THE UNIVERSITY OF MICHIGAN INDUSTRY PROGRAM OF THE COLLEGE OF ENGINEERING HELA, MONKEY KIDNEY, AND MONKEY TESTICULAR TISSUE CULTURES Normand Robert Goulet A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the University of Michigan 1958 June, 1958 IP-297

EnaprN Doctoral Committee: Professor Gordon C. Brown, Chairman Assistant Professor Kenneth W. Cochran Professor Thomas Francis, Jr. Resident Lecturer Pearl L. Kendrick Assistant Professor Donald J. Merchant

ACKNOWLEDGMENTS I wish to express my sincere gratitude to Dr. G. C. Brown for his unusually effective and heuristic teaching, to Dr. T. Francis, Jr. for making available the facilities of the Department of Epidemiology, to Dr. K. W. Cochran for his invaluable advice and guidance, to Dr. P. L. Kendrick who imparted the value of critical evaluation, and to Dr. D. J. Merchant for his interest in my work. Appreciation and thanks are also due to Mr. J. H. Schieble for his cooperation, time, and friendship. To the Faculty and students of the Department of Epidemiology I give thanks for the warm welcome, advice, and assistance extended to a "visiting" New Englander. I heartily thank the personnel of the Industry Program of the College of Engineering for their part in the preparation of this manuscript. And without fear of being accused of extreme sentimentality, I would like to express my everlasting gratitude to my parents and to my wife who have borne the brunt of my scholarly endeavors for lo these many years. -ii

TABLE OF CONTENTS Page ACKNOWLEDGMENTS...................................... ii LIST OF TABLES................................................ v LIST OF FIGURES............................................... vii INTRODUCTION................................................... 1 HISTORICAL SURVEY.............................................. 5 MATERIALS AND METHODS.................................... 13 Tissue Culture Techniques......................... 13 HeLa (Human Cervical Carcinoma-Gey)............. 13 Monkey Kidney Epithelium....**,....O.... 13 Monkey Testical Fibroblasts.......~... 13 Antibiotics..................................... 14 Drugs................................................ 14 Selection and Preparation................ 1,l Toxicity Criteria............................. 15 Viruses.............................................. 15 Viruses Employed............................ 15 Preparation and Identification of Virus Pools... 16 Titration of Viruses................ 17 Evaluation of Virus Inhibition....................... 8 18 EXPERIMENTAL RESULTS.......................................... 21 Studies on the Toxicity of Compounds................. 21 Cinephotomicrographs...................... 21 Effect of M-8X50 on the Continuous Growth of HeLa Cells................................ 22 Comparative Inhibitory Studies in HeLa Cultures...... 24 Differential Inhibition of Poliomyelitis and Coxsackie Viruses............................... 24 Effect of Benzimidazole and Cysteic Acid on the Growth Curves of Poliomyelitis and Coxsackie viruses............................... 25 iii

TABLE OF CONTENTS CONT'D Page Selected Inhibitors............................. 26 Analytical Centrifugation...................... 28 Effect of Removing the Antibiotic from Treated Cultures................................ 30 Effect on Adsorption and Attachment of Virus.... 31 Effect of Medium........3....................... 32 Attempts to Recover Antigen from Treated Cultures................................. 33 Differential Inhibition in Monkey Kidney Epithelial Cultures............................................. 35 Effectiveness of HeLa System Virus Inhibitors... 35 Effectiveness of other Experimental Compounds... 36 Studies on the Activity of the Plant Extract M-2............................................ 37 Extent of ECHO-4 and 11 Inhibition...@.......... 37 IN VITRO Effect Upon ECHO-4 and 11 Viruses...... 38 Concurrent Inhibition and Growth of Viruses..... 39 Effect on the Antibody Response of Mice Inoculated With ECHO-4 and 11 Viruses................ 41 Inhibition of Poliomyelitis and Coxsackie Viruses by M-8450 in Monkey Testicular Cultures................. 42 DISCUSSION......................................... 43 SUMMARY AND CONCLUSIONS.o.............................. 47 BIBLIOGRAPHY.................................................. 75 iv

LIST OF TABLES Table Page 1 Hypothetical Results of Cytopathology of Virus Control and Drug-Treated Cultures.................... 49 2 Compounds Assayed for Inhibition of One Hundred Tissue Culture Doses of Poliomyelitis, Coxsackie, and Vaccinia Viruses in HeLa Cultures.............. 50 3 Comparative Toxicity of Compounds in Tissue Culture............................................. 56 4 Effect of M-8450 on the Multiplication of HeLa Cells in Continuous Culture.......................... 58 5 Compounds Demonstrating Inhibitory Activity Against the Viruses of Poliomyelitis and Coxsackie Grown in HeLa Cultures....................................... 59 6 Therapeutic Indices of Selected Virus Inhibitors in HeLa Culture s......................... 62 7 Therapeutic Indices of Fractions of Ultra-centrifuged M-8450 Tested Against One Hundred Tissue Culture Doese of Poliomyelitis Virus in HeLa Cultures........ 62 8 Protection of M-8450 Treated and Washed HeLa Cultures from the Cytopathology of Poliomyelitis and Coxsackie Viruses.............................................. 63 Effect of 1/10 M-8450 on Poliomyelitis Virus Adsorption and Attachment onto HeL. Cultures............... 64 10 Effect of Medium Upon the Inhibition by M-8X50 of One Hundred Tissue Culture Doses of Poliomyelitis Virus in HeLa Cells.................5......... 11 Effect of Eagle's Medium Upon the Inhibition by M-8450 of Poliomyelitis Virus in HeLa~ Cultures...... 65 12 Cytopathology and Virus Isolations from M-8+50Treated Virus-Infected HeLa Cultures.......,.,,..... 66 13 Antigenicity of M-81450- Treated, PoliomyelitisInfected Culture Fluids in Mice......6.o..6....... 6 v

