequence of earlier infestation by the infective parents. The newborn nymphs were removed to fresh healthy plants by means of a sharpened pencil, the tip of which was slightly moistened. These nymphs were confined singly t o successive new healthy plants. This procedure required great patience and skill because the tiny nymphs were very sensitive and leaped with surprising agility. When the nymphs had grown to maturity, females were paired with virus-free males. No instances were observed in which viruliferous insects emerged from eggs deposited in infected plants by virus-free females. None of the insects derived from infective females infected plants earlier than 9 days after hatching. This information was important because in his most significant experiment Fukushi kept newly hatched nymphs for periods as long as 5 days on the first plant and then transferred them to fresh young rice plants on each succeeding day as long as they lived. Thus no virus could have been introduced into the first plant on which each insect fed for 5 days, nor could virus be recovered from that plant. Because rice stunt virus has an incubation period of at least 6 days in the plant and the average incubation period in Fukushi’s tests was between 9 and 13 days, it seems most unlikely that virus could have been obtained from any of the plants supporting the insects for a single day. It is the general experience (see, for instance, Maramorosch, 1952a) that leafhoppers can recover virus from plants only a day or so before the appearance of first disease signs. After 5 years of effort Fukushi demonstrated that the virus from a single female could be passed through the egg to 6 succeeding generations without replenishment from plants. The most significant single experiment in Fukushi’s work (Fig. 1) lasted 374 days,

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during which time 82 infective leafhoppers, in 6 generations derived from a single original viruliferous female, infected about 1200 plants. A calculation carried out by Black (1953b) showed that the dilution of original virus, if no multiplication occurred, would have reached at least 1:563,000. Black's careful analysis of Fukushi's experiments showed that an important underestimation resulted from Fukushi's experimental procedure; only the numbers of progeny picked up a t the time of hatching were reported and

mQ

( P P ? b t ? x - 0 0

g o b o o ?

()0 0 000 0 0 0 0 OiVIl Infective leaf hoppers

0 Noninfective leaf hoppers

8 FIQ.1. Diagram showing the descent of infective and noninfective leafhoppers in Fukushi's experiment No. 6. From Fukushi, 1940.

these numbers were much lower than the number of eggs one would expect a single female to lay. It is therefore quite likely that the actual dilution attained by Fukushi in the sixth generation surpassed l&*. However, the ability of the insects to infect plants showed no progressive decline. The data on this point are excellent because Fukushi transferred each insect to a fresh test plant each day as long as it lived. When the experiments were terminated, the infectivity of the tested leafhoppers was not less than a t their beginning; instead, the percentage of infective leafhoppers in late generations was similar to the percentage in the first generation. Fukushi fully realized the significance of his findings and concluded that the virus of rice stunt multiplied in the leafhopper vector. This interpretation was

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OF PLANT VIRUSES IN INSECT VECTORS

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so novel, however, that it met with adverse criticism from some workers (Bawden, 1950). 111. ASTER YELLOWSVIRUS The relationship of aster yellows virus to its vector, the aster leafhopper, has been studied for many years. I n 1924 Kunkel discovered that the virus is transmitted by Macrosteles fascifrons Stbl (known earlier as Cicadula seznotala Fall. or Macrosleles divisus Uhler). The relationship of the virus t o the insect vector has been comprehensively studied and today it is probably the best known virus-vector relationship in this group. Kunkel (1926), in his classical study of aster yellows, showed that the vector insect became infective only after the completion of an incubation period of 9 or more days. The virus was not transmitted through the egg of the progeny in this case, but it was retained during consecutive molts. Once the leafhopper became able t o transmit virus, it usually retained this ability for life without the necessity of fresh acquisition of virus. The relatively long incubation period and protracted period of retention of virus led Kunkel (1926) to suggest that the aster yellows virus might reproduce in the aster leafhopper.

A . Heat-Induced Incubation Period Kunkel (1937a) presented the first experimental evidence that aster yellows virus multiplies in its vector insect. Infective colonies of leaf-hoppers lost their ability to transmit yellows within 24 hr. after being placed in a thermostatically controlled hotroom that was kept a t about 32°C. The exposed insects did not regain infectivity while kept at that temperature. If, however, they were removed from the room after a 24-hr. treatment, they invariably regained ability to transmit during the succeeding 24-hr. period. If kept in the hotroom for 2 days they sometimes regained ability to transmit during the day following their removal. Sometimes, however, several days elapsed before they became infective. They never regained ability to transmit within a 24-hr. period when heat-treated for 3 days or longer. As a rule, the longer colonies were kept in the hotroom, the longer they took to recover their ability to transmit after removal from the room (Table 1). All colonies kept in the hotroom for 12 days or longer lost permanently their ability to transmit yellows. No evidence was obtained that heat treatments affected the ability of insects to become viruliferous when subsequently confined on yellows plants. Colonies in which the virus had not completed its incubation period lost permanently the ability to transmit after shorter heat treatments than colonies that were infective a t the time of treatment. Heat-treated colonies, instead of transmitting typical severe yellows, frequently transmitted mild strains of yel-

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lows. These findings indicated that long heat treatments caused inactivation of all the virus carried by infective insects and short heat treatments caused inactivation of a part only. The time required for insects to regain infectivity wm called the heat-induced incubation period. During this period that portion of the virus not inactivated by the treatment multiplied sufficiently to render the insects infective. The fact that heat treatments had a more marked effect on colonies in which virus was undergoing natural incubation than on colonies already infective was interpreted m being due to a lower concentration of virus in the former. Kunkel described also unusually long incubation periods in plants exposed to colonies immediately after the insects recovered ability to transmit. This suggested that these TABLE 1 EFFECTOF HEATTREATMENT O N ASTERYELLOWS VIRUSI N Macrosteles fascifrons Stal.* Temperature of treatment

Days of heat treatment

0

36°C. 36°C. 36°C. 36°C. 36°C. 36°C. 36°C. 36°C. 36°C.

* Compiled from data of

Days of heat-induced incubation period 1 3 3 6 8 13 23 4)

Kunkel, 1941.

insects carried less virus than those that had been infective for some time. According to this view, treatment lasting only 1 day would inactivate sufficient virus to render previously infective insects noninfective, but insufficient to prevent the insects from regaining infectivity after a few hours in which virus concentration would be increased by multiplication. Long heat treatments would inactivate more virus than short heat treatments, and any that might remain in the insects after long exposures to heat would require relatively long periods in which to multiply before it could reach a concentration that would render the insects infective. A still longer heat treatment would be expected to inactivate all the virus carried by the insects and to make loss of infectivity permanent. If the natural incubation period is a period during which virus multiplies in the insects, then the concentration during early stages of incubation would be less than the concentration on completion of incubation. Insects bearing virus at subinfective

MULTIPLICATION OF PLANT VIRUSES IN INSECT VECTORS

227

concentrations might be expected to lose it more readily when heat-treated than insects bearing virus a t higher concentrations. The results presented by Kunkel supported these views. These interpretations were strengthened in a number of ways. It was shown (Kunkel, 1941) that heat treatments were affecting the virus and not the insects. The virus could be inactivated in Vinca rosea L. plants by treatment in a hotroom a t 3842°C. for 2 weeks, or by immersion of plants in a water bath a t 4045°C. for a few hours. The aster leafhoppers, however, could be grown through their complete life cycle a t 35OC. (Kunkel, 1938). Leafhoppers that lost their transmitting ability by long heat treatments could be rendered infective readily by letting them feed on diseased plants.

B . Assay of Virus in Insect Vectors An entirely different type of experiment, carried out by Black in 1941, produced further evidence that aster yellows virus multiplies in its insect vector. A large virus-free colony of aster leafhoppers was maintained for 6 successive days on 6 healthy China asters. The exposed plants remained healthy. The insects were then caged on diseased plants that had been stripped of all leaves but those showing pronounced signs of aster yellows. Twenty-four hours later the colony was transferred to large healthy aster plants and transferred daily to fresh healthy aster plants for 30 days. The colony infected none of the plants for 16 days, but infected each of 2 plants on which it fed on each of the following 14 days. Counting the day on which the insects fed on diseased plants as the first day, 2 samples of 50 insects each were withdrawn a t random from the colony on the 2nd, 4th, Sth, 12th, and 16th days and tested for virus by subinoculation to virus-free aster leafhoppers. Uninoculated insects and insects inoculated with juice from insects which had been fed all their lives on asters with yellows were included as controls. I n order to test for virus, the following procedure was carried out a t 0°C. The sample of source insects was weighed and ground in a mortar. Sufficient neutral 0.85% NaCl solution was added to dilute the sample as desired. The suspension was cleared by low-speed centrifugation and minute quantities from each dilution were injected by glass capillaries into 120 young virus-free adults. These were then placed on immune rye plants and maintained in the greenhouse for 3 weeks while the virus passed through the incubation period. Surviving injected insects were then tested in colonies of 5 on aster plants; each colony fed on 1 aster plant for 1 week, and on a second aster plant for 2 weeks. The noninoculated control insects did not transmit the virus, whereas almost 100 % of the surviving inoculated control insects became infective. Titrations made on the second day of incubation were entirely negative, and the max-

228

KARL MARAMOROSCH

imum virus concentration was observed on the 12th day, that is, 6 days before the completion of the incubation period. Increasing amounts of virus were detected on the 4th and 8th day, and somewhat less virus on the 16th day. The comparative measurements of virus concentration were based on the ratio of insects rendered infective to the number of insects tested. The data (Table 2) indicated multiplication of the virus in the insects a t least a hundredfold between the 2nd and 12th days of the incubation period. During the incubation period the source insects were maintained on susceptible aster plants, none of which became diseased. It was impossible, therefore, for the insects to have introduced virus into these plants and to have withdrawn virus from them after its multiplication in the plant host. The increase of virus occurred solely in the insect vectors. TABLE 2 RECOVERY O F ASTERYELLOWS VIRUSDURINGINCUBATION PERIOD* Day of injection

Ratio of number of colonies infective to number of colonies tested

2

0/20

4 8 12

1/19 3/20 13/14

Control insects Uninoculated

Inoculated

0/17 0/18 0/19 0/18

17/19 18/18 19/19

20/20

* Colonies of five leafhoppers were inoculated with a 10-9 dilution of juice from insects fed on diseased aster plants for one day. Compiled from data of Black, 1941. Three additional experiments were carried out by Black shortly after the above results were published. The results of these supplementary experiments were consistent with earlier data and strengthened the evidence obtained previously (Black, 1953b). Black (1941), in his original publication, expressed the view that the only explanation other than multiplication for the results of these experiments was that the virus might be present in more readily dispersed form at one time in the incubation period than a t another. There is, however, no evidence to support this alternative interpretation, while all experiments of Black as well as those of other workers support the evidence for multiplication. As has been stated, the increase of aster yellows virus in vectors during the natural incubation period was demonstrated by using the number of insects rendered infective as a measure of virus concentration. Maramorosch ( 1 9 5 3 ~ )using ~ as an assay method the minimal lengths of incubation periods, confirmed Black’s findings. Maramorosch showed also that a

MULTIPLICATION OF PLANT VIRUSES IN INSECT VECTORS

229

similar increase of aster yellows virus occurred in insects which were inoculated by injection.

C . Correlation of Incubation Periods In 1948 Kunkel pointed out that the incubation period of aster yellows virus was of similar length in insects and plants at optimal temperature. Maramorosch (195313) found that this correlation holds also at temperatures lower than the optimal. This correlation was also explained on the basis of virus multiplication in the plant and insect hosts. Viruses with long incubation periods in plants have also long incubation periods in their leafhopper vectors, while those with shorter incubation periods in plants are characterized by shorter incubation periods in their respective vectors. D . Dosage Eflect Maramorosch (1950b) injected aster leafhoppers with varying dilutions of virus-containing insect juices. With a dilution of 1W1, the shortest incubation periods were 11, 14, and 15 days, respectively. With a dilution of 10-3 the minimal incubation periods were 24, 28, and 38 days, respectively. These results were interpreted on the same basis as those with animal viruses (Bryan and Beard, 1940; Card, 1940), that is, on the supposition that multiplication of a small amount of virus would take a longer time t o render the insect infective than multiplication of a greater amount. It was of considerable interest to find whether or not changes in the volume of inoculum without changes in concentration would influence the length of incubation period of aster yellows virus in injected insects. When tobacco mosaic virus is inoculated mechanically into a tobacco leaf, differences in concentration can be detected easily by the local-lesion test, but differences in the volume of inoculum have no appreciable influence on the number of lesions. However in an animal virus, that of rabbit papilloma, the length of incubation period, which can be used as a measure of active virus, varies both with concentration and with volume of the inoculum (Bryan and Beard, 1940). Aster yellows virus can be considered as both a plant and an animal virus because the virus multiplies in both hosts. Experiments with varying volumes of inoculum (Maramorosch, 1953d) gave results similar to those of earlier tests with varying concentrations. In both cases higher doses resulted in shorter incubation periods.

E . Eflect of Low Temperature The shortest incubation periods of aster yellows virus have been obtained a t 25"C., which can be considered as the optimal temperature for multiplication of this virus. At lower temperatures the length of incubation increases considerably. The writer (1953b,d) inoculated leafhoppers at 25°C.

230

KARL MARAMOROSCH

and placed them after 2 days in an icebox maintained at 4°C. Every week groups of 10 to 15 insects were removed from storage and tested at 25°C. in temperature control chambers. An average of from 6 to 9 days elapsed before the leafhoppers became infective, irrespective of the time spent at 4°C. Exposure to 4°C. had apparently arrested or interrupted the processes necessary for the completion of incubation. It seemed unlikely that movements of virus were influenced to such an extent by low temperature. It was concluded that the low temperature had a direct effect on arresting the multiplication of the virus in the inoculated insects. F . Serial Passage The evidence adduced above was derived from a variety of approaches, some of which were of an indirect nature. In the past the interpretation of certain of these experiments has met with adverse criticism (Bawden, 1950). It should be kept in mind that interpretation on some other basis than multiplication would have been feasible in the absence of additional, direct evidence. It seemed that the question of whether or not any given plant virus multiplied in its vector could easily be settled if the virus were carried in serial passages from insect to insect with the inoculum adequately diluted at each passage. Such a test had been made earlier for the animal virus of equine encephalomyelitis (Merrill and TenBroeck, 1934) but no similar evidence was available at that time for any plant virus. The discovery by Black (1940) that aster yellows virus could be transmitted by needle inoculations to aster leafhoppers made it possible to obtain such evidence for aster yellows virus in its carrier insect. Black had already, in 1939-1942, attempted such passages (Black, 195313) but high mortality of insects invariably encountered in the second group of injected insects prevented the successful outcome of these earlier tests. In 1951 Maramorosch applied the mechanical inoculation technique to a serial passage experiment and succeeded in carrying the virus serially through 10 groups of leafhoppers (1952a). The experiment (Table 3) was started with 100 viruliferous aster leafhoppers, weighing altogether 140 mg.; a microsyringe was employed to inject virus-free insects with measured amounts of known dilutions of the source leafhoppers. It was calculated that the original virus would be diluted approximately to lW4 at each passage and that it would become less concentrated in successive passages unless it multiplied to a like extent. Between passages the injected insects were kept on caged plants at 25°C. This was probably important for the success of the experiment. During each of the first 6 passages the injected insects were kept for 30 days on rye, which is immune to infection by the virus. The survivors were then tested individually on susceptible aster plants for 2 days before being used as a source of virus for subsequent pas-

231

MULTIPLICATION OF PLANT VIRUSES IN INSECT VECTORS

sages. During the last 4 passages the insects were kept for long periods on asters. They were transferred to fresh, healthy plants 3 times each week in some cases, and every 5 days in other cases. Virus could not have been replenished from the rye plants on which the insects fed because rye is immune to aster yellows. The immunity of rye to the virus, which was established by Kunkel in 1926, has been repeatedly tested and was also retested by the writer during TABLE 3 SERIALPASSAGE OF ASTER YELLOWS VIRUSTHROUGH

ASTER LEAFHOPPER!

Days after injection on:

Passage*

No. of injected insects

1 2 3 4 5 6

200 300 150 300 100 200

30 30 30 30 30 30

7 8 9 10

100 240 50 50

6 40 19 30

Rye

Asterst

A

B

THE

Survivors$

Calculated dilution 10-a .a 10-8 10-12 10-10 10-20 10-24

54'7 25 13" 20

10-*8 10-32

10-36 10-40

* The diluted insect juice in the fourth passage was filtered through sintered glass. Penicillin was added in the fourth, fifth, sixth, and seventh passages. t Individual insects were transferred t o individual aster plants : a three times weekly; * every 5 days. $ Number of infective survivors in parentheses. In series A, all 100 control insects for each passage survived, and in series B all 20 control insects for each passage survived. From Maramorosch, 1952a. the serial passages. Rye plants never develop yellows and virus cannot be recovered from rye plants on which infective insects have fed. Virus could not have been replenished from the aster plants fed on by the insects in the series because Kunkel (1948) and Maramorosch (1952a, 1953d) showed that the minimum incubation period in asters was 9 days. Maramorosch (1952a) found that insects could not recover virus from plants until the 7th day after inoculation. The dilution of original virus used for injection in the 10th passage would have reached yet tests indicated that there was just as much virus present at this passage as in the 1st passage (Maramorosch, 1952~). The successful inoculation, therefore, attained in the

232

KARL MARAMOROSCH

10th passage with a final dilution of in terms of the original virus provided conclusive evidence that the virus had multiplied in the insect. It should be pointed out that the actual dilution of the virus during serial transfers was unknown because the virus did multiply. Evidence for multiplication has been strengthened further by more recent experiments (Maramorosch, 1955a). Although it seemed highly improbable that the insects in Maramorosch’s experiment obtained aster yellows virus from plants during the course of the tests, Storey (1928), working with maize streak virus, was able t o recover virus by nonviruliferous vector insects feeding on the same leaf as infective insects during periods of 1 or 2 days. Therefore, 4 additional serial passages of aster yellows virus were carried out by a somewhat different experimental plan in which susceptible aster plants were completely eliminated. It was found that aster yellows virus can be carried from insect to insect in a series without access of inoculated leafhoppers to any plants that are susceptible to the virus. At each transfer the dilution of insect pulp by weight was 1:100. The dilution endpoint of virus in leafhopper juices has been found to be below W4(Black, 1941). It was concluded, therefore, that the transfer of virus for more than 2 passages could succeed only in the event of virus multiplication in the insect vector. Success in carrying the virus through 4 successive passages was, therefore, highly significant.

G . Measurement of Virus Concentration Further, additional evidence for the multiplication of aster yellows virus in vector insects came from measurements of relative virus concentration in the inocula of leafhoppers during serial passages (Maramorosch, 1952~). Measurements of virus concentration were made in samples of insect juices used for the injection of the lst, 7th, and 9th passages. These measurements employed a method, based on the relationship between the amount of injected virus and the length of incubation period, which had been tested previously under controlled environmental conditions (Maramorosch, 1950b). The volume of inoculum, 1/8000 ml. per insect, was delivered by a calibrated microsyringe. Individual leafhoppers, fed through screens of small cages fastened to leaves, were transferred to fresh sets of young test plants at daily intervals or 3 times weekly. Inocula for the 7th and 9th passage were made from insects maintained on test plants for periods up to 60 days. The concentrations of virus in the inocula were found to be similar to the concentration of the original inoculum. Measurements of virus concentration were made also in 4 more recent serial transfers in which the aster leafhoppers were maintained solely on immune rye plants (Maramorosch, 1955a). The concentration of virus in the inoculum used for the original injection was somewhat higher than in

MULTIPLICATION OF PLANT VIRUSES IN INSECT VECTORS

233

those used in later injections, presumably because a greater number of insects used in the preparation of the inoculum for the 1st transfer were infective than was the case among those used for inocula in later transfers. These recent measurements provided additional evidence for the multiplication of this plant pathogen in its insect vector. IV. CLOVER CLUBLEAF VIRUS I n 1943 Black (1944) collected a small number of Agallian leafhoppers in the vicinity of Washington, D. C. When the insects were tested in a greenhouse a t the Rockefeller Institute for Medical Research, it was found that they carried 2 strains of potato yellow dwarf virus and 2 other, previously unknown, viruses. One of the newly discovered viruses was named clover club leaf virus (Aureogenus clavijolium Black), and the other wound tumor virus (Aureogenus magnivena Black). Subsequently both were found to multiply in their respective vector insects. Black (1948) found that the clover club leaf virus passes through the egg of its vector, Agalliopsis novella (Say), in a high proportion of the progeny. This significant finding was followed up by Black, who immediately decided to carry out a n experiment on the serial passage of this virus through many generations of vectors. His experiments resembled Fukushi’s tests with rice stunt. However, Black was able to profit by the earlier work of Fukushi in planning the clover club leaf passages. I n particular, an attempt was made to determine the total number of progeny from each female and the proportion of progeny that was infective. Since the critical information desired was whether or not each insect was infective, no tests were necessary to see how many plants could be infected by any particular insect. Precautions were taken against the accidental termination of the experiment. Additional colonies were maintained as a reserve in each generation, and the viruliferous females were mated with virus-free males from a stock colony to prevent possible deleterious effects of inbreeding. The experiment (Table 4) was begun by Black in 1945 by the mating of a single viruliferous female leafhopper with a virus-free male. The pair was caged on Grimm alfalfa, immune to the clover club leaf virus. Of 42 nymphs from the progeny, 21 were tested individually on crimson clover (Trijolium incarnatum L.) plants and 15 produced infections. When the nymphs changed into adults, the females were mated to virus-free males. The pair that produced the greatest number of progeny was always chosen to continue the main line of descent, thus providing a basis for selection independent of virus concentration. This method also secured a record of the greatest possible dilution of the original virus. Supplementary families from additional pairs were held in reserve and discarded as soon as the main line of descent proved infective. Samples removed from the progeny

234

KARL MARAMOROSCH

and tested on susceptible plants permitted the determination of the fraction that was infective. The experiment was continued for more than 5 years TABLE 4 O F CLOVER CLUB LEAFV I R U S D U R I N Q P A S S A G E CALCULATED MINIMUM DILUTION THROUGH THB EGGO F ITS INSFCT VECTORFOR 21 GENERATIONS* Reciprocal of dilution

Generation

Number of progeny

Infectivity tests

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

42 101 35 54 60 89 104 106 64 173 88 31 52 59 129 60 114 50 107 128 105

15/21 6/15 3/15 9/15 3/16 9/15 6/15 15/20 3/20 5/11 6/21 2/11 0/15 1/10 1/5 1 1 /20 2/15 2/15 1/10 5/10 1/10

Each generation

30 40 7 32 12 53 42 80 10 79 25 6 0 (l>t

6 26 33 15 7 11 64 11

Total

30 12 x 101 84 X 10' 2688 X 104 32,256 X 102 1,709,568 X lo* 71,801,856 X lo4 574,414,848X lo8 574,414,848X 10* 453,787,729X loE 113,446,932X 108 680,681,594X 108 680,681,594X 108 408,408,956X lo9 106,186,328X 1Ol1 350,414,885X loL4 525,622,327X loL* 367,935,629X 404,729,192X lot6 259,026,683X loL7 284,929,351X 10l8 or 2.8 X loz6

From Black, 1950. ?This was the only point where the absence of virus in the family with the largest number of progeny necessitated shifting the main line of descent t o the family with the second largest number of progeny. It is an anomaly that the test of this latter family happened to be negative and that of the abandoned family (not shown) happened t o be positive in this generation. In spite of this negative test a t this point it is permissible to multiply the reciprocal of the dilution by 1 instead of 0 because the occurrence of infective descendants means that at least 1 of the progeny in this family waa infected.

through 21 generations of insects grown only on immune alfalfa plants. Infectivity was retained throughout the experiment. Thus it was demonstrated that the virus can be maintained essentially indefinitely in its insect vector. The dilution of the original virus exceeded 1:2.8 X lo2' on a most

MULTIPLICATION

OF PLANT VIRUSES IN INSECT VECTORS

235

conservative estimation (Black, 1950). Actually, Black’s calculation purposely did not take into account systematic errors underestimating the dilution of the virus. So, for instance, tests revealed only a part of the insects which were viruliferous and no provisions were made to account for virus left in the mother and for virus in insects of the progeny that died prematurely. Excellent controls were provided in these tests. Alfalfa plants were grown in steamed soil to kill weed seeds. Virus-free insects were obtained by selection from insects collected in the field, as well as by heat treatment. It was concluded that the persistence of infectivity through 21 generations must mean that the virus multiplied in the leafhopper vectors. Recently clover club leaf virus was mechanically transmitted to clover leafhoppers (Maramorosch, 1955~). The incubation period in injected insects was comparatively long, which indicated a rather slow multiplication of the virus in its vector. This extended period of multiplication might perhaps have been responsible for the lack of virus transmission by a few insects in Black’s experiment; although these insects carried the virus to their progeny, they were not themselves infective. VIRUS V. WOUNDTUMOR Indirect evidence of the multiplication of this virus in an insect vector was first obtained by Maramorosch (1950a). The evidence was based on three findings. The first concerned lengths of incubation period. The wound tumor virus can be transmitted mechanically to its vector (Maramorosch et al., 1949) and this technique was used to test the effect of dosage on the length of incubation period. A dilution of 10-’ resulted in a surprisingly short incubation period of only 7 days, whereas dilutions of and 10-3 gave minimum incubation periods of 11 days, and required a 19-day minimum period. Thus this virus also reached an infective concentration in a shorter time when larger amounts of virus were injected. The second indirect approach to the problem was made by exposing insects, after they acquired virus by feeding, to a temperature of 0°C. Insects were removed from the icebox after 4-, 5-, and 6-week periods. When tested a t 25°C. they were unable to infect healthy plants immediately but became infective during subsequent weeks. The incubation periods were lengthened by a time equivalent to the duration of exposure to 0°C. Here again, as in the case of aster yellows virus, it seemed unlikely that differences in osmotic and diffusion processes a t lower temperatures could have accounted for the observed lengthening of the incubation period and the results suggested virus multiplication in the insect (Maramorosch, 1949). The third finding indicating multiplication in leafhopper vectors was derived from a study of incubation periods in plants and insects a t various

SERIAL TRANSMISSION OF WOUNDTUMOR VIRUSTHROUGH

TABLE 5 INSECT VECTOR (Agallia constricta) GROWNO N IMMUNE ALFALFA*

AN

Infectivity of insects inoculated with insect juice at indicated dilutions

Passage

10-7

10-6

10-5

10-4

0/24t 0/24

0/21 -

0/17 0/28 1/16 1/61 1.6

1/20 1/28 3/20 0/20 1/27 0/25 2/30 8/170 4.7

10-3

10-2

10-1.5

Fraction of original virus used Uninoculated as source for each control dilution series insects (minus log)

~~

1 2 3 4 5 6 7

Total infective Percentage infective

0

-

0/21 0

1/20 5/27 4/20 3/27 5/29 8/28 8/28 34/179 19.0

6/20 11/27 11/30 15/28 17/30 60/135 44.4

9/30 6/28 4/10 -

19/68 27.9

* From Black and Brakke, 1952. t Numerator is the number of insects infective; denominator is the number of insects tested.

0.00 3.06 6.05 8.76 12.13 15.24 18.32 -

0/30 0/27 0/20 0/24 0/25 0/27 0/28 0/181

0

K! Ti

MULTIPLICATION

OF PLANT VIRUSES IN INSECT VECTORS

237

controlled temperatures (Maramorosch, 1950a). A correlation in length, similar to that described above for aster yellows virus, was found to occur with wound tumor virus. Convincing evidence for the multiplication of the wound tumor virus in an insect vector was obtained in 1952 by Black and Brakke, who confirmed the usefulness of Maramorosch’s serial passage method but by means of a somewhat different experimental plan (Table 5 ) . The original source of virus consisted of juices of infective adult Agallia constricta Van Duzee leafhoppers. The needle inoculation technique (Maramorosch et al., 1949) was used, with certain improvements, for the mechanical transmission of the virus to insects. Various dilutions were injected into virus-free leafhoppers kept later on immune Grimm alfalfa plants. Two weeks after inoculation most of the injected insects were tested for infectivity on susceptible crimson clover plants. The remaining insects were maintained on immune plants for one month and used afterwards as a source of virus for succeeding passages. The dilution of the original virus was calculated and titrations of virus concentrations were made a t each transfer. Eventually the original virus source mas diluted to approximately without loss of infectivity, although actually wound tumor virus was never recovered from insect juices a t dilutions beyond lW5. Besides the usual, conventional controls, an ingenious and novel method was introduced for the first time in these experiments. Genetically marked color mutants (Teitelbaum and Goulet, 1950) of infective vectors were kept on the same alfalfa plant on which virus-free females of a different marking were maintained. Although the infective and virus-free females as well as their progeny fed on the same plant, only the progeny of infective females were infective (Black, 1953b). These experiments proved conclusively that alfalfa was immune in the sense that it would not acquire the virus and return it to vector insects. The serial passage of wound tumor virus provided direct evidence for the multiplication of this virus in one of its insect vectors. Incidentally, it was shown that a small percentage of progeny received wound tumor virus from their mothers through the egg (Black, 1953~). VI. CORNSTUNTVIRUS The fifth virus requiring discussion in this group is the virus causing corn stunt disease. Kunkel found in 1946 that in the United States corn stunt virus is transmitted by the leafhopper Dalbulus maidis Del. and Wol. The incubation period of the virus in the insect is at least 14 days. I n 1948 Kunkel pointed out the correlation between the lengths of incubation periods of this virus in corn plants and in corn leafhoppers. The present writer (1954) found that this correlation holds at various temperatures.

238

KARL MARAMOROSCH

In 1951he transmitted corn stunt virus to corn leafhoppers by needle inoculations, thus providing a technique for serial transfers from insect to insect. Although experiments carried out by the writer (195213) were not specifically aimed to demonstrate virus multiplication, the data from 3 serial passages (Table 6) indicated that this virus, like aster yellows and wound tumor viruses, can be carried from insect to insect indefinitely due to its multiplication in the vector. At each transfer a 1: 100 dilution of insect pulp was prepared. Some injected insects in all 3 groups became infective, while SERIAL PASSAGE OF

TABLE 6 Dalbulus maidis

CORN STUNT V I R U S I N

BY

MEANSO F

INSECT

INJECTIONS* Transmission record of injected insects transferred individually once every Total of Total of week t o fresh plants1 Passage exposed infected No.? plants plants Weeks

Control insects$ Weeks 1st

6th

0/50 0/50

0/29 0/32 0/41

___-__

0/26 0/32 0/29

0/21 0/26 0/24

1/17 4/25 0/20

3/17 6/18 2/17

112 114 122

4 10 2

0/50

* From Maramorosch, 1952b. t Diluted juices from 20 viruliferous insects were used as source of virus for the first group. The inoculum for the second and third group was obtained by maceration and dilution to 1: 100 of all insects of the first and second group, respectively, which survived 30 days. 1The numerator denotes the number of infected plants, the denominator the total number of tested plants. 0 The numerator denotes the number of infective insects, the denominator the total number of tested insects.

all controls proved virus-free. In each group approximately 6 weeks elapsed between the day of mechanical inoculation and the day the insects were rendered infective. These incubation periods were indicative of the virus concentration, which apparently reached the same level in each group of the 3 serial passages. VII. CURLYTOPVIRUS Most leafhopper-borne viruses are considered to have substantial incubation periods in their vectors. Absence of such a period might indicate lack of virus multiplication in the leafhopper, while presence of an incubation period of considerable length might be presumed to indicate multi-

MULTIPLICATION OF PLANT VIRUSES IN INSECT VECTORS

239

plication. There are, .however, a few leafhopper-borne viruses characterized by rather short incubation periods. An element of uncertainty about these viruses may be justified, because the evidence that they do not multiply in their vectors is, of necessity, negative. One of the most thoroughly studied viruses in this group is the virus causing curly top of sugar beets. For many years the possible multiplication of this virus in its vector, Circulifer tenellus, has been a topic of speculation. I n 1915 Smith and Boncquet found that virus-free leafhoppers were unable to transmit curly top immediately after feeding on diseased plants; 1 or 2 days elapsed before the beet leafhoppers could transmit the causal agent of the disease. Severin (1921) found that at a temperature of 100°F. (38°C.) the incubation period of the virus in the vector was occasionally as short as 4 to 6 hr., whereas at lower temperatures the incubation periods were considerably longer. In 1924 Carsner and Stahl pointed out that most insects became able to transmit the virus after a longer incubation period than 24 hr. although occasional insects transmitted it after shorter periods. Although Severin (1931) reported that curly top virus could be transmitted in as short periods as 20 min., 30 min., 1 hr., 1% hr. etc., these findings have not been confirmed by other workers. The minimal incubation period of approximately 4 hr. in the beet leafhopper was confirmed in 1938 by Bennett and Wallace. I n 1936 Freitag, and in 1938 Bennett and Wallace, reported that infective beet leafhoppers frequently lost the ability to infect plants. Former transmitters could reacquire virus from plants and transmit it once again. Longer acquisition feeding periods increased the efficiency and duration of transmission. Giddings (1950) found that an individual insect could lose and reacquire several strains of curly top virus independently. It is obvious that the relationship of curly top virus to its vector differs in many respects from the relationship of several other leafhopper-borne viruses to their respective carriers. The gradual ceasing of transmissions by infective insects, the relation of the length of feeding time on diseased source plants t o the length of virus retention by beet leafhoppers, the ability of insects which have lost infectivity to become reinfected by feeding on diseased plants, and the independent acquisition and loss of different strains of the curly top virus by individual insects has led almost all workers in the field to accept the view that curly top virus does not multiply in its vector. Lack of multiplication has, however, never been proved crucially despite many statements in the pertinent literature. Gradual loss of transmitting ability is not uncommon with other leafhopper-borne viruses, for instance, in aster yellows virus (Kunkel, 1926, 1954). Despite such losses, virus may well have multiplied and it may have been present even in leafhoppers that became unable to infect plants. Fukushi (1940) demonstrated that some leafhoppers, although carrying

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rice stunt virus during their entire lives and passing the virus through the eggs to the progeny, never transmitted it to plants. Bennett and Wallace (1938) presented evidence that infectivity of insects originally transmitting curly top virus decreases with the passage of time and that the virus may actually be lost from some lots of beet leafhoppers. This evidence was derived from results of attempts to recover virus from extracts of leafhoppers that fed for periods of different durations on immune plants. These tests constitute a valuable contribution to the problem of virus retention in the beet leafhopper but the findings by no means prove lack of multiplication. Insects are small animals and the pattern of infection in animal virus diseases often differs from that of plants. In the majority of animal virus diseases no recovery of the infective agent is possible after the acute stages of infection are over, whereas in diseased plants such infective agents can usually be recovered throughout the life of the host. Although there is no evidence of antibody formation against viruses in arthropods (Bernheimer et al., 1952), such antibodies are produced in higher animals; however, the disappearance of viruses seems often to be unrelated to antibodies. For instance, herpes simplex virus in man, vaccinia virus in rabbits, and equine encephalomyelitis virus in horses have been recovered in the presence of high antibody titers. In fact, the high titers of antibodies in certain animal virus diseases like ectromelia, yellow fever, or poliomyelitis are probably due to the constant presence of these viruses in the infected host. The eventual “disappearance” of most viruses from animals, for example influenza and smallpox viruses from infected human beings, certainly does not indicate lack of virus multiplication in such hosts. If an accurate quantitative titration of curly top virus were available, the question of multiplication could easily be solved. In many animal virus infections it is possible to follow the rise to a peak concentration, which is not related to the size of the initial dosage, although the time the peak is reached may depend on this dosage. In the beet leafhopper there is no evidence of multiplication to a peak concentration unless it is assumed that the peak concentration coincides with the end of the incubation period. The ability of the beet leafhopper to remain infective for longer or shorter periods depending on the length of acquisition feeding time has been interpreted on the basis of “charging” of vectors with larger or smaller doses of virus (Bennett and Wallace, 1938). This explanation was critically discussed by Kunkel(l954) in a recent paper. The existence of a relationship between acquisition feeding time and the retention of the curly top virus does not deny the possibility of multiplication in the vector. The relationship of the curly top virus to its insect vector presents many unsolved problems. Recently a new technique for the study of this relationship was developed (Maramorosch, 1955b). The curly top virus was

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transmitted mechanically to its insect vector by needle inoculation, using an improved microinjector. It was found that mechanically inoculated insects could transmit curly top virus only after an incubation period, which varied with the dose of inoculum. The introduction of virus-containing juices a t a 1:30 dilution resulted in incubation periods of from 1 to 9 days, while injection of a 1:300 dilution was followed by incubation periods of from 5 t o 20 days. Although there were considerable variations in the incubation periods of the virus in insects injected with one as well as the other concentration and the mean incubation did not differ significantly, the value for the shortest periods in the 2 groups indicated a n effect of dosage similar t o the one found earlier with aster yellows and wound tumor viruses. These minimal values in the curly top virus were 1 day for the 1:30 dilution and 5 days for the 1:300 dilution. It is hoped that future tests with groups rather than individual insects will permit use of lengths of incubation periods as comparative measures of curly top virus concentration in cell-free extracts. The results of the experiments suggest that curly top virus may multiply in its vector just as do the viruses of wound tumor or aster yellows in their respective vectors. Other explanations are possible but this simple one seems the most logical interpretation of the findings. Although curly top virus, injected into leafhoppers, persisted for long periods, the incidence of infection for mechanically inoculated insects was much lower than would be expected with insects that acquired their virus by feeding a relatively short period on curly top plants. It is hoped that eventually the technique of curly top virus transmission will be used for a serial transfer of this virus from insect to insect, similar to earlier transfers of aster yellows virus and wound tumor virus. This type of experiment could provide the most conclusive evidence for or against multiplication. MULTIPLICATION OF VIRUSESI N NONVECTOR VIII, POSSIBLE SPECIES OF LEAFHOPPERS Attempts t o transmit specific viruses by leafhopper species known to transmit other viruses have been undertaken repeatedly by many investigators. It has been found that leafhopper-borne viruses are usually highly specific and ofteii only one or a few closely related species are able to transmit a given virus. Severin (1934, 1945, 1846, 1947a, b, 1948, 1950) described several leafhopper species in a number of different tribes of the Cicadellidae as vectors of California aster yellows virus, but only a few species have proved to be efficient vectors. It seems possible that leafhopper vector specificity is mainly due to the ability of a given virus to multiply only in certain vector insects. An experimental attempt to test

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this working hypothesis was made by Maramorosch (195213). He tried to find whether or not the differential transmission of aster yellows and corn stunt viruses by certain vectors could be explained on the basis of virus multiplication in vectors and lack of it in the nonvectors. The findings indicated that aster yellows and corn stunt viruses either were unable to multiply in nonvector leafhoppers or multiplied to a very limited extent. Serial passages in nonvectors were tried without success. However, the unstable aster yellows virus could be recovered after a long interval from corn and clover leafhoppers. Perhaps some barrier prevented passage of virus from the blood to the stylets. In more recent experiments (Maramorosch, unpublished data) a comparison was made of virus retention by aster leafhoppers which acquired aster yellows virus while feeding simultaneously with the nonvector leafhoppers. It was not possible to recover aster yellows virus during the first few days after the insects acquired it by feeding. There was a slow loss of virus from nonvector leafhoppers, whereas it persisted in the body of vector insects throughout life. This could be seen from titration of virus recovered after 42 days and after 21 days. Five out of 5 colonies became infective after injection of insect juice from the 21-day and from the 42-day group of aster leafhoppers. Three out of 5 colonies and 1 out of 5 became infective after the injection of juice from corn leafhoppers which retained the virus for 21 and 42 days, respectively. In addition, the incubation period of the virus in aster leafhoppers injected with virus from aster leafhoppers varied in both groups from 20-28 days, as compared to 30-38 days for the 21-day group and 66 days for the single colony rendered infective in the 42-day group of insects injected with corn leafhopper juice. The dilution of insect pulp in all transfers wae 1:300. The long incubation period of 66 days indicated that the amount of aster yellows virus retained for 42 days in the nonvector species was extremely small. Aster yellows virus was recovered from its vector up to a dilution of 1:1O00, whereas from nonvector species it could be recovered only up to a dilution of 1:100. The lower concentration in nonvectors, resulting from little or no multiplication, seemed to be responsible for the inability of these species of insects to transmit this virus. It seems that the amount of virus acquired by nonvectors is at first adsorbed to some tissues of the insect body and later gradually released into the blood. The lack of viral antibodies in insects may explain the long retention. An alternative explanation for the disappearance of virus in nonvectors was also considered. The virus may perhaps undergo a change from infective to noninfective (Maramorosch, 1953c) and subsequently mature; however, in contrast to the virus in its proper vector, multiplication may be lacking or may be very limited.

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The possibility of a mechanical barrier in the gut wall of insects to the penetration of virus was investigated. Puncturing was applied to the nonvectors which acquired corn stunt and aster yellows virus by feeding. Nonvectors could not be changed into vectors by needle puncturing. These findings confirm earlier observations by Storey (1933) and also support the conclusion that biologically transmitted viruses can be transmitted only by insects that are susceptible to them, that is, by insects in which the viruses find suitable conditions for multiplication. It is not yet known how viruses penetrate through the gut wall of vector leafhoppers. It is possible that the viruses play an active role; the infective particles of some viruses seem too large to diffuse through semipermeable membranes (Black, 1955; Black el al., 1948; Brakke et al., 1951, 1953, 1954). Perhaps the ability to pass through the gut wall plays a part in determining vector specificity. It is well-established that nonvector species of insects acquire viruses while feeding on diseased plants. Some of the more stable viruses, like maize streak virus (Storey, 1933) and curly top virus (Bennett and Wallace, 1938; Smith, 1941) have been recovered from nonvector species as late as 14 days after acquisition. Also the virus of aster yellows, although very unstable in vilro, was recovered from nonvector species of leafhoppers after considerable lengths of time (Maramorosch, 1952b). Permeability of the gut wall plays a role in the case of active and nonactive races of Cicadulina mbila (Storey, 1933) but puncturing of the gut wall has not transformed nonvector species into vectors (Storey, 1933; Maramorosch, 1952b). These negative results indicate that other requirements in addition to gut wall permeability have to be fulfilled. One of these necessary requirements seems to be the ability to multiply in the insect body.

IX. POSSIBLE VIRUSMULTIPLICATION IN OTHERGROUPSOF ARTHROPOD VECTORS At present, experimental evidence for multiplication of plant viruses in insect vectors is limited to a single group of Cicadellid leafhoppers. I n addition to the cases discussed above, multiplication of viruses in many other leafhoppers is likely to be established in the future, particularly in instances of transovarial virus transmission, where proof of multiplication is relatively easy. Transmission through the egg may be a much more common phenomenon in leafhopper-transmitted viruses than previously thought, as indicated by recent findings of Black (1953~)and Grylls (1954). Aphids transmit the largest number of plant viruses but only a few viruses spread by aphids are transmitted biologically. The majority of aphid-borne viruses are “nonpersistent” (Watson and Roberts, 1939; Bawden, 1950), but a few are known to have incubation periods in their vectors

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and these viruses also persist in aphids. Pea enation mosaic virus, strawberry yellow etch virus, lily symptomless virus and potato leafroll virus are in this small group. It seems probable that these viruses may multiply in their vectors but as yet no experimental evidence has been reported in favor of this assumption. In a recent paper, Day (in press, personal communication) reports experiments which are interpreted on the basis of multiplication of potato leafroll virus in its vector Myzus persicae. Only one virus, that of spotted wilt, has been demonstrated beyond doubt to be transmitted by thrips (Bald and Samuel, 1931). The incubation period of this virus is of considerable length, at least 5 days. The virus persists in the vector for life. Among many species of thrips only 3 have been found capable of acting as vectors. The possibility of multiplication of tomato spotted wilt virus in its vector has never been tested experimentally. In tropical and subtropical areas of the world, white flies (Aleyrodidae) play an important role as vectors of plant viruses. The white flies acquire a number of viruses which are spread biologically by the adult form, while nymphs remain stationary, attached to plants. Among the white-fly-borne viruses is the causative agent of infectious chlorosis. This disease was long considered a mystery and geneticists cited it as an example of a plasmagene originating de novo as a result of union of related proteins following grafting of two species (Darlington, 1944). I n 1946 Orlando and SilberSchmidt in Brazil found the vector, Bemisia tuba&, and described the biological relationship of the virus to this white fly. Another virus, causing Euphorbia mosaic in Brazil (Costa and Bennett, 1950), and one causing Bhendi mosaic in India (Varma, 1952) are also transmitted by species of white flies. These are only a few examples of white-fly-transmitted viruses that are characterized by an intrinsic incubation period in the vector and seem to be retained in the arthropod for considerable time. No work on the possible multiplication of plant viruses in white flies has been published. Mites (Acarina) were known as vectors of animal viruses and of rickettsiae for many years but none were proven as vectors of plant viruses until 1953. In that year Slykhuis (1953a, b) presented evidence that a mite, Acem'a lulipae, transmits the virus of wheat streak mosaic. Apparently wheat streak virus can be transmitted after very short periods (Slykhuis, 1955). There are no indications that multiplication occurs in the mite vector. It seems likely that certain viruslike diseases of plants earlier reported as due to feeding or association with mites are caused by viruses. Recently Flock and Wallace (1955) reported the transmission of fig mosaic virus by the citrus mite Acem'aJicus. It remains to be learned whether mite-borne plant viruses do, or do not, multiply in their vectors.

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X. CONCLUSIONS A . Significance of Plant Virus Multiplication in Insects The direct and indirect evidence for the multiplication of 5 plant viruses in their respective vectors summarized in Table 7 leaves no doubt that these viruses reproduce themselves in both plant and arthropod carriers. TABLE 7 EXPERIMENTAL EVIDESCEFOR MULTIPLICATION O F PLANT VIRUSESI N LEAFHOPPER VECTORS Virus (common name) Rice stunt Aster yellows Aster yellows Aster yellows Aster yellows Aster yellows Aster yellows Aster Aster Aster Aster

yellows yellows yellows yellows

Clover club leaf Wound tumor Wound tumor Wound tumor Wound tumor Corn stunt Corn stunt Corn stunt

Method of demonstrating viral multiplication Transovarial passage (7 generations) Heat-induced incubation period Titration of virus, acquired by feeding, during incubation Correlation of incubation period in plant and insect Correlation of incubation in plant and insect a t various temperatures Effect of concentration on length of incubation Titration of virus acquired by feeding as well as by injection Serial passage (10 transfers) Serial passage (4 transfers) Effect of volume on incubation Arrested incubation period a t 0°C. Transovarial passage (21 generations) Arrested incubation period a t 4°C. Effect of dosage Correlation of incubation in plants and insects at various temperatures Serial passage (7 transfers) Correlation of incubation in plant and insect Correlation of incubation a t various temperatures Serial passage (3 transfers)

Reference Fukushi (1940) Kunkel (1937a, 1941) Black (1941) Kunkel (1948) Maramorosch (1954) Maramorosch (1950a) Maramorosch (1953~) Maramorosch Maramorosch Maramorosch Maramorosch

(1952a, c) (1955a) (1953d) (1953d)

Black (1950) Maramorosch (1949) Maramorosch (1950a) Maramorosch (1950a) Black and Brakke (1952) Kunkel (1948) Maramorosch (1954) Maramorosch (1952b)

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There is no reason to assume that the number of viruses in this category is limited to the 5 that have been studied in detail. Nor is there any reason to suspect that multiplication of plant viruses is limited to vectors among the Cicadellidae. Very likely many other plant viruses that are transmitted biologically also multiply in their insect vectors. The evidence presented over the past 20 years has a bearing on our ideas not only about virus multiplication but also about classification and origin. These viruses cannot be considered as solely plant viruses; they constitute a close link between plant and animal viruses. The ability to multiply in such diversified hosts as plants and animals strongly suggests that they are living organisms. The hypothesis of a large molecule precursor responsible for virus multiplication can hardly account for multiplication of the plantanimal viruses. It is inconceivable that such a large molecule precursor would occur in two hosts that are serologically and otherwise so unrelated. The morphology of viruses transmitted by leafhoppers shows that they are complex in their nature (Black, 1955). Presumably some virus particles are surrounded by a membrane (Black et al., 1948; Brakke et aZ., 1951). Their morphology and the complex biological behavior seem to place these viruses in the category of microorganisms. They are not only capable of multiplication, but they also mutate and their mutations can be induced by physical means like mutations in other microorganisms (Kunkel, 193713). Multiplication is an intrinsic character of a functional organism in which like produces like. Viruses, probably, are unable to sustain independent metabolism and in this characteristic may represent an extreme form of parasitism (Green, 1935; Laidlaw, 1938). Retrograde evolution in parasites might explain the existence of the highly specialized plant-virusarthropod relationship of biologically transmitted viruses. The viruses do not interfere with the life functions of their animal vectors. Should the viruses in some way endanger the life span of the arthropod vectors or interfere with their feeding, opportunities to transmit the infective agent would be diminished. Is it proper for us to apply the term vector to arthropods and the term host to plants? If we look at the problem from the standpoint of disease in plants, the arthropods play merely the role of carriers. But from the virus aspects and its requirements for survival in nature, arthropods transmitting plant viruses often constitute better hosts or reservoirs than the plants. Arthropods seem to constitute an important reservoir of plant pathogenic viruses and plants may serve only as incidental hosts for some of the viruses harbored by insects (Maramorosch, 1953a, e, 1954). Similarly, man and many other species of vertebrates serve as incidental hosts for the viruses of yellow fever, louping ill, and Colorado tick fever (Meyer, 1953; Schlesinger, 1952). The plant and the warm-blooded animal hosts may be re-

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garded, in these cases, as only temporary repositories and the disseminators of viruses to the main, mobile reservoirs-the arthropod vectors. The life cycles of viruses, alternating between plants and arthropods, could have arisen either by very simple nutritive requirements or by a well-organized adaptation of all metabolic activities. Our present knowlsdge of biologically transmitted plant viruses shows that, with certain exceptions, they have a fairly large host range among plant species but a narrow one among the arthropod vectors. This favors the assumption that their adaptation to the animal host is well-organized and, from an evolutionary point of view, probably also of much longer existence. It, therefore, seems conceivable that these viruses were originally arthropod viruses and, because of the long association between the host and parasite, ideally balanced in their almost symbiontlike relationship. This explanation could account for the complete tolerance of the otherwise highly virulent plant viruses in respect to the “susceptible” arthropods. The view of the origin of biologically transmitted viruses in arthropods seems further supported by the finding that several viruses have the capacity of passing through the egg of the vector to the progeny and of being thus maintained indefinitely in animals without the necessity of alternating plant hosts. Others are only occasionally transmitted transovarially, and still others have to rely on the complicated life cycle, imposed by alternation between plants and arthropods. Biologists tend to think of viruses as being organisms, just as chemists tend to think of them as being chemicals. There is no reason to assume that all viruses, or even all viruses causing plant diseases, multiply in a similar fashion. In some fungi a great variety of developmental stages occurs, both sexually and asexually, and in certain highly developed groups, such as the rusts, there are life cycles alternating between different and highly specific hosts. It seems reasonable to assume that various groups of viruses, like different groups of fungi, multiply differently from each other; for instance, tobacco mosaic virus may multiply in an entirely different way from that characteristic of aster yellows virus. The evidence for the existence of a group of viruses that are able to multiply both in plants and animals and that require both hosts for their maintenance in nature has been well-established. The old controversy of whether viruses are able to multiply in such diversified hosts has been settled and the balance of evidence points towards the view that these viruses are parasitic organisms. The still prevailing and very convenient division of viruses into bacteriophages, animal, plant, and insect viruses may have to be abandoned in the future; like bacteria, which are no longer divided according to the hosts they attack, viruses will come to constitute a unified field of study.

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REFERENCES Bald, J. G., and Samuel, G. (1931). Australia Council Sci. Znd. Research Bull. 64, 24. Bawden, F. C. (1950). “Plant Viruses and Virus Diseases,” 3rd ed. Chronica Botanica, Waltham, Mass. Bennett, C. W., and Wallace, H. E. (1938). J. Agr. Research 66,31. Bernheimer, A. W., Caspari, E., and Kaiser, A. D. (1952). J. Exptl. 2001.119, 23. Black, L. M. (1940). Phytopalhology 30, 2. Black, L. M. (1941). Phytopathology 31, 120. Black, L. M. (1944). Proc. Am. Phil. Soe. 88, 132. Black, L. M. (1948). Phylopathology 38, 2. Black, L. M. (1950). Nature 168, 852. Black, I,. M. (1953s). Advances i n Virus Research 1, 69. Black, L. M. (1953b). Ann. N . Y. Acad. Sci. 66, 398. Black, L. M. (1953~). Phylopalhology 43, 9. Black, L. M. (1954). Exptl. Parasitol. 3, 72. Black, L. M. (1955). Phylopalhology 46. 208. Black, L. M., and Brakke, M. K. (1952). Phytopalhology 42, 269. Black, L. M., Mosley, V . M., and Wyckoff, R. W. G. (1948). Biochim. et Biophys. Acta 2, 121. Brakke, M. K., Black, L. M., and Wyckoff, R. W. G. (1951). Am. J. Botany 38,332. Brakke, M. K., Maramorosch, K., and Black, L. M. (1953). Phytopalhology 43,387. Brakke, M. K., Vatter, A. E., and Black, L. M. (1954). Brookhaven Symposia Biol. 6, 137. Bryan, W. R., and Beard, J. W. (1940). J. Infectious Diseases 66, 245. Carsner, E., and Stahl, C. F. (1924). J. Agr. Research 28,297. Costa, A. S., and Bennett, C. W. (1950). Phylopathology 40, 266. Darlington, C. D. (1944). Nature 164, 164. Day, M. F., and Bennetts, M. J. (1954). “A Review of Problems of Specificity in Arthropod Vectors of Plant and Animal Viruses,” p. 172. Australia Commonwealth Sci. Ind. Research Organization, Div. of Entomology, Canberra. Day, M. F. (1955). Exptl. Parasitol. 4, 387. Flock, R. A., and Wallace, J. M. (1955). Phylopalhology 46, 52. Freitag, J. H. (1936). Hilgardia 10, 305. Fukushi, T. (1933). PTOC.Imp. Acad. (Tokyo) 9. 457. Fukushi, T. (1940). J. Fac. Agr. Hokkaido Univ. 46, 83. Card, S. (1940). J. Exptl. Med. 72, 69. Giddings, N . J. (1950). Phytopalhology 40, 377. Green, R. G. (1935). Science 82, 443. Grylls, N. E. (1954). Australian J. Biol. Sic. 7 , 47. Kunkel, L. 0. (1924). Phytopathology 14, 54. Kunkel, L. 0. (1926). A m . J. Botany 13, 646. Kunkel, L. 0. (1937a). A m . J. Bolany24. 316. Kunkel, L. 0. (1937b). J . Bacteriol. 34, 132. Kunkel, L. 0 (1938). J . Econ. Entomol. 31, 20. Kunkel, L. 0. (1941). Am. J. Bolany28, 761. Kunkel, L. 0. (1946). Proc. Null. Acad. Sci. (U.S . ) 32, 246. Kunkel, L. 0. (1948). Arch. ges. Virusjorsch. 4, 24. Kunkel, L. 0. (1954). i n “The Dynamics of Virus and Rickettsia1 Infections”

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(F. W. Hartman, F. L. Horsfall, Jr., and J. G. Kidd, eds.), p. 150. Blakiston, New York. Laidlaw, P. P. (1938). “Virus Diseases and Viruses.” Cambridge Univ. Press, New York. Maramorosch, K. (1949). Phytopathology 39, 14. Maramorosch, K. (1950a). Phytopathology 40, 1071. Maramorosch, K. (1950b). Proc. SOC.Exptl. Biol. Med. 76, 744. Maramorosch, K. (1951). Phytopathology 41, 833. Maramorosch, K. (1952a). Phytopathology 42, 59. Maramorosch, K . (195213). Phytopathology 42, 663. Maramorosch, K. (1952~).Nature 169, 194. Maramorosch, K. (1953a). Sci. American 188, 78. Maramorosch, K. (1953b). 6th Intern. Congr. Microbiol., Rome, Riassunti delle Commun. 2, 163. Maramorosch, K. (1953~). Cold Spring Harbor Symposia Quant. Biol. 18, 51. Maramorosch, K. (1953d). Am. J. Botany 40, 797. Maramorosch, K. (1953e). Plant Disease Reptr. 37, 612. Maramorosch, K. (1954). Trans. N . Y . h a d . S c i . 16, 189. Maramorosch, K. (1955a). Am. J. Botany 42, 676. Maramorosch, K. (1955b3. Virology 1, 286. Maramorosch, K. (1955~). Bull. Torrey Botan. Club 8a. 339. Maramorosch, K., Brakke, M. K., and Black, L. M. (1949). Science 110, 162. Merrill, M. H., and TenBroeck, C. (1934). Proc. SOC.E s p t l . Biol. Med. 32, 421. Meyer, K. F. (1953). Am. J. Trop. Med. Hyg. 2, 757. Orlando, A., and Silberschmidt, K. (1946). Arquiu. biol. Siio Paulo 17, 1. Schlesinger, R. W. (1952). in “Monographs in Medicine” (W. B. Bean, ed.), Ser. 1, p. 380. Williams and Wilkins, Baltimore. Severin, H. H. P. (1921). Phytopathology 11, 424. Severin, H. H. P. (1931). Hilgardia 6, 263. Severin, H. H. P. (1934). Hilgardia 8, 339. Severin, H. H. P. (1945). Hilgardia 17, 23. Severin, H. H. P. (1946). Hilgardia 17, 141. Severin, H. H. P. (1947a). Hilgardia 17, 197. Severin, H. H. P. (194713). Hilgardia 17, 511. Severin, H. H. P. (1948). Hilgardia 18, 203. Severin, H. H. P. (1950). Hilgardia 19, 357. Slykhuis, J.. T. (1953a). Can. J. Agr. S c i . 33, 195. Slykhuis, J. T. (195313). Phytopathology 43, 484. Slykhuis, J. T. (1955). Phytopathology 46, 116. Smith, K. M. (1941). Parasitology 33, 110. Smith, R. E., and Boncquet, P. A. (1915). Phytopathology 6,103. Storey, H. H. (1928). Ann. A p p l . Biol. 16, 1. Storey, H. H. (1933). Proc. R o y . SOC.B 113, 463. Storey, H. H. (1939). Botan. Revs. 6, 240. Teitelbaum, S. S., and Goulet, P. (1950). Proc. Entomol. SOC.W a s h . 62, 269. Varma, P. M. (1952). Zndian J. Agr. Sci. 22, 75. Watson, M. A., and Roberts, F. M. (1939). Proc. Roy. Soe. B 127,543.

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Cross Protection Between Strains of Yellows-type Viruses L. 0. KUNKEL The Rockefeller Institute f o r Medical Research, New York, New York

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ .. . . . . . . . . . . . A. Natural and Acquired Immunity. . . . , . , . . . , . . . , . . . . . . . . . . . . . . . . . . . . . . B. The Cross Protection Reaction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Mechanism of Cross Protection.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Interference Between Viruses.. . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Cross Protection Experiments with Yellows-Type Viruses. . . . . . . . . . . . . 11. Identification of California Aster Yellows. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Host Range of California Aster Yellows Virus.. . . . . . . . . . . . . . . . . . . . . . . B. California Aster Yellows in the East.. . . , . . . , . . . . . . . . . . . . . . . . . . . . . . . . . C. California Aster Yellows in Vinca Tosea Cured by Heat. . . . . . . . . . . . . . . D. Symptom Differences Between Aster Yellows and California Aster Yellows.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Aster Yellows Transmitted to Zinnia elegans and Celery.. . . . . . . . . . . . . 111. Cross Protection Experiments with Vinca rosea P l a n t s . . . . . . . . . . . . , . . . . IV. Cross Protection in Leafhoppers.. . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . , , A. Cross Protection Against Acquisition of Infectivity. . . . . . . . . . . . . . . . . . . B. Effects of Varying Exposure Periods ............................ C . Subinoculations from Plants Expose rotected Leafhoppers. . . . . . . V. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary.. . . . . . . . . . . . . . _................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

251 251 251 252 252 253 254 254 255 255 256 260 262 263 263 265 266 269 272 272

I. INTRODUCTION A . Natural and Acquired Immunity Natural immunity is used extensively in breeding economic plants for resistance to diseases of many kinds (8, 14, 15). Acquired immunity, on the other hand, is not generally effective for control of plant diseases (9). There is, in fact, some doubt as to whether plants acquire immunity from any disease because of having had a previous attack of the disease, or because of having or having had an attack of a closely related disease. It is because of this doubt that the phenomenon known as cross protection from virus diseases of plants, which resembles cross immunization in animals in some respects, deserves attention (1, 19, 25, 4 6 4 8 , 52, 54, 67, 68).

B . The Cross Prokction Reaction Cross protection depends on the reactions of infected plants to new infections. Plants invaded by one virus are generally protected against other 251

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closely related viruses but are not protected against distantly related viruses. There is considerable variation in the degree of cross protection afforded by plant viruses that are believed to be closely related. However, the variation is no greater than that shown by serological cross reactions between closely related animal viruses. The reaction includes protection from infection as well as protection from invasion after infection, and may be either partial or complete. It is this reaction that prevents most of the numerous mutants of tobacco mosaic virus from surviving and multiplying extensively in nature (18, 32, 37). The ordinary strain of tobacco mosaic virus overgrows them and limits their multiplication to small bits of tissues immediately surrounding their points of origin. It is this reaction also that has enabled virologists to identify easily naturally occurring variant strains of the viruses causing tobacco mosaic, cucumber mosaic, potato X disease, and many other similar maladies (26, 49).

C . Mechanism of Cross Protection How protection is afforded is not presently known but several theories have been advanced to account for it (2,3). One that fits the known facts rather satisfactorily assumes that closely related viruses require the same host cell materials for multiplication. A strain of a virus that invades a plant cell first uses up these materials and without them a second strain of the same virus cannot multiply. It also assumes that distantly related viruses use different host cell materials for multiplication. In that case the virus that invades a cell first uses up the materials required for its multiplication but leaves untouched materials required for multiplication of the second virus (51). However, a theory assuming production of unstable immune bodies in infected cells would seem to fit the known facts equally well (69). The concentrations reached by some plant viruses during chronic stages of the diseases they cause are rather definitely limited under any given set of environmental conditions (50). If it is assumed that virus multiplication is limited because of production of immune bodies in infected cells, these same bodies might prevent multiplication of a second closely related virus but not prevent multiplication of a distantly related virus. Regardless of what the correct explanation may be, the cross protection reaction is highly specific and effective. In these respects it is equivalent to cross immunity from virus diseases in animals. For example, a tomato plant infected by a lethal strain of tobacco mosaic virus is sure to die unless given protection by a nonlethal strain. When so protected, the plant lives. It cannot be saved by use of an unrelated virus or in any other way (31, 35). D. Interference Between Viruses The cross protection reaction to virus diseases of plants should not be confused with reactions in which a disease caused by one virus is modified

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through infection by a second, unrelated or distantly related virus, or the disease caused by the latter is modified by the former, or the two together cause a disease markedly different from that produced by either alone. Interference of this type does not ordinarily prevent multiplication of either virus though it may modify multiplication rates and final concentrations (53).

E. Cross Protection Experiments with Yellows-Type Viruses Most viruses known to protect plants from invasion by other viruses are transmitted manually by means of juices from diseased plants. A desire to study cross protection with viruses of the yellows group, where manual transmission by means of infective plant juices has not been achieved, arose soon after the phenomenon of cross protection was discovered. In 1936 it was shown that peach rosette virus invaded peach seedlings affected by either peach yellows or little peach when transmitted by grafting (27). On the other hand, when seedlings having yellows were grafted with tissues bearing little peach virus, they did not come down with little peach, and when seedlings having little peach were grafted with tissues carrying yellows virus, they did not come down with yellows. From these results i t was concluded that peach rosette virus was not closely related t o either peach yellows or little peach virus, but that the latter were closely related to each other. Cross protection tests with these viruses, using leafhopper inoculations, were theoretically possible for it had been shown that the vector of peach yellows virus spreads little peach (24, 41). However, Macropsis trimaculata (Fitch), the leafhopper concerned, was not successfully cultured over long periods of time in cages. Hence, it was not used in cross protection experiments. The aster leafhopper, Macrosteles fascijrons (Uhl.) , that transmits aster yellows and had been cultured continuously in cages for several years, was suitable for use in cross protection experiments with this virus but variant strains that could be readily distinguished were not available (20, 21). I n 1937 mild strains of aster yellows virus were obtained by heat-treating viruliferous aster leafhoppers (28, 29). One of these was employed in combination with typical aster yellows virus in cross protection tests on asters by means of leafhopper inoculations. The mild strain seemed to protect against the ordinary strain in some instances but the symptoms produced by these viruses in plants kept for several months were too much alike to give a convincing demonstration. Hence, work with these strains was not continued. It was hoped that a strikingly different naturally occurring variant of aster yellows would be found. Although a considerable number of yellows-type diseases have been obtained and cultured in Vinca rosea L. plants since 1937, none proved to be closely related to aster yellows. A yellows disease occurring on carrots in Texas could not be distinguished from aster yellows in carrot and V . rosea plants (36).

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However, it was readily transmitted to red clover by dodder, whereas aster yellows was difficult to transmit to this host. It also differed from aster yellows in not being spread by the aster leafhopper.

11. IDENTIFICATION OF CALIFORNIA ASTERYELLOWS A . Host Range of California Aster Yellows Virus For many years it was assumed that a yellows-type disease affecting China asters, celery, carrots, and other species in California was aster yellows. The chief justification for the assumption was that the California disease was transmitted by the same aster leafhopper that transmits aster yellows, and that it caused symptoms in asters and other plants that were much like those of aster yellows. Also, the two diseases were known to have overlapping plant host ranges that included a considerable number of species. There was no reason for believing that the California disease was not identical with typical aster yellows of the East, which was first described from Massachusetts in 1902 (64). But in 1929 Severin found that the California disease was readily transmitted to Zinnia elegans Jacq. and celery that were then thought to be immune from aster yellows of the East (55).

A large plot of Zinnia elegans plants was grown beside plots of asters at Yonkers, New York, in 1926 (22). By the end of July the incidence of yellows in the aster plants was above 90 %, but the disease had not appeared in the zinnias nor did it appear in them up to the end of the growing season. Z . elegans plants in cages were exposed to colonies of infective aster leafhoppers that would have transmitted aster yellows virus to every aster plant on which they fed. The zinnia plants developed no symptoms of aster yellows and virus-free aster leafhoppers allowed to feed on some of them did not pick up the virus. On this evidence Z . elegans was considered immune from aster yellows. Celery, also, was believed to be immune because young celery plants on which infective aster leafhoppers were confined remained healthy indefinitely and no field-grown celery plants could be found that showed symptoms of aster yellows (22). Moreover, there were no reports of yellows on celery from any of the eastern states in which celery was grown commercially. Hence, Severin’s observation that the California disease occurred abundantly in zinnia and celery plantings seemed to distinguish it sharply from New York aster yellows. In order to study this host range difference under comparable environmental conditions, California aster yellows was obtained through the kindness of Professor H. H. P. Severin in 1931. The behavior of this disease was compared with that of New York aster yellows in transmission tests with celery and zinnia plants a t the Boyce Thompson Institute in Yonkers, New York (23). The western virus was easily transmitted to celery but the eastern virus

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was not. In experiments using small numbers of plants neither disease was transmitted to zinnia. It was deemed unwise to maintain the western virus at the Boyce Thompson Institute for further tests because of the possibility that it might escape and damage eastern celery crops. Hence, it was destroyed. The Yonkers tests proved conclusively that the two viruses differed in their ability to infect celery. This finding was confirmed by Severin in transmission experiments with aster yellows from a number of different sources (56). Using infected plants obtained from California, Idaho, Indiana, Maine, New York, and Wisconsin as sources of aster yellows viruses, he found that celery was resistant to all except that from California. Severin did not observe symptom differences between the diseases from eastern and western states. Since publication of these observations, eastern and western aster yellows have been classified as closely related strains of one disease. Holmes named the eastern virus, Chlorogenus callistephi H., and the western, Chlorogenus callistephi variety Californicus H. (16).

B . California Aster Yellows in the East In 1952 Magie, Smith, and Brierley found California aster yellows in gladiolus plants growing in the East (40). The western virus thus became available to easterners without importing it intentionally from the West. In the spring of 1953 a celery and an aster plant carrying California aster yellows virus, with gladiolus as the source, were received from Dr. Floyd F. Smith, who suggested that the eastern and western forms be compared as to heat-tolerance (33, 34). This gave an opportunity to study the two viruses in more detail than had been possible previously. The California aster yellows virus used in studies to be reported here was taken from the celery plant to Vinca rosea plants by means of dodder, Cuscuta campestris Yunckers, and to aster plants by means of the aster leafhopper. The disease it produced was compared with eastern aster yellows for the purpose of confirming or refuting the assumption that the two were closely related. This seemed necessary because in recent years Severin had found that the California virus was transmitted by 20 different species of leafhoppers in addition to the one that transmits the eastern virus (38, 57-63). At least one of the 20 species was unable to transmit the eastern form and most of the others were not shown to do so. The differences in plant host ranges and insect vectors suggested that the two viruses might not be as closely related as the Holmes classification indicated (66).