LIST OF TABLES CONT'D Table Page 14 Lowest Inhibitory Concentration (Mg/ml) of Compounds Assayed for Inhibition of One Hundred Tissue Culture Doses of Virus in Monkey Kidney Cultures............. 7 15 Therapeutic Indices of Selected Virus Inhibitors in Monkey Kidney Cultures............................. 68 16 Reciprocal of the Lowest Inhibitory Dilution of Plant Extracts Assayed for Virus Inhibitory Properties in Monkey Kidney Cultures..................... 69 17 Inhibition of Cytopathology of M-2 Treated Monkey Kidney Cultures Infected with ECHO-4 and 11 Viruses.. 70 18 Inhibition of Cytopathology of Monkey Kidney Cultures Treated with M-2 at Time of ECHO-4 Infection and One Hour Later......,.......7....................... 71 19 Therapeutic Indices for M-2 Treated Monkey Kidney Cultures...o.......o..o... o..... 71 20 Inhibition of Cytopabhology of M-2 Treated Monkey Kidney Cultures after Double Virus Exposures......... 72 cl Cytopathology of M-2 Treated Monkey Kidney Cultures after Double Virus Exposures....................... 73 22 Rtciprocal of the Antibody Titers of Sera Obtained from Mice Inoculated with ECHO-4 and 11 Viruses and Treated with M-2 Fraction..................... 74 vi

LIST OF FIGURES Figure Page 1 Effect of Benzimidazole (0.1 mg/ml) and Cysteic Acid (1.0 mg/ml) on Poliomyelitis (Mahoney) Virus Grown in HeLa Cultures......................... 60 2 Effect of Benzimidazole (O.1 mg/ml) and Cysteic Acid (1.0 mg/ml) on Coxsackie (Conn. -5) Virus Grown in HeLa Cultures...................... 61 vii

I. INTRODUCTION Two points of prime concern to tnose studying virus inhibitors are (1) the role of "tissue toxicity" and (2) the relative ineffectiveness, in vivo, of inhibitors selected on the basis of tissue culture studies (Pearson, 1953; Burnet, 1955; Matthews and Smith, 1955; Tamm, 1956b Hurst and Hull, 1956). Successful chemoprophylaxis and chemotherapy appear to be a matter of relative toxicity; i.e., the host and parasite do not seem to represent two entirely different and independent metabolic economies, and the problem is to find a substance which, as used, is not harmful to the host but which will be toxic to those portions of the host's metabolism which are essential for virus replication. The experimental demonstration in tissue culture of latent or masked viral infections (Ackermann and Kurtz, 1955; Ginsberg and Boyer, 1956; Karzon and Barron, 1957) demonstrates the propagation of virus without observable damaging effects to the host cultures, and suggests that the host and parasite may not always utilize an identical metabolic economy, and that the different economies should provide sites vulnerable to the antiviral agents without undue damage to the host. Several theories can be expounded to explain the relative ineffectiveness of tissue culture inhibitors in the chemotherapy and chemoprophylaxis of experimental animals infected with the smaller viruses, but it must be noted that studies of the inhibition of Rickettsiae and the larger viruses in tissue culture correlate quite well with in vivo experiments. The high degree of specificity required of -1

-2a substance in order that it may prevent the production of virus nucleoprotein without interfering with the normal nucleoprotein synthesis of the host, suggests that some of the reported tissue culture inhibitors of the smaller viruses might be "false positives" and should not be expected to inhibit virus in the intact animal. It appears reasonable to assume that following the multiplication process the released virus is a product of a replicating mechanism which is peculiar for each virus entity capable of growth within that host. This highly specific mechanism suggests that the reactions contributing to one virus entity differ from those contributing to other virus entities at some point in the replication process. It should be possible to inhibit one of these viruses without affecting other replicating mechanisms. Differential virus inhibition within a particular tissue culture would suggest a direct relationship between an inhibitor and one of the viruses. Since viruses can re-direct the synthesizing abilities of more than one host to produce specific virus, it should be possible to demonstrate the same direct virus-inhibitor relationship in another host, though possibly to a varying degree. Demonstration of such specific action would appear to rule out the possibility of drug-incited host injury. A second assumption, the concurrent inhibition and growth, respectively, of two distinct viruses within the same tissue, also appears feasible. That is to say, that within a particular tissue culture, it should be possible to inhibit a virus with a compound while simultaneously allowing another virus not so affected to complete

-3its entire infectious cycle. This manipulation, demonstrated herein, presents conclusive evidence that the inhibition of virus multiplication can be specific and may occur without undue damage either to the host tissue or even to other reactions leading to the reproduction of' another virus.