C . California Aster Yellows in Vinca rosea Cured by Heat The first experiments with the new sample of western virus were for heattolerance in Vinca rosea plants. In a preliminary experiment a few mature

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plants affected by the western disease and similar plants with the eastern disease were held in a hot room at about 42°C. for varying periods of time. Those with the western disease were killed by treatments causing little damage to plants with aster yellows. When V . rosea plants that were about 8 months old from cuttings were given the western disease and held a t 42°C. for 8 days or longer, they died. When similar plants were given the same treatment for 6 days or less, they survived but were not cured. In further tests V . rosea plants that were more than 2 years old from cuttings were given the western disease by inserting 2 scions in the branches of each plant. After the disease had started to spread in the plants but before it had caused much damage, they were given the following treatments: 3 were held a t about 42°C. for 10, 13, and 14 days, respectively; all survived. Four were held a t 41 to 42°C. for 14 days; of these 2 lived and 2 died. Four were held a t 42 to 44OC. for 14 days; of these 1 lived and the others died. The virus survived in all plants that lived but only in the roots, main stems, and large branches of those treated for 14 days. It was inactivated in all small branches. This was shown by the healthy-appearing new growth they made before virus invaded them from other parts of the plant. Some of the original scions that had been used in inoculating the treated plants were removed after the heat treatments and regrafted in healthy young V . rosea plants. The scions grew and in a period of 8 months produced only healthy-appearing new growth. The plants on which they were grafted remained healthy, thus proving that virus originally carried by the scions had been inactivated. The experiment showed that the western virus could be inactivated in V . rosea tissues by treatments at about 42OC. for as short a period as 14 days. Because of a difference in the severity of the two diseases, heat treatment experiments did not bring satisfactory evidence as to whether the viruses causing them differ in heat-tolerance. All that can be said is that they are similar in this respect. Comparative studies on heat-tolerance of the two viruses in their insect vector should give more definite results. These have not yet been made (28).

D . Symptom Diferences Between Aster Yellows and California Aster Yellows California aster yellows virus was transmitted to several different plants in the hope of finding symptom and host range differences. Young celery plants were exposed to colonies of aster leafhoppers carrying the western virus and similar plants to colonies carrying the eastern virus. Again, as in 1931, the western virus was readily transmitted to celery but the eastern was not. Asters seemed definitely more resistant to the western than to the eastern disease but actual tests for comparing their susceptibility to the two diseases were not made. Each virus was transmitted to a number of

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common plant species. It was soon noted that in general they produced somewhat different symptoms. The differences were especially striking in Vinca rosea and Nicotiana rustica L. Even in the China aster the two diseases produce symptoms sufficiently different to permit their identification in plants held for a month or longer after becoming diseased. The difference is shown by the chronically diseased plants presented in Fig. 1. The plant a t the left has aster yellows, that at the right has California aster yellows. The western virus was considerably more severe and produced more

FIG.1. China ast,er plants of the same age. The plant at the left has aster yellows; the plant at the right has California aster yellows. They were exposed to viruliferous leafhoppers 63 and 72 days, respectively, before the photograph was taken and show typical symptom differences. (Photograph by J. A . Carlile.)

stunting of growth than the eastern virus in all plants to which it was taken. It tended to cause production of short fleshy side shoots (rosettes), while the eastern virus tended to cause production of spindly side shoots (witch's brooms). The differences in type of growth are strikingly shown by the secondary shoots of Nicotiana rustica pictured in Fig. 2. The shoot on the left was taken from a plant one month after it became infected with the western virus, while the shoot on the right was taken from a plant that had had eastern aster yellows for the same period of time. Two N . rustica plants of the same age and affected for the same period of time as those from which the branches were taken are presented in Fig. 3. On the left in Fig. 4 is shown a cabbage-like tip from a N . rustica plant that had been affected by the western disease for about 2 months, while on the right is shown a tip from a similar plant t'hat had had the eastern disease for 2 months. The

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differences in symptoms produced by the two diseases in V i m rosea were not aa striking a~ in N . rustica but were equally constant and distinct. Branches from 3 different V . rosea plants of the same age are presented in Fig. 5. At the left is shown a branch from a healthy plant, in the middle a branch from a plant with the eastern disease, and at the right a branch from a plant with the western disease. Such distinctly different symptoms

FIG.2. Side shoots from two yellows Nicotiana rustica plants showing characteristic symptom and growth-habit differences. The shoot at the left has California aster yellows; the shoot at the right has aster yellows. (Photographby J. A. Carlile.)

produced under identical environmental conditions indicated that the two viruses were suitable for use in cross protection tests. However, before undertaking such testa an attempt was made to find still other plant host range and symptom differences. When the diseases were taken to carrots, they produced early symptoms that were indistinguishable. However, carrots with the western disease were more severely stunted and died sooner than carrots with the eastern disease. Both diseases were easily taken to flax and to Thlaspi arvense L. In these species they produced similar but not identical symptoms. Symp-

FIQ.3. Two Nicoliana rustica plants of the same age. The plant at the left has California aster yellows, while the plant a t the right has aster yellows. The photograph was taken one month after plants were inoculated. The diseases produce strikingly different symptoms in this host. (Photograph b y J. A. Carlile.)

FIG.4. Tip shoots from two Nicoliana rusliea plants. The shoot at the left has California aster yellows, while that a t the right has aster yellows. The photograph was taken about two months after plants became infected. The symptoms shown are typical of chronic &ages of the diseases in this host. (Photograph by J. A. Carlile.)

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toms also were similar in Nicotiana glutinosa L. but here again plants with the western disease died sooner than those with aster yellows. Also, on tomato, symptoms were not very different but plants with the western disease were more severely stunted arid died earlier than plants with aster yellows. Turkish tobacco seedlings on which N . rustica scions carrying the western virus were grafted and Turkish tobacco seedlings on which N . rustica scions carrying aster yellows virus were grafted were held under observation in a greenhouse for 3 months. The scions lived and produced many new branches but neither disease was transferred to tobacco.

FIG.5. Branches from three Vincu roseu plants. The branch at the left ia from a healthy plant, that in the middle from a plant with aster yellows, and that at the right from a plsnt with California aster yellows. Aster yellows causes production of thin, elongated side shoots, while California aster yellows causes production of short, swollen side shoots in this host. (Photograph by J . A. Carlile.)

E. ‘4 strr Yellows Transmitted to Zinnia elegans and Celery When efforts to broaden the known host range differences of the two viruses failed, an attempt was made to narrow the host range differences by further efforts to transmit aster yellows virus to celery and Zinnia eleyans plants. Experience has taught that there are many different degrees of susceptibility to aster yellows. One infective aster leafhopper feeding for 1 day on a young aster plant transmits with a high degree of certainty (39). The aster is the most susceptible plant known. One infective aster leafhopper feeding for 1 day on a young lettuce or carrot plant also transmits with considerable certainty but not with the same degree of certainty as in the case of the aster. The incidence of aster yellows i n lettuce and carrot fields usually does not exceed 10 %, while in aster plantings it frequently exceeds 90%. Aster yellows is readily transmitted to

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buckwheat but seldom affects more than 5 % of the, plants in buckwheat fields. The disease also is easily transmitted experimentally to African marigolds, but appears sparingly on this host in gardens. It is difficult but by no means impossible to transmit aster yellows to young tomato plants experimentally. Its occurrence in tomato fields is extremely rare. We have learned that aster yellows can occasionally be taken t o species that

FIG.6. Side shoots from two yellows Zinnia elegans plants. The shoot at the left is from a plant with California aster yellows; that at the right, from a plant with aster yellows. California aster yellows causes production of thick, stubby side shoots, while aster yellows causes production of thin, elongated side shoots. (Photograph by J . A. Carlile.)

were thought t o be immune if persistent efforts are made by exposing very young plants of such species t o large numbers of infective leafhoppers. When very young plants were given severe exposures, aster yellows was taken t o both Zinnia elegans and celery. I n zinnias the symptoms produced differed markedly from those caused by the western disease, a s may be seen in Fig. 6. I n celery, aster yellows was not readily distinguishable from California aster yellows. With plant host range differences eliminated, it seemed likely that the two diseases were caused by closely related strains of the same virus, even though they went to celery and Z . elegans

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plants with different degrees of readiness, produced markedly different symptoms, and had different insect host ranges. The evidence supported Holmes’ conclusion regarding the close relationship of the two viruses. But whether the viruses were closely or distantly related, they fulfilled the requirements that had long been sought and were necessary for cross protection tests with insect-transmitted yellows-type diseases. The symptoms were sufficiently different to provide an easy means of determining which virus was present in any given plant. The aster leafhopper was an efficient vector of both. A number of different plants were sufficiently susceptible to both.

111. CROSSPROTECTION EXPERIMENTS WITH V I N C A ROSEA

PLANTS

Vinca rosea plants were chosen for use in the first experiments, Being grown from ruttings, they are free from the variability of plants, such as asters, that are grown from seeds. Also, they are good food plants for the aster leafhopper. Six healthy young Vinca rosea plants were raged for 3 days with colonies of infective leafhoppers carrying aster yellows virus, and G similar plants were (+agedwith colonies of infective leafhoppers rarrying California aster yellows virus. Earh rolony consisted of about 25 adult insects. Another set of G healthy young V . rosea plants that were not caged with insects served as controls. As was expected, the plants on which the insects fed became diseased while the control plants remained healthy. About G weeks after symptoms first appeared, 5 of the G plants that had aster yellows were exposed for 3 days to colonies of infective leafhoppers carrying California aster yellows virus, and 5 of the 6 that had California aster yellows were exposed to colonies of infective leafhoppers carrying aster yellows virus. The other plant in each set was not re-exposed. At the same time, 2 of the control plauts were exposed for 3 days to similar colonies carrying aster yellows virus and 2 for 3 days to similar colonies carrying California aster yellows virus. The other 2 control plants were not exposed to insects. In due course the 2 control plants exposed to insects that were infective for aster yellows virus came down with aster yellows, and the 2 exposed to insects that were infective for California aster yellows virus came down with California aster yellows. The 5 plants affected by aster yellows that were exposed to insects infective for California aster yellows virus and the 5 affected by California aster yellows that were exposed to insects infective for aster yellows virus continued to show symptoms of aster yellows only and California aster yellows only, respectively, during the 5 months that they were kept under observation. The 5 plants in each of these sets looked exactly like the Gth which had not been re-exposed to infective leafhoppers.

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The 2 remaining control plants that were not exposed to insects remained healthy. The experiment brought evidence that the viruses protect against each other in Vinca rosea plants exposed to insect inoculations. Other tests with V . rosea plants confirmed these results. Comparable tests in which aster and Nicotiana rustica plants were substituted for V . rosea also confirmed them. A considerable number of aster plants affected by each disease were exposed to colonies of leafhoppers that were infective for the virus of the other, in connection with experiments on cross protection in leafhoppers. None of these developed symptoms caused by the viruses to which the diseased plants were exposed. The cross protection tests in plants supported fully the view that aster yellows and California aster yellows are caused by closely related strains of the same virus.

IV. CROSSPHOTECTION IN LEAFHOPPERS A . Cross Protection Against ilcpuisition of Infectivity That the virus of aster yellows multiplies in its insect vector, the aster leafhopper, is a well-established fact (5, 28, 30, 42-44, 52). If, as is believed, California aster yellows virus is closely related to aster yellows virus, it probably multiplies in the aster leafhopper also, for it undergoes a similar incubation period in and is retained about as long by the vector (23). Neither virus is known to cause disease in the leafhopper (11) but if both are capable of multiplying in leafhopper cells, which seems likely, they might well be expected to immunize against each other i n the insect and thus cross protect plants exposed to the insect. Plants so exposed would, of course, be infected by the virus affecting the plant on which the insect had first fed but not by the other virus even though the insect had subsequently also fed on a plant carrying the other virus. In order to investigate this possibility cross protection tests were undertaken with the aster leafhopper. Three colonies, consisting of about 30 virus-free young aster leafhoppers each, were placed on 3 healthy young aster plants in lantern globe cages. A similar colony was put on a n aster plant affected by aster yellows in a fourth cage, and another similar colony on an aster plant affected by California aster yellows in a fifth cage. The colonies were kept on the plants for 2 weeks. At the end of this period, 1 colony that had been confined on a healthy aster plant was transferred to a cage containing an aster plant affected by aster yellows. Another was transferred t o a cage containing an aster plant affected by California aster yellows. The third was transferred to a cage containing a healthy aster plant. This colony was to serve as a control. The colony that had been confined on a n aster plant affected by aster yellows was transferred to a cage containing a n aster plant affected by California aster yellows; and the colony that had been confined on an aster plant affected by California aster

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yellows was transferred to a cage containing a plant affected by aster yellows. The colonies were kept on these plants for 2 weeks also. Then each colony was transferred daily to a series of healthy young aster plants for 4 days. The colonies were next kept on healthy young aster plants for 3 days, and finally each was again transferred to a healthy young aster plant daily for 4 days. The 45 aster plants on which the colonies fed during the 1I -day period were kept under observation for 3 months. The plants exposed to the control colony that had fed on healthy aster plants during both 2-week intervals remained healthy. Those exposed to the colony that had fed on a healthy aster plant during the first 2-week interval and on an aster plant affected by aster yellows during the second 2-week interval came down with aster yellows. Eight of the 9 of those exposed t o the colony that had fed on a healthy aster plant during the first 2-week interval and on an aster plant affected by California aster yellows during the second 2-week interval came down with California aster yellows. One plant in this group died without showing symptoms of yellows. The plants exposed to the colony that had fed on an aster plant affected by aster yellows during the first 2-week interval and on an aster plant affected by California aster yellows during the second 2-week interval came down with aster yellows, while those exposed to the colony that had fed on an aster plant affected by California aster yellows during the first 2-week interval and on an aster plant affected by aster yellows during the second 2-week interval came down with California aster yellows. The virus-free leafhoppers that were allowed to feed for 2 weeks on ail aster plant with aster yellows acquired and transmitted aster yellows virus, but the leafhoppers that were infective for California aster yellows virus before they were allowed to feed, during the same 2-week period, on an aster plant with aster yellows did not apparently transmit aster yellows virus. The virus-free leafhoppers that were allowed to feed for 2 weeks on an aster plant with California aster yellows acquired and transmitted California aster yellows virus, but leafhoppers infective for aster yellows virus before they were allowed to feed during the same 2 weeks on a plant with California aster yellows did not apparently transmit California aster yellows virus. The experiment described above, without modification as well as modified in several different ways, was repeated many times. Almost invariably plants exposed to insects that had fed during a 2-week period on plants affected by one of the viruses and during a second 2-week period on plants affected by the other virus came down with the disease aflicting the plant on which the insects first fed. The only exception to this type of behavior occurred in an experiment carried out during the month of July when greenhouse temperatures were very high. In that experiment healthy aster

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plants were .exposed to leafhoppers that had fed for 2 weeks on an aster plant with aster yellows and then for 2 weeks on an aster plant with California aster yellows. Several of these plants came down with aster yellows in some branches and California aster yellows in others. It is presumed that high temperatures prevented acquisition of aster yellows virus by some of the insects, and that these later picked up and transmitted California aster yellows virus. The cross protection was complete during other seasons of the year.

B . Effects of Varying Exposure Periods In a number of experiments the lengths of time of the first and second feeding periods were varied. In some instances the first period was shortened to 12 or 13 days and the second kept a t 14 days, while in others the first period was kept a t 14 days and the second shortened to 12 or 13 days. In still other experiments both periods were shortened or increased in length by a day or two. I n one experiment the first period was shortened to 1 and 2 days, respectively, while the other was kept at 14 days. The variations had no effect on results except in cases where the first feeding period lasted only 1 or 2 days. Plants exposed to colonies so treated plainly showed mixed infections. Combinations of feeding periods of intermediate lengths were not tested. Neither were experiments with single insects made. The diseased plants on which colonies were kept during the 2 feeding periods were asters in all cases, but test plants other than asters were also used. In Vinca rosea and Nicotiuna rmstica symptoms are much more distinct than in asters. However, these plants are not highly susceptible to either disease, and this is a disadvantage. When they are used, the insect colonies must be rather large. Otherwise the colonies do not infect regularly even when the exposure period is lengthened to as long as 5 days. Nevertheless, both species were used as test plants in several experiments. They always came down with the disease afflicting the plant on which the colonies had first fed. A side shoot from a N . rustica plant that was exposed during 5 days to a colony of aster leafhoppers kept on an aster plant with California aster yellows for 2 weeks and then on an aster plant with aster yellows for 2 weeks is shown a t the left in Fig. 7. A comparable side shoot from a N . rustica plant exposed during 5 days to a colony of aster leafhoppers kept on an aster plant with aster yellows for 2 weeks and then on an aster plant with California aster yellows for 2 weeks is presented on the right in this figure. The photograph was taken 64 days after the plants were exposed. It shows clearly the symptoms of the disease on which the insects that infected the plants first fed in each case. No symptoms of the disease affecting the plant on which the insects fed last are shown. This of course does not prove that no virus whatever was transmitted from the

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plants on which the insects fed last. However, if any was transmitted it did not alter the symptoms produced and subinoculations to test plants did not bring evidence of its presence. When subinoculations were made from N . rustica and V . rosea test plants by means of the aster leafhopper, grafts, or dodder, or from aster test plants by means of the aster leafhopper, they invariably produced symptoms like those of the test plants.

FIQ.7. Side branches from two Nicotiana rustica plants. The branch a t the left is from a plant that was exposed for 5 days to a colony of aster leafhoppers that had fed for 2 weeks on an aster plant with aster yellows but were protected by the virus of California aster yellows. The branch shows symptoms of California aster yellows only. The branch a t the right is from a plant that was exposed for 5 days to a colony of aster leafhoppers that had fed for 2 weeks on an aster plant with California aster yellows but were protected by the virus of aster yellows. This branch shows symptoms of aster yellows only. (Photograph by J. A. Carlile.)

C . Subinoculationsfrom Plants Exposed to Protected Leafhoppers Two plants of Nicotiana rusticu that were subinoculated from Vinca rosea test plants by means of aster leafhoppers are shown in Fig. 8. The plant on the left was exposed to insects that were confined for 19 days on a V . rosea plant that had become diseased through exposure to an insect colony held for 2 weeks on an aster plant with aster yellows and then for 12 days on an aster plant with California aster yellows. The V . rosea plant from which the subinoculation was made showed the symptoms of aster yellows

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and no symptoms of California aster yellows. It will be seen that the N . rustim plant also developed symptoms of aster yellows only. The N . rustics plant on the right in Fig. 8 was infected by exposure to aster leafhoppers that were confined for 19 days on a V . rosea plant that became diseased through exposure to a leafhopper colony held for 15 days on an aster plant with California aster yellows and then for 14 days on an aster plant with

FIG.8. Two yellows Nicotiana rustica plants of the same age. The plant a t the left was subinoculated by means of aster leafhoppers from a Vinca rosea plant that had been exposed t o a colony of aster leafhoppers kept for 2 weeks on a n aster plant with aster yellows and then for 12 days on an aster plant with California aster yellows. The l'. rosea plant from which the subinoculation was made showed symptoms of aster yellows only, just as does the N . rustica plant. The plant on the right was subinoculated by means of aster leafhoppers from a V. rosea plant t h a t had been exposed t o a colony of aster leafhoppers kept for 15 days on an aster plant with California aster yellows and then for 2 weeks on an aster plant with aster yellows. T h e V . rosea plant showed symptoms of California aster yellows only, just as does the N . rustica plant. Cross protection in the leafhopper seems to have been complete. (Photograph by J. A. Carlile.)

aster yellows. The V . rosea plant from which the subinoculation was made showed symptoms of California aster yellows only. The N . rustica plant to which the subinoculation was made likewise shows symptoms of California aster yellows only. Similar subinoculations from Vinca rosea plants to Nicotiana rustica plants by means of the dodder Cuscuta campestris gave similar results. In Fig. 9 is shown a N . rustica plant to which virus was transmitted by means of dodder from a V . rosea plant that became diseased through exposure to a colony of aster leafhoppers held for 14 days on an aster plant with aster yellows and then for 12 days on an aster plant with California aster yellows.

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The V. roseu plant showed symptoms of aster yellows only and it will be seen that the N. rustica plant likewise shows symptoms of aster yellows

FIG.9. A Nicotiana rustica plant subinoculated by means of Cuscuta campeslris from a Vinca rosea plant that had been exposed to aster leafhoppers that had fed on an aster plant with California aster yellows for 12 days but after being protected by the virus of aster yellows. The Y . rosea plant showed symptoms of aster yellows only, just as does the N. rustica plant. Protection in the leafhopper seems to have been complete. (Photograph by J. A. Carlile.)

only. The photograph was taken 100 days after dodder was placed on the plant. A N. rusticu plant subinoculated by means of dodder from a V. rosea plant that became diseased through exposure to a colony of aster leafhoppers held for 15 days on an aster plant with California aster yellows and then for 14 days on an aster plant with aster yellows is presented in Fig. 10. This plant shows symptoms of California aster yellows only, just

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as did the V . rosea plant from which the subinoculation was made. The photograph was taken 81 days after dodder was placed on the plant. Subinoculations carried out by means of grafts from Vinca rosea plants exposed to leafhopper colonies that had fed for two successive 2-week periods on aster plants affected by aster yellows and California aster yellows, respectively, and showed symptoms of aster yellows only, were made to 3 healthy young V . rosea plants. These plants developed symptoms of

FIG.10. A Nicoliana rustica plant t h a t was subinoculated by means of Cuscuta canipestris from a Vinca rosea plant exposed t o a colony of aster leafhoppers kept for 2 weeks on an aster plant with aster yellows but after protection by the virus of California aster yellows. The V. Tosea plant showed symptoms of California aster yellows only, just as does the N. rustica plant. Protection seems t o have been complete. (Photograph by J. A. Carlile.)

aster yellows only. Similar subinoculations carried out by means of grafts from V . rosea plants that had been exposed during two successive 2-week periods to leafhoppers that had fed on aster plants affected by California aster yellows and aster yellows, respectively, were made to 3 healthy young V . rosea plants. They transmitted a disease causing symptoms of California aster yellows only. V. DISCUSSION The experiments reported above demonstrate for the first time that two strains of yellows-type virus, namely, the ordinary aster yellows strain and

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the California aster yellows strain, when transmitted by their leafhopper vector, protect against each other in asters and other plants just as do strains of tobacco mosaic virus when manually transmitted to tobacco by means of infective plant juices. The experiments also demonstrate that the strains protect against each other in the insect vector as effectively as they do in plants. Since neither strain causes disease in the vector, neither can be said to protect the vector in the way it protects plants. The strains do, however, cross protect against acquisition of infectivity by the vector and thus protect plants on which the vector feeds. It has not been proved that aster leafhoppers which are infective for one of the viruses do not pick up the other, but only that they are prevented from becoming transmitters of the other. It is presumed that infective leafhoppers are infected and that when infected by one of the viruses they become immune from infection by the other. It also is presumed that this cross reaction in the insect results from the viruses being closely related. It would be desirable to determine whether viruses that are obviously unrelated fail to cross protect in the leafhopper. Unfortunately experiments that might bring evidence for or against that possibility cannot be made a t this time because no virus that is not closely related to that of aster yellows is now known to be transmitted by the aster leafhopper. Warm-blooded animals in which an animal virus multiplies usually acquire lasting immunity from the disease caused by the virus and its closely related strains. This immunity develops regardless of whether the disease produced is severe or mild, and also, apparently, regardless of whether it is quickly lost or long retained. Not much is known regarding immunity from virus diseases of insects (4, 12, 17). Nothing whatever is known regarding immunity in insects infected by insect viruses that multiply but cause no disease, if indeed there are such viruses. The mechanism by which strains of aster yellows virus protect against each other in the aster leafhopper is unknown but may be more amenable to solution than the mechanism of cross protection in plants. It also is not known whether loss of one of the aster yellows viruses through heat inactivation or otherwise would restore the leafhopper’s ability to become infective for the other, since experiments designed to settle this point have not yet been made. A considerable number of plant viruses require an incubation period in their insect vectors and are long-retained by the vectors. Of these only a few have been proved to multiply in the insects concerned (6). It is probable that eventually many more will be found to do this. Such viruses furnish a fertile field for studies on cross protection in insects and in plants. There is some evidence of cross protection between strains of maize streak virus in maize (45, 65) and between strains of cacao swollen shoot virus in cacao (10) when the viruses are transmitted by their vectors, the leafhopper Cicadulina mbila Naude and the mealy bug Pseudococcus njalensis Laing,

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respectively. It has not been possible to demonstrate cross protection between strains of curly-top virus in sugar beets (13) when transmitted by the beet leafhopper, Circulijer tenellus (Baker). Using the graft method of transmission, Wallace (69) showed cross protection between strains of curly-top virus in tomato plants originally infected by scions from tobacco plants that had acquired immunity to the different strains. None of the strains of these or other insect-transmitted viruses have been shown to cross protect in their vectors. Aster yellows and California aster yellows are rather severe diseases. Hence, there is no great advantage in protecting plants against either when this involves infection by the other. Still, California aster yellows is considerably more severe in most plant hosts than aster yellows. If a n aster plant were in danger of contracting California aster yellows, it would be an advantage i n so far as length of life of the plant was concerned to have the protection afforded by infection with the virus of aster yellows for this virus prolongs rather than shortens length of life of the aster (64). One of the most striking differences between plant viruses as a group and the viruses of animals is that all of the latter are transmissible manually by means of juices from diseased tissues, whereas many of the former cannot be transmitted in this way. Whether or not the difference is real or only due to lack of knowledge is not presently known. Perhaps plant pathologists will at some future time learn to transmit all plant viruses manually, or perhaps animal pathologists will eventually find animal viruses that are not manually transmissible and cause obscure diseases. Plant viruses that are not manually transmissible have received far less study and attention than the others, largely because the others are easier to manipulate experimentally. Animal viruses that are riot manually transmissible, if there are any, have received no attention whatever. Some pathologists that work with virus diseases of man and animals tend to avoid the conclusion that plant and animal viruses are essentially alike (7). They believe that viruses having animal hosts are microorganisms, whereas viruses having plant hosts are nucleoproteiri molecules and, hence, belong in a separate category. If this viewpoint is maintained, where are the viruses that multiply in both animals and plants to be placed? Are they to be considered microorganisms in animals and nucleoproteins in plants? It seems unlikely that such a view will long prevail. Most of the reasons cited for believing animal viruses to be microorganisms apply equally well t o the viruses of plants (7). Animal viruses have not yet been crystallized but there is no obvious reason why, under appropriate conditions, some at least should not crystallize. Most plant viruses have not yet been crystallized. Since aster yellows virus is more widely distributed and seems better adjusted to its plant hosts than California aster yellows virus, it is presumed

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that the latter was derived from the former through one or more mutations. Whether both strains will spread to all parts of the United States is not known. There is no apparent reason why they should not. Whenever they overlap in geographical range, cross protection in plants and in the aster leafhopper may he expected to operate in determining the incidence of each disease.

VI. SUMMARY Cross protectioii reactions between closely related viruses of plants and original experiments on cross protection between yellows-type viruses are discussed in this chapter. California aster yellows produces symptoms in several different plants that distinguish it from aster yellows, hut is cured by heat treatmeiit,s similar to those that cure aster yellows. The California strain of aster yellows virus and ordinary aster yellows virus protect against each other in asters and other plants. They also protect against each other in the aster leafhopper which, when infective for one, apparently becomes immune from iiifection by the other. REFERENCES Bnwderi, F. C., and Kassanis, B . (1945). Ann. Appl. B i d . 32. 52. Bennett, C. W. (1951). Ann. Rev. Microbial. 6 , 295. Bennett,, C. W. (1953). Advances in Virus Research 1, 39. Bernheimer, A. W., Caspari, E . , and Kaiser, A . D. (1952). J . Ezptl. Zool. 119, 23. 5. I3l:tck, I,. M . (1941). Phytopalhology 31, 120. 6 . Black, I,. M . (1953). Ann. N . Y . Acad. Sci. 66, 398. 7. Hurnet, F. M . (1953). “Viruses and Man,” Penguin Books, Baltimore., 197 1111. 8. Carsner, E. (1933). U . S . Dept. Agr. Tech. Bull. No. 360, 68 pp. 9. Chester, B.S. (1933). Quart. Rez,. Biol. 8 , 129, 275. 10. Crowdy, 8 . II., and Posnette,A. F. (1947). Ann. Appl. Biol. 34, 403. 1 1 . Dobrosrky, I . D. (1929). Phytopathology 19, 1009. 12. Glnuer, It. W . (1918). Psyche 26, 39. 13. Gidtlings, N. .J. (1950). Phytopathology 40, 377. 14. Holmea, F. 0. (1934). Phytopathology 24, 984. 15. Holmes, F. 0. (1938). Phytopathology 28, 553. 16. Ilolmes, F . 0. (1948). in “Bergey’s Manual of Determinative Bacteriology,” 6th ed., Suppl. 2, pp. 1146, 1147. Williams & Wilkins, Baltimore. 17. Huff, C . G . (1940). Physiol. Revs. 20, 68. 18. .Jetisen, J . H . (1933). Phytopathology 23, 964. 19. Iiiihler, E. (1943). Anpew. Botan. 26, 313. 20. Iiunkel, J,. 0. (1924). Phgtopalhology 14, 54. 21. Kunkel, L. 0. (1926). Am. J . Botany, 13. 646. 22. Kunkel, L. 0. (1931). Contribs. Boyce Thompson Znsl. 3, 85. 23. Iiunkel, I,. 0. (1932). Contribs. Boyce Thompson Znst. 4, 405. 24. Iiunkel, I,. 0. (1933). Ponlribs. Boyce Thompson Znst. 6 , 19. 25. Iiunkel, I,. 0. (1934). Phytopalhology 24,437. 26. Icunkel, I,. 0. (1935). Botan. Rev.1. 1. 27. Iiunkel, L. 0. (1936). Phylopathyology 26, 201. 1. 2. 3. 4.

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Kunkel, L. 0. (1937a). Am. J . Botany 24, 316. Kunkel, L. 0. (1937b), J. Bacteriol. 34, 132. Kunkel, L. 0. (1938). J. Econ. Enlomol. 31,20. Kunkel, L. 0. (1939). “Science in Progress,” Chapter IV, p. 112. Yale Univ. Press, New Haven. 32. Kunkel, L. 0. (1940). Publ. Am. Assoc. Advance. Sci. No. 12, 22. 33. Kunkel, L. 0. (1941a). Phylopathology 31, 14. 34. Kunkel, I,. 0. (1941b). Am. J. Botany28,761. 35. Kunkel, L . 0. (1944). in “Handbuch der Virusforschung” (R. v. Doerr and C. Hallauer, eds.), Suppl. 1, p. 473. Springer-Verlag, Vienna. 36. Kunkel, I,. 0. (1945). J . Baeteriol. 60,238. 37. Kunkel, I,. 0. (1947). Ann. Rev. Microbiol. 1, 85. 38. Kunkel, I,. 0. (1951). Bull. Torrey Botan. Club 78, 269. 39. Kunkel, L. 0. (1954). in “The Dynamics of Virus and Rickettsia1 Infections” (F. W. Hartman, F. L. Horsfall, Jr., and J. G. Kidd, eds.) p. 150. Blakiston, New York. 40. Magie, R. O., Smith, F. F., and Brierley, P. (1953). “The Gladiolus,” 28th annual ed., p. 93. New England Gladiolus Society, Boston. 41. Manns, T. F., and Manns, M. M. (1935). Ann. Rept. Director Univ. Delaware Agr. E z p . Sta. Bull. No. 192, 40. 42. Maramorosch, K. (1952a). Phytopathology 42, 14. 43. Maramorosch, K. (1952b). Phytopathology 42, 59. 44. Maramorosch, K. (1952~). Nature 169, 194. 45. McClean, A, P. D. (1947). Union S . Africa Dept. Agr. and Forestry, Sci. Bull. No. 266, 39 pp. 46. McKinney, H . H. (1929). J . Agr. Research 39, 557. 47. McKinney, H. H. (1935). Science 82, 463. 48. Price, W. C. (1932). Contribs. Boyce Thompson Znsl. 4, 359. 49. Price, W. C. (1935). Phytopathology 26, 947. 50. Price, W. C. (1936). Phytopathology 26, 503. 51. Price, W. C. (1940a). Am. Naturalist 74, 117. 52. Price, W. C. (1940b). Quart. Rev. Biol. 16, 338. 53. Ross, A. F. (1950). Phytopathology 40, 24. 54. Salaman, R. N . (1933). Nature 131, 468. 55. Severin, H. H. P. (1929). Hilgardia 3, 543. 56. Severin, H. H. P. (1934a). Hilgardia 8, 305. 57. Severin, H. H. P. (1934b). Hilgardia 8, 339. 58. Severin, H. H. P. (1945). Hilgardia 17, 21. 59. Severin, H. H. P. (1946). Hilgardia 17, 139. 60. Severin, H. H. 1’. (1947a). Phytopathology 37, 364. 61. Severin, H. H. P. (1947b). Hilgardia 17, 197. 62. Severin, H. H. P. (1947~). Hilgardia 17, 511. 63. Severin, H. H. P. (1948). Hilgardia 18, 203. 64. Smith, R. E. (1902). Hatch Expt. S f a . Mass. Agr. College Bull. N o . 79, 26 pp. 65. Storey, H. H., and McClean, A. P. D. (1930). Ann. A p p l . Biol. 17,691. 66. Storey, H. H. (1931). Compt. rend. commun. 8nd Congr. intern. pathol. comp. Paris, p. 471. 67. Thung, T. H. (1931). Handel. 6th Ned. I n d . Natuur. Congr. Bandoeng, Java, p. 450. 68. Thung, T. H. (1947). TKjdschr. Plantenziekten 63,43. 69. Wallace, J. M. (1944). J . Agr. Research 69, 187. 28. 29. 30. 31.