II. HISTORICAL SURVEY Prior to 1955 positive achievements in the field of chemotherapy of infectious diseases were, by modern standards, relatively modest. Domagk's discovery of the sulfonamides radically altered the situation. Even more important than their intrinsic therapeutic value, was their effect in directing chemical effort and research into this new and hitherto inadequately explored channel. When a year or two later, Florey was able to develop an interesting laboratory phenomenon with Penicillium notatum into an outstandingly active therapeutic remedy of the lowest conceivable general toxicity, he instigated an additional vast amount of research into chemotherapeutic substances produced by living organisms. The result was an impressive degree of control over many bacterial infections achieved in a total period of little more than a single decade. The chemotherapy of virus diseases is a more recalcitrant problem. With the exception of the largest viruses no practical means of influencing virus diseases by chemotherapy yet exists. This is perhaps not unexpected when we reflect on the differences between the bacteria, larger viruses, and the smaller viruses. The problem of chemotherapy of the diseases caused by the smaller viruses is that of selectively influencing the processes near to those essential to the life of the host without endangering its safety -- an objective once widely held to be unattainable even in the case of the relatively'5 -

-6independent pathogenic bacteria, but one now realized for these and for the Rickettsiae and the larger viruses. As facts accumulate, it would not seem unreasonable to harbour a restrained optimism regarding the future. Although no substance of practical value against the virus diseases of man or domestic animals has yet emerged, inhibition of viru growth in particular complexes of host cell and virus is known. These instances have been recently reviewed by Matthews and Smith (1955) and by Hurst and Hull (1956). Although there are few, if any, reports on the inhibition of the Coxsackie or Enteric Cytopathogenic Human Orphan (ECHO) viruses, the literature is not wanting with respect to the inhibition of the viruses of poliomyetitis and vaccinia. Several aliphatic compounds have been found to inhibit the aforementioned viruses. Thus, Thompson (1947) has shown that iodoacetate and sodium malonate prevented the multiplication oi vaccinia in chick embryonic tissue. Benzaldehyde thiosemicarbazone, its pacetamide, p-amino-p-methoxy, p-propoxy, and p-ethylsulfonyl analogues were reported as active inhibitors of vaccinia virus grown in chick embryonic tissue and in the embryonated egg. There was also a marked degree of protection with mice inoculated intracranially with the virus when benzaldehyde thiosemicarbazone was fed to the mice in diet form (Thompson, et al, 1951; Hamre, Bernstein, and Donovick, 1950; Hamre, Brownlee, and Donovick, 1951).

-7An ester, glycerylmonoacetate, was found to inhibit the growth of poliomyelitis virus in monkey testicular tissue (Brown, 1952). A number of aromatic compounds have been found to interfere with the growth of viruses in their tissue culture medium. Phenols and their oxidation products, the quinones, and related compounds have been tested for their effect on poliomyelitis virus in roller-tube cultures of monkey testicular tissue. Only fifteen of these compounds were capable of inhibiting virus-induced degeneration of fibroblasts over a wide range of concentrations. Most of the inhibitors were equal in their effect against Types 1, 2, or 3 poliomyelitis virus. When tested against the three types of virus in monkey kidney cultures, some of the compounds were less effective against Type-1 virus or were not effective at all (Kramer, Robbins, and Smith, 1955; Smith, Knox, and Hollinshead, 1955; Hollinshead and Smith, 1956). The growth of vaccinia virus in chick embryonic tissue has also been found to be inhibited by 2,4-dinitrophenol. (Thompson, 1946 and 1947). Many of the heterocyclic compounds which have been used in the inhibition of virus growth in tissue culture are structurally related to the vitamins, components of nucleic acid, and other naturally occurring substances. Knox, Robbins, and Smith (1957) studied the possible antiviral effects of one hundred and sixty-three analogues

-8of the pyrimidine moieties oi nucleic acid against Type-2 poliomyelitis virus in cultures of monkey testicle. Forty-three were found to be inhibitory and were further tested against the three types of poliomyelitis viruses in monkey kidney cultures. Only eight of these compounds, the pyrimidine analogues thiouracil, 5-methyl thiouracil, hypoxanthine,, 4, 5, 6-tetramino pyrimidine, o-thio-4phenyl-6-oxypyrimidine, and the barbiturates pentobarbital and butethal were found to inhibit in both monkey testicular and monkey kidney cultures. Four of these compounds had a direct in vitro virucidal action. When the chemicals were tested for their effects on the three types of poliomyelitis viruses, no apparent differences in virus susceptibility were noted in the kidney cultures, while in the testicular cultures higher concentrations were sometimes required to inhibit Type-1 virus. All the inhibitors gave evidence of complete suppression of growth for a period up to twelve days in monkey testicular cultures, while in the kidney tissue, growth of virus was observed after a delay of twenty-four hours and then approached the titers obtained in the untreated cultures. Analogues of other basic components of nucleic acid, the purines, interfere with virus proliferation. Thus, 2, 6 - diaminopurine will inhibit poliomyelitis virus grown in monkey testicular cultures

-9(Brown, 1952). Gifford, Robertson, and Syverton (1954) reported that 2,6-diaminopurine did not inhibit poliomyelitis virus except at concentrations which were markedly inhibitory to the respiration of the host cells. That the mechanism of action of 2,6-diaminopurine is not clear is further demonstrated by the following observations: its inhibition of Russian spring-summer encephalitis virus in cultures of mouse and chick embryonic cells, Crocker Sarcoma 180, and Carcinoma 1025 (Friend, 1951); its injurious effects in Sarcoma 180 tissue cultues at concentrations which have no effect on the same tumor in vivo (Stock et al, 1950; Biesele et al, 1951); its prolongation of the survival time oI mice infected with transplantable leukemia (Burchenal et al, 19-9); its serious injurious actions on the hematopoietic system of the dog (Phillips and Thiersch, 1949), swine (Cartwright et al, 1950), and of the chick embryo (Karnofsky et al, 1949), and finally its reduction of the effective content of Kappa particles in Paramecium aurelia (Stock et al, 1951). A number of cQ-haloacyl derivatives of various 5-aminopyrimidines inhibited the proliferation of vaccinia in chick embryonic tissue (Thompson et al, 1949 a,b). Halogenated analogues, for example 2,6,8-trichloropurine, were inhibitory against vaccinia virus (Thompson et al, 1950). These authors pointed out that the compounds studied were all toxic for other experimental animals, and the concentrations required to produce injury were of the same order of magnitude as those required to inhibit the multiplication of vaccinia virus. Since mice were not protected, the authors concluded that the action of the compounds was directed

-10primarily toward the embryonic tissue substrate. Vitamin analogues have been used in attempts to interfere with virus proliferation. Thus, benzimidazole has proved effective as an inhibitor of poliomyelitis virus (Brown, 1952; cf. Gifford, Robertson, and Syverton, 1954) and vaccinia virus (Thompson, 1946 and 1947), and homobiotin inhibited the growth of Lansing poliomyelitis virus in monkey testicular tissue (Brown, 1952). The enzyme inhibitors atabrine and proflavine were effective against vaccinia virus in chick embryonic tissue (Thompson, 1946). Briody and Stannard (1951) demonstrated that proflavine inhibited the growth of vaccinia on the chorio-allantoid membrane whereas the growth of mumps and Newcastle disease viruses was unaffected. The dosage of proflavine used was close to the toxic level, and the authors' dilemma appeared to be the explanation for the differential activity with respect to toxicity for the host. Proflavine is also effective against poliomyelitis virus (Brown, 1952). Natural and synthetic amino acids and analogues of amino acids have been tested against viruses in tissue culture. Thus, the importance of methionine has been demonstrated in the biosynthesis of the Lansing strain of poliomyelitis virus in human brain and monkey testicular cultures by inhibition with ethionine (Brown and Ackermann, 1951; Brown, 1952; cf. Gifford, Robertson, and Syverton, 1954). B-2-Thienylalanine has also been found to be inhibitory to poliomyelitis virus (Brown, 1952) and to vaccinia virus (Thompson and Wilkin, 1948). Inhibition of vaccinia virus in chick embryonic tissue cultures has also been demonstrated by the use of aC-amino-methane sulfonic acid, ct-amino-isobutane sulfonic acid,

-11 - and a-amino-phenyl-methane sulfonic acid (Thompson, 1947). Inhibition of the multiplication of poliomyelitis virus in HeLa cultures was demonstrated with 4-fluorophenylalanine (Ackermann, Rabson, and Kurtz, 1954). An interesting feature of this inhibition was the apparent dissociation o' virus multiplication and cytopathogenic effect, with destruction of the host cells apparently unimpeded although virus multiplication was prevented. Fluorophenylalanine also inhibited the multiplication oi the host cells; however, the efiect upon the uninfected cell was reversible after three days, as indicated by the viability after treatment. A few studies have been done with carbohydrate inhibitors. Robertson, Gif'ord, and Syverton (1956) presented evidence that exposure of HeLa cells to autoclaved solutions of D-ribose prior to infection with poliomyelitis virus reduced the rate of virus synthesis, but not the final yield. The inhibitory effect was correlated with the furiural content; furfural also inhibited virus when added twenty hours after virus inoculation. Ginsberg and Horsfall (1949) showed that the replication of mumps virus and pneumonia virus of mice (PVM) in the embryonated egg was inhibited by the injection of small quantities of the type-specific capsular polysaccharide of Freidlanders bacillus whereas large quantities of the polysaccharide will not affect the multiplication oi' influenza A or B or Newcastle disease viruses. That the three viruses were capable of unrestricted multiplication whereas multiplication of either PVM or mumps was inhibited suggested that the two groups of viruses might require different host metabolic systems for their replication.

-12Little antibiotic activity has been reported against the smaller viruses in tissue culture. Phagopedin sigma was found to inhibit the growth of poliomyelitis in monkey testicular cultures (Brown, 1952). Hull and Lavelle (1953 and 1954) reported on the inhibition of the cytopathogenic effect of the poliomyelitis viruses in tissue cultured monkey testicular cells with a penicillium mold filtrate designated as M-8450. The authors concluded that the antibiotic exerted its effects upon the cells rather than on the virus, as pretreatment of the cells was necessary, and viable virus could be demonstrated in the undamaged treated cultures as long as five days after inoculation. Inhibitory studies are yielding valuable information regarding the metabolic requirements of virus growth in cellular environments. Once again, however, but little of the accruing knowledge has yet led to advances toward an effective chemotherapy of virus diseases.