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Current Status of Bacterial Transformations HARRIETT EPHRUSSI-TAYLOR Laboraloire de GLnClique de la Facullb des Sciences, Paris, France, el du Centre Nationale de la Recherche Scientijique I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 11. General Biological Description of Transformation. . . . . . . . . . . . . . . . . . . . . . . . 276 111. Chemical and Physical Properties of Transforming Agents. . . . . . . . . . . . . . . . 278 A. Fractionation Studies on D N A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 B. Molecular Weight of a Transforming Factor. ......................... 281 IV. Mechanism of Transformation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 A. Phases of the Transformation Process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 B. Quantitative Kinetic Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 V. Genetic Recombination between Transforming Factors. . A. Allogenic Transformation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. Linked Capsular Agents in Hemophilus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 C. Linkage of Streptomycin Resistance and Mannitol Factors.. . . . . . . . . . . 301 D. Significance of Recombination Data . . . . . . . . . . . . 303 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307

I. INTRODUCTION When it became clear that a number of plant viruses are crystallizable macromolecules composed of ribonucleic acid and protein it was possible to attribute to a particular sort of macromolecule the striking biological property of autoreproduction. It could be imagined that this extraordinary property was a consequence of the particular arrangement of the atoms forming the nucleoprotein. Since the nucleoprotein nature of genes had been suspected for some time, it became only natural to consider that the activities of genes, too, were natural consequences of a nucleoprotein structure. In the past, a very high degree of autonomy has been assigned to both genes and viruses. Thus, the very fact of a particle being composed of nucleoprotein could be considered to result in a high degree of autonomy in the cell. Today, two very important modifications are taking place in our thinking about autoreproducing particles. I n the first place, it is becoming more evident that the autonomy of any element of a living cell is only very relative. This is true of the nuclear gene whose function and perhaps even structure may be modified by conditions prevailing in the cell as a whole (see Ephrussi, 1953, for a discussion of this question). It is equally true for viruses, for we are beginning to recognize real restrictions of the autonomy of viruses. One such restric275

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tion is clearly demonstrated by host specificity; a virus may penetrate into a cell but fail to reproduce itself, even though the cell is synthesizing the nucleotides and amino acids presumably necessary for reproduction of the virus. Another such restriction can be seen in the recent demonstrations of host-induced modifications of viruses (Luria, 1953). A second modification in our thinking about autoreproducing particles has occurred as a result of the work of Hershey and Chase (1952) upon the relative importance of protein and deoxyribonucleic acid in the reproduction of bacteriophage. The idea is now being entertained that the element of a virus which actually penetrates the living cells and which causes the formation of new virus is the nucleic acid component, and that virus protein plays only an accessory role, analogous to that of a seed coating, and functioning to preserve the essential nucleic acid during its extracellular existence, and/or to assure its reintroduction into a new host cell. Consequently, that autonomy which remains after we have taken into account the restrictions imposed by the host cell now is becoming invested essentially in the nucleic acid alone. Assigning to nucleic acid the key role in autoreproduction is an extreme hypothesis, in which a minimum number of components is postulated: a nucleic acid molecule on the one hand, and a living cell on the other. However, the hypothesis is hardly a simplification over its predecessor, for the most complicated part of the virus-host complex is, in fact, the host cell, not the virus. How true this proves to be can be amply seen from studies of a system in which we are in point of fact observing the reactions of a cell with molecules of nucleic acid; namely, in studies upon induced transformation in bacteria. Consequently, it is the purpose of the present article to summarize in general our present knowledge of transformation and transforming factors, and then to enter in detail into the evidence indicating the complex nature of the contribution of the cell to the reproduction of nucleic acid introduced into the cell in the form of a transforming agent.

11. GENERALBIOLOGICAL DESCRIPTION OF TRANSFORMATION The phenomenon with which we shall be dealing can be described as a permanent, hereditary change induced in certain individual bacteria as a result of their coming in contact with deoxyribonucleic acid extracted from a closely related but differing bacterial strain. Not only is a new hereditary character acquired, but also the transformed bacterium propagates the specific deoxyribonucleate responsible for the change. In a sense, this phenomenon resembles virus infection in that a particle of heterologous origin establishes itself and is perpetuated by a living cell, as a consequence of which cell properties are altered. I n the realm of viruses, it is lysogeny

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which is most similar. However, there are several important differences between the two phenomenon: insofar as evidence goes today, it does not seem that the process of transformation is associated with any deleterious effects at the level of the individual cell. It is possible to induce in populations of bacteria transformations to cell types which compete unfavorably with untransformed sister-cells, but here the deleterious effect is at the level of population dynamics. The failure to observe deleterious effects at the level of the individual cell may be due, of course, to the difficulty of detecting them if they are rare. Hence, this difference may be quantitative rather than qualitative. Perhaps more important is the fact that transforming agents seem devoid of any special structure to insure their uptake by the bacterium, in contrast to bacteriophages, which are so admirably adapted in this respect. It is only thanks to the very arduous years of trial and error that investigators today can perform transformations with a high degree of success. Both of these considerations lead to the notion that transforming agents are the least specialized of all isolated particles endowed with genetic continuity, a notion which is confirmed by the fact that they are the simplest in terms of chemical structure. The types of characters for which transformations are reported seems to be limited only to the variety of stable properties which the experimenter is able to recognize and maintain. Their variety grows daily. Highly specific serological characters were the first for which transformations were induced, and remain the most prevalent, being recognized in Pneumococcus (Griffith, 1928; Avery, MacLeod, and McCarty, 1944), Hemophilus (Alexander and Leidy, 1951), Escherichiu (Boivin et ul., 1945), Shigella (Weil, 1947), and Meningococcus (Alexander and Nedman, 1953). In addition, transformations have been found which involve changes in colony morphology unassociated with any known serological change (Taylor, 1949a), resistance to various antibiotics (Hotchkiss, 1951 ; Alexander and Leidy, 1953), alteration of metabolism of lactic acid (Ephrussi-Taylor, 1954), carbohydrate fermentation (Austrian and Colowick, 1953; Hotchkiss and Marmur, 1954), and growth in the presence of an amino acid analogue, canavanine (Ephrussi-Taylor, unpublished data). It has proven possible to transform not only for characters which differentiate naturally occurring races of the same species, but also for characters which have been deliberately selected by the experimenter. In the former instance, the strain donating the nucleic acid and that reacting with it may be supposed on phylogenetic grounds to have arisen, at some remote past time, from a common ancestor. In the latter instance, however, it is clear that donor and receiver strain are very closely related, the one being derived from the other by mutation. It is of interest to note that while it is possible to transform a mutated

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property of a cell back to normal (as in the case of capsular transformations), it is equally possible to transform a normal character into a mutant one (as in transformation to streptomycin resistance). Even more significant is the fact that in a few instances, it is possible with pairs of strains, one normal and the other mutant, to perform transformations in either direction (Taylor, 194913; Hotchkiss and Marmur, 1954). These observations have led to the notion that in transformations we are dealing with pairs of homologous agents; or in other words that, in the bacterial cell exists a genetic entity composed of DNA, or the specificity of which is determined by its DNA component, and that this entity is capable of existing in two or more relatively stable alternate conditions, each characterized by a particular phenotypic expression, usually the presence or absence of a property. In transformations converting a mutant character to normal, under the action of a DNA extracted from the nonmutant cell, we are apparently observing the substitution of the normal DNA homologue for the mutant one in the cell, whereas in transformations operating in the opposite direction, it is just the reverse substitution which is occurring. From these early experiments we discern the emergence of two important ideas which are in harmony with classical genetics. First, the discovery that the synthesis of capsular polysaccharide is determined by a specific deoxyribonucleate proves the separate existence in bacteria of character and hereditary factor. Second, the existence of reciprocal transformations shows that the disappearance or alteration of a cell character is not necessarily correlated with a loss of a genetic determinant; that the latter may simply be present in a modified or mutant form. These few remarks serve to indicate that today it is possible to regard transforming factors as having the same degree of autonomy as the Mendelian gene, and viruses, possess. This is not surprising if these latter entities owe their special properties to the nucleic acid they contain. In all three instances it remains to define the nature and extent of this autonomy, which will be completely achieved, no doubt, only when the process of self-duplication will be understood. 111. CHEMICAL AND PHYSICAL PROPERTIES OF TRANSFORMING AGENTS When it was proposed in 1944 that the capsular transforming factor was composed exclusively of DNA (Avery, MacLeod, and McCarty, 1944), legitimate doubts were raised that this could be the case, for such biological specificity of nucleic acids was unheard of. The question of the chemical identity of transforming agents has subsequently been dealt with in various studies (Hotchkiss, 1948; Zamenhof, Alexander, and Leidy, 1953), which will not be entered into here. The purity of a number of prepara-

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tions of transforming agents was investigated, as well as the sensitivities of transforming activity to various chemical and physical treatments, and in every instance results were concordant with the view that the active principles have the chemical properties of DNA. It consequently seems likely that the origin of biological specificity of transforming agents must be sought in specific structural properties of this type of substance. However, to demonstrate that agents having different specificities also have distinctive chemical structures may prove exceedingly difficult, for it is now quite clear that a deoxyribonucleate prepared from a single bacterial strain must be composed of an unknown number of different transforming factors, and that analytical data on even highly purified extracts represents the average composition of the active factors. Bacterial strains differing by one or a few factors can hardly be expected to form deoxyribonucleates differing significantly in chemical composition. It was encouraging to discover with the extension of modern analytical techniques to DNA prepared from widely different species that the ratios of the bases were not always the same (Chargaff, 1951 ;Wyatt, 1952). But the challenging task of separating in pure form a transforming agent, and assigning to it a spec& structure remains to be performed. Indeed, we do not even know whether such separation is possible, for this will depend on how the agents are organized on the DNA particles.

A . Fractionation Studies on DNA However, toward this end, new methods of study have been developed by which highly polymerized DNA obtained from a given animal (or bacterial) source may be separated into fractions having different ratios of purine and pyrimidine bases, and at present these methods are being applied in an effort to bring definite proof of the chemical basis of biological specificity (Chargaff, Crampton, and Lipschite, 1953 ; Brown and Watson, 1953). To the author’s knowledge, no one has as yet succeeded in separating in distinct fractions transforming factors having different specificities. It is, however, opportune to discuss very briefly a typical result of a fractionation of pneumococcal DNA by the method of Brown and Watson (Brown and Ephrussi-Taylor, unpublished data), for it has furnished another strong presumption in favor of the view that transforming factors are deoxyribonucleates. In the method of Brown, as originally published, a column is constructed of kieselghur upon which purified histone has been adsorbed. This column has the property of adsorbing both RNA and DNA from dilute sodium chloride solutions (below 0.5 M). Washing the column with solutions of sodium chloride containing more and more salt results in €he elution of a series of fractions. The fi s t material eluted is composed of RNA, and any DNA which has been denatured. As the

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molarity of salt increases, DNA begins to be eluted. A sharp separation of RNA from native DNA is achieved. The elution curve of DNA from the column is skewed, as may be seen in Fig. 1, a small amount of material requiring very high salt concentrations for its elution. Analyses of the base ratios of the material in the different fractions shows that the ratio of adenine to guanine increases progressively from left to right. If now we look at the distributions of transforming factor in these fractions, the one shown in Fig. 1 being the factor which confers a high level of resistance to streptomycin upon sensitive pneumococci, we find that a very similar profile is obtained, except that it is displaced slightly to the left. In other x

o

CPticol density Transforming activity

-. 5 z - 6x10)

-. i 0,

9

Y l a

3 5 X V

-/*< a60 0.70

0.72

0.74

CkiE

0.78

Y

P

- +

Q00 0.02

0.S4

a86 0.80

a90

Fro. 1. Fractionation of a pneumococcal deoxyribonucleate containing the Sr transforming agent by the method of Brown and Watson (1963). Data from Brown and Ephrussi-Taylor, unpublished.

words, the DNA which has the highest specific activity is the first to be eluted. In experiments in which there has been very little inactivation, the most active fraction may be as much as five times more active than unfractionated DNA. The two factors (streptomycin and canavanine resistance) which have thus far been subjected to fractionation by this method prove to give very similar distributions. At present, from these experiments two conclusions may be drawn: (1) the chemical identity of transforming agents with DNA seems more than ever indicated, and (2) high specific activity seems to be closely associated with a high content of guanine. This is of particular interest in connection with a recent discussion of the origins of specificity in DNA (Feughelman et.aZ., 1955) in which it is concluded that the free amino group of guanine probably plays a very important role. The second conclusion is also in harmony

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281

with the observation that very little deamination suffices to completely inactivate a transforming agent of Hemophilus.

B . Molecular Weight of a Transforming Factor Although the past decade has seen considerable evolution of our ideas concerning the chemical composition of nucleic acid, as a consequence of which, in part, it has been possible to formulate a structure for DNA (Watson and Crick, 1953), the exact dimensions of molecules in their native state remain undefined. The literature abounds in estimations of molecular weight of DNA by several physical methods, but in view of the tremendous assymetry of the particles, and the absence of proof of the homogeneity of the samples studied, the values obtained must be considered as subject to very great error. It is accordingly of interest to note that, in a recent study of the sensitive volume of a transforming factor (streptomycin resistance) inactivated by X-rays, it is concluded that the molecular weight of the active particle is equal to, or smaller than, 7 X 105 (Ephrussi-Taylor and Latarjet, 1955). This is smaller than the molecular weight currently assigned to highly polymerized DNA by a factor of 10 (Reichman, et al., 1954). Furthermore, evidence was obtained which indicated that the particles bearing a single specific activity might be of several sizes. Thus, the inactivation curve is exponential until only 5 to 10% of the initial activity remains at which point there is a fairly sharp break in the curve so that the remaining activity disappears only very slowly, as a function of X-ray dose. This result is compatible with two interpretations : either the active particles are clumped, resulting in aggregates which require more than one “hit” for inactivation to occur, or one may suppose that the specific activity studied exists effectively in the form of particles of two distinctly different sizes. Experiments were undertaken in an effort to distinguish between these two possibilities (Ephrussi-Taylor, Latarjet, and Thomas, unpublished data). Strong urea solutions are known to greatly decrease intermolecular interactions. Solutions of the transforming agent which confers streptomycin resistance were therefore subjected for 8 hr. at 25°C. to a concentration of 5 M urea, and the X-ray inactivation curves obtained upon such treated material were compared with those found with untreated solutions. It should be noted first of all that the treatment resulted in no inactivation whatsoever of the transforming agent, and secondly, that both treated and untreated solutions gave strictly identical inactivation curves when subjected to X-irradiation, the “resistant particles” being present in both instances. It seems all the more likely that these particles do in fact represent active material of lower molecular weight. It should be added that these particles are in no way different genetically from the more sensitive ones, and that bacteria

282

HARRIETl’ EPHRUSSI-TAYLOR

transformed with them synthesize DNA upon which one again obtains the same kind of broken inactivation curve. There remains, nonetheless, a possibility of reconciling these observations with the measurements of molecular weight made by centrifugation or by light scattering methods. The sensitive volume of a transforming factor represents only that region within which a hit must occur for biological activity to disappear. It is possible that this region is only part of a very large molecule. This amounts to saying that what is being measured is the distance over which the effects of a hit travel. The particles which appear to be “more resistant” may, by this reasoning, be molecules in which, for some unknown cause, the energy of a hit is conducted only a relatively short distance. If this sort of a situation were the real explanation of the results, it is clear that the sensitive volume in no way corresponds either to molecular weight, nor necessarily to the actual size of the specific region which determines resistance to streptomycin. If a physical separation of “big” and “small” particles could be effected, it would be possible to determine whether their different sensitivities to X-rays actually resulted from a difference in size.

IV. MECHANISM OF TRANSFORMATION One of the great mysteries of the transformation phenomenon is that of how the very large molecule of DNA succeeds in reaching a deep enough site in the cell so as to become integrated into its genetic apparatus. The picture which we can make today of this process is more a description of the phases of the reaction, under conditions which have been found by trial and error to be effective, than it is a definition of the reaction in terms of its essential components. Nonetheless, there can be little doubt that, through the analysis of these phases, a definite progress is being made toward a more fundamental approach, in particular in the work of Hotchkiss (1954) and Thomas (1955), each working with pneumococcus, but employing to different advantages two entirely different sets of conditions.

A . Phases of the Transformation Process It has been known for some time that in cultures in which conditions will lead to transformation, cells capable of reacting with a transforming agent are only present at certain moments of logarithmic growth of the inoculated cells (McCarty, Taylor, and Avery, 1946). Roughly, the period during which they are present corresponds to the latter half of the logarithmic phase. However, once such cells appear in the culture, only a very short exposure to nucleic acid su5ces to induce transformations. With the development by Hotchkiss of transforming systems in which the numbers of transformed cells can be easily determined, it has been possible

CURRENT STATUS OF BACTERIAL TRANSFORMATIONS

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to describe with considerable precision the various phases of the reaction between DNA and bacterial cells. Thus, using the transforming agent which confers a high level of streptomycin resistance (the Sr agent) upon normally sensitive pneumococci, the moment of appearance of resistant cells and their number can be determined as a function of a large of number of variables. As the result of such experiments (Hotchkiss, 1954; Thomas, 1955) it is possible to divide the transformation reaction into several phases, the recognition of these phases being an essential preliminary to truly kinetic analysis. The phases which may be recognized are the following: (1) a preliminary period of growth in the special transforming medium, which may take place equally well in the presence or in the absence of DNA; (2) a period during which reactive cells make their appearance. By exposing reactive cells t o transforming factor for only a very brief period of time and following development of resistance, it can be seen that (3) a short period is necessary for the uptake of the active agent which, for all of a sizeable population of reactive cells to be transformed, is of the order of 10 min.; (4)that the resistant phenotype is developed gradually by the transformed cells, and attains full expression only after about 45 min. to an hour; (5) and that the transformed cells appear to give rise to resistant progeny only after still another hour or two. Thomas (1955),in addition, found evidence suggesting that during period (3)two phases can be recognized, one consisting of the formation of a complex between bacterium and nucleic acid in which the DNA can still be attacked by deoxyribonuclease, and the second corresponding to a state in which the transforming factor becomes inaccessable to the enzyme. The two stages require only about 10 min. time. The recognition of these various steps in the transformation process has permitted a large variety of new experiments to be performed. One of the fortunate consequences of the sequence of events in time is that although it is necessary to allow an hour for the resistant phenotype to develop, during this time the total number of transformed cells will not increase by transmission of the new agent to new infective centers. Consequently, counts of colonies in streptomycin-containing plates need no correction, even though one must wait 1 hr. after exposure to the Sr agent before submitting the cells to the selective action of streptomycin. This may be due either to a failure of the newly acquired agent to be reproduced at once, or to the growth of the cocci in small chains. It is possible today to confine experimental attack to any one of these phases, each of which probably corresponds to a distinct set of biological and biochemical events. Thus far, the majority of experiments have dealt with phases (1)-(3), but phases (4)and (5) certainly concern the most important and most intriguing steps of transformation. It is in

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understanding these latter phases that we may hope to learn a vast amount about the relation between a character and its determinant, and about autoreproduction itself. It is obvious that the study of any phase prior to the completion of phase (4) depends upon the bacteria being able to go through to the end of phase (4) before encountering streptomycin, and to the end of phase ( 5 ) in its presence. We shall see in a moment that the fact that we are obliged to study the process in terms of the very end product may complicate somewhat the kinetics obtained.

B . Quantitative Kinetic Experiments The very first quantitative experiments upon phases (2) and (3) are those of Hotchkiss (1954). I n the media which he employed, reactive cells make a sudden appearance in growing populations after about 3% hr. of incubation, during which time logarithmic growth has been occurring. The rate at which these cells appear is much faster than multiplication of the population as a whole. From this moment on, the absolute number of reactive cells remains approximately constant until shortly before the slowing down of growth of the culture, at which time the reactive cells disappear. The question arises as to whether these cells constitute a physiologically distinct fraction of the population which fails to increase with the population as a whole. A direct experiment can be performed to test this point. If this were so, once reactive cells appeared, the number of transformed cells should be identical, whether one exposed the population for a short or for a long time to the transforming agent. This proves, however, not to be the case. Roughly speaking, the longer the exposure, the more transformed cells appear. By comparing the numbers of cells transformed as a result of exposures of either 10- or 20-min. intervals, the conclusion was drawn that the relatively constant number of reactive cells is the resultant of a constant appearance and disappearance of reactive cells in the population, the average time during which a given bacterium remains reactive being less than 20 min. This point could be more directly demonstrated by means of interference experiments using calf thymus nucleic acid as an interfering agent. If the inhibiting DNA is added a few minutes before the transforming agent, and the latter allowed to act for only 1 min. inhibition is complete. However, if as little as 7 min. more is allowed for the transforming factor to act, inhibition falls to about 70 %, which is the level obtained when both are added simultaneously. This is interpreted as indicating that, in the additional 7 min., new sensitized cells appear, and the level of inhibition falls to that of simple competition between the two substances for the limited new sites available. We shall see in a moment that the experiments of Thomas fully justify this interpretation.

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285

Having arrived at the conclusion that reactive cells are constantly appearing and disappearing, and that this, at the level of the individual cell, seems cyclical, it was natural to wonder whether a correlation did not exist between some phase of the bacterial division cycle, and the ability to react with DNA. Hotchkiss consequently tried to see whether by a temperature shock, a degree of synchronization of division might not be produced, and whether, simultaneously, the appearance and disappearance of reactive cells might not be made to occur in a demonstrably wavelike fashion. Accordingly, he applied a cooling period of 15 min. to a culture in which a large number of reactive cells had already made their appearance, and after returning the culture to 37°C. followed the evolution of the number of reactive cells in the culture. Effectively, a very pronounced wavelike behavior was obtained. Immediately upon being returned to 37"C., the number of reactive cells dropped precipitously, only to rise to a new high level and fall again, within an interval of 40 min. Several such cycles could be obtained. As for cell division, growth curves done on such temperature-shocked cultures indicated that division, too, had been synchronized. However, the time interval between successive divisions was of the order of 30 min. The temperature treatment seems, therefore, to introduce a definite measure of synchronization both in bacterial multiplication, and in the appearance and disappearance of reactivity toward the transforming agent. In spite of the fact that the two cyclical phenomena exhibit slightly different timing, it is tempting to suppose that the transforming agent is taken up as a function of the mitotic cycle. The experiments of Thomas, performed in different culture media (Ephrussi-Taylor, 1951) began with the formulation of a simple question. It has been evident for some time that, in the medium used, the period during which reactive cells are found is very restricted. The question was then as follows: just as there is a period of logarithmic growth during which it is too early to add transforming agent and obtain rapid reaction of agent and bacteria, is there also a period in which it is too late? The experiment set up to test this point is a very simple one (Experiment I). A large series of replicate cultures in transforming medium is set up, each containing lo6bacteria per milliliter. Each culture will make 7 to 8 generations of bacteria before growth ceases, each generation requiring 30 min. One-half of these tubes receive the Sr agent at time 0, and at 10-min. intervals deoxyribonuclease will be added to one tube of the series. The other half of the tubes form a second series, in which the Sr agent will be added to one tube every 10 min. All of the cultures will be poured in streptomycin-blood agar plates after about 6 generations have elapsed. Since the expression of the resistant phenotype requires a little less than 1 hr., the picture obtained describes events occurring between 0 and 120-

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140 min. The first series of tubes describes how soon reactive cells make their appearance, while the second series of tubes tells us whether there is a period during this interval when it is too late to add the Sr agent and obtain transformations. The results obtained by such experiments are

A FIQ.2. Study of the reactive period of a culture of pneumococcus, using the Sr transforming agent. 0 :Numbers of bacteria transformed in a series of cultures t o which transforming agent was added at time t . x: Numbers of bacteria transformed in a series of cultures in which the transforming agent was added at time zero, and deoxyribonuclease added a t time t . Data from Thomas, 1955. shown in Fig. 2. It is quite clear from this figure that reactive cells make their appearance some time after growth has begun, that their number increases very rapidly for about 20 min., following which they disappear with equal rapidity. This can be demonstrated in another way (Experiment 11). A single culture can be made, containing the same number of pneumococci, but in which the Sr agent is omitted (“sensitization culture”). At various times after initiating growth from a young, but stationary,

CURRENT STATUS OF BACTERIAL TRANSFORMATIONS

287

stock culture, small samples of the sensitization culture can be transferred into transforming medium containing the Sr agent. Ten minutes later the enzyme deoxyribonuclease is added, and 1 hr. later the entire culture, or a small sample of it, is plated in blood agar containing streptomycin. Thus, one can plot, as a function of time of incubation of the sensitization culture, the appearance and disappearance of reactive cells, which proves to have the shape expected from Fig. 2; that is, when the logarithm of the number of transformable cells is plotted against the age in minutes of the sensitization culture, a sharp peak in the number of reactive cells is observed at about 90 min. The ascending and descending slopes are linear. If one assumes that the peak is the result of the virtually synchronous appearance of reactive cells, each of which remains reactive a short time, the average time each cell remains in this state is determined from the time elapsing between the moment when 50% of these cells have attained this state, and when 50% have lost it. This interval is 16 min., which corresponds very closely with Hotchkiss’ estimation that the reactive state lasted, in each cell, a little less than 20 min. What is most interesting, and exceedingly useful to know, is that Experiment I1 can be done, omitting the addition of the enzyme, and the result obtained is essentially the same. This shows that the addition of deoxyribonuclease is unnecessary, and that the mere transfer of the cells from the sensitization culture into the fresh medium containing the DNA is sufficient to “freeze” the sample of bacteria in the state in which they were at the moment of transfer for a long enough period to permit uptake of the Sr agent. No new reactive bacteria make their appearance, and it proves to be true that those bacteria which were not reactive at the moment of transfer must start all over again at the beginning of the process leading to the reactive state. This can be seen experimentally by transferring a population of cells containing reactive ones into fresh medium to which no Sr agent has been added. The disappearance of reactive cells can be followed by adding the DNA at various moments after transfer. This turns out to be a t the same rate as the down slope, shown in Fig. 2. Finally, it should be added that, if the inoculum has not been too dense at the start of the sensitization culture, a second wave of reactive cells makes its appearance 45 min. after the first. Before descrihing any further experiments by Thomas, it should be pointed out, that t,he “peaks” which describe the appearance and disappearance of reactive cells in the experiments just mentioned resemble in a remarkable way the waves of disappearance, reappearance, and disappearance of reactive cells in the experiments of Hotchkiss, in which he has synchronized the culture by a temperature shock. However, when viable counts are made upon cultures undergoing a cycle in the medium

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used by Thomas, no indication can be found to suggest that division is synchronized (Ephrussi-Taylor, unpublished data). It is clear that something in the cultures of Thomas is synchronized, and it may be asked whether this is not nuclear division, rather than actual fission. Thomas has performed an experiment which is very difficult to reconcile even with this notion. I n all of the transformation media used with pneumococcus, it is necessary to add serum albumin to the basal culture medium. Provided the culture has reached a sufficient density, the minimum time required for the albumin to play its role is of the order of 40 min. By adding albumin at later and later moments after initiating the culture, Thomas TABLE 1

EFFECTS OF INOCULUM SIZEON TIMEOF APPEARANCE OF FIRSTWAVE OF REACTIVE CELLS* Dilution of inoculum culture 1 14 118 1/10 1/16 1/32 1/100 1/1000 1/10,000

Time elasping between inoculation and first peak (min.) 40 45 50

60 65 90 140 220

* A standardinoculum culture containing on the average 3 X 108 bacteria per milliliter is diluted in sensitizing medium, and the time of appearance of the maximum of the first wave of sensitization is determined. The wave is displaced in a continuous fashion as fewer cells are added. From Thomas, 1955. was able to retard the appearance of the peak in a continuous fashion. Consequently, if one supposes synchronized mitosis in the culture, it does not exist prior to the addition of the albumin, and one must now suppose that the addition of albumin rapidly reduces all of the cells of the culture to the same mitotic stage. While this is not excluded, it would seem to be asking a great deal. For some time it has been known that the time required for sensitized cells to make their appearance varies inversely with the size of the inoculum. In the media employed by Thomas, in which, for unknown reasons, reactive cells appear in synchronous fashion, the effect of inoculum size on the time of appearance of the first wave of reactive cells is shown in Table 1. It is evident that the time during which albumin acts on the cells is not the only factor determining the appearance of reactive bacteria, but

CURRENT STATUS OF BACTERIAL TRANSFORMATIONS

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that the cells must interact in some way with the medium and reach a certain state in order for the albumin to have its effect. Although all of these facts do not permit us to formulate a mechanism of transformation which can be generalized for application to other organisms, they have given such a degree of control to the investigator that kinetic investigations are possible. One such investigation is of the rate at which a transforming factor becomes inacessible to deoxyribonuclease. A sensitization culture is set up and, at a time when reactive cells are known to be present in large number, aliquots are transferred rapidly to a series of tubes containing transforming factor. At various times enzyme is added to destroy all transforming factor still accessible. After time has been dlowed for expression of the phenotype the transformed cells are assayed in streptomycin agar. The curve obtained is sigmoid (Stocker, Krauss, and MacLeod, 1953; Thomas, 1955) indicating the complexity of the process. Another kind of investigation which has become possible is a study of the quantitative relationships between the amount of DNA added and the numbers of bacteria transformed. It has been known from studies performed with the capsular transforming agent that within certain limits the number transformed is a linear function of the amount of nucleic acid added (Stocker, Krauss and MacLeod, 1953; Ravin, 1954). This suggests that the reaction is bimolecular. In a study of transformation of Hemophilus, Zamenhof, Alexander, and Leidy (1953) have calculated the number of molecules of DNA required to transform one bacterium to streptomycin resistance, and have come to the conclusion that it is of the order of 108. If the calculation is correct, it might seem necessary to suppose that only one molecule out of lo3actually carries the streptomycin resistance factor. However, it is unlikely that this calculation is correct in view of the assumptions involved, but one of the most obvious ways of testing its validity is to determine to what extent the transformation reaction can be treated as a bimolecular reaction, and in particular, to see whether a stoichiometric relationship can be demonstrated to exist between the transformed bacterium and added DNA. Thomas obtained data for testing this relationship in the following way. A sensitization culture is prepared (the transforming agent omitted), in which reactive bacteria are going to appear and disappear in a wavelike fashion. At several moments during the wave, aliquots of culture are rapidly transferred to a series of tubes containing varying amounts of the Sr agent, dissolved in medium. Time is allowed for the phenotypic expression of resistance by the bacteria which will be transformed, and the numbers of transformed bacteria assayed. I n this way a titration is performed with a k e d number (concentration) of reactive bacteria, for the

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transfer into fresh culture medium arrests the development of new reactive bacteria. Those that were reactive at the moment of transfer remain so for a few minutes before becoming unreactive. Table 2 shows two such titrations performed; in the one, the concentration of reactive bacteria is 617 per milliliter, while in the other the concentration is 155 per milliliter. What appears to remain constant, in comparing the two sets of data, is the per cent of reactive cells transformed by a given amount of nucleic acid. In other words, a fixed amount of nucleic acid is required, not to TABLE 2 TITRATIONS OF SR-TRANSFORMINQ ACTIVITYPERFORMED WITH Two POPULATIONS OF TRANSFORMABLE CELLS* CONTAININQ DIFFERENT NUMBERS ~

~

A . Actual numbers of resistant colonies per plate Number of bacteria in a standard sample of culture transformed Age of by the following amounts of DNA sensitization 50 mg./ml. 5 0.5 0.05 0.005 0.0005 culture

60 min. 60 min.

685 143

546 181

621 182

358 91

59 27

8 3

B . Same titration expressed i n per cent of transformable bacteria transformed by each quantity of D N A Per cent of transformable cells transformed by the following Age of amounts of DNA sensitization 50 mg./ml. 5 0.5 0.05 0.005 0.0005 culture ~~

60 min.

80 min.

88

111 92

104

58 58

101 104

9.5 17

1.3 2

* Data kindly provided by R. Thomas. transform an absolute number of reactive bacteria, but in order to transform a given proportion of the reactive members of the population. A formulation of the kinetics of the reaction was attempted by taking into consideration the fact that one of the reactants-the competent bacteria-is highly unstable. The appearance and disappearance of such bacteria can be expressed tw follows: Growing bacteria

-

Reactive bacteria

-<

Unreactive bacteria Transformed bacteria

The first step of this chain of events can be blocked simply by transferring into fresh medium a population containing some reactive bacteria.

CURRENT STATUS OF BACTERIAL TRANSFORMATIONS

29 1

This leaves us with two routes by which the reactive bacteria will disappear: by reacting with transforming factor, and by losing the competent state before encountering transforming factor. The first of these reactions can be assumed to be formally analogous to a bimolecular reaction, in view of the linear relationship which exists between the number of bacteria transformed and the concentration of DNA at low values of the latter. The second of these reaction, Thomas showed, can be considered to follow the kinetic behavior of a first order reaction. Thus, it is possible to derive an equation describing the resultant of the two reactions, which proves to have the following form: [Transformed bacterial = [Reactive bacteria]

DNA1 k1

-

k2

+ DNA1

In this equation, the concentration of reactive bacteria is the number present at the moment of transfer into the presence of DNA; k* is the constant of the first order reaction describing the decay of the reactive state, and can be readily measured; k2 is the constant of the affinity of DNA for the reactive bacteria. When half of the reactive bacteria are transformed, k1/lc2is equal to the concentration of DNA. Figure 3 shows graphically the data of Table 2 along with the theoretical curve obtained from the equation in which the value of k1/k2is taken from Table 2. The equation would seem to fit the data satisfactorily.’ The solution of the two simultaneous equations by Thomas involved one further assumption : that the total number of DNA molecules is at all times so much greater than the number of bacteria, that the concentration of DNA does not appreciably change as a result of the reaction. If Thomas’ analysis is correct, then the data of Zamenhof, Alexander, and Leidy (1953) most likely mean that, out of lo3 molecules of DNA, only one has entered into effective contact with a reactive bacterium and, therefore, their result cannot be taken as a measure of the heterogeneity of DNA. Finally, Thomas’ study resulted in the formulation of an entirely new interpretation of the curve describing the rate at which a transforming Since writing this, the author has obtained five additional sets of data, in which the numbers of reactive bacteria mixed with the range of nucleic acid concentrations varied from 23,230 to 382 per milliliter. The five new titrations were all performed in a single, new batch of medium, and, therefore, not under absolutely identical conditions a8 maintained in Thomas’ titrations (Table 2). Nonetheless, the relationship found between DNA concentration and the per cent of reactive bacteria transformed is identical, except that the value of kl/k*is ten times greater in all the five titrations. Since no great differences have yet been observed in the rates of disappearance of reactive cells (k’), the results suggest that the greatest variable from one preparation of transforming medium to another affects k’, the affinity constant.