III. MATERIALS AND METHODS Tissue Culture Techniques The choice of the appropriate tissue culture has been intimately related to the availability of the source materials, technical flexibility and adaptibility, and the virus spectrum of these potential hosts. HeLa (Human Cervical Carcinoma - Gey) These cultures, originally obtained from Dr. J. T. Syverton, were grown and maintained in as close an approximation as possible to the methods described in detail by Syverton, Scherer, and Elwood (1954). Monkey Kidney Epithelium The procedures employed for the growth and maintenance of these cultures were essentially similar to the quantitative methods reported by Youngner (1954 a,b), modified by the use of a lactalbumin medium introduced by Melnick (1955 b), and further modified by the substitution of horse serum for the calf serum component. Buifering of the mediums was accomplished by reinforcement of the bicarbonate and phosphate with O.O0M "Tris" (tris hydroxymethyl aminomethane). Plaque Cultures —The techniques employed in the use of plaques were essentially those reported by Dulbecco (1952) and Hsiung and Melnick (1955) modified by the incorporation of the desired concentration of drugs into the agar overlay as well as into the liquid phase. Monkey Testicle Fibroblasts The monkey testicular cultures were prepared by the plasmaclot roller-tube methods described in detail by Robbins, Weller, and -13 -

-14Enders (1952). In addition, several experiments were performed with trypsinized (0.5s trypsin, Ditco, 1:250) cells grown directly on the glass surface without the aid of a supporting plasma-clot. Antibiotics Penicillin, streptomycin, and mycostatin were incorporated into all tissue culture media. Penicillin (500,000 units) and streptomycin (one-half gram) were dissolved in 50 ml. Hanks balanced salt solution or Earle's saline. Mycostatin (500,000 units) was suspended in the above solution which was distributed into 1 ml. aliquots and frozen until ready for use. One ml. of the antibiotic mixture was used per 100 ml. of tissue culture medium, giving a final concentration of 100 units penicilli~i, 0.1 mg. streptomycin, and 100 units mycostatin. Drugs Selection and Preparation The compounds used in the study were either metabolic analogues reported in the literature as inhibitory to a particular virus, experimental drugs and antibiotics which had in some cases been defined, or unknown compounds. Most of the drugs were prepared in a routine manner. If the compound_ was a powder its solubility was attempted in 1) the culture medium bathing the tissue in question, 2) sterile distilled water, 3) sterile distilled water to which AN acid or 1N base had been added, or 4) propylene glycol. The drug was then diluted so

-15 - that a final concentration of 2.5 rag/ml of culture fluid originated a two-fold dilution series. If the drug was liquid in nature, a 1/2 dilution originated the two-fold dilution series. Toxic levels were then determined by introducing 1 ml. of each dilution of drug into replicate tube cultures. Toxicity Criteria The compounds were tested for toxicity in each tissue culture system in which the virus inhibitory studies were to be performed. Microscopic Evidence —Certain gross cytological changes, such as distortion, pyknosis, granularity of the cytoplasm, swelling or bubbling of the cytoplasm, sloughing, or complete destruction of the cultures were the conditions associated with toxicity for a given tissue in culture. Cinephotomicrographic Evidence —A limited number of selected compounds were also employed with time-lapse photomicroscopy. The toxic properties which can be determined are associated with abnormal pinocytosis, cessation of the normal undulation of the cellular membranes, cessation of Brownian movement within the cytoplasm, or inhibition of the migratory activity of the cells. Viruses Viruses Employed Type-1 poliomyelitis virus (Mahoney) was employed throughout the study with the exception of one experiment wherein Type-2 poliomyelitis virus (MEF) was employed. Both strains of virus were obtained from the Connaught Laboratories, Canada.

A representative of the Coxsackie group of viruses, Type B-1 (Conn.-5), was also included in the study. Vaccinia virus, obtained from the Michigan Department of Health, was also employed. The study also included some of the Enteric Cytopathogenic Human Orphan (ECHO) viruses which were originally obtained from Dr. J. L. Melnick, Yale University. The viruses employed were ECHO-1 through 9, and ECHO-11 through 14. Preparation and Identification oi Virus Pools Pools of virus were prepared in the following manner: one ml. or undiluted virus was inoculated into bottle cultures of the appropriate tissue which contained 9.0 ml. of' nutrient fluid. The bottles were incubated at 57~C and observed daily for cytopathology. Upon destruction of most of the cultured cells, the fluids were pooled and centrifuged in 40 ml. conical centrifuge tubes at 2000 rpm for 15 minutes. The supernatant fluids were distributed in 1 ml. aliquots into glass tubes. The tubes were then stored at about minus 20"C. Aseptic technique was used throughout and the pools were tested for bacterial contamination by inoculation into thiolycolate and beef infusion broths. Each virus type (except vaccinia) was identified by employing hyperimmune sera representative of the types of virus employed in the tests. For control purposes normal sera from the appropriate animal species were employed. In vitro neutralization was employed for type determinations.