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agent becomes inaccessable to the enzyme deoxyribonuclease, following the contact of the DNA with reactive bacteria (see p. 283). What Thomas noticed was that the development of the protected state of the transforming factor coincides absolutely in time and degree to the disappearance of the ability of the bacteria to react with DNA. Both of these processes

01%

50

5

05

a05 a005 a0005 Qo0005 Concentration of DNA

FIG.3. The per cent of the transformable bacteria actually transformed as a function of concentration of the Sr agent. The points are from two experiments. The solid line is a theoretical curve calculated from the equation: DNA1 [Transformed Bacteria] = [Reactive Bacteria] k1 DNA1

kz

+

in which k'/k' is the concentration of DNA yielding 50% transformation. The value of kl/k' is taken from the experimental points. Data from Thomas, 1955.

can be studied by transferring two samples of a single population containing competent bacteria into medium containing DNA, on the one hand, and medium lacking DNA, on the other. In the first subculture, the evolution toward insensitivity to deoxyribonuclease can be followed while, in the second, the disappearance of the reactive state can be determined. As a result of such experiments, Thomas suggests that the reactive state corresponds to a moment when the bacteria become generally permeable to large molecules; until the competent state disappears, not only can DNA enter the cell, but so, also, can the enzyme which destroys it. According

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to this hypothesis, the sigmoid curve described elsewhere is simply a description of the way in which the abnormally permeable state disappears in the bacterial population. The relatively complex relationship described by Thomas may result from the fact that the reaction between the Sr agent and bacteria is described solely in terms of the bacteria which have not only fixed the Sr agent, but gone on through to the completion of phase ( 5 ) . It must be pointed out that by “unreactive,” we can, owing to the limitations of the experimental method, only mean those bacteria which have not completed phase (5) of the transformation process. “Unreactive” does not mean that the bacteria so classified are not also taking up the nucleic acid in an irreversible fashion. They may indeed be doing so, but fail to incorporate the Sr agent as a genetic entity. How they complicate the kinetic picture remains to be determined, and could profitably be studied with the aid of marked transforming DNA.

V. GENETICRECOMBINATION BETWEEN TRANSFORMING FACTORS If it proves true that every transformation consists essentially in the substitution of a transforming agent introduced into the environment for one already existing in the inoculated cells, and the reasons for believing that this is the case have already been discussed then it is clear that transformation is one more kind of genetic recombination. In genetic recombinations involving chromosomes, as well as recombinations of determinants in bacterial viruses, it has been possible to discern recombination at two levels of the genetic material: first, between determinants which are situated upon separate physical structures, or at least which behave as though they were, and second, between determinants which must be presumed to be situated upon a single organized structure. Insofar as chromosomal inheritance is concerned, the reasons for this are clear, for recombination involves on the one hand reassortment of entire chromosomes, and on the other, reassortment of genes as a result of crossing over between pairs of homologous chromosomes. As for bacteriophage, it is more difficult to assign a physical picture to the genetic organization. Recombination may occur at two levels because there are several linkage groups, or may be the result of a lack of markers in certain regions of a very long, single, chromosomelike structure. Today, it is evident that the situation found in bacteriophage is essentially duplicated in bacterial transforming factors. What are the methods which should be applied ideally in a study of the structural relationships between different transforming factors? In principle, this should consist of determining the frequency with which two given factors are acquired simultaneously by the transformed cell under conditions in which the total incidence of transformation is limited by the nucleic

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acid concentration. If this frequency is in appreciable excess of that expected from the random distribution of each of the agents considered independently, then it may. be supposed that physical association exists between them. In fact, these conditions have not been fulfilled in any one of the three cases of linkage of transforming factors about to be described. However, special considerations leave little doubt that in all three examples linkage between genetically independent loci is involved.

A . Allogenic Transformation The first instance of apparent recombination between loci situated 011 a single physical particle waa reported in detail in 1951 (Ephrussi-Taylor, 1951). A series of mutant forms of Type I11 pneumococci were isolated which differed from normal in secreting diminished amounts of polysaccharide. The question arose as to what had mutated. A priom', two situations were expected: first, a mutation of the Type I11 capsular transforming agent itself, or second, the mutation of some other genetically independent element of the bacterial genome having as a consequence a supression of the activity of the Type I11 agent. These two alternative situations can be distinguished in transformation experiments by preparing a DNA from each of the mutant strains, and testing activity upon an unencapsulated race. If the first situation is the real one, inductions should result only in the formation of bacteria having the mutant character. If the second situation is the true one, transformation should, by and large, produce only normal Type I11 bacteria, and very few mutant ones, for only rarely should two factors be acquired simultaneously by a single bacterium. Experiments showed that nucleic acid isolated from each mutated race gave transformations only to the mutated capsular state. It seemed clear, therefore, that the mutations had occurred only in the Type 111capsular agent. Two categories of mutant forms were recognized. In one, capsule secretion is highly reduced, and bacteria propagating such a mutant agent secrete too little polysaccharide to entirely cover their somatic antigens. Consequently, such bacteria can be recognized by the fact that they do not form a mucoid colony, and can be agglutinated both by Type 111 antibodies and antibodies directed against the somatic antigens. This class of mutants is called the SIII-1 class. In the second category, capsule secretion is reduced, and the colony less mucoid than normal, but somatic antigens are entirely covered by polysaccharide. Agglutination is observed only in the presence of Type I11 antibody. This class of mutants is called SIII-2. A study was performed upon four mutated agents of the first category and one of the second. Each represented an independent mutation of the Type I11 agent. All of the strains of the SIII-1 category can undergo transformation, and it is thus possible to test the action of normal and mutated cap-

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sular agents upon these strains, which already are propagating a mutated capsular agent. The results of such a study can be summarized in the following way: if one treats a pneumococcus containing a mutated capsular agent with another mutated agent of independent origin, there may occur transformations leading to the complete restoration of normal capsule synthesis. The result actually obtained depends upon which pair of agents is being confronted in the pneumococcus, and is, therefore, specific and dependent upon the intrinsic properties of the agents involved. With certain pairs, restoration appears to be total, while with other pairs it is partial. Close examination of the transformed bacteria reveals that not only is capsule synthesis partially or totally restored, as the case may be, but also that the transforming agent in such bacteria is, correspondingly, either entirely normal or at least approaches normal. That is to say, when these agents are tested upon unencapsulated pneumococci, inductions are either to the normally encapsulated state, or to a state approaching it. Consequently, the confrontation of the two mutated capsular agents seems to have led not only to the elimination of all or most of the mutant character of capsule synthesis, but also to have led to an exactly parallel restoration of the agent responsible for this synthesis. When the pair of agents confronted is on the one hand an SIII-1 agent and on the other the SIII-2 agent, it can be seen that two kinds of inductions can occur, resulting in the appearance of (a) normally encapsulated bacteria, and (b) bacteria resembling the SIII-2 donor strain (the SIII-1 strain being, as always, the recipient or transformed strain). The recipient strain seems thus to be able to react in two ways: by incorporating the SIII-2 agent unchanged, or by forming a normal agent out of it. Transformations of the first sort are the familiar ones (autogenic) in which the agent formed by the transformed cell has the same specificity as the inducing agent, while transformations of the second sort were called allogenic to indicate that the transformation results in the formation of an agent having properties which are not those of the inducer. The explanation offered for these results is that allogenic transformations are the consequence of a recombination occurring between the mutated capsular agent in the bacterium, and the mutated agent with which the induction was performed. The recombination is presumed t o lead to the elimination of mutated areas of each of the agents involved. This can be represented diagrammatically as shown in Fig. 4. The fact that, with certain pairs of agents, only partial restoration ensues is explained by supposing that the mutated regions in the two agents overlap (Fig. 4B). This picture is strongly supported by the fact that allogenic transformations of this second sort occur only with a low frequency, which would be predicted from the hypothesis, in view of such transformation requiring recombination to takelplace within a very limited region.

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If one accepts the idea that an allogenic transformation results from recombination of the normal parts of two mutated agents, and consequent elimination of a defective part of each agent, we arrive at a picture of the DNA particle as a structure containing subunits capable of independent mutation and of recombination. If this is so, why then does the normal Type I11 agent ever behave as a unit? Why, on the contrary, does it not give a whole range of transformations when acting on a rough strain? Here it is only possible to suggest a reason. If we suppose that the rough bacterium contains a very highly mutated capsular agent so that all expression of capsule synthesis has been suppressed, then it may be that recombinations between this hypothetical agent and a normal one lead only to recombinants in which no phenotypic effect is discernable. Or it may be that owing to the A

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FIG.4. Diagram of pairs of mutated Type I11 capsular agents: (a) in which recombination occurring between the mutated regions (thickenings of the lines) leads t o complete restoration of capsular function b y elimination of the mutated regions; and (b) in which mutated regions overlap. In this latter instance, recombination within the indicated region can eliminate part of the mutated region but not all, leading to partial restoration of the capsular function and the capsular agent.

anomalies of the rough bacterium, recombination within the region determining capsule synthesis is suppressed. The same question may be asked in considering the action of the normal Type I11 agent upon any one of the mutants of the SIII-1 series. Why have recombinants not been isolated? Such recombinants might be supposed to be less mutant in character than SIII-1 without, however, being quite normal. Here an explanation is easily envisaged; such recombination would resemble that shown in Fig. 4B, and would be rare, occurring, however, under the same conditions which produce large numbers of autogenic transformations. To detect this event would probably require special selective procedures. It has proven possible to study the relative frequencies of allogenic and autogenic transformations induced by a single transforming agent-the SIII-2 agent acting upon an SIII-1 strain (Ravin, 1954). This was done by an ingenious but laborious method, in which transformations were performed in a semisolid culture medium containing known amounts of transforming agent. In these conditions, each inoculated bacterium gives rise

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to a colony in which competent cells appear and become transformed. Colonies are isolated individually, dissociated in liquid, and samples of them streaked on a solid medium which permits the identification of the two kinds of transformed cells: the normally encapsulated ones resulting from allogenic transformation, and the mutant SIII-2 ones resulting from autogenic transformation. The number of colonies containing one or more transformed bacteria is scored as a function of the amount of DNA added to the semisolid medium. This number proves to be a linear function of the concentration of transforming DNA added until about 40-50 % of the colonies are scored as positives. This is the range which may be expected to be linear in such a method.2 The curve relating DNA concentration to the number of colonies containing transformed cells passes through the origin of the plot, and it can be inferred that very few or no transformations are undetected. Since the method devised by Ravin satisfied the elementary requirements of a quantitative method, it was applied to study the relative frequencies of autogenic and allogenic transformations, under several conditions. The conclusions of these experiments are as follows: (1) The two kinds of transformed cells are produced by independent events. This follows from the fact that, throughout a wide range of DNA concentrations, the frequency of colonies containing both kinds of transformed cells is equal to the product of the over-all frequencies of colonies containing allogenic transformations on the one hand and autogenic on the other. (2) Under one set of conditions, autogenic transformations can be induced in 100 % of the colonies if enough DNA is added, whereas allogenic transformations can be induced only in a maximum of 70 % of the colonies. (3) The proportion of colonies in which allogenic transformation can be induced is not a clonal property, for by altering experimental conditions (reducing slightly the amount of agar added to solidify the medium) it is possible to obtain both kinds of transformations in all colonies. It is supposed that special physiological conditions determine the incidence of cells capable of giving rise to allogenic transformations, and hence that the “decision” of whether the transforming agent will produce an allogenic transformation or not depends upon some physiological state of the pneumococcal cell. (4)Under conditions in which allogenic transformations can be induced

* In this method, as long as a sizeable proportion of the colonies still contain no transformed cells, each increment of DNA will produce an increment of “positive” colonies which will be proportional, for all practical purposes, to the DNA increment. As soon as many colonies become positives, an increasing proportion of the transformations will be occurring in colonies which are already scored as positives. Hence the deviation from linearity.

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in only a fraction of the colonies by the SIII-2 agent, with another mutated agent it is possible to induce allogenic transformations in all of the colonies. This emphasizes again the specific nature of the reaction between each pair of mutated agents. As has been pointed out just above, it appears that whether or not a recombination occurs when a reactive bacterium encounters an agent is not a simple matter of chance determined by the properties of the agent in the bacterium and that introduced into the medium. A particular physiological state of the bacterium would seem also to be involved. Until the observations of Ravin are more fully understood, it would seem most unsound to try to construct maps of the mutant regions, based upon the hypothesis that the probability of recombination is a simple function of the distances between the two mutated regions. A last, and most interesting point which emerges from Ravin’s study is that allogenic transformation, when compared with autogenic transformation induced by the same agent, is very fequent. Indeed, under some conditions, allogenic transformations are as frequent as autogenic. Consequently, we must conclude that even though the mutated regions involved in SIII-1 and SIII-2 transforming agents affect one single character, it is extremely easy to obtain recombination between them.

B. Linked Capsular Agents in Hemophilus The second instance of physical association between genetic units in a deoxyribonucleate possessing transforming activity was found by Leidy, Hahn, and Alexander (1953), also in connection with capsular transformation. Three distinct serological types are involved, a, b, and d. In one series of experiments, bacteria with Type b capsules (and transforming agents) are treated with a transforming extract prepared from Type a cells. There results the formation of cells synthesizing both a and b antigens and containing the corresponding agents. However, the remarkable fact is that transforming extract prepared from the ab cells confers the double antigenic property upon unencapsulated cells, with what must be presumed to be a high frequency. In the same cultures in which transformations to ab occur, one also obtains transformations to a alone and to b. The exact frequencies of the single and double transformations could not be estimated, but there can be little doubt of a physical association between the a and b agents in the ab cells; in control experiments in which a DNA from a cells is mixed with a DNA from b cells, no double transformations ensue. The incidence of bacteria transformed by two physically distinct agents must be so low as to be undetectable by the methods employed. In a second series of experiments, a DNA extract of Type a cells is introduced into cultures of rough, unencapsulated bacteria derived originally

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from a Type b strain. A very rare transformation is observed to take place giving rise again to ab cells. Again, in these cells the ab agents appear to be linked together upon a single physical particle, as judging from the ease with which a DNA extract prepared from the transformed cells gives rise to ab bacteria when tested on a strain lacking both agents. The interesting aspect of this experiment is that the transformation by the a extract seems to reactivate the b agent, lying dormant in the rough cells. In a third group of experiments, the Type d agent, prepared from Type d cells, was introduced into ab cells in which a mutation had previously occurred, diminishing the quantity of polysaccharides secreted. The transformed cells isolated now form the d antigen in the place of the b, and both a and d are secreted in normal amounts. Transforming extracts prepared from the ad cells have the property of conferring both a and d characters upon an appropriate test strain. Thus, a appears to be linked with d. It is unfortunate that capsular agents do not lend themselves to easy quantitative study, for the Hernophilus capsular antigens would seen to offer a rich field for the investigation of the genetic structure of DNA. The experiments just cited strongly suggest that antigenic variation of Hemophilus has taken place through the localized differentiation of subunits of a primitive agent determining capsule secretion; and that the factors a, b, and d are analogous to the subunits described in pneumococcus with the exception that, in pneumococcus, mutation of subunits thus far described has given rise only to quantitative variation in antigen synthesis, while in Hemophilus, mutation has produced detectable, qualitative antigenic differences. In effect, the experiments described by Leidy, Hahn, and Alexander can be fitted into a coherent picture by supposing that four recombineable subunits or loci are involved: an a locus, a b locus of which one allele, d, is known, a suppressor locus closely linked to b, and a partial suppressor locus which may or may not be closely linked to b. Fig. 5 shows how these subunits may be visualized. In Fig. 5A we see how an ab factor can be formed by a recombination between a and b agents. I n Fig. 5B,the formation of an ab factor is shown as it may have occurred in the transformation of rough cells derived from Type b with an a transforming extract. It is supposed that the rough cell contains the b agent plus a closely linked mutated region, to the right of b which acts as a suppressor. A recombination which eliminates the suppressor region will give rise to a normally active b locus, but now the a region is linked to b. Such a recombination would be rare owing to the smallness of the region within which it must occur, and would be entirely similar in this sense to the recombination shown in Fig. 4B. Finally, we come to the transformation in which mutated ab cells are transformed to ad, by the Type d agent. Here, it suffices to suppose that the mutation in the a b cells, which partially suppresses antigen secretion, is either closely

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HARRIETT EPHRUSSI-TAYLOR

linked to the right of the b locus, or that it lies to the left of b, and, further, that d is an allelic form of b. Recombinations in which b is replaced by d will usually eliminate the suppressor region, if the suppressor is to the right and closely linked to b, and always eliminate the suppressor if it is to the left of b. Figure 5 is only one of several possible models which might be made. Detailed experiments of a more quantitative nature would be of greatest value in connection with this series of observations and not only would provide A Type b ogent

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interesting data upon DNA structure, but also might well be of very wide immunogenetical interest, in view of the evidence that R h antigens in higher organisms are determined by complex loci. Finally, it should be noted that the essentially new feature introduced into our picture of transforming agents by the experiments under discussion is that the recombineable subunits of a DNA particle can involve regions which are differentiated with respect to biochemical function. While this might prove true also of the mutant SIII agents of pneumococci (were the mechanism of polysaccharide synthesis to be understood) it is not evident from a study of the final polysaccharide product, which in all cases seems seems t o be the Type I11 antigen.

CURRENT STATUS OF BACTERIAL TRANSFORMATIONS

30 1

C . Linkage of Streptomycin Resistance and Mannitol Factors For the first time, in the work of Hotchkiss and Marmur (1954), linkage between two transforming factors has been postulated as the result of a quantitative analysis. As was seen t o be true of the Hemophilus antigenic factors, the loci in question not only recombine and/or mutate as distinct genetic units, but again have distinctive physiological effects. The experiments of these authors strongly indicate that the factors determining streptomycin resistance (Sr) and the ability to ferment mannitol (M) are located on a single particle, for when a DNA is prepared from a pneumococcal strain carrying both .factors, it has the special property of transforming for both characters a high proportion of pneumococci lacking either. The data also suggest a similar linkage between the Sr agent and an agent determining resistance to sulfanilamide, but the results thus far published are less convincing for these latter two factors. Since the experiments of Hotchkiss and Marmur demonstratethe feasability of quantitative work in pneumococcus on an extensive scale, it becomes important to examine critically what kind of experiments are most suited for linkage studies, and least likely t o lead us astray. As was pointed out before, ideally speaking, linkage should be considered as demonstrated between two agents if the frequency of double transformation is in excess of that expected from the random distribution of each of the agents considered independently. The determination of transformation frequency should be undertaken at concentrations of DNA known to be limiting; that is, the population of transformable bacteria should not be saturated with transforming agents. This was the approach adopted in the experiments of Hotchkiss and Marmur, except that DNA concentration was not deliberately limited. Instead, multiple encounters between bacterium and transforming factors were cut down in some experiments by limiting the duration of the contact of bacteria with DNA. It is clear that a high frequency of multiple encounters will distort calculations in favor of the hypothesis of linkage, for the only multiple encounters t o be detected will be those in which particles possessing different specificities are acquired. Bacteria picking up two M or two Sr particles will be scored as having only one, and the total frequency of encounters with M and Sr, treated individually, will be underestimated. For this reason, it is the feeling of the writer that the data upon which the relative frequencies of single and double transformations with M and Sr are calculated are, by themselves, insufficient t o prove linkage. The strongest evidence in favor of linkage comes from the control experiment in which the two agents were introduced as separate particles; that is, when the test bacteria were treated with a mixture of equal amounts of DNA containing only the Sr factor and DNA containing only the M

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factor. A very small proportion of bacteria react with both agents following an exposure of as little as 5 min. In these experiments, the incidence of double hits is lower than expected on the basis of random distribution of two particles, a fact which the authors attribute to interference. There can be no question but that the mixture of the two DNAs, each contributing one of the two markers, behaves quite differently from a DNA prepared from a doubly marked strain. The argument for a structural difference, in the two situations, is thus rendered very strong. A second hazard in the kind of experiments performed by Hotchkiss and Marmur arises from the difficulty of being certain that the selection employed for each marker, and upon which the quantitative results depend, is equally efficient under all conditions. The published data show a very wide variability in the incidence of transformation for mannitol fermentation, indicating a variable recovery of cells transformed for this character (confirmed by Hotchkiss in personal discussion). A failure, in a given experiment, to detect all of the transformations for this character is of no consequence provided the same low efficiency prevails in selection of the doubly transformed cells. However, if in the latter the efficiency of the screening for mannitol is somehow increased, there will be an apparent excess of double transformations. This is probably not the case in the experiments reported, but should be recognized as a hazard in methods employing selection. To avoid the sources of error just discussed, it might be advantageous to adopt quite different methods of analysis. As soon as preliminary experiments suggest a physical association between two factors, as indicated by their being acquired often by a single bacterium, the most rapid fashion to to settle the point might well be simply to determine the quantitative relationship between the absolute number of double transformations and DNA concentration, starting with low concentrations of nucleic acid. If one particle is involved, a linear relationship will be observed; if two are concerned, the relationship will be exponential. Selection will be homogeneous, and no assumptions need be made concerning its efficiency. Such a titration need extend over only a ten-fold range of concentration to give completely conclusive results. A most interesting aspect of the experiments of Hotchkiss and Marmur arises from their being able to do experiments with linked factors in both the coupling and repulsion phase. All of the experiments mentioned above were done in coupling phase, with both mutant markers on the transforming particle. However, it is possible to have one mutant marker on the transforming particle, and the other in the bacterium undergoing the transformation. Selection is made for the marker in the transforming particle, and the transformed bacteria thus selected are then screened for the second

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marker. In about one-fourth of the clones examined, no recombination has occurred between the two markers, and the normal factor of the transforming particle is found to be incorporated into the transformed bacterium along with the selected mutant marker. The mutant marker which was in the bacterium prior to its transformation thus disappears. By this method of selection, it can be seen that transformations are reciprocally possible between streptomycin resistance and sensitivity and between mannitol fermentation and its absence. It is in experiments of this type that a linkage between Sr and sulfanilamide resistance seems especially likely, for by treating a sulfanilamide-resistant strain with a DNA isolated from a streptomycin resistant, sulfanilamide-sensitive strain, it is possible to recover, by selecting for streptomycin resistance, cells transformed to sulfanilamide sensitivity. Here a t last, come three more instances in which transformations are reciprocally possible between alternate cell-states, joining the one previously known case, and thus reinforcing and generalizing the genetic conclusions which had been drawn from its discovery. The average frequency with which the Sr and M agents remain associated is of the order of one-fourth of the time the particle is acquired by a pneumococcus. However, the actual frequencies from one experiment to another seem quite variable. This may possibly be due to sampling error, but may reflect the phenomenon found by Ravin (1954); namely, that the frequency of recombination can be influenced by environmental conditions. Neglecting this obstacle to comparison, one may note that whereas, on the average, the Sr and M factors undergo recombination about 75% of the time, recombination between the SIII-1 and SIII-2 loci occurs less frequently, occuring, on the average, a little less than 50% of the time.

D. Signifiance of Recombination Data To explain the observations gathered in all three of thesets of experiments just discussed, it seems necessary to suppose that the individual particles of DNA are composed of subunits which can mutate independently of each other, which can be recombined within the bacterium, and which may or not have differentiated physiological function. These properties are, presumably, reflections of the structural organization of the DNA molecule, and it is worthwhile considering briefly what this might imply. Accepting the Watson-Crick model of the structure of DNA, the results suggest that the sequence of bases along the length of a single molecule varies to such an extent that different regions can have different functions. Further, a certain discreteness of these regions must exist, for mutation at one point need not affect the activity at another point. Obviously, the exact limits in space which are involved in determining a single genetic character cannot be de-

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fined by defining the size of the DNA particle. However, provisionally, we may suppose that these limits can be defined as that region within which any mutation has an effect upon a single character. If we take as an example of such a series of mutants the mutant Type I11 agents of pneumococci, then it is clear that recombination can also occur within the limits of of the region involved in the determination of a single character. This amounts to saying that recombination can probably occur anywhere,along the nucleotide chain, and that recombination studies will not define the locus which determines a character. Such an image of the molecular organization of genetically active DNA has very definite repercussions upon what to expect in fractionation studies designed to separate particles endowed with different specific activities. We have arrived a t the conclusion that it is probably incorrect to think of one particle of DNA as being the site of only one specific biological activity, but what are native DNA molecules like? How many loci are there on a single fiber of native DNA? In any organism having more than one kind of chromosome, the minimum number of kinds of DNA fibers would be equal to the number of nonhomologous chromosomes, and the true number is probably very much greater. In the bacteria in which transformations can be induced, there may be only a single giant fiber along which all loci are located, or there may be several smaller fibers, each composed of a particular array of loci. If there is only one filament, fractionation studies will prove ineffective, and only end group analyses will provide information on the nature of the chemical differences responsible for biological specificity. If there are a few medium-sized filaments, each representing many characters, probably neither fractionation nor end group analyses will be of use. I n this instance, fractionation is likely to fail because the differences along each type of filament may well be sufficient to equalize the average composition of bases from one type of filament to another, thus leaving inadequate differences in over-all compositionfor the molecules to have distinctive adsorption characteristics. If either of these situations describes native DNA, then fractionation may prove effective only if it is feasable te prepare small fragments of molecules which still are endowed with specific biological activity . The three recombination studies just discussed have also significance in connection with the mechanism of the transformation reaction. It seems fairly clear that in allogenic transformations, and in the transformations in which recombination occurs between physiologically differentiated markers linked on a single particle, the transformed bacterium propagates a DNA molecule in which part of the specific pattern has been determined by the inducing DNA, and part by the bacterium undergoing the transformation. Rather than postulate that allogenic transformations differ funda-

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mentally from autogenic, or that, in certain transformations a DNA molecule is acquired only in part rather than as a whole, it seems preferable to suppose that only one basic process is going on: that all transformation consists of the incorporation of greater or smaller regions of molecules. By incorporation we shall mean the substitution, by an unknown mechanism, of a given region of the inducing molecule for a corresponding region of a very similar (homologous) molecule already possessed by the recipient cell. We observe transformation t o occur because small, heritable differences occur in the substituted regions. Transformations for a single character occur when the region happens to include a single marker. Two markers are observed t o be linked if they are situated closely enough together so as to fall often within a single incorporated region, and as unlinked if they are too far apart, or located on separate particles. Bearing in mind this image of transformation, let us now return t o the most interesting question of all: what is the degree of autonomy of DNA in the cell? If our picture of transformation is correct, the perpetuation of DNA introduced from outside of the cell depends upon the presence in the cell of a homologous molecule, or region of a molecule, for which the exogenous DNA becomes substituted. Depending on how this substitution occurs, and on how the fixed DNA of the cell functions and reproduces, we shall or not consider DNA as a substance having a high degree of autonomy. We have no idea of the mechanism by which the transforming DNA is substituted for endogenous DNA. As has been pointed out, only one fact is established: the mechanism can lead to the formation (in daughter cells of the transformed one) of a DNA molecule, part of the specificity of which comes from the transforming agent and part from the endogenous DNA. It is tempting to imagine that this results from some simple physical contact between transforming agent and endogenous DNA, but this is by no means demonstrated. If recombination involves only such a direct interaction between the two DNA molecules in question, then we may say that successful transformation is dependent essentially upon the presence in the cell of a particular previously existing DNA molecule. DNA remains, in this picture, a substance endowed with a high degree of autonomy. However, if this interaction is not direct, but is mediated by additional structures, and in particular, if such structures also have a template function, the autonomy of DNA becomes highly restricted. The likelihood that subsidiary structures essential to the duplication process have themselves a template function may seem small if one accepts the Watson-Crick model of the structure of DNA. In this model, composed of two helical chains of nucleotides, if one chain is given, the sequence of nucleotides in the second is rigorously determined by the sequence in the first. At first sight, it might seem that no other structure having the spa-

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tial requirements of a template need be invoked. However, one must then suppose that in the duplication process, the mechanism by which the chains are separated involves no cell site having a specific and complementary structure. One must further suppose that, once separated, each chain will by itself bind the complementary nucleotides from which the complementary chain will be built. This phase of duplication, as pointed out by Watson and Crick (1953), is particularly difficult to reconcile with the idea that a single, separated chain serves, by itself, as the template for the attraction of the appropriate nucleotides and the formation of the new chain, since there are no longer steric limitations to oblige a given free nucleotide to attach to a particular exposed base of the given chain. Also, the single chain must somehow be prevented from getting tangled. There remains, after these two steps, the task of linking up the nucleotides. Is this done by a soluble, diffusing, esterase, or is it done by a series of fixed enzymatic sites properly spaced? Cytochemical studies of chromosomes could be interpreted in favor of this second view. The first two postulated stages of DNA duplication may well be accomplished through the action of highly specific structures, in some way complementary to the DNA molecule. Of course, if such structures exist, they may be synthesized on the surface of the DNA molecule itself, and the DNA may still retain a unique template function. I n this event, the entire question of the uniqueness of the role in DNA in determining specificity of hereditary characters would revolve, then, upon whether such subsidiary structures could in turn influence the specificity of newly formed DNA. Another image of such structures is that they constitute in themselves an autoreproducing system; that they constitute a self-perpetuating stroma to which DNA must be attached in order to execute its specific functions. In this event, DNA would be autonomous insofar as determination of specificity is concerned, but entirely dependent upon pre-existing, self-perpetuating structures insofar as function is concerned. So far, evidence that protein and RNA are capable of inducing hereditary changes in cells is negative, but this does not exclude both substances from having a template function. Negative evidence may result from the impossibility of reintroducing these substances into the cell in an active state, or the impossibility of obtaining soluble native material, for example. It is in this connection that the comparative study of viruses may prove to be of greatest importance, for these entities are, in many instances, highly efficient agents for the introduction of autoreproducing material into the cell. It is established today that DNA-containing bacteriophages can induce permanent genetic changes in host bacteria without necessarily inducing their own perpetuation (Zinder, 1953), thus indicating that DNA brought in by the virus can intervene in a very intimate way in the duplication, by the host, of its own DNA. Can RNA-containing viruses do the

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same, and are their activities manifested in the nucleus as well as the cytoplasm? Study of host-virus relationships in material in which the genetic composition of the host can be controlled and analyzed may well lead to information of highest significance and utility in defining the structures and substances having template functions in autoreproduction of normal as well as pathological cell constituents. REFERENCES Alexander, H. E., and Leidy, G. (1951). J . Exptl. Med. QS, 345. Alexander, H. E., and 1-eidy, G. (1953). J . Exptl. Med. 97, 17. Alexander, H. E., and Redmnn, W. (1953). J . Exptl. Med. 97, 797. Austrian, R., and Colowick, M. F. (1953). Bull. Johns Hopkins Hosp. 92,375. Avery, 0. T., MacLeod, C . M., and McCarty, M. (1944). J . Exptl. Med. 79,137. Brown, G. L., and Watson, M. A. (1953). Nature 172, 339. Chargaff, E. (1951). Federation Proc. 10, 654. Chargaff, E., Crampton, C. F., and Lipschitz, R. (1953). Nature 172, 289. Ephrussi, B. (1953). “Nucleo-Cytoplasmic Relations in Micro-Organisms.” Oxford Univ. Press, New York. Ephrussi-Taylor, H . (1951). Exptl. Cell Research 2, 589. Ephrussi-Taylor, H. (1954). Expll. Cell Research 6, 94. Ephrussi-Taylor, H., and Latarjet, R. (1955). Biochim. et Biophys. Acta 16. 183. Feughelman, M., Langridge, R., Seeds, W. E., Stokes, A. R., Wilson, H. R., Hooper, C. W., Wilkins, R.I. H . F., Barclay, R. I<., and Hamilton, I,. D. (1955). A’ature, 176,834 Griffith, F. (1928). J . Hyg. 27,113. Hershey, A. D., and Chase, M. (1952). J . Gen. Physiol. 36, 39. Hotchkiss, R. D. (1948). Colloq. Intern. Centre Natl. Recherche Sci. (Paris). Unites Biologiques Dou6es de Continuit6 GCnBtique,.p. 56. Hotchkiss, R. D. (1951). Cold Spring Harbor Symposia Quant. Biol. 16, 457. Hotchkiss, It. D. (1954). Proc. Natl. Acad. Sci. ( U . 8.) 40,49. Hotchkiss, R . D., and Marmur, J. (1954). Proc. Natl. Acad. Sci. (V. S.) 40, 55. Leidy, G., Hahn, E., and Alexander, H . E. (1953). J . Ezptl. Med. 97,467. Luria, S. E. (1953). (‘old Spring Harbor Symposia Quant. Biol. 18, 237. McCarty, M., Taylor, €I. E. and Avery, 0. T. (1946). Cold Spring Harbor Symposia Quant. Biol., 11, 177. Ravin, A. W. (1954). Exptl. Cell Research 7, 58. Reichman, M. E., Rice, S. A., Thomas, C. A., and Doty, P. (1954). J . Am. Chem. SOC.76, 3047. Stocker, B. A. D., Krauss, M. R., andMaeLeod, C. M. (1953). J . Pathol. Bacterio2. 66, 330. Taylor, H. E . (1949a). J . Exptl. Med. 89, 399. Taylor, H . E. (1949b). Compt. rend. 228, 1258. Thomas, R. (1955). Biochim. et Biophys. Acta, in press. Watson, J. D., and Crick, F. H . C. (1953). Cold Spring Harbor Symposia Quant. Biol. 18, 123. Weil, A. J (1947). Proc. SOC.Exptl. Biol. Med. 64, 349. Wyatt, G. R. (1952). in “Chemistry and Physiology of the Nucleus” (V. T. Bowen, ed.), p. 201. Academic Press, New York. Zamenhof, S., Alexander, H. E., and Leidy, G. (1953). J . Exptl. Med. 98, 373. Zinder, N. D. (1953). Co2d Spring Harbor Symposia Quant. Biol. 18, 261.

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Author Index Numbers in parentheses are reference numbers and are included to assist in loeating references in which the authors’ names am not mentioned in the text. Numbers in itslies indicate page on which the referenca ia listed.