-17Hyperimmune antipoliomyelitis serum was obtained from Dr. H. Wenner, University of Kansas. Hyperimmune antiCoxsackie serum was obtained by inoculating mice intraperitoneally with infected HeLa tissue culture fluid with bi-weekly injections for a period of four weeks. The mice were then bled, the serum pooled, and the neutralizing ability tested in vitro. Hyperimmune antiECHO sera were obtained by extensive intravenous and subcutaneous inoculations of young rabbits with high titered monkey kidney tissue culture antigens. The in vitro neutralizing titer of the rabbit sera was determined periodically. A titer of 1/1024 to 1/2048 was considered adequate. Typing of the vaccinia pools was not attempted since the unique cytopathology caused by this virus served as the indicator of the virus used. Titration of Viruses Virus was estimated by determining the limiting dilution which could initiate infection in 50% of the tube cultures inoculated. Serial half-log dilutions of virus were prepared and 0.1 ml. aliquots of each dilution were added to 4 tube cultures. The cultures were incubated at 357C and examined for cytological changes characteristic of infection each day for seven days. The 50% endpoints were calculated by the method of Reed and Muench (1938). The dilutions recorded were always the initial rather than the final dilutions. Since all the cultures contained 1 ml. of fluid, all titers were expressed as the number of tissue culture infectious doses (TCID50) per ml. of sample.

In the earlier experiments, 10, 50, and 100 TCID50 were employed in the studies, but later all tests were done with virus doses ranging from 52 to 320 TCID50. Since this variation represents only one-half log over or under 100, the dosage was recorded as approximately 100 TCID50. Results obtained with virus dosages above or below this range were discarded and the tests were repeated. Evaluation of Virus Inhibition The inhibitory phenomena observed included any delay in the time of appearance of cytopathology, partial inhibition of cytopathology, or complete inhibition of characteristic virus-induced changes with no evidence of virus infection. Preliminary scoring of the inhibition was done on a plus or minus basis, these designations representing the degree of involvement of the cultures; when all the cells were destroyed the plus designation was given. Later, the effectiveness of the compounds was judged by the value obtained in calculating the therapeutic index (Kramer et al, 1955). The therapeutic index is a ratio of the maximum tolerated dosage of the compound to the minimal virus suppressive dose: Maximum Tolerated Dose = Therapeutic Index. Minimal Virus Suppressive Dose This ratio reflects any observation of inhibition which occurs only on the first day in which the virus control cultures show complete degeneration. As an example, Table 1 represents the scoring of the cytopathology obtained with virus-infected control and drug-treated

-19virus-infected cultures. It can be seen from the table that on the third day the virus control cultures were completely degenerated. On that same day, the drug-treated virus-infected cultures were protected at 1/320 but not at the 1/640 drug dilution. The 50% endpoint calculated by the method of Reed and Muench (1938) is 1/442, and this value represents the minimal virus suppressive dose. The previously determined maximum tolerated dose was 1/10. The therapeutic index would then be equal to 44.2 (1/10 divided by 1/442). The reproducibility of the evaluation was demonstrated by the repeated testing of an inhibitory compound, the antibiotic M-8450, used as a control drug in approximately twenty separate tests. The reliability of the evaluation was further demonstrated by the use of Dulbecco's plaque technique in the inhibitory studies. The therapeutic indices so obtained were essentially identical to those obtained in the tube culture studies.

IV. EXPERIMENTAL RESULTS Studies on the Toxicity of Compounds Preliminary to the present study, experiments were carried out to determine the toxicity oi the experimental compounds for the cultured tissue (n.b., Materials and Methods). One hundred and twenty compounds have been studied in either HeLa, monkey kidney, or monkey testicular cultures. The highest non-toxic concentrations for the compounds tested in HeLa cultures can be found in Table 2. Comparative studies have been completed for thirty-five of the aforementioned compounds which were selected mainly because of some known virus inhibitory potency. It can be seen from the data given in Table 3 that thirteen of the thirty-five compounds had different toxic levels when inoculated into various tissue cultures, and that these levels sometimes varied by as large a factor as ten or twenty-five. It would appear that the compounds must be assayed for toxicity in each tissue before the drug-tissue complex can be employed in virus inhibitory studies. Cinephotomicrographs Two compounds, the antibiotic M-8450 and methionine sulfoximine, selected because of their effectiveness as virus inhibitors in HeLa cultures were employed at their maximum non-toxic concentrations in time-lapse photographic studies. These cinephotomicrographs appeared to be similar in every respect (n.b., Materials and Methods) to those obtained when normal -21

-22HeLa cultures were employed as controls. These experiments provided additional evidence that the maximum tolerated dose of M-8450 and methionine sulfoximine did not alter the morphological appearance of the HeLa cultures. Effect of M-8450 on the Continuous Growth of HeLa Cells Subsequent inhibitory results suggested that concentrations of M-8450 near the maximum tolerated dose might still be toxic to the cultures in a manner not readily apparent morphologically. It was of interest to determine whether the antibiotic affected the continuous growth of HeLa cells. Accordingly, HeLa cells were cultured (n.b., Materials and Methods) in continuous contact with various concentrations of M-8450 diluted in the normal growth medium. The cultures were discarded after five weekly passages, or earlier if the results were not significantly different from the control cultures. It can be seen from Table 4 that HeLa cells exposed to 1/40, 1/80, and 1/160 dilutions of M-8450 multiplied at approximately the same rate as did the control cultures. These cultures behaved normally as indicated by their morphology, pH changes, and general cultural appearance. However, the cells exposed to a dilution of 1/10 M-8450 appeared to multiply at a slower rate. Although displaying normal pH changes, these cells were noticeably more rounded, were not in a continuous sheet, and had fewer processes than seen in the normal cultures. In a subsequent experiment, the growth of cells in a medium containing a 1/20 dilution of the antibiotic closely approximated the behavior of the control cultures.

-23The results obtained with 1/10 M-8450 treated cells are considered significant. If we ascribe 100% growth to the average foldincrease for the control cultures, the antibiotic inhibited 60% of the growth in the 1/10 treated cultures. It would appear from the above results that a 1/10 dilution of the antibiotic either reduces the rate of multiplication of the HeLa cells and/or selects out the M-8450 resistant cells. An attempt was made to determine whether a 1/10 dilution of M-8450 was selecting resistant cells. If one assumes such a selection, it then appears reasonable to assume that the resistant cells may demonstrate an altered virus susceptibility. Accordingly, normal and 1/10 M-8450-treated cells from the third passaged cultures were seeded in tubes and allowed to multiply in the presence of growth medium devoid of M-8X50. These tube cultures were then tested for their virus susceptibility by subjecting them to parallel titrations of poliomyelitis virus (Type-l, Mahoney). The results indicated no significant difference in virus titer. -5.2 The titer in the control cells was 10, while the titer in the M-8450 treated cells was 10'. As can be seen in Table 4 the M-8450 treated cultures also regained their normal reproductive activity when replaced in the normal growth medium. Either the progenies of M-8450 treated cultures are as fully susceptible to the growth and multiplication of virus as are the normal cells or they have reverted from insusceptible to susceptible in a single generation, a premise which seems highly unlikely. It can be concluded then, that a 1/10 dilution of M-8450 does not bring about selection of resistant cells but does reduce the rate of

-24multiplication of' HeLa cells by virtue of its toxicity for these cells. Comparative Inhibitory Studies in HeLa Cultures Upon completion of the toxic endpoint determinations, the compounds were used in attempts to demonstrate differential virus inhibitory activity. Differential Inhibition oI Poliomyelitis and Coxsackie Viruses Twenty-nine compounds were screened for their ability to inhibit the cytopathology caused by the viruses of poliomyelitis and Coxsackie. In this pilot study, the maximum non-toxic concentration of the compounds was added to three pairs of replicate tube cultures simultaneously with virus inocula containing 10, 50, and 100 tissue culture doses. As seen in Table 5 twenty-six of the compounds tested inhibited the cytopathology attributable to poliomyelitis or Coxsackie virus infection. Six of these inhibitors showed a differential activity in that they inhibited only one of the viruses. An isocytosine, 2-amino4-hydroxy-5,6-dimethylpyrimidine, and two compounds supplied by Abbott Laboratories, dimethyl glycine and 2-thio-4,6-diaminopyrimidine, inhibited the virus of' poliomyelitis only. Hydroxylamine hydrochloride and 2-amino4-chloro-5-methyl-6-phenylpyrimidine appeared to selectively inhibit Coxsackie but not poliomyelitis virus. The antibiotic M-8450, though it inhibited both viruses, showed differential activity in that it completely inhibited the cytopathology caused by poliomyelitis but merely delayed that caused by the Coxsackie virus.

-25 - Methionine sulfoximine and the benzimidazoles seemed to be the most active of the remaining compounds. Just how much inhibition resulted with any of these compounds could not be ascertained at this time since a delay in the time of cytopathology was the only index of inhibition used. Effect of Benzimidazole and Cysteic Acid on the Growth Curves of Poliomyelitis and Coxsackie Viruses In order to determine the extent and duration of the inhibition produced by the aforementioned group of compounds, the effect of two representative compounds upon the growth curves of Type-1 poliomyelitis and Coxsackie viruses was studied. Benzimidazole and cysteic acid were selected primarily because of their effectiveness reported herein and by others (Thompson, 1946 and 1947; Brown, 1952). In the first experiment, three parallel bottle cultures of seven-day old HeLa cells were treated with normal maintenance containing 0.1 mg/ml of benzimidazole, and maintenance containing 1.0 mg/ml of cysteic acid. One hundred tissue culture doses of poliomyelitis virus per ml of culture fluid were immediately added to each bottle. The bottles were incubated at 37~C for two hours. The cells were then washed five times (to remove virus) with normal maintenance medium and the appropriate maintenance solutions were reintroduced into the bottles. Samples of the last wash were saved for titration of virus content. The bottles were further incubated at 37~C and samples were taken from each bottle at two hour intervals. The volume was equated after each sampling by replenishment with the appropriate maintenance solution. The experiment

-26was terminated when there was maximum degeneration in the control culture. The above experiment was subsequently repeated against Coxsackie virus. The samples taken in both experiments were titrated (n.b., Materials and Methods) and the results are presented graphically in Figures 1 and 2. It can be seen from these growth curves that the control virus cannot be detected before eight hours. There is a slight delay in the appearance of virus in the drug-treated cultures. Maximum titers are attained at forty-six hours in the case of poliomyelitis infection, and at thirty-eight hours with Coxsackie virus. The drugs retard the appearance of infective virus during the entire growth cycle and the final titers in the drug-treated cultures differ from the control by as much as one to two logs. Selected Inhibitors Since the use of compounds at or near the maximum tolerated dose does not indicate relative degrees of effectiveness, and since the time involved in growth curve studies of large numbers of compounds is prohibitive, an extension of the former technique was employed in an attempt to quantitate the relative efficacy of the compounds. The HeLa cultures were now routinely pre-treated with the compounds for 24 hours at 37'C prior to infection with one hundred tissue culture doses of virus. Table 2 lists the minimal suppressive concentrations for the viruses of poliomyelitis, Coxsackie, and vaccinia. The inhibition resulting with each compound-virus complex was then