Barner, H. D., 42(7,40),44 Barrington, L. F., 33W,43 Ackermann, W. W., 111, 112, 113, 132, Barth, L. G., 164(78), 196 133,142,143 Barry, G.T.,9(9), @ Ada, G . L., 65,142 Bawden, F. C., 54,61,82,84,142, 192(7), Adams, M. H . , 60,119,142 193,222,225,230,243,.24e, 25i(i), 272 Ainslie, J. D., 132,142 Beale, H. P., 54, 129,148 Alexander, H. E., 277,278,289,291,298, Bean, C . w., 155(92), 158(92), 196 300,307 Beard, D., 15(45), 16(125), 19(1% 165, Allen, E. G., 31(19), 44 166), 44, 46, 47, 154(58, 59,661, 1% Altenbern, R.A . , 130,142 (59),156(29), 158@ 64,67,127,152, Amies, C. R., 167,183(2), 193 157), 159(129, 156), 160(152, 170), Amos, H . , 30(2),@ 161(8,152), 162(51,66,152,160,1611, Anderegg, J. W . , 12(102), 46 163(126, 152), 164(59, 65,66,67,125, Anderson, T. F., 15(3, 281, 21(133), 37 127, 152, is), 165(63, 66, 67, 1551,

A

109, 110, (28), 40(28), @ I 44, i66(86, 124), 167(61, 62, 65, 66), 142,I @ 168(66), 169(65,66,152), 170(65, 66), Andervont, H. B . , 150(3), 193 173(57, 59), 176(57, 59, 68, 130), Andrewes, C. H . , 128,142 178(57), 179(59), 180(59), 181(59,68), Angier de Montgremier, H . , 131,142 182(60, 129), 189(171), f94, 196, l M , Arbogast, R.,15(33), 26(33), 44. 197 Arley, N . , 189,196 Beard, J. W., 15(45), 16(125), 18(11), Arnold, W., 7(4), @ 19(125, 165, 166), 29(11), @, 44, &, Arvy, Lucie, 211,219 47,150(12,140,141,142),151(12,140, Asheshov, I . N.,131,142 141), 152(32), 154(58, 59,661,155(11, Austrian, R., 277,307 59, 153), 156(11, 29, 1291, 157(159), Avery, 0 . T., 277,278,282,307 158(8,64,67,127,151, 152, 157,158, 159), 159(13, 129, 156, 1581, 1m(9, B 11, 13,152,170), 161(8, 150,152),162 (51,66,151, 152, 158, 160, 1611,163 Babbitt, D., 125,144 (126,150,152), 164(59, 65,66,67,88, Bachrach, H . L., 19(5), 43 125, 127,150,151,152,154,1&), 165 Backus, R.C . , 163(119), I96 (10,63,66,67,87, 155), 166(86, 88, Bailey, W. T., 60,143 124), 167(61, 62,65,66), 168(66), 169 Bald, J. G., 189(4,5,144),193,197,244,248 (65,66, 150, 1521, 170(65, 66), 173 Bang, F. B . , 162(6), 193 (29-31,57, 59), 174(31), 176(57, 59, Bang, O . , 152(71), 196 68,130),178(57), 179(59), 180(59), 181 Barbier, M., 6,43 (59,68), 182(30, 32,60,129),183(31), Barclay, R . K . , 280,307 184(31), 185(29-31),189(31, 171); 191 Barlow, J . L., 15(67), 4.6,86,1-44 (lo), 193, 194, 196, 196, 197,229,948 Barnam, C. P.,107,142 309

3 10

AUTHOR INDEX

Bedson, S. P., 21(12), 44 Beeman, W. W., 12(102),46, 63, 147 Behrens, B., 175(14), 199 Belding, T . C., 154(43), 194 Bendet, I. J., 12(100),46 Bennett, C. W., 239, 240, 243, 244, 248, 252(2, 3), 872 Bennetts, M. J., 222,248 Bergold, G. H., 20, 25(15), 44, 199, 201, 203, 206, 207, 209,214, 210 Bernheimer, A. W., 240,24&,270(4), 272 Beumer, J., 120, 148 Beumer-Jochmans, M. P., 120,142 Bird, F. T., 203, 219 Bittner, J. J., 150 (15-17), 199 Black, L. M., 12(20), 4.4, 222, 224, 227, 228, 230, 232, 233, 234, 235, 236, 243, 245, 246, 8&,263(5), 270(6), 872 Blank, H., 10(147),47 Bliss, C. I., 175(18-20, 22), 177(18-20), 180(21), 181, 183(18-20), 190, 199 Bond, H. W., 86,90,92, 132, 148 Bonner, D., 7(16), 44 Bonner, J., 54, 142 Boncquet, P. A., 239, 2@ Bourke, A. R., 86, 125,148 Bovarnick, M. R., 31(17-19), 4.4 Boaeman, F. M., 130, 143 Brachet, J., 107, 146 Brakke, M. K., 12(20), 44, 235, 236, 237, 243, 245, 246, 248 Brandenberger, H., 104, 1.43 Breese, 5. S., 18(176),48 Breindl, V., 199, 2lB Brierley, P., 255(40), 273 Briody, B. A., 127, 14.9 Brockman, R. W., 129, 146 Bronson, L. H., 174(134), 175(134), 189 (134), 186 Brown, D. M., 91, 106, 1.43 Brown, F. B., 103, 143 Brown,G. C., 111,112, 113,116, 117, 127,

26,28,33-36), 187(27,28,35), 188(27), 189(31, 38), 103, 194, 107, 229, 248 Bueding, E., 4(21), 44 Buffa, P., 138,146’ Bunting, H., 159(123),196 Burgoon, C. F., 10(147),47 Burmester, B. R . , 152, 153(40, 41), 154 (4143), 155(40,153),156(39),176(68), 181(68), 194, 196, 197 Burn, J. H., 175(44), 194 Burnet, F. M., 60, 148, 160(45), 194, 271(7), 272 Burnside, C. E., 214,919 Butler, C. G., 214, 819 C

Cain, J. C., 103,l49 Calkins, D. G., 103, 143 Calnan, D., 173(35), 174(35), 186(34, 35), 187(35), 194 Calvin, M., 7(22), 44 Capell, L. T., 102,146’ Carnelly, H. L., 167(115), 186 Carlo, P.-E., 97, 14s Carpenter, F. H., 13(23), 44 Carr, J. G., 30(24), 44, 167, 174(46), 183(2), 188, 193, 194 Carrel, A., 153, 194 Carsner, E., 239, 248, 251 (8), 272 Caspari, E., 240,248, 270(4), 278 Chambers, L. A., 21(133), 46’ Chargaff, E., 68, 144,279, 907 Chase, M., 16, 17(73), 39(72), 46, 62, 120, 1.64, 276, 907 Cheener, F. S., 135, 1.43 Chester, K. S., 251(9), 272 Chu, C. M., 162(69), 196 Ciaccio, G., 125, 149 Ciotti, M. M., 139, 148 Clark, P. F., 113, 146 Claude, A., 150, 194 Cline, J., 119, 146 Cochran, K. W . , 129,143 1 4 4 144 Cohen, 5. S., 4(38), 7(34, 37, 39), 8(38), Brown, G. L., 279, 280, 907 9(36), 13(25), 15(28, 29, 183), 18(26), Brownlee, K. A., 133, 14 21(32, 183), 26(33, 183), 36(182, 183), Bryan, W. R., 152(32), 153(45), 155(11), 37(27,28,36,42), 39(30,31,38,41,98, 156(11, 29), leO(ll), 173(25-31, 35, 174), 40(28, 36), 41 (36, 41), 42(7), 43, 37), 174(25, 26, 28, 31, 35, 37, 38), 44, M, 47,48,89,99,109,110,115,1.43, 175(27, 37, 38), 181, 182(30, 32), 144, 148, 167, 168, 194 183(31), 184(31), 185(29-31), lsS(25, Colowick, M. S., 277,907

311

AUTHOR INDEX Colowick, S. P., 139, 148 Common, I. F. B., 201, 219 Commoner, B., 23, 47, 54, 64, 82, 83, 84, 143, 146, 147 Cooper, G. R., 12(124), 16(125), 19(125), 46, 156(129), 159(129), 182(129), 196 Cooper, W. D., 24(43), 44 Coriell, L. L., 10(147), 47 Cornuet, P., 131, 146 Cosslett, V. E., 12(44), 44 Costa, A. S., 243, 248 Cottral, G. E., 153, 154(41), 194 Craig, D. E., 117, 1-43 Crampton, C. F., 279, 307 Crick, F. H . C., 23, 28(173), 47, 68, 1-43, 147, 281, 306, 307 Crowdy, S. H., 270(10), 272 Csaky, T. Z., 15(45), 44, 158(8), 161(8), 193 Culbertson, C. G., 129, 146 Cunha, R., 162(51), 194 Cushing, R. T., 90, 91, 111, 116, 14.9

Doyle, L. P., 152(102), 196 Dubos, R . J., 5, 4.4 Dubuy, H. G., 9(49, 179), &,@ Dudgeon, J. A., 162(69), 196 Dulbecco, R., 1, 34(52), 44, 59, 1-43 Dunn, D . B., 87,88,93, 99, 105, 114, 1-43 Duran-Reynals, F., 150,194 Dutky, S. F., 214, 219 du Vigneaud, V., 13(23), 44

E

Eurle, W. R., 153(145), 197 Euton, M. D., 127, 135, 14.9 Ebeling, A. H., 153,194 Eckert, E. A., 154(58, 59, 66), 155(59, 153), 158(8,64,67,127,152), 160(152), 161(8, 152), 162(66, 152), 163(126, 152), 164(59, 65-67, 125, 127, 152, 155), 165(63, 66, 67, 155), 166(86), 167(61, 62, 65, 66), 168(66), 169(65, 66, 152), 170(65, 66), 173(57, 59), 176(57, 59, 68, 130), 178(57), 179(59), 180(59), 181(59, 68), 182(60), 193, 194, D 196, 196, 197 Edlinger, E., 130, 131, 14.9, 1.6.6 Danauskas, J. X., 130, 1-43 Edney, M., 34(80), 46 Darlington, C. D., 192(52), 194,244,248 Elford, W. J., 130, 1.6.6, 162(69), 196 Davis, B. D., 7(46), 44 Dawson, I. M., 162(69), 170(53), 194, 196 Elion, G. B., 87,90,91, 104, 105, 116, 117, Day, M. F., 201, 219, 222, 248 144, 147 Ellermann, V., 152(70, 71), 154(70), 158 de Haan, P. G., 87, 114, 148 (70), 196 Dekker, C. A., 68,72,14.9 de Lind van Wijngaarden, C., 175(54), Ellis, E. L., 61, 1.64 Ellison, S. A., 10(122), 19(122), 20(121), 194 46, 217, 819 Delbriick, M., 33, 47, 60, 61, 72, 110, 14.9, Elson, D., 68, 1.66 144 Elvehjem, C. A., 113, 1.6s Dellweg, H., 87, 148 Engel, 1'. L., 18(53), 44 de Mars, R. I., 60, 126, 1-43 Engelbreth-Holm, J., 153(72), 196 Dewey, D. L., 7(48), 44 Engelman, M., 103,144 Dewey, V. C., 96,146 Englander, S. w., 14(55), 46 Dice, J. R., 146 Ephrussi, B., 275,307 Dietrich, L. S., 102, 14.9, 147 Ephrussi-Taylor, H., 277, 281, 285, 294, Dillon, E. S., 15(45), 44 307 Dingle, J. H., 189(171), 197 Epstein, H. T., 14(55), 46 Dion, H. W., 103, 1-43 Ercoli, N., 125, 1-43 Dixon, J., 39(72), 46 Evans, E. A., 39(92, 93), 42(93), 46 Dmochowski, L., 150(55), 194 Dobroscky, I. D., 263(11), 272 F Dolby, D. E., 165(120), 196 Fabrant, J. L., 201, 819 Donker, H. J. L., 3(87), 46 Faguet, M., 130, 131, I&, 146 Donovick, R., 133,144 Fagraeus, A., 176(73), 188,196 Doty, P., 17(139), 47,281, 307

312

AUTHOR INDEX

Falco, E. A., 58,87,105,116,134,1~, 147 Feemster, R . F.,175(74), 189(74), 196 Feller, H. E.,189(171), 197 Feughelman, M.,280,307 Fildes, P.,119, 144 Finney, D.J., 175(75), 180(75), 196 Fischer, A.,153(76), 196 Fisher, H., 60, 126, 149, 146 Fitagerald, R.J., 125, 144 Fleisher, M.S., 58,124,144 Flock, R. A.,221, 244, 948 Folkers, K.,58, 117, 147 Foster, R.A. C., 126,14 Foulds, L., 150,153(77), 196 Fowler, C.B.,109, 110,14.9,144 Francis, T.,Jr., 129, 133,1.@,1.6-6 Frank, S.,164(78), 106 Frankel, J. W., 129, 1.66 Franklin, R.E.,68, 14.4 Fraser, D., 14(57), 25(56), 46 Freitag, J. H., 239,948 Friedkin, M., 97,98, 14.4 Friend, C.,90,91,1&, 1.46 Fuerst, C.R., 14,47 Fukushi, T.,223,224,239,245,948 Fulton, F.,59,144, 162(69), 196 Fulton, R.W.,129, 146 Furth, J., 18(84), 46,152,153,154(80,81), 156(80, 103), 158(81), 167, 168, 196, 196 G

Gaddum, J. H., 175(82), 177(82), 183(82) 196 Gant, D. E., 103,149 Gard, S.,176(83), 196,229,948 Garen, A.,119, 146 Gaaparini, G., 125,14.9 Gebhardt, L.P.,133,147 Geller, D.M.,4(62), 46 Gey, G. O., 59, 148 Gentry, R.F., 154(42), 194 Giddings, N.J., 239,948,271(13), 979 Gifford, G. E.,91, 111, 112, 14.4 Gillespie, H. B., 103, 144 Gilpatrick, J. D., 118, 122, 123, 1.48 Glaser, R.W.,270(12), 979 Gocke, I. M.,135,149 Goebel, W.F., 15(81), 16(82), 46, 63,1.46 Golub, 0. J., 176(84), 196 Gooden, E. L., 214, 819

Gorodskaja, 0. S., 118, 146 Gosling, R.G., 69, 144 Goatling, J. V. T., 21(12), 44 Gottschalk, A,, 29(58), 35(58, 59), 46 Gottschalk, R.G., 156(85), 196 Goulet, P.,237, 949 Gr&, S.,103, 144 Graham, A. F., 19(60), 46 Green, D.E., 30(109), 46 Green, I., 154(66), l62(66), 164(65, 66, 88), 165(66, 87), 166(86, 88), 167(61, 62, 65, 66), 168(66), 169(65, 66), 170 (65,66),194, 196 Green, R. G., 246,948 Green, R. H., 174(134), 175(134), 189 (134), 196 Greenwood, M., 173(89), 196 Gregoire, C.,213,919 Griboff, G., 87, 102, 105, 1.48 Griffith, F., 277, 307 Grimaon, H., 13(128), 46 Group4, V., 129, 1.66 Grylla, N.E.,243,248 Gunsalus, I., 4(61), 46 Gutmann, A., 73,146

H Haddow, A., 168(90, 91), 192(90, 91), 196 Hager, L. P.,4(62), 46 Hahn, E.,298,300,307 Hall, E.A., 131, 14.9 Hall, W.J., 155(92), 158(92), 196 Halvorson, H. O.,173(93), 196 Hamilton, L. D.,280, 307 Hamre, D.,133, 144 Harris, J. I., 64,14.4 Harris, R. J. C., 150,168(94), 174(46,94), 188, 104, 196 Hartman, R.E.,18(63), 46 Henle, W.,18(64), 34(65), 35(104), 46, 46 Herriot, R. M., 15(66,67), 16(68), 33(68),

46, 86, 144

Herrmann, E. C., Jr., 129, 144 Hershey, A. D., 16, 17(73-75), 23(74), 39(70-72,74),46,60,62,120,1 4 , 276, 907 Heston, W. E., 150(95), 186 Hewitt, L. F., 137, 144 Heyl, D.,117, 147 Higginbotham, R.D.,133, 147 Himmelweit, F., 130, I&

313

AUTHOR INDEX

Hirst, G. K., 160(96), 196 Hitchings, G. H., 58, 87, 90,91, 104, 105, 116, 117, 134, 135, 144, 147 Hoagland, C. L., 19(151),30(76, 151), 46, 47, 165(97),196 Holmes, F. O.,54, 140, 1.64,251(14, 15), 255(16), 272 Holmes, W. I,., 104, 146 Hooper, C. W., 280, 307 Hopps, H. E., 130, 143 Hook, A. E., 157(159), 158(159), 197 Hooker, C. W.,156(111), 196 Horsfall, F. L., Jr., 35(164), 47, 58, 117, 132, 246, 147, 174(98), 196 Hotchkiss, R. D., 277, 278, 282, 283, 284, 301, 307 Houlahan, M. B., 7(77), 46 Hoyle, L., 34(78), 35(79), 46, 124, 1.64 Hudis, J. D., 17(73), 46 Huff, C. G., 270(17), 272 Hughes, D. E., 138, 1.64 Hughes, K. M., 207,209,216,219 Hull, R. N., 129, 1.64 Hurst, E. W., 125, 127, 128, 1.64 Huseby, R. A.,107, 14.2 Huybers, K., 120,142

I Ipsen, J., Jr., 180(99), 181, 196 Isaacs, A., 34(80), 46, 59, 1.64 Ishii, M., 60, 64, 147 Iversen, S., 189, 196

J Jacob, F., 136, 1.64 Jacobson, W., 91,148 Jeener, R., 63, 64, 83, 84, 107, 146 Jeffries, J., 18(176),48 Jensen, J. H., 252(18), 272 Jerrel, E. A., 25(56), 46 Jesaitis, M. A., 15(81), 16(82), 17(83), 38(83), 46, 63,146 Johnson, E. P., 152(102), 196 Johnson, R. B.,133, 142 Joklik, W. K., 119, 1.64 Jones, C. R., 54, 129, 14.2 Jungherr, E., 152(101, 102), 153(101), 196

K Kabat, E. A., 18(84), 46, 154(80), 156(80, 103), 167, 168, 196, 196

Kaesberg, P., 12(102), 46, 63, 147 Kaiser, A. D., 240, 248,270(4), 272 Kalmus, H., 122, 146 Kandel, A,, 117, 133, l4S, 1.64 Kaplan, A. S., 136,146 Kaplan, N. O., 139, 148 Karibian, D., 127, 143 Karlin, R., 105, 1 4 Karp, A., 31(85), 46 Kassanis, B., 9(86), 46, 54, 82, 84, 122, 140, 142, 146 Kay, D., 119, 14.1 Kidd, J. G.,150(108), 151(108, 110, 143, 164), 152(105-107, 109, 164), 196, 197 Kidder, G. W., 96, 146 Kirkpatrick, H. C., 54, 130, 1.46 Kirschbaum, A., 156(111, 166), 157, 196, 197 Kluyver, A. J., 3,46 Knight, C. A., 11, 13(91), 18(90), 19(90), 22, 23(91), 24(91), 25(91), 28(91), 46, 64, 14.4, 146, 155(114), 160(113, 114), 167, 168, 196 Knowlton, K., 39(93), 42(93), 46 Koch, A. L., 39(92), 46 Kogl, F., 103, 146 Kohler, E., 251(19), 272 Komarek, J., 199,219 Kozloff, L. M.,33(8), 39(93, 94), 42(93),

M, .46

Krampitz, L. O., 42(95), 46 Krauss, M. R., 289, 307 Kream, J., 102, 147 Krebs, H. A.,4(96), 46 Kunkel, L. O., 225,226,227,231,237,239, 240, 245, 246, 248, 251(25), 252(26, 31, 32, 35, 37), 253(20, 21, 24, 27-29, 36), 254(22, 23), 255(33, 34, 38), 256(28), 260(39), 263(23, 28,30), 272,273 Kutsky, R. J., 54,130,131,146

L Lagerborg, D. L., 110,111,146 Laidlaw, P. P., 246,249 Langridge, R., 280, 307 Lanni, F., 33(97), 46, 164(154), 165(63), 194, 197 Lanni, Y. T., 33(97), 46, 164(154),197 Lanning, M., 39(98), 46 Lasnitrki, I., 86,146 Laster, W.R., 129,146

314

AUTHOR INDEX

Latarjet, R., 121, 146,281,907 Lauffer, M. A., 12(100, 144), 18(63, 99), 46, 46, 47, 167(115), 174, 176(116), 182, 189, 10.4, 196 Lavelle, J. M., 129, 14.4 Leben, C., 129,146 Lechevalier, M. P., 129, 144 Lederer, E., 6(101), &,& Lee, H. H., 33(136), 47 Leidy, G., 277,278,289,291, 298,300,307 Lemoine, P., 64,146 Leonard, B. R., 12(102), .@ Levenson, C. G., 135, 14.9 Levieil, F., 131, 1.46 Levine, A. S., 129, 1& Levine, S., 33(143), 34(143), 47 Levinthal, C., 60, 126, 1.43, 146 Leyon, H., 9(103), 46 Likely, G. D., 163(145), 197 Limasset, P., 131,146 Lind, P. E., 60,1.69 Linder, R. C., 64,130, l& Lindhorst, T. E., 82,83,84,146 Linet, N., 107,146 Lipmann, F., 4(62), 46 Lipshits, R., 164(78), 196,279,307 Liu, 0. C., 35(104), 46 Locke, S. B., 131,146 Loring, H. S., 24(43), 44 Ludford, R. J., 153(117), 106 Luria, S. E., 25(105), 35(106), aS, 60,120, 126, 1.65, 146, 163(119), 174(118), 106, 276,907 Lwoff, A., 8(107), 14,15(107), 43(107), .@, 73, 136, 144, 146 Lynch, V., 7(22), 44

Maganaaik, B., 127, l& Magie, R. O., 265(40), 673 Mahler, H., 30(109), .@ Mandel, H. G., 97, 14.9 Manil, P., 129, 1-46 Manns, M. M., 253(41), 973 Manns, T. F., 263(41), 679 Manson, L. A., 39(110), .@ Markham, R., 11, 12(44), 13(111-113), 24(113-115), 25, 44, 46, 60, 63, 65,67, 93, 94, 106, 1-46 Maramorosch, K., 176(22), 106, 222, 223, 228, 229, 230, 231, 232, 235, 237, 238, 648, 940,263(4244), 679 Marks, H. P., 190, 109 Marmur, J., 277, 278,301, SO7 Marshak, A., 36(116), 46,94, 146 Martignoni, M. E., 214,960 Maver, M. E., 186(33, 34), lo4 Matthews, R. E. F., 24(116), .@, 54, 67, 74,77,78,79,80,81,82,83,86,93,96,

121,146 Mayers, V. L., 17(117),46 Melnick, J. L., 159(123), 108 Melvin, P., 125, 127, 128,144 Mercer, F. L., 54,82,83, 84,1&, 1-46 Merrill, M. H., 230, 640 Meyer, K. F., 222,246,848 Miller, E. M., 9(118), 46 Miller, G. L., 64, 146 Miller, H. K., 154(81), 168(81), 106 Miller, J. C., 31(18), 44 Minton, S. A., Jr.,58,90,91,117,133,134, 145, 147 Mitchell, H. K., 7(77), 46 Mogabgab, W.J., 132,148 Moloney, J. B., 173(35), 174(35), 186(33M 36), 187(35), 194 Mommaerts, E. B., 168(67,127), 163 M a a l e , O., 39(108), 46 (126), 164(67, 126, 12'7, 155), 165(67, Maasab, H. F., 112, 113, 1-42 156), 166(124), 106,106, 107 McCarty, M., 277,278, 282, SO7 Monod, J., 40(120), .@ McClean, A. P. D., 270(45), 679 Moore, A. E., 91,146 McClement, W.D., 11,M Moore, D. H., 10(122), 19(122), 20(121), McClelland, L., 136, 146 &, 217, $10 MacDonald, E., 167(116), 198 Moore, J. A., 148 McFarlane, A. S., 170(53),104 Morel, G., 131, 1.48 MacFarlane, M. G., 165(120), 108 Morenne, P., 121,146 McKinney, H. H., 251(46), 873 McLean, I. W., Jr., 162(160, l6l), 189 Morgan, C., 10(122), 19(122), 20(121), 46, 217, 610 (171), 107 Morgan, H. R., 90,91,111,116,1.65 MacLeod, C. M., 277, 278, 289, 907

315

AUTHOR INDEX

Mosley, V. M., 243, 246, 248 Moulder, J. W., 21(123, la),31(23), 46,

48 Moulton, F. R., 150(128), 196 Murphy, J. B., 150, 194 Murphy, M. K., 86, 90, 115, 148 Muscatine, N. A., 15(3), 4.9

N Neurath, H., 12(124), 16(125), 19(125), @,156(129), 159(129),182(129),196 Newton, G. W., 176(130),196 Nichols, C. W., 132, 146 Nickell, L. G., 54, 125, 131, 146 Nicolaides, E. D., 146 Nicolle, P., 130, 1.46 Niven, J. S. F., 128,149 Norris, D., 54, 56, 125, 146 Northrop, T. G., 13(126), 46

Price, M., 58, 90,91, 117, 133, 134, 147 Price, W. C., 174, 175(116), 182, 189, 189(135, 165), 190, 196, 197, 251(48, 49, 50,51), 263(52), $73 Price, W. H., 27(134), 31(134), 46 Prickett, C. O., 154(43), 194 Prusoff, W. H., 104,146 Puck, T. T., 33(135-137,143), 34(143),47, 119, 146 Pugh, L. H., 129,144 Putnam, F. W., 14(138), 15(138),26(138), 29(138), 39(92, 93), 42(93), 46, 47, 62,

146 R Rafelson, M. E., Jr., 90,92,110,111,117,

146

Randall, R., 18(53), & Rappaport, C., 15(3), 43 Rasmussen, A. F., Jr., 113, 123, 127, 146 Ravin, A. W., 289, 296,303,307 0 Rawlins, T. E., 54, 55, 84, 113, 118, 128, Officer, J. E., 133, 134, 146, 147 130, 131, 146, 146 O’Kane, D. J., 4(127), 46 Redman, W., 277,307 Olson, C., Jr., 152, 196 Reeve, R. H., 158(67), 164(67), 165(67), Oppenheimer, J. R., 7(22), 4.9 196 Orlando, A., 244, 249 Reichmann, M. E., 17(139), 47, 281,307 Oster, G., 13(128),46 Rennie, J., 203, 919 Rice, S.A., 281, 307 P Rice, S.E., 17(139), 47 Rich, A., 28(140), 47, 73, 146 Paillot, A., 207, 219 Richkov, V. L., 113, 118, 128, 146 Pardee, A. B., 42(129), 46 Riley, V. T., 186(36),194 Parker, L. F. J., 103, 14.9 Parker, R. F., 173(132, 133), 175(132, Ria, H., 22(141), 47 Rita, E., 136, 146 134), 189(132,134),196’ Rivers, T. M., 30(76), 46, 165(97), 196 Patterson, A. M., 102, 146 Robbins, M. L., 86, 90,92, 111, 112, 116, Pauling, L., 100, 146 125, 127, 132,136,149, 147 Pearson, H. E., 90,92, 110, 111, 117, 146 Roberts, D., 97, 98, 1.64 Perrin, J., 31(130),46 Roberts, F. M., 243,249 Perrine, T. D., 86,90,92, 132, 1.48 Robertson, H. E., 91,111, 112,144 Perry, B. T., 65, 14-9 Robinson, R. H. M., 151(147),197 Perry, M. E., 127, 135, 14.9 Rogers, S., 151(136), 186 Peters, J. M., 125, 127, 128, 144 Rose, H. M., 10(122), 19(122), 20(121), Peters, R. A., 138, 146 46, 217, 219 Pfiffner, J. J., 103, 14.9 Ross, A. F., 253(53), 273 Pirie, N. W., 11, 46 Rosseels, J., 83, 146 Plotz, H., 21(133), 46 Rothe Meyer, A., 153(72), 196 Pollard, M., 155(92), 158(92), 196 Roth, L., 7(37), 4 Posnette, A. F., 270(10), 279 Rotman, R., 60,1.64 Potter, C., 201,819 ROUE,P., 150(12, 137-142), 151(12, 110, Powell, H. M., 129, 146

316

AUTHOR INDEX

136-141, 143, 164),152(164), 193, 196, 197 Rumley, G. E., 122,146 Rues, 5. B.,18(176), 48 Russell, P. B.,105,116,135,1#, 147 Rutten, F. J., 114, 146

S Salaman, M. H.,165(121), 196 Salaman, R. N.,251(54), 873 Salemink, C.A., 103, 1&5,146 Samuel, G.,l89(44), 197, 244,248 Sanford, K.K.,153,197 Schabel, F.M.,129,146 Schachman, H. K.,12(144), 47, 68, 72, 143, 155(146), 159(146), 197 Scherer, W.F.,59, 1.46, 147 Schlegel, D.E.,54, 55, 84, 113, 118, 128, 130, 146 Schlesinger, R. W.,35(145), 47,240,249 Schmidt, J. R. A., 123, 1.6s Schmidt, P.,63, 147 Schneider, I. R., 54,55,80,81,119,147 Schoenbach, E. B.,29(158), 47 Schwerdt, C.E.,19(146), 47 Scott, T.F.M., 10(147),47 Sechet, M.,131, 146 Seeds, W.E., 280,SO7 Selbie, F.R., 157(147), 197 Severin, H.H.P.,239, 241, 249,254(55), 255(56-63),273 Shapiro, D. M.,102,143,147 Sharp, D. G., 16(125), 18(148), 19(125, 165, 166), 46, 47, 154(66), 155(153), 156(129), 157(159), 158(8, 64,67, 127, 151, 152, 157-159), 159(129, 156, 158), 160(149, 152, 1701, 161(8, 150, 152), 162(51, 66, 151, 152, 158, 160, 161), 163(126, 150, 152), 164(65-67, 125, 127, 148, 150-152, 154, 155), 165(66, 67, 155), 166(62), 167(61, 85, 66), 168(66), 169(65, 66, 150, 152), 170(65, 66), 176(130), 182(129), 189(171), 193, 194,196, 196, 197 Shaw, E.,159(123), 198 Sherwood, M.B.,105,144 Shimkin, M.B.,150(162), 173(37), 174(37, 38), 175(37,38),189(38), 194,197 Shope, R. E.,129, 147, 151(147, 163), 197 Shulman, S.,12(102), 46 Shunk, C.H., 117,147

Sigik, B.,33(143), 34(143), 47 Silberschmidt, K.,244,9@ Siminovitch, L.,136,144 Simmons, N.,13(150), 38(150), 47 Singer, S.,104, 105, 14.6 Sinsheimer, R. L., 13(126), 37(149), 38 (149)146, 47, 99, 115,147 Skipper, H.E.,129,146 Slykhuis, J. T.,221,244, 8 4 Smadel, J. E., 19(151), 21(133), 30(76, 1511, &, 46, 47,165(97), 196 Smiles, J., 162(69), 196 Smirnova, V. A., 118,128, 1.4s Smith, E.L.,103, 143 Smith, F. F.,255(40), 973 Smith, J. D.,13(113), 22(152), 24(113, 115), 27, 46, 47, 65, 67, 86, 87, 88,93, 94, 96,99, 105, 106, 114, 143, I& Smith, K. M.,lO(153, 154), 20(153, 154, 156), 21(165), 46, 47, 208, 207, 819, 220, 214, 216, 217, 243, $49 Smith, L.,6(157), 47 Smith, P. K., 86,90,92,111,112,116,125, 127, 132, 136, 143, 147 Smith, R. E., 239, 2@, 254(64), 271(64), 973 Smith, S. C.,113, 148 Smith, W.E., 151(164), 152(164), 197 Snyder, J. C.,31(17), .6.6 Spencer, E. L.,189(165), 190, 197 Spizizen, J., 17(117), 48, 110,147 Spooner, E.T.C., 11,M Sprince, H., 29(158), 47 Stahl, C. F.,239,948 Stanier, R. Y.,3(159), 4(159), 5(159), 33(159), 47 Stanley, W. M., 11(160), 13(25), 44, 47, 64, 146 Stannard, C., 127, 143 Steiner, D.L.,120,146 Steinhaus, E.A.,207,209,819 Stekol, J. A.,138,147 Btent, G.S.,14,47 Stern, K.G.,156(111, 166), 157,196,197 Stevens, C.,13(23), 4.6 Stock, C.C., 91, 1.48 Stocker, B. A. D.,289,307 Stoddard, E.M.,54, 122,147 Stoker, M.G.P., 22(152), 27,47 Stokes, A. R., 68,148,280,307 Stokes, J. C.,113, 127,146

317

AUTHOR INDEX

Storey, H. H., 222, 232, 243,248, 255(66), 270 (65), 275 Strandskov, F. B., 105, 147 Strauss, M. J., 159(123), 196 Strelitz, F., 131, 142 Syverton, J. T.,59,91, 111, 112, 14.4,I&, f47, 150(67), 151, 152(167), 191(167), 197 Szaforz, D., 107, 146

Volkin, E., 17, 37(170), 38(170), 47, 99, 115, 147

W

Wacker, A., 87, 148 Waddell, J. G., 138, 147 Wakelin, R. W., 138, 1.46 Waksman, S. A., 129,1& Wallace, H. E., 240,243, ,948 Wallace, J. M., 221,244,248,252(69), 271, T 273 Wang, T., 23,47,64, 147 Takahashi, W. N., 60,64, 125, 147 Takemoto, K. K., 90, 92, 111, 112, 116, Ward, S. M., 30(76), 46, 165(97), 196 Warren, J., 18(176), 48 125, 127, 132, 136, 147 Wasser, H. B., 20(172), 47, 209, 210, 219 Tamm, I., 35(164), 47, 58, 117, 118, 147 Watanabe, T., 133, 147 Tanada, Y., 209, 219 Taylor, A. R., 16(125), 19(125), 46, 47, Waters, N. F., 176(68), 178(173, 174), 181(68), 196, 197 156(129, 169), 157(159), 158(157-159), 159(129, 156, 158), lSO(168-170), 162 Watson, J. O., 23, 28(140, 173), 39(108), 46, 47, 68, 73, 143, 146, 147, 281, 306, (51, 158, 160, 161), 164(155), 182(129), 507 189(171), 196, 197 Watson, M. A., 243, 249, 279, 280, 307 Taylor, H. E., 277, 278, 282,307 Weed, L. L., 39(41, 174), 41(41), 4.4 Teitelbaum, S. S., 237, 249 Weidel, W., 33(175), 48 TenBroeck, C., 230, $49 Weil, A. J., 277, 507 Theorell, H., 30(168), 47 Weil, M., 18(176), 48, 162(51), 194 Thomas, C. A., 17(139), 47, 281, 5G7 Thomas, R., 282: 283, 286, 288, 289, 292, Weintraub, M., 118, 122, 123, 148 Weiser, J., 213, 280 307 Weiss, K., 138, 147 Thomas, W. D., 122, 1.46 Welch, A. D., 104, 1.46 Thompson, C. G., 207, 209,219 Thompson, R. L.,58,90,91,111,112,116, Wellington, Eunice F., 20, 25(15), 44, 201, 220 117, 123, 127, 133, 134, 135, 145, 147 Wells, W. F., 175(74), 189(74), 196 Thung, T. H., 251(67, 68), 273 Westland, R. D., 146 Todd, A. R., 91, 106,143 Weygand, F., 87, 148 Tokuyasu, K., 209, 219 Whalen, M. M., 203, 219 Tolmach, I,. J., 33(137), 47 White, C. L., 186(33,34), 194 Trevan, J. W., 175(172), 197 Whittle, E. L., 146 Tyrrell, D. A. J., 58, 147 Wilkin, M. L., 111, 116, 135, 147 Wilkins, M. H. F., 68, 148, 280,307 U Wille, H., 214, 290 Umbreit, W. W., 138, 147 Williams, I., 42(129), 46 Williams, R . C., 14(57), 15, 46, 48, 163 V (119), 196 Williamson, M., 91, 14.8 van der Want, G. M., 103, 146,146 Vanderwerff, H., 105, 144 Willison, R. S., 118, 122, 123, 148 van Rooyen, C. E., 135, 1.46 Wilmer, H. A., 30(178), 48 Wilson, H. R., 68, 148, 280, 307 Varma, P. M., 244, ,949 Winkler, K. C., 87, 114, 148 Vatter, A. E., 243, 248 Winder, R. J., 90,92, 110, 111, 117, 146 Vogel, H. J., 94, 146 Wisseman, C. L., 130, 143 vogt, M., 59,145

318

AUTHOR INDEX

Wollrnan, E.,40(120), 46, 136, 144 Wood, J. L.,13(23), 44 Wood, M.T., 186(33,34),194 Woods, M.W., 9(49), 44,9(179), 48 Wooley, J. G.,86,90,92,115,132,148 Woolley, D.W., 42(95), 46 Wolff, R.,105,148 Work, E.,7(48), ~$4 Wyatt, G. R., 15(183), 21(183), 26(180, 183), 27(181), 36(182, 183), @,76,89, 94,95,99, 115,l&, 201, 220,279, 507 Wyckoff, R. W. G.,12(20), 44, 155(11), 156(11), 159(13), 160(ll, 13), 193,205, 206, $10, 243, 246, $48 Wyss, O.,105, 147

X Xero8, N.,20(156), 21(155), 47, 201, 203, 204, 205, 206, 207, 210, 214, 216, 217, 860

Y Yaaofsky, C., 7(184), 48 Yarwood, C.E., 54,123,148 Yule, G.U.,173(89), 196 2

Zahler, S.A., 21(185), 48 Zamenhof, S.,87, 102, 105, 148, 278, 289, 291,307 Zatrnan, L. J., 139,148 Ziegler, N.R.,173(93), 106 Zinder, N.D., 306,507

Subject Index A Abraxae grossulariata, virus of, 201, 206, 218 Acarina, virus vectors, 221, 244 Aceria $CUB, virus vector of, 244, Aceria tulipae, virus vector, 244 Acetate, bacterial conversion of, 7 Acetyl CoA, from acetate, 7 Acetylglycine, virus inhibitor, 114 Acetylmethionine, virus inhibitor, 114 Acetyl phosphate, in acetate conversion, 7 3-Acetylpyridine, and virus inhibition, 118 Acetyltryptophan, virus inhibitor, 114 Achromobacler zeroeis, antibiotic from, 129 Aconitase, and citrate, 138 Actinomycetin, virus inhibitor, 129 Actinomycin, virus inhibitor, 129 Acriflavine, virus inhibitor, 128 Adenine, and 8-azaguanine activity, 76 and virus inhibition, 82, 86,91, 92, 103 growth requirements, 105 in nucleic acids, 2&28,65,68,69,71,72, 77, 88, 99 Adenine nucleotide, in virus, 25 Adenosine, and virus inhibition, 82, 92, 118 Adenosine diphosphate, and enzyme activity, 166 Adenosinetriphosphatase (ATPase) in birds, 164, 165 in virus, 29, 31, 133 Adenosine triphosphate (ATP), 138 dephosphorylation Of 164-166, 170 Adenylic acids, and 8-amguanine activity, 76 and enzyme activity, 166 and virus inhibition, 82,91,92, 110,118 in virus nucleic acids, 93 African marigolds, yellows virus in, 261

Agallia constricla, virus transmission by, 236, 237 Agalliopsis novella, virus multiplication in, 233 Agar sedimentation technique, 161 Alanine ethyl ester HCl, virus inhibitor, 114 Aleyrodidae, virus vectors, 221,244 Alfalfa caterpillar, virus of, 216 Alfalfa mosaic virus, characteristics of, 11 Algae, cytochemistry of, 6 a,r-diamino-pimelic acid in, 5 , 6 metabolic systems of, 6 Allogenic transformations, 294-298, 304 Allothreonine, virus inhibitor, 114 Aluminum ion, and virus growth, 122 Amidase, action on mucoprotein, 35 Amino acids, in viruses, 23, 25 virus inhibitors, 108-114, 141 5-Aminoacridine, virus inhibitor, 128 9-Aminoacridine, virus inhibitor, 127 a-Aminoadipic acid, lysine precursor, 7 Aminoadipic acid, virus inhibitor, 111 4-(7)-Aminobenzimidazole,103 p-Aminobenzoic acid, and virus, 31 a-Aminobutanesulfonic acid, virus inhibit,or, 112 4-Aminofolic acid, virus inhibitor, 116 5-Amin0-7-hydroxy-(3,1,2) oxadiazolo (5,4d)pyrimidine, virus inhibitor, 01

OI

~-Amino-4-hyd~~xybenzimidazo~e, 103 4-(5) #mino- 1 ~ - 1 , 2 , 3 , t r i a z o l e - -5(4) carboxyamide, effect on virus, 7577, 79, 80,96, 106 4 - (5) -Amino - 1H- 1,2,3-triaeole - 5 - (4) carboxylic acid, effect on viruses, 75, 77, 79, 96 5 -Amino-7 - hydroxy - 1,2,4,6-tetraazaindene, 81 4 - (5) - Aminoimidazole - 5(4) - carboxyamide, purine precursor, 106

319

320

SUBJECT INDEX

5-Amino-5-imidazolecarboxyamide, in nucleic acids, 97 4-Amino-5-iodopyrimidineI virus inhibitor, 85 Aminomethanesulfonic acid, virus inhibitor, 110, 112 a-Amino-p-methoxyphenyl - methanesulfonic acid, virus inhibitor, 112 a-Amino-B-phenylethanesulfonic acid, virus inhibitor, 112, 113 a-Aminophenylmethanesulfonicacid, virus inhibitor, 112 2-Aminopurine, growth inhibitor, 105 5-Aminouracil, in nucleic acid, 98 virus inhibitor, 92,97,104,105 6-Aminouracil, virus inhibitor, 92 Angleshades moth, virus of, 202,206,216 Animal viruses, adsorption sites, 34 amino acids of, 26 biological properties of, 3 effects of metal ions and chelating agents, 123, 124 effects of purine analogues, 90-92 effects of pyrimidine analogues, 92 enzymatic activities, 29, 30 form and composition, 17-22,62 inhibition of, 110-113, 115-118, 123125, 127-129, 141 nucleic acids of, 26, 62

Antibiotics, 5 and virus, 31, 128-131 resistance to, 277 Aphididae, virus vectors, 221 Aphids, virus vectors, 74, 221, 243, 244 Atabinopyranosyl, virus inhibitor, 118 D-Arabinose, in mycobact,eria, 6 Arctia caja, virus of, 205 Arctia villica, virus of, 205, 206 Arginine, in bacteriophages, 25 virus inhibitor, 110 Argyrataenia velutinana, virus of, 209 Arsenite, and bacterial lysogenization, 137

Arthropods, plant virus vectors, 221 determination of, 221,222 Ascorbic acid, and virus inhibition, 118 Asters, yellowsvirusin, 254,255,260,262271

Aster leafhoppers, see also Leafhoppers cross protection in, 263-270

virus vector, 226-233,242,253-257,260, 262, 263

Aster yellows, symptoms of, 256-260 Aster yellows virus, 255-271 multiplication in leafhopper vector, 225-233, 237-239, 241-243,245, 247, 253, 254 assay of virus in insect vectors, 227229 correlation of incubation periods, 229 dssage effect, 229 effect of heat, 226-227 effect of low temperature, 229, 230 heat-induced incubation period, 225227

measurement of virus concentration, 232, 233

serial passage, 230-232 Atebrin, virus inhibitor, 127, 128 Aureogenue clavifolium, see Clover club leaf virus Aureogenue magnivena, see Wound tumor virus Aureomycin, in virus therapy, 128-131 Australian pasture caterpillar, virus of, 201

Autogenic transformations, 295-298,305 Avian erythromyeloblastic leucosis, ATPase in, 29 and normal particles, 18 antigenic constitution, 167-173, 192, 193

B.A.I.A. strain, 155 chemical constitution, 163 classification of, 152 dose response to, 177, 185 enumeration of virus particles, 163,164 enzyme activity, 164-166 host-virus relation, 174, 176182, 188, 191

lymphomatoais in. 153, 154 malignancy of, 160, 191 origin of, 192 physical properties, 160-163 purification of virus, 156-158 R.P.L. 3 strain, 155 stability of, 166, 167 titration of, 173 virus of,150, 151-155, 192 size and shape, 18

SUBJECT INDEX

2-haadenine, virus inhibitor, 55,80,86 g-Azaadenine, incorporation in virus nucleic acids, 96, 107 virus inhibitor, 55,7677, 79,80,86,91 8-Azaguanine, incorporation in virus nucleic acids, 76-78, 92, 93, 96, 97, 100, 101, 106 metabolism in plant tissues, 79,80,102 virus inhibitor, 53,56,74-76,82,85,86, 90, 91, 95, 96, 141 mechanism of action, 76-80, 141 8-Azaguanosine phosphates, 93 8-Azaguanylic acid, in virus nucleic acid, 77, 80, 93, 94, 96 8-Azahypoxanthine, effect on E. coli, 97 effect on viruses, 75, 77, 79, 86, 90, 96 8-Azaisoguanine, effect on virus, 75, 77, 79, 96 2-Azapurines, effects on plant viruses, 80 8-hapurines, effects on viruses, 74-80, 90,96 metabolism in plant tissues, 79, 80 4-Azathymine, 104 8-haxanthine, effect on virus, 75,77,79, 80, 90, 96, 102 Azide, and bacterial lysogenization, 136, 137 and virus multiplication, 31 Azoquinone-imide dyes, virus inhibitors, 124

B Bacillus cereus, and virus inhibitors, 86, 100 Bacillus coli, see Escherichia coli B . megaterium, cytochrome system of, 6 lysogenic strain, 120 B . sublilis, glucose metabolism in, 7 inhibition of, 134 niacin derivation in, 7 Bacitracin, virus inhibitor, 130 Bacteria, acetate conversion in, 7 cytochemistry of, 5 cytochrome content of, 6 a,€-diamino-pimelicacid in, 5, 6 enzymes of, 6 lysogenization of, 136, 137 metabolic systems of, 6 Bacterial transformations, 275-307 biological description of, 276-278

321

chemical and physical properties of transforming agents, 278-282 fractionation studies on DNA, 279281 molecular weight of a transforming factor, 281, 282 genetic recombination between transforming factors, 29S307 allogenic transformation, 294-298, 304 linkage of streptomycin resistance and mannitol factors, 301-303 linked capsular agents in Hemophiius, 298-300 significance of data, 303-307 mechanism of, 282-293 phases of process, 282-284 quantitative kinetic experiments, 284-293 effects of inoculum size, 288 effect of temperature, 285,287 effect of time, 284, 287 Bacterial viruses, see also Bacteriophage amino acids of, 25,26 and bacterial penetration, 33-35 biological properties of, 3 form and composition, 14-17,22 liberation of, 34,63 lysogenic systems, 8,10,73 metabolites in, 29 temperate, 8,9,136 virulent, 8,9 Bacterial virus T2,biology of, 2, 3, 8, 9, 11 Bacterial virus T4, biology of, 2, 8,9, 11 Bacterial virus T6, biology of, 2, 8, 9, 11 Bacteriophage, see also Bacterial viruses antigens of, 9 dose-response to, 177,189, 190 effect of metal ions and chelating agents, 119-121 effects of purine analogues, 85-87 effects of pyrimidine analogues, 87-90 enzyme-resistant coat, 9 form and composition, 21,22 genetic recombination, 60, 306 genetics of, 293 incomplete viruses, 60,63 inhibition of, 95,108-110,114,115,119121, 125-127, 130, 131, 141 nuclease-resistant coat, 9

322

SUBJECT INDEX

nucleic acid of, 62,66,66,99,100 protease-resistant coat, 9 purification of, 161 Bacteriophage A, , in Salmonella, 14,16 Bacteriophage lambda (X), 8,14,16,121 Bacteriophage methods, and animal viruses, 1 Bacteriophage reproduction, deoxyribonucleic acid in, 276 protein in, 276 Barium ion, and virus growth, 122 Bean plants, virus diseases of, 121-123 Bemisia tabaci, virus vector, 244 Bemaldehyde thiosemicarbazone, virus inhibitor, 133, 134 Benzimidasoles, virus inhibitors, 103, 116, 117-119, 142 Beneoylalanine, virus inhibitor, 113, 114 c-Benzoyllysine, virus inhibitor, 114 Bemtricteoles, 103, 104 2-Bemyl-4,6-dihydroxy-l ,3,li-triaeine, virus inhibitor, 86 Betaine, and vim inhibition, 112 Bhendi mosaic, insect vector of, 244 Big leg, in chickens, 163 Biochemistry, and virology, see also Vgrology, 1-43 unity)doctrine, 3-6,36,36 Biotin, and virus inhibition, 116 in tobacco mosaic virus, 29 in vaccinia virus, 30 Biotin analogues, 30 Black currant sawfly, virus of , 203 Blue disease, Japanese beetle, 214 Bombyx mori, virus of, 201,206,218 Brilliant p e n , virus inhibitor, 126 Broad bean, virus@ of, 82 Broad bean mottle virus, inhibition of, 82 6-Bromodeoxyuridylic acid, 96 Bromophenol blue, virus stain, 206 5-Bromouracil, in DNA, 94, 96,98, 100102

in RNA, 99 virus inhibitor, 61, 86, 87-91, 96,101 6-Bromouracil deoxyriboside, in DNA, 102 Buckwheat, yellows virus in, 261 Bunyamwere virus, inhibition of, 91 Bupalw piniarius, virus of, 208,214 Butyrate, virus metabolism of, 31

Butyryl-coenzyme A, and vaccinia virus, 30 C

Cacao swollen shoot virus, cross protection in cacao, 270 Cacoecia muranana, virus of, 207,209 Caesium ion, and virus growth, 122 Caffeine, and 8-azaguanine activity, 76 Calcium, and virus growth, 119,120, 122 Calcium chloride, virus inhibitor, 122, 123 California aster yellows, 264-262, 270-272 cross protection in leafhoppers, 263-269 cross protection in Vinca rose0,262,263 heat-treated in Vinca rosea, 265, 266 host range, 264, 265 insect vectors of, 241, 242 in the east, 266 symptom and those of aster yellows, 266-260 Camptochironomue tentana, virus of, 213 Canavanine, and bacterial growth, 277, 280 Cancer cell tissue culture, 91 Capsular transforming factor, 278, 289, 294300 Capsule viruses, of insects, 27, 200, 207210 Carbohydrate fermentation, 277 Carboxypeptidase, and tobacco mosaic vim protein, 64 2-p-Carboxyphenyl-5,7-diamino -u - tria eolo (d) pyrimidine, virus inhibitor, a7 2-Carboxypyrrole, 36 Carcinogens, 174, 175, 189 Carnation mosaic virus, chemotherapy of 122 Carrots, yellowsvirusin, 263,264,268,260 Casein hydrolyzate, and oxygen uptake, 31 Catalaae, in virus, 31 Caterpillar, viruses of, 206, 210, 211 Cations, virus inhibitors, 141 Celery, yellows virus in, 25+266,260-262 Chelating agents, effects on viruses, 119.124 Chemotherapy, 6 of viruses, 49-142 Chick embryos, nucleic acids of, 27, 28

SUBJECT INDEX

Chicken, neoplasia of, 149 viruses of, 176-182, 186, 187 Chicken sarcoma, virus of, 150, 160 antigen of, 167-170 Chicken sarcoma I, see Rous tumor China aster, virus of, 227 yellows virus in, 254, 257 Chinese cabbage plants, virus in, 74, 76 5-Ch1oroacetamidouraci1, virus inhibitor, 135,136 5-Chlorodeoxyuridylic acid, 95 2-Chloro-4-dimethylaminopyrimidine, virus inhibitor, 92 Chlorogenus callistephi, varieties of, 255 Chloromycetin, and bacterial lysogenization, 136 virus inhibitor, 129, 130 Chlorophyll a, in chloroplasts, 6 , 7 Chloroplasts, in plants, 6 5-Chlorouracil, in DNA, 94, 95, 98, 100 virus inhibitor, 85, 87-89 5-Chlorouridine, virus inhibitor, 92 Choline, and virus inhibition, 112, 116 in tissues, 138 Chorioallantoic membrane, virus culture medium, 58,59 Chromatography, of virus nucleic acids, 92-95 Chromatophores, in microorganisms, 6 Cicadellidae, virus vectors, 221 virus multiplication in, 223-247 Cicadulina nibila, virus vector, 243, 270 Circulifer tenellus, virus vector, 239, 271 Cirphis unipuncta, virus of, 210 Citrate, and virus multiplication, 119, 120, 123, 124, 141 metabolism of 31, 138 Citric acid, and bacterial lysogenizations, 136, 137 effect on virus lesions, 121, 122 oxidation of, 132 Clavicin, virus inhibitor, 130 Clover club leaf virus, multiplication in insect vectors, 233-235, 245 Cobalt ion, and virus growth, 120, 123 Cobalt salts, and bacterial lysogenization, 137 Coccidae, virus vectors, 221 Cockchafer, virus disease of, 214 Cocoecia murinana Hb, capsule virus of, 27

323

Coenzyme A, 31 Coenzyme I, and virus inhibition, 91, 118 Coliaa philodice, eurytheme, virus of, 216 Coliphages, characteristics of, 14-17 5-hydroxymethylcytosine in, 35-43 inactivation of, 15, 16 Colorado tick fever, hosts of, 246 Columbia SK virus, treatment of, 129 Congo red, virus inhibitor, 125 Copper, in vaccinia virus, 30 Copper salts, and bacterial lysogenization, 137 Corn stunt virus, multiplication in insect vectors, 237,238, 242, 243, 245 Corynebacterium diptheriae, phages of, 137 Cosmopolitan armyworm, virus disease of, 20,210 Cottontail rabbit virus, see Rabbit papilloma Coxsackie virus, size and shape, 18 Cozymase synthesis, 138,139 Crane fly, virus of, 203-205, 210-213, 215, 216 Creatine, and virus inhibition, 110, 112 Creatinine, in tissues, 138 Cresyl violet, virus inhibitor, 124 Crimson clover, in virus study, 233 Crystal violet, virus inhibitor, 124, 125 Cucumber 3 virus, characteristics of, 11 Cucumber 4 virus, characteristics of, 11 ribonucleic acid composition, 25 Cucumber mosaic virus, amino acids of, 23 treatment of, 74, 75,80-82, 84, 85, 114, 118, 130 variants of, 252 Curly top virus, cross protection in sugar beets, 271 multiplication in insect vectors, 238241, 243 Currant moth, virus of, 201, 206, 218 Cuscuta campestria, virus vector, 255, 267,269 Cyanide, and bacterial lysogenization, 136 and virus multiplication, 32 L-Cysteine, and virus inhibition, 110,112, 113,121,123 Cysteine, in tobacco mosaic virus, 23 L-Cystine, and virus inhibition, 110, 121

324

SUBJECT INDEX

Cytidylic acid, in virus nucleic acid, 93 Cytochrome c oxidase, in bacteria, 6 Cytochromes, in bacteria, 6 in vaccinia virus, 30 Cytosine, in virus nucleic acids, 25-28, 36-43, 65, 68, 69, 71, 72, 77, 88 and virus inhibition, 83,84 Cytosine deaminase, of E. coli, 41 Cytosine deoxyriboside, from DNA, 37

D DDT, 5 Dahlia violet, virus inhibitor, 124 Dalbulus maidis, virus vector, 237, 238 1-Deazaadenine, virus inhibitor, 103 3-Deazaadenine, virus inhibitor, 103 Deoxycytidine, 40,41 Deoxycytidine deaminase, of E. coli, 41 Deoxypyridoxine, and virus inhibition, 115,116,138 Deoxyribonuclease (DNAase), and bacterial transformations, 283, 285-287, 289, 292 and virus DNA, 15, 37-39, 41, 42, 95, 139 Deoxyribonucleates, in bacterial cells, 276,279,280,282,298 Deoxyribonucleoprotein, synthesis of, 29 2-Deoxy-~-ribose,in virus nucleic acid, 65 Deoxyribonucleic acid (DNA), and bacterial transformations, 282-285, 287 289-294,297-303 and virus inhibiton, 118,126 composition of, 65, 66, 106 degradation in host cells, 39 fractionation studies on, 279-281 5-halogenated uracil8 in, 93-95, 100 5-hydroxymethylcytosine conent, 37, 61,66,68,69,99,108 in bacterial cell, 276, 278, 279, 282, 303-306 in vaccinia virus, 10 in viruses, 12-17, 19, 21-23, 27, 38-41, 62,63,6649,71-73, 86,88,106,107 composition of, 2628,36, 115 transfer to virus progeny, 39 DNA-avidin complex, in egg white, 106 Deoxyuridine, 40,41 Detergents, and DNA preparations, 17 m-Dethiobiotin, virus inhibitor, 116

Diamidines, virus inhibitors, 135 2,4-Diamino-5,6-dimethylpyrimidine, virus inhibitor, 116 2,4-Diamino-6 - hydroxy -5 - (p - carboxy amidopheny1)-pyrimidine, virus inhibitor, 90 4,5-Diamino-2-mercaptopyrimidine,virus inhibitor, 84 2,4-Diamino-5-nitroso-6-hydroxypyrimidine, virus inhibitor, 92 a,c-Diamino-pimelic acid, in algae and bacteria, 5,6,25 lysine precursor, 7 2,6-Diamino-4-propoxy-s-triazine, virus inhibitor, 92 2,6-Diaminopurine, growth inhibitor, 55, 82,91,92, 105 5,6-Diaminouracil, virus inhibitor, 92 Diazouracil, virus inhibitor, 55,85 N-Dichloroacetyl-ru-(p-nitrophenyl) dycine, virus inhibitor, 111 5,6-Dichlorobenzimidazole, virus inhibitor, 140 2,6-Dichloro-8-hydroxypurine, virus inhibitor, 90 2,6-Dichloro-7-methylpurine,virus inhibitor, 91 Dichlorophenoxyacetic acids, virus inhibitors, 131, 132 2,4-Dichlorophenoxypropionic acid, virus inhibitor, 132 (Dichlorophenoxy)thiouracil,virus inhibitor, 134 Diesterase, and DNA hydrolyzates, 37, 95 5,6-Diethylbenzimidazole,virus inhibitor, 117 Digitalis, host response to, 175, 191 3,5-Dihydroxy-6-methyl-l, 2,4-triazine, 104 3,4-Dihydroxyphenylelanine, virus inhibitor, 114 2,4-Dihydroxypyridine, virus inhibitor, 85 Dihydroxy-s-triazine, 104 Dimethylamino-8-azaguanine,effect on virus, 75, 77, 79 5,6-Dimethylbenzimidazole, virus inhibitor, 103, 117, 118, 139 5,6-Dimethyl-1-8- D - ribofurenosyl - ben -

325

SUBJECT INDEX

zimidazole (a-ribazole), virus inhibitor, 117, 118 Dinit.ropheno1, and virus multiplication, 31, 32, 133, 141 2,4-Dinitrophenol, and bacterial lysogenization, 136,137 Diphosphopyridine nucleotidases, 139 Diphosphopyridine nucleotide, 31 Diprion hercyniae, virus of, 203 Diptera, viruses of, 200, 203-205,210,216 Dithiothymine, virus inhibitor, 92 2,4-Dithiouracil, virus inhibitor, 84 Dodder, virus vector, 32, 254, 255, 266269 Dyestuffs, effect on viruses, 124, 125, 141

E Eastern equine encephalomyelitis virus, inhibition of, 127, 128, 133 Ectromelia, antibody titer, 240 Electrophoresis, of virus nucleic acids, 92-95 Embden-Meyerhof scheme, of glucose metabolism, 7 Embryonated egg, virus culture medium, 57, 58 Encephalomyocarditis virus, and normal particles, 18 size and shape, 18 Enzymes, in virus, 29-32 in virus penetration and liberation, 32-35 Equine encephalomyelitis virus, 240 and normal particles, 18 lipid in, 22 nucleic acids of, 19 serial passage, 230 size and shape, 18 Erlichin, in virus therapy, 128,129 Erythroblastosis, 153-155 Erythroleukosis, 150 antigen of, 167 Erythromyeloblastosis, see Avian erythromyelobastic leukosis Escherichia, transformations in, 277 Escherichia coli, 3 carbohydrate metabolism in, 7 cystosine deaminase, 41 cytosine-requiring strain, 40 deoxycytidine deaminase of, 41 a,c-diaminopimelic acid in, 25

effects of 8-azapurines, 96,97 enzymes of, 6,38 5-hydroxymethylcytosine in, 26,36,37, 42 inhibition of, 108, 109, 114 lysine derivation in, 7 lysogenic effects, 120 6-methylaminopurine in, 108 niacin derivation in, 7 nucleic acid of, 115 phages of, 8,14,15,38,39,86,121 RNA synthesis, 39 sterols in, 6 T phage adsorption on, 119, 120 thymine-requiring strain, 40,42,87,99, 101,102,105 uracil-requiring strain, 40 virus penetration of, 33,34 Esterase, in virus, 31 DL-Ethionine, virus inhibitor, 112-1 14 Ethionine C14 labeled, 138 2-Ethyl-5-methylbenzimidazole, virus inhibitor, 117 5-Ethyluracil, B . coli inhibitor, 105 Euphorbia mosaic, insect vector of, 244 European pine sawfly, virus of, 203 European spruce sawfly, virus of, 203

F F at t y acids, and virus inhibition, 115 in mycobacteria, 6 Feline pneumonitis virus, multiplication of, 31 nucleic acids of, 21 treatment of, 129 Feulgen stain, for virus, 205 Fibroblast cells, sarcoma host, 153 Fibroma virus, dose-response, 190 Fig mosaic virus, insect vectors of, 244 Flavine adenine dinucleotide (FAD), in vaccinia virus, 30,32 Flavotin, and 8-azaguanine activity, 102 Flax, yellows virus in, 258 Fluoride, and virus multiplication, 31 Fluoroacetate, synthesis of, 138 Fluorocitrate, 138 5-Fluoronicotinic acid, 138, 139 Fluorophenylalanines, virus inhibitors, 111 Folk acid, and virus inhibiton, 116, 118 Foot and mouth disease, inhibiton of, 125

3%

SUBJECT INDEX

Guanine analogues, in nucleic acids, 100, Forest tent caterpillar virus, 27 101, 280 Formic acid, in DNA analysis, 36 6-Formylpteridine, and 8-azaguanine Guanosine, and virus inhibition, 118 Guanylic acid, 76,91,93,94,96,118 activity, 102 Guinea pigs, virus disease of, 125 Forssman antigen, and virus, 167-171 Gypsy moth virus, 27 Fowl pox virus, development of, 217 elementary bodies of, 19 H Fowl sarcoma, dose-response, 176 a-Haloacylamides, virus inhibitors, 105, French bean, viruses of, 82 135, 136 Fucose, 36 5-Halogenated uracils, in nucleic acids, Fumarate, virus metabolism of, 31 93-95,100,101,106,141 Fumaric acid, and bacterial lysogenizaHeat, and bacterial lysogenization, 136, tion, 136 137 ~~-@-Furylalanine, virus inhibitor, 111 HeLa cells, virus culture medium, 59 G Helenine, virus inhibitor, 129 Hemin, in tumor viruses, 30 Gaddum precision index, 181 Hemophilus, and DNA preparations, 17 Galactose, 36 Hemophilus, linked capsular agents in, Genes, nucleoprotein nature of, 276 298301 Genetic recombination, and bacterial transformations in, 277,281,289 transformation, 293-307 Henbane mosaic virus, treatment of, 74, Giemsa stain, for virus, 206 80-82, 85 Gladiolus, virus of, 256 Herpes febrilis virus, in mice, 125 Glucosamine, 35 inhibition of, 128,134 Glucose, and virus inhibiton, 116 Herpes simplex virus, 240 in T-phage DNA, 37, 38 and enzymes, 30,32 virus metabolism of, 31 development of, 217 Glucose hydroxymethylcytosine, in nuelementary bodies of, 20, 22 cleic acids, 100 multiplication of, 10 Glucose metabolism, pathways of, 7 transmissible form, 73 Glucose-6-phosphate, and virus inhibiHexamidine, virus inhibitor, 136 tion, 116 Hexokinase, in virus, 31 Glucose-6-phosphate metabolism, 7 5-Glucosyl hydroxymethyl cytosine de- Hexosamine, 35 Hexose phosphate, and vaccinia virus, 30 oxynucleotide, 38 Histidine, and virus inhibition, 123 Glutamate, virus metabolism of, 31 in bacteriophages, 26 Glutamic acid, and oxygen uptake, 31 in tobacco mosaic virus, 23 and virus inhibition, 109,113 virus inhibitor, 110 Glycine, in bacteriophages, 26 DL-Homobiotin, virus inhibitor, 116 Glycolytic pathway, of glucose metaboHomocysteine, and virus inhibition, 112 lism, 7 Homofuchsin, virus inhibitor, 126 Granuloses, 20,200,207-210 Honeybeee, virus paralysis of, 210, 214 Gray lung disease, treatment of, 128 Honeybee larvae, sacbrood of, 210, 214 G r i m alfalfa, in virus study, 233-237 Host response, and virus infectivity, 173Oryllulus domesticus, virus of, 213, 214 191 Guanase, 102 latent period, 173,176,178-181,184-187 Guanine, and 8-maguanine activity, 76 quanta1 response, 173, 175, 176, 178, and virus inhibition, 82, 91 181, 184-186, 188, 189 in virus nucleic acids, 26-28,37,39,66, House crickets, virus of, 213.214 68,69,71,72,76,77,88,88,93,

94,96

4-Hydroxy-6-aminobenztriazole,104

SUBJECT INDEX

5-Hydroxy-lH-1,2,3-triazole, effect on viruses, 76,79,96 4- (5)-Hydroxy-1H-1 ,2,3-triazole-5-(4) carboxylic acid, effect on viruses, 75, 77 Hydroxymethylation, 40-42 5-Hydroxymethylcytoeine, in virus nucleic acids, 26, 28, 37, 61, 66, 68, 69, 88,95,99,108,115 metabolism in T-even phages, 35-43 nucleotides of, 38 5-Hydroxymethylcytosine deoxyriboside, 38, 40 5-Hydroxymethylcytosine glucoside, 39, 108

5-Hydroxy-7-methyl-tetrazolo(A) pyrimidine, virus inhibitor, 87 5-Hydroxymethyluracil, 40-42 6-Hydroxynicotinic acid, and virus inhibition, 118 2-p-Hydroxyphenyl-5-amino7- hydroxy0-triazolo (d) pyrimidine, virus inhibitor, 87 4 - Hydroxy - 2,5,6 - triaminopyrimidine, virus inhibitor, 90 5-Hydroxyuracil, 105 Hymenoptera, viruses of, 200,203 Hypoxanthine, and 8-azaguanine activity, 76 and virus inhibition, 91 I Infectious chlorosis, virus vector of, 244 Ilheus encephalitis virus, inhibition of, 91 4-(Imidazolidone-2)-caproicacid, virus inhibitor, 116 6-1mino-5-nitrosohydroxyuraci1, virus inhibitor, 92 Indene derivatives, effects on plant viruses, 80 Indolacetic acid, virus inhibitor, 131 Indole, 110 niacin precursor, 7 virus inhibitor, 141 Indolebutyric acid, virus inhibitor, 132 Influenza virus, 240 and normal particles, 18 antigens of, 167, 168 biology of, 3 chemotherapy testing, 57-59

327

composition of, 10, 19 dose-response to, 177,181,189 enzymes in, 32 enzyme activity of, 165 genetic recombinations, 60 hemagglutination in, 166 infectivity of, 64,65 inhibition of, 90-92, 110-113, 118-118, 123-125,127,129,132-136,139,141,

142 liberation of, 34,35 lipid in, 22 mucinase in, 29,30,3p nucleic acid content, 19,62, 65 penetration of, 34,36 receptor-destroying enzyme of, 35 sedimentation constant, 162 size and shape,. 18 titration of, 173,188 Inosine triphosphatase, in virus, 166 Inosine triphosphate, dephosphorylation of, 164 Inosinic acid, 76 Insect vectors, and plant viruses, 221-247 virus-host relationship, 222 Insect viruses, and lysogenic system8,lO DNA content, 12 development of, 29 enzymes in, 32 form and composition, 12-14, 22 morphology and development, 199-218 apparent viruses, 200, 211-214 development, 214-218 granuloses or capsular, 200, 207-210 no intracellular inclusion, 200, 210, 211 polyhedral virus diseases, 200-207 cytoplasmic type, 205-207,218 lepidoptera, 205-207 nuclear type, 200-205,216, 218 diptera, 200,203-205 hymenoptera, 200,203 lepidoptera, 200-202,205 nucleic acids of, 26,27,62 polyhedral bodies, 20,21,27 size and shape, 20,21 5-Iododeoxyuridylic acid, 95 5-Ioduracil, in DNA, 94,95,98,100 virus inhibitor, 87,88,89 Isatin thiosemicarbozone, virus inhibitor, 134

328

SUBJECT INDEX

Isobarbituric acid, virus inhibitor, 92 Isocitrate, 138 DL-Isoleucine, and virus inhibition, 110, 113

Isonicotinic acid hydrazide, and virus inhibition, 118, 139

Lipocarbohydrate, and virus DNA release, 16, 17, 63 Lipoic acid, and pyruvate oxidation, 4 Lipoic acid dehydrogenases, in a-keto acid oxidases, 6 Lipomucoprotein antigen, in Shigella, 15, 16

J Janus green, virus inhibitor, 58,124 Japanese beetle, blue disease of, 214 Junonia coenia, virus of, 209

K Kappa factor, in Paramecium, 91 a-Keto acid oxidases, in mammals, 6 Ketoadipic acid, virus inhibitor, 111 a-Ketoglutarate, virus metabolism of, 31 Krebs cycle, in rickettsiae, 31

L Lactic acid, and bacterial lysogenization, 136

bacterial metabolism of, 277 Lactobacillus casei, effect of 5-bromouracil, 87 glucose metabolism in, 7 growth of, 104, 105 Lactobacillus delbrueckii, pyruvic oxidase of, 4 Leafhoppers, cross protection in, 263-269 against acquisition of infectivity, 263-265

effects of varying exposures, 265,266 subinoculations from plants exposed to protected leafhoppers, 2 6 6 269

nonvector species, 241-243 virus vectors, 221,253,254,271 virus multiplication in, 223-247 Leatherjacket, virus of, 212, 217 Lepidoptera, viruses of, 200-202,205-207 Lettuce, yellows virus in, 260 Leucine, in tobacco mosaic virus, 23 L-Leucine, virus inhibitor, 110,111 Leuconostoc mesenteroides, glucose metabolism in, 7 Leukosis virus, dose response to, 177,189 Lily symptomless virus, insect vector of, 244

Lipids, in viruses, 22,62

Little peach virus, 253 Liver cells, 3 Louping ill virus, hosts of, 246 inhibition of, 91,127,128 Lucerne mosaic virus, in tobacco, 53 inhibition of, 74-76, 82, 114, 118, 130 Lymantria dispar, virus of, 201 Lymantria monacha, virus of, 201,202,218 Lymphocytic choriomeningitis, inhibition of, 128 Lymphogranuloma-psittacosis viruses, 51

Lymphomatosis, 153, 154 Lysine, in bacteriophages, 25 precursors of, 7 virus inhibitor, 110, 111, 113

M MM virus, treatment of, 129,135 Macropsis trimaculata, virus vector, 253 Macrosteles fascifrons, virus vector, 225, 226, 253

Magnesium, and virus growth, 120,122 Magnesium nitrate, and virus growth, 122, 123

Maize streak virus, cross protection in maize, 270 insect vectors, 232,243 Malachite green, virus inhibitor, 125 Malacosoma neustria, virus of, 211, 213 Malate, virus metabolism of, 31 Malic acid, and bacterial lysogenization, 136 Malonic acid, and bacterial lysogenization, 136 Mammals, enzymes of, 6 Manganese, and phage growth, 120, 121 Mannitol and streptomycin resistance factors, linkage of, 301-303 Mannose, 35 Marblebone in chickens, 153 Mealybugs, virus vectors, 221,270 Melolonlha vulgaris, virus disease of, 214 Meningococcus, transformations in, 277

SUBJECT INDEX

329

Meningo-pneumonitis virus, 5-hydroxy- Mites, virus vectors, 221, 244 methylcytosine in, 37 Monkeys, poliomyelitis therapy in, 117, nucleic acid, 21 133 Mepacrine, see Atebrin Mouse fibroblasts, virus culture medium, 2-Mercapto-4-methylamino-pyrimidine, 59 virus inhibitor, 84 Mouse mammary carcinoma, 150 6-Mercaptopurine, growth inhibitor, 105 Mucinase, in virus, 29,30,35 Merfene, and virus growth, 121 Mucoprotein, composition of, 35 Metal ions, and enzyme activity, 165,166 Mumps virus, inhibition of, 90, 91, 110, effects on viruses, 119-124 111,116,118, 127,136 Methionine, and virus inhibition, 110- Mustards, and tobacco mosaic virus, 13 112, 114 Mycobacteria, chemical components of, 6 in tobacco mosaic virus, 23 Mycolic acids, in mycobacteria, 6 Methionine sulfoxide, virus inhibitor, Myeloblast,osis, virus of, 150, 153-155 109, 110 Myxoma virus, dose-response, 173, 174, Methionine sulfoxime, virus inhibitor, 176, 182, 190 113 Myzus persicae, virus vector, 244 DL-Methoxinine, virus inhibitor, 111, 112 N p-Methoxyphenylmethanesulfonic acid, virus inhibitor, 113,141 Napthaleneacetic acid, virus inhibitor, 131, 132 2-Methyladenine, 103 6-Methylaminopurine, in DNA, 99, 105, Napthoxyacetic acid, virus inhibitor, 131 108 2-Methyl-4-chlorophenoxyacetic acid, DL-1-Napthylalanine, virus inhibitor, 111 virus inhibitor, 131 Natada nararia, virus of, 207-209 Methylcholanthrene, host response to, Nematus oljaciens, virus of, 203 176,177 Neodiprion serlijer, virus of, 203 5-Methylcytosine, in microorganisms, 6, Neoplasia, virus of, 149 27, 28, 36, 41, 65, 66, 68, 69, 71 Nephotettix apicalis, virus transmission, 228 in nucleic acid, 98 2-Methyl -4,6-dihydroxy - 1,3,5 triazine, Netropsin, in virus therapy, 129 virus inhibitor, 85 Nettle grub, virus of, 207-209 Methylene blue, virus inhibitor, 125 Neurolymphomatosis, i n chickens, 1531-Methylguanine, 105 155 7-Methylguanine, 100 Neurospora, lysine derivation in, 7 niacin derivation in, 7 2-Methyl-5-hydroxymethyl-6-aminopyrimidine, 42 Newcastle disease virus, form of, 162 inhibition of, 127 5-Methyl-4-hydroxypyrimidine, in L. casei growth, 105 New York aster yellows, 254-256 2-Methylnapthoquinone, virus inhibitor, Niacin, derived from indole, 7 116 tryptophan precursor of, 7 2-Methylthioadenine, virus inhibitor, 81 Nicotiana glutinosa, virus lesions in, 53, 4-Methyl-2-t hiouracil, virus inhibitor, 84 54, 84 5-Methyltryptophan, and bacterial lysovirus treatment in, 74,75,85, 113, 118, genizat,ion, 136, 137 123,125,128 virus inhibitor, 108, 109, 110 Nicotiana rustica, yellows virus, 257-260, 263,265-269 6-Methyltryptophan, virus inhibitor, 113 5-Methyl-uracil, see Thymine Nicotinamide, 138, 139 and virus inhibition, 118 1-Meth ylxanthi ne, 105 Mice, poliomyelitis therapy in, 117, 132 Nicotinamide ribonucleotide, 138 virus diseases in, 125, 127-129, 133, 135 Nicotinamide riboside, 138 I

330

BUBJECT INDEX

Nicotinic acid, and virus inhibition, 116, 118 cozymase synthesis, 138,139 Nile blue A, virus inhibitor, 124 Nitroakridin 3582, virus inhibitor, 127 2-Nitro-6-sminoacridine, virus inhibitor, 127 6-p-Nitrobenzamidouracil, virus inhibitor, 92 3-Nitro-6,7-dimethoxy-9-(2-hydroxy-3diethylamino-propylamino)-acridine, 127 3-Nitro-6,7-dimethoxy-9(2-phenyl-4diethylamino-butylamino) -acridine, 127 Nitrogen, of T phages, 14,15 Nitrogen excretion, and water balance, 4 5-Nitrothenaldehyde thiosemi-carbazone, virus inhibitor, 134 5-Nitrouracil, virus inhibitor, 85,92, 105 Nocardia formica, antibiotic from, 130 D-Norbiotin, virus inhibitor, 116 DL-Norleucine, and virus inhibition, 110, 113 Normal particles, and viruses, 18, 19 Nucleic acid, see also DNA and RNA Nucleic acids, incorporation of analogues into, 92-95 in virus multiplication, 62-65 Nucleosides, in viruses, 66 Nucleoside diphosphates, in virus nucleic acids, 93 Nucleotides, in viruses, 66, 69, 71, 72 0

Omnivorous looper, virus of, 207,209 Organic acids, and virus multiplication, 119,121,123,124 Ornithine, virus inhibitor, 110,111 Orotic acid, in nucleic acid, 98 Osteopetrosis, in chickens, 153,154 Oxadiaaolo-pyrimidines, effects on plant viruses, 81 Oxalacetate, virus metabolism of, 31 Oxalate, and virus multiplication, 120 Oxythiamine, virus inhibitor, 116

P Panaxia dowiinula, virus of, 201,218 Pantothenate, in tobacco mosaic virus, 29

Pantothenic acid, and virus inhibition, 116 Pantoyltaurine, virus inhibitor, 116 Papain, and phage protein, 17 Papilloma virus, see Rabbit papilloma Paramecium, Kappa factor of, 91 Pea enation mosaic virus, insect vector of, 244 Peach rosette virus, cross protection, 263 Peach X disease, chemotherapy of, 122 Peach yellows virus, 253 Peas, “purple” and “blue” metabolites in, 79,80 Pea mosaic virus, treatment of, 74 Peas, virus diseases of, 74 Penicillin, 5 in virus assay, 231 virus inhibitor, 129,130 Penicillium funiculosum, antibiotic from, 129 Penicillium stoloniferum , antibiotic from, 129 2,4,5,6,7-Pentamethylbeozimidazole, virua inhibitor, 117 Pentamidine, virus inhibitor, 135 Pepsin, and vaccinia virus, 170 Perchloric acid, in DNA analysis, 36,37, 94,95 Peridroma margaritosa, virus of, 209 5-Phenoxythiouracils, virus inhibitors, 58, 105, 134, 136 Phenylacetic acid, virus inhibitor, 132 Phenylalanine, and virus inhibition, 111113 2-Pheny14,6-dihydroxy-lI 3,5-triazine, virus inhibitor, 85 p-Phenylenediamine, and vaccinia virus, 30 Phenyl mercury borate, and virus growth, 121 Phenylpropionic acid, virus inhibitor, 132 2-u-Phenylpropyl-4,6-dihydroxy-l,3,5triazine, virus inhibitor, 85 Phenylvaleric acid, virus inhibitor, 132 Phlogophora meticulosa, virus of, 202,206, 216, 218 Phospliagens, in muscle, 4 Phosphatase, and virus nucleic acid, 13, 39 in vaccinia virus, 30

SUBJECT INDEX

Phosphate, in virus nucleic acid, 65, 66, 68,69.72 Phosphatides, in mycobacteria, 6 Phosphogluconate pathway, of glucose metabolism, 7 Phosphorus, in plant viruses,l2 in tobacco mosaic virus, 29 in virus nucleic acids, 93 of T phages, 14,15,39,40 Phycobilins pigment, in blue-green algae, 7 Phycocyanins, in algae, 7 Phycoerythrins, in algae, 7 Pieris rapae, virus of, 209-211 Pine looper caterpillar, virus of, 208,214 Pine sawfly virus, 27 Plant cells, 3 Plant viruses, biological properties of, 3 characteristics of, 11, 12 effects of metal ions and chelating agents, 121-123 effects of purine analogues, 7482 effects of pyrimidine analogues, 82-85 form and composition, 12-14, 21, 22 hydration of, 12,14 incomplete, 60 inhibition of, 113,114,118,119,121-123, 125, 129, 130, 141 metabolites in, 29 multiplication in insect vectors, 221247 multiplication of, 10, 32 nucleic acids of, 12-14,23-25,28,62 phosphorus content of, 12 titration of, 189,190 transmission of, 32 Plaque counting technique, and animal viruses, 1 Plasmodesmata, and virus transfer, 32 Plastids, and virus synthesis, 9 Pneumococcal DNA, fractionation of, 279, 280 Pneumococcus, and DNA preparations, 17 transformations in, 277, 282, 283, 286, 288, 294-297, 299-301, 303 Poisson’s binomial theorem, 174 Poisson curve, 173,177, 183-185, 189, 190 Poisson distribution, 174, 182, 183, 185, 187,189,190 Poliomyelitis virus, antibody titer, 240

331

chemotherapy testing, 57,59 composition of, 10 inhibition of, 91, 111-113, 116,117, 127, 129, 132, 133 nucleic acids of, 19 pathology of, 160 size and shape, 18 tissue culture of, 1 Poliomyelitis virus vaccines, production of, 1

Polyhedra, virus particles, 199, 200, 215218 Polyhedral bodies, composition of, 200 Polyhedral diseases, 199 Polyhedral silkworm virus, amino acids of, 25, 27 Polyhedral viruses, see also Insect viruses Polyhedral virus, 5-hydroxymethyl-cytosine content, 37 Polyhedroses, 20,21 Polymerized benzoid sulfonic acids, virus inhibitors, 136 Polysaccharides, in viruses, 22, 62 Papilla japonica, virus disease of, 214 Potassium ion, and virus growth, 122 Potato leafroll virus, insect vector of, 244 Potato virus B, ribonucleic acid composition, 25 Potato X virus, characteristics of, 11 nucleic acids of, 24,62 treatment of, 74,82,125,131 variants of, 252 Potato virus XL, ribonucleic acid composition, 25 Potato virus Y, treatment of, 74, 82, 131 Potato yellow dwarf virus, in insects, 233 hydration of, 12,13 particles in, 12 treatment of, 129 Potatoes, virus chemotherapy studies, 56 Probits, in virus study, 175,177,180,181, 183,184,186 Proflavine, virus inhibitor, 61, 126, 127, 141 Proline, in tobacco mosaic virus, 23 Propamidine, virus inhibitor, 135 Prophage, 73, 120,137 4-Propyl-2-thiouraci1, virus inhibitor, 84 Proteins, in viruses, 22,6143 in virus multiplication, 62-65

332

SUBJECT INDEX

Proteus OX-19, enzymes of, 6 Proteus vulgaris, pyruvic oxidase of, 4 Pseudococcus njalensk, virus vector, 270 Psittacosis virus, enzymes in, 30, 31 form and composition, 21,22 Psittacosis-lymphogranuloma viruses, treat,ment of, 128 Pterolocera amplicornis, virus of, 201 Purine analogues, effects on animal viruses, 90-92 effects on plant viruses, 74-82 Purine bases, in virus nucleic acid, 65, 66, 68, 69, 71 virus inhibitors, 52, 54, 55 Purple bacterium, 3 Pyridine-3-sulfonic acid, virus inhibitor, 116, 118 Pyridoxal, 138 and virus inhibition, 116 Pyridoxal phosphate, 138 Pyridoxine, and virus inhibition, 115 Pyrimidine analogues, effects on viruses, 82-85, 96 Pyrimidine bases, in virus nucleic acid, 65,66,68,69,71 virus inhibitors, 52,55 Pyrimidine-N-deoxyriboside,38 Pyrimidine of thiamine, 42 Pyrophosphate, and virus DNA release, 16 Pyruvate, and virus growth, 123 bacterial oxidation of, 4 virus metabolism of, 31 Pyruvic acid, and bacterial lysogenization, 136 and oxygen uptake, 31 and virus inhibition, 115 Pyruvic oxidase, 4

Q Quick decline, in citrus, 140

R R.P.L. 12 tumor, 154 Rabbit papilloma virus, 150-152, 154, 157 host-virus relation, 174, 176, 182-186, 189-191 lipid in, 22,160 malignancy of, 151-153, 191 nucleic acids of, 19

physical and chemical properties, 158160. purification of, 155,156, 158, 161 sedimentation constant, 159, 162 size and shape, 18,19 titration of, 173, 229 Range paralysis, in chickens, 153 Red clover, virus diseases of, 254 Reducing sugar, 35 R h antigens, 300 a-Ribazole, virus inhibitor, 118 Riboflavin, and virus inhibition, 118 in tobacco mosaic virus, 29 Ribofuranoside, virus inhibitor, 118 Ribofuranosylbenzimidazole, virus inhibitor, 118 Ribonuclease, and virus nucleic acid, 13 in amino acid analyses, 25,106 8-azaguanine in, 93,94,96 Ribonucleic acid, and virus inhibition, 110, 124 composition of, 65, 105, 106 in bacterial cells, 279,280,306 incorporation of pyrimidine analogues, 98, 99 in viruses, 11-15, 19, 21-24, 39, 40,62, 65-68,73,84,86,92, 107 nucleotide composition, 24,25,28 Ribopyranoside, virus inhibitor, 118 D-Ribose, in virus nucleic acid, 65, 106 Rice stunt virus, multiplication in arthropod vectors, 223-225, 233,240, 245 Rickettsia burneti, nucleic acids of, 22, 27, 28 Rickettsia prowazeki, metabolism of, 31 nucleic acid of 21, 2 3 28 Rickettsia rickettsii, metabolism of, 31,32 nucleic acids of, 28 Rickettsiae, enzymes in, 30, 31 form and cornposition, 21, 22 nucleic acids of, 27, 28 Rift Valley fever, inhibition of, 128, 135 Rothamsted tobacco necrosis virus, inhibition of, 82 Rous tumor, 152 Rous tumor virus, and normal particles, 18 host cell, 153 host-virus relation, 174, 182, 186-188

SUBJECT INDEX

size and shape, 18 titration of, 173 Rumez acetosa, virus tumors from, 125, 131 Russian spring and summer encephalitis virus, inhibition of, 90, 91 Rutenol, virus inhibitor, 127 Rye, in virus assay, 227, 230-232

S Sabulodes caberta, virus of, 207, 209 Sacbrood, of honeybees, 210, 214 Safranine 0, virus inhibitor, 125 Saframine-pyrazolon-sulfonamide,virus inhibitor, 124 St. Louis encephalitis virus, inhibition of, 92, 128, 132, 134 Salmonella typhimurium, A, phage in, 14, 15, 136 inhibition of, 141 Salmonella typhosa, inhibition of, 134 Sarcoma 13 leukosis, antigen of, 167 Sarcoma 537, of mice, 97 Sawflies, virus of, 200,203 Scarlet tiger moth larva, virus of, 201, 218 Semliki Forest virus, treatment of, 129 Serine, 6, 41 in bacteriophages, 25 L-Serine, virus inhibitor, 110, 111 Serum albumin, and bacterial transformation, 288, 289 Severe etch virus, in solanaceous plants, 9 Shigella, transformations in, 277 Sheep, louping ill in, 128 Shigella sonnei, lipomucoprotein antigen in, 15, 16 Silkworm jaundice, virus of, 200 Silkworm virus, 27, 214, 217, 218 Silkworm virus disease, 20 Skatole, 110 Smallpox viruses, 240 Snake venom 5’ nucleotidase, 95 Sodium ion, and virus growth, 122 Sodium monofluoroacetate, virus inhibitor, 124, 132, 133 Sodium succinate, and virus growth, 123 Sodium thioglycolate, and virus growth, 123

333

Solanaceous plants, and severe etch virus, 9 Southern bean mosaic virus, characteristics of, 11 Spotted wilt, insect vector of, 244 Spruce budworm virus, 27 Sr agent, and bacterial transformations, 280, 283, 285-290, 292, 293, 301-303 Staphylococcus, inhibition of, 130, 131 Sterols, in bacteria, 6 Stilbamidine, virus inhibitor, 135 Stone-fruit trees, virus diseases of, 222 Strawberry yellow etch virus, insect vector of, 244 Streptococcus faecalis, 104 effect of 5-bromouracil, 87 enzymes of, 138 pyruvate oxidation, 4 Slreptomyces lavandulae, antibiotic from, 128 Streptomyces netropsis, antibiotic from, 129 Streptomycin, and bacterial lysogenization, 137 bacterial resistance to, 278, 280, 281, 283-285, 287, 289 virus inhibitor, 129, 130, 131 Streptomycin resistance and mannitol factors, linkage of, 301-303 Streptothricin, virus inhibitor, 130 k-Strophanthin, host response to, 175 Subtilin, virus inhibitor, 130 Succinate, virus metabolism of, 31 Succinic acid, and bacterial lysogenization, 136 and oxygen uptake, 31 effect on virus lesions, 121, 122, 123 Sugar, in virus nucleic acid, 65,66,68,69, 71, 72 Sugar beets, curly top virus, 239 Sugar beets, yellows disease of, 140 Sugar beet latent virus, 73 Sulfanilamide, 5 in E. coli culture, 87-89, 101, 114 virus inhibitor, 114, 115 Sulfanilamide resistance factor, 301, 303 Sulfonamides, and virus, 31 Sulfonic acids, virus inhibitors, 112, 113 Swine influenza virus, sedimentation constant, 162 treatment of, 129

334

SUBJECT INDEX

Swollen shoot disease, in cocoa, 140 Sym-triazines, 104 Systemic necrosis, in tobacco, 140

T Taurine, virus inhibitor, 113 Terramycin, in virus therapy, 128-131 1,2,4,6-Tetrazaindene, virus inhibitor,

80 Tetrahymena, inhibition of, 96 Theiler’s GD VII virus, inhibition of, 90, 92, 110,111, 117 Theobromine, and 8-azaguanine activity, 76 Theophylline, and 8-azaguanine activity, 76 Thiaminase, 42 Thiamine, and virus inhibition, 118, 118, 119 @S-Thienylalanine,virus inhibitor, 111, 114 a-Thioadenine, growth inhibitor, 105 2-ThiocytosineJ virus inhibitor, 84 Thioglycollic acid, and phage production, 121 Thioguanine, virus inhibitor, 81 2-Thio-4-n-propyluraci1, 105 2-Thioorotic acid, virus inhibitor, 84 Thiopurines, effects on plant viruses, 81 Thiopyrimidines, plant virus inhibitor, 82-84 Thiosemicarbazones, virus inhibitors, 133, 134 2-Thiothymine, virus inhibitor, 84, 105 2-Thiouracil, 97, 105 in nucleic acid, 98, 107 Thiouracil, virus inhibitor, 54-56, 82-85, 135, 140, 141 Thiouridylic acid, 83, 84 2-Thioxanthine, virus inhibitor, 81 Thlaapi arvense, yellows virus in, 258 Threonine, in tobacco mosaic virus, 64 virus inhibitor, 113 Thrips, virus vectors, 221,244 Thymidine, 104 in B. coli, 101, 102 Thymine, and 8-azaguanine activity, 76 and virus inhibition, 83, 84,87,114,115 antagonists of, 104, 106 in B. coli, 99, 101, 102, 105 in coliphsges, 15, 26, 42, 88-90, 101

in viruses, 12, 27, 28, 39, 40,65, 66, 68, 69, 71, 72, 89, 94, 98 Thymus DNA, hydrolysis of , 37 Thymus nucleic acid, and bacterial transformations, 284 Thysanoptera, virus vectors, 221 Tiger moths, virus of, 205 Tipula paludosa, virus of, 203-205, 210213, 215,216, 218 Tipulidae, virus of, 200 Tobacco, virus diseases of, 74,75,270,271 Tobacco mosaic virus, 270 amino acids of, 23 composition of, 9 characteristics of, 11 estimation of, 52, 53, 229 hydration of, 12,13 I8 fraction, 64 inclusion bodies, 73 inhibition of, 5456, 74-76, 78, 80-85, 95, 96, 102, 113, 114, 118, 119, 123, 125, 128-132, 140 local lesions, 53, 64 metabolites in, 29 multiplication of, 32, 247 mutants of, 252 nucleic acid of, 13, 14, 23, 24, 62, 67, 68, 76, 77, 93 penetration of, 35 protein fraction from, 64 reaction with mustards, 13 ribonucleic acid composition, 25 threonine residues in, 64 Tobacco necrosis virus, characteristics of, 11 effect of organic acids and metal ions, 121-123 nucleic acid of, 62 Tobacco ring spot virus, characteristics of, 11 inhibition of, 130 Tomato, virus diseases of, 74, 252, 271 yellows virus in, 260, 261, 271 Tomato bushy stunt virus, characteristics of, 11 hydration of, 12 nucleic acids of, 24, 62 ribonucleic acid composition, 25 Tomato mosaic virus, ribonucleic acid composition, 25

SUBJECT INDEX

335

Tomato spotted wilt virus, insect vecU tors of, 244 Ultraviolet light, and bacterial lysogenitreatment of, 74 sation, 136 Triazoles, virus inhibitors, 75, 77 and phage development, 120, 121 2,6,8-Trichloro-purine , virus inhibitor, and virus-host relation, 8 90 Uracil, and 8-asaguanine activity, 76 4,5,6-Trichloro-l-j3-~-ribofuranosylben- and virus inhibition, 83, 84, 91 aimidasole, virus inhibitor, 118 antagonists of, 105 Triethylcholine, virus inhibitor, 116 in coliphages, 15, 40, 42 Trifolium incatnatum, in virus study, 233 in virus nucleic acids, 25,65,68,77,83, 2,3,5-Triiodobensoic acid, virus inhibi94, 98 tor, 131 Urea, and intermolecular interactions, Tropaeolum virus 11, treatment of, 74 281 Trypaflavin, virus inhibitor, 128 effect on phage protein, 16, 17 Trypan red, virus inhibitor, 125 y - (3,4-Ureylene cyclohexyl) -butyric Trypsin, and virus culture, 59 acid, virus inhibitor, 116 Tryptophan, and growth inhibition, 109, Uric acid, and 8-asaguanine activity, 76 110, 113 Uridine, and virus inhibition, 92 niacin precursor, 7 Uridine diphosphate, 107 Tubercle bacillus, inhibition of, 134 Uridylic acid, 83, 93 Tumor viruses, 14%193 V and hemin, 30 physical and chemical properties, 158- Vaccinia virus, biology of, 3 chemotherapy testing, 57, 58 173 deoxyribose nucleic acid content of, erythromyeloblastosis virus, 160-173 10,28 papilloma virus, 158-160 development of, 29,217 purification, 155-158 dose-response, 173, 174, 176, 177, 181, erythromyeloblastosis virus, 166-158 182, 185, 189, 190 papilloma virus, 155, 156 elementary bodies of, 19 rabbit papillomatosis and avian leuenzymes in, 30, 32 kosis, 151-155 form and composition, 21 titration of, 173-191 growth of, 10, 240 virus infectivity and host response, 5-hydroxymethylcytosine content , 37 173-191 inhibition of, 90-92,111,112,116,117, Turnip yellow mosaic virus, and 8-asa123, 127, 129, 133-135 lipids in, 22 guanine, 78, 79 metabolites in, 30 characteristics of, 11 nucleic acid of, 20, 22 hydration of , 12 penetration of, 34 inhibition of, 95 pepsin treatment, 170 nucleic acid of, 13,24,25,28,62,63,67, titration of, 173, 188 77 DL-Valine, and virus inhibition, 110, 114, treatment of, 74, 76 116 ribonucleic acid composition, 25 Vector, definition of, 221 Typhoid bacteriophage, effects of metal Vinca rosea, cross protection experiions, 119 ments, 262, 263, 265-269 Typhus vaccine, 21 virus inactivation in, 227 Tyrosine, and virus inhibition, 111 yellows virus in, 253,256-258,260 Tyrosine decarboxylase, 138 Virology, and comparative biochemistry, Tyrothricin, virus inhibitor, 129 1-43

336

SUBJECT INDEX

biochemical variability, 2-8 fine structure of viral constituents, 22-28 metabolism of 5-hydroxymethyl cytosine in T-even phages, 35-43 relation between virus composition and entrance and release, 32-35 virus form and composition, 11-22 animal virus, 17-22 bacterial virus, 14-17 plant virus, 12-14 virus-infected cells, parasitic patterns in, 8-11 virus metabolic equipment, 29-32 Virology, and genetic determination, 23, 24 Virus chemotherapy, 49-142 diamidines, 136 2,2,4-dinitrophenol, 133 effect of acridine derivatives, 126-128 animal viruses, 127, 128 bacteriophage, 126, 127 plant viruses, 128 effect of antibiotics, 128-131 animal viruses, 128, 129 bacteriophage, 130, 131 plant viruses, 129, 130 effect of dyestuffs, 124, 125 animal viruses, 124, 125 bacteriophage, 125 plant viruses, 126 effects of metal ions and chelating agents, 114-124 animal viruses, 123, 124 bacteriophage, 119-121 plant viruses, 121-123 effect of plant growth regulators, 131, 132 effects of purine and pyrimidine analogues, 74-108 animal viruses, 90-92 bacteriophages, 85-90 2-azaadenine, 86 8-aaadenine, 86 8-aaguanine, 85 8-azahypoxanthine, 86 &halogenated pyrimidines, 87-90 incorporation into bacterial DNAs, 87,88 incorporation into phage nucleic acids, 88, 89

properties of phage containing, 89,90 demonstration of analogue incorporation into nucleic acids, 92-95 design of new analogues, 102-106 ring modifications, 103, 104 substituted purines and pyrimidines, 104-106 sugar component, 106, 106 factors affecting incorporation, 98102 host metabolism, 101,102 conversion of analogue to inactive material, 102 conversion of analogues to nucleosides and nucleotides, 102 rate of synthesis of nucleic acid, 101, 102 route of incorporation, 101 structural features of analogues, 98-101 growth inhibition by, 95-97 absence of competitive inhibition, 96,97 delay in inhibition, 96 inhibition and incorporation, 96 virus infectivity, 95,96 plant viruses, 74-85 2-azapurines, 80 8-azapurines and related compounds, 74-80 indene derivatives, 80 oxadiazolo-pyrimidines, 81 substituted pyridines, 86 substituted pyrimidines, 84,85 substituted triazines, 85 thiopurines, 81 thiopyrimidines, 82-84 incorporation phenomena and antimetabolite action, 137-140 inhibition by amino acids, 108-117 animal viruses, 110-113 bacteriophage, 108.110 plant viruses, 113, 114 inhibition by vitamin analogues and related compounds, 114-119 animal viruses, 115-118 bscteriophages, 114-1 15 plant viruses, 118, 119

SUBJECT INDEX

337

lysogenic bacteriophage-host relationanimal viruses, 64, 65 ship, 136, 137 bacteriophages, 62, 63 phenoxythiouracils, 134, 135 plant viruses, 63, 64 polymerized benzoid sulfonic acids, 136 virus composition, 61, 62 sodium monofluoroacetate, 132, 133 Viruses, see also animal viruses, bacterial testing compounds for virus inhibition, viruses, bacteriophage, coliphage, 52-61 insect viruses, plant viruses and speagainst animal viruses, 57-59 cific viruses mode of action of compounds, 58, carcinogens, 149, 150 59 definition of, 61 measurement of, 59 host-induced modifications, 276 screening tests, 57, 58 lysogenic systems, 3,8,10 against bacteriophages, 59-61 oxygen uptake catalysis, 31 intracellular phage development, plaque counting techniques, 1 61 ribose nucleic acid content, 11-13 latent period and burst size, 60,61 Virus-infected cells, parasitic patterns plaque count, 60 in, 8-11 production of immature or non- Virus infection, and glucose metabolism, infective phage, 61 7 screening tests, 59 Virus multiplication, and nucleic acid against plant viruses, 52-57 metabolism, 52, 62-65 application of compounds, 54-56 Virus nucleic acid, 9 choice of virus and host, 56 Vitamin B1z, 103,107, 115,117 estimation of virus, 52, 53 and virus inhibition, 118 mode of action, 56,57 Vitamin K, and virus inhibition, 116 reduction in number of local le- Vitamins, virus inhibitors, 114-119 sions, 53, 54 W virus tumors, 54 thiosemicarbazones, 133, 134 Watson-Crick structure for DNA, 68-73, 99, 281, 303, 305, 306 virus structure and multiplication, 6173 West Nile encephalitis virus, inhibition lysogenic virus-host relationship, 73 of, 91 nucleic acid structure, 65-73 Western equine encephalomyelitis, inbranching of chains, 66 hibition of, 128, 129 components of, 65,66 Wheat streak mosaic, insect vectors of, 244 linkage between nucleotides, 66,67 proportions of bases in nucleic White flies, virus vectors, 221, 244 acids, 68 Wound tumor virus, multiplication in relation t o reduplicating genetic insect vectors, 233, 235237,238, 241, material, 68 245 genetic implications WatsonX Crick structure, 72, 73 RNA structure, 73 X-ray, and phage development, 121 reduplication of DNA, 69-72 inactivating transforming factors, 281, Watson-Crick DNA structure, 282 68-71 Xanthene dyes, virus inhibitors, 124 repeating unit, 66 Xanthine, and 8-azaguanine activity, 78, size of nucleic acids, 6 M 8 102 and virus inhibition, 91, 114 persistence of virus infections, 73 protein and nucleic acid in virus in E. coli culture, 87 multiplication, 62-65 Xanthopterin, guanase inhibitor. 102

338

SUBJECT INDlX

Xanthine oxidase, inhibition of, 102 Xerosin, virus inhibitor, 129

Y Yeast, cytosine deaminase, 41 nucleic acid of, 24 and virus inhibition, 91 nucleotide composition, 26 Yellow fever, antibody titer, 240 hosts of, 246 Yellows-type viruses, cross protection between, 261-2'72 cross protection reaction, 251,252 experiments, 253,264 experiments with Vinca rosea, 262, 263

identification of California aater yellows, 254-282 in leafhoppere, 263-269 interference between viruses, 252, 253

mechanism of cross protection, 252 natural and acquired immunity, 251 Z

Zinc chloride, virus inhibitor, 122 Zinc ions, and virus growth, 120,122, 123 Zinc nitrate, virus inhibitor, 123 Zinc sulfate, virus inhibitor, 122, 123 Zinnia elegans, yellows virus in, 264,255, 280-262

ADVANCES IN VIRUS RESEARCH VOLUME I HERMAN T . EPSTEIN. The Properties of Bacteriophages . . . . . . . . . . . . . 1 C. W . BENNETT. Interactions between Viruses and Virus Strains . . . . 40 L . M . BLACK. Transmission of Plant Viruses by Cicadellids . . . . . . . . . . 69 G . H . BERQOLD. Insect Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 WERNERHENLE. Multiplication of Influenza Virus in the Entodermal Cells of the Allantois of the Chick Embryo . . . . . . . . . . . . . 142 JOSEPH L . MELNICK, Poliomyelitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 D . GORDON SHARP, Purification and Properties of Animal Viruses . . . . 277 ROYMARKHAM, Virus Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 VOLUME I1 FRANCIS 0. HOLMES,Inheritance of Resistance to Viral Diseases in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 F. C . BAWDEN, Inhibitors and Plant Viruses . . . . . . . . . . . . . . . . . . . . . . 31 PREBEN VON MAGNUS, Incomplete Forms of Influenza Virus . . . . . . . . 59 W. WILBURACKERMANN AND THOMAS FRANCIS, JR., Characteristics of Viral Development in Isolated Tissues ..................... 81 ERNEST POLLARD, The Action of Ionizing Radiation on Viruses . . . . . . 109 C. A. KNIGHT,The Chemical Constitution of Viruses . . . . . . . . . . . . . . . 153 ROBLEY C. WILLIAMS, Electron Microscopy of Viruses . . . . . . . . . . . . . 183 MAXA. LAUFFER AND IRWIN J . BENDET,The Hydration of Viruses . . 241

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