-27evaluated by the use of the therapeutic index (n.b., Materials and Methods). Through these techniques certain compounds have emerged as having potential significance, and they are listed in Table 6. The antibiotic M-8450 completely inhibits the virus of poliomyelitis with no evidence of virus infection appearing in dilutions of the antibiotic up to and including 1/160. Coxsackie virus infection, however, is merely delayed for twenty-four to seventy-two hours with all inhibitory dilutions. The antibiotic does not seem to have a significant effect on vaceinia infection. Two other antibiotics, Helenine and M5-11496, were selected because of their known inhibitory activity against poliomyelitis infection in monkeys and mice (Cochran and Francis, 1956, Cochran, 1956). It can be seen that Helenine was active against poliomyelitis virus only if the cultures had been pre-treated for forty-eight hours. Similar activity against Coxsackie virus was obtained with pre-treatment Oi either twenty-four or forty-eight hours. M5-11496 appeared to have some activity against the virus of poliomyelitis. Neither compound inhibited the cytopathology of vaccinia virus. Methionine sulfmximine completely inhibited the cytopathology of all three viruses at the maximum tolerated dose of 0.5 mg/ml while its effect at lower concentrations was merely dilatory. Benzaldehyde thiosemicarbazone appeared to be a more specific inhibitor of vaccinia virus than of the other two agents.

-28Diazouracil, another compound which was inhibitory for poliomyelitis in mice (Cochran, 1957), failed in repeated attempts to inhibit this virus in tissue culture. In the preliminary experiments the benzimidazole derivatives seemed to be good inhibitors when employed near the maximum tolerated dose. However, calculation of the therapeutic indices revealed only small amounts of activity against these viruses. One derivative, 5, 6-dichloro-l-D-ribosylbenzimidazole, appeared to be specific in its inhibition of poliomyelitis virus. Many of the compounds having therapeutic indices of two, four, or eight are not shown in Table 6. The author considers these low indices as probably reflecting a non-specific inhibitory phenomenon. This refinement of the data would leave but four significant inhibitors in the HeLa tissue culture system, M-8450, methionine sulfoximine, benzaldehyde thiosemicarbazone, and 5, 6-dichloro-l-D-riboSylbenzimidazole. Studies on the Activity of the Antibiotic M-8450 —Since M-8450 was the only compound to completely inhibit the cytopathology of poliomyelitis over a wide range of concentrations, it was adjudged most promising, and concurrent studies were initiated to elucidate its mode of action. Analytical Centrifugation Since the antibiotic preparation was a crude filtrate of the mold Penicillium stoloniferum, it was of interest to determine if the active principle could be separated by ultracentrifugation. The biophysical preparation of th filtrate fractions was performed by Dr. R. Hartman, Department of Epidemiology, University of

-29Michigan. Ten ml of the filtrate were subjected to a gravitational force of 150,000g in a Spinco analytical centrifuge (Model E) for ten hours. Examination of the Schlieren photographs revealed that the sedimenting component of the filtrate appeared to separate as a single boundary which was composed of two closely associated peaks. In the present experiment the filtrate was centrifuged until the material represented by the two peaks was sedimented into a single pellet. The supernatant was removed from the analytical cell and saved for virus studies. The pellet was washed with one ml of culture fluid and saved, and then reconstituted with another ml of culture fluid. A control tube containing the crude filtrate was kept at room temperature throughout the centrifugation and reconstitution. The samples were then tested for toxicity in HeLa cultures and then assayed for inhibitory activity against Type-l poliomyelitis virus. It can be seen from the indices given in Table 7 that the inhibitory principle in the filtrate was clearly associated with the pellet fraction. It also appeared that the control sample left at room temperature lost some activity. Since reconstitution of the pellet with one ml represented a ten-fold concentrate of the starting material, considerable activity was lost in the centrifugation and reconstituting processes. It was also of interest that the concentrate was no more toxic to the cultures than was the original filtrate. Since centrifugation for ten hours at 150,000g was sufficient to separate the active principle present in the crude M-8450 filtrate, it can be concluded that the inhibitory principle is probably a large molecule or is at least associated with a large molecule.

-30Effect of Removing the Antibiotic from Treated Cultures Hull and Lavelle (1953 and 1954) reported that the protection afforded monkey testicular cultures by M-8450 could not be removed by extensive washing of the cultures. An experiment was designed to confirm these findings in HeLa cells. Tube cultures were pre-treated with a 1/20 dilution of M-8450 for twenty-four hours at 37~C. The cultures were then washed five times with Hanks balanced salt solution. One ml of maintenance solution was added and one hundred tissue culture doses of either poliomyelitis or Coxsackie virus were inoculated into the tubes. Control cultures were similarly pre-treated with hyperimmune antipoliomyelitis monkey serum (1/80) and antiCoxsackie mouse serum (1/40). These cultures were also washed and infected with the appropriate viruses. Normal cultures, and M-8450 treated cultures in which the antibiotic was allowed to remain throughout the experiment, were also included as controls. The results are presented in Table 8. It can be seen that treating the cultures with a 1/20 dilution of M-8450 protected them from the cytopathology caused by poliomyelitis virus and delayed infection with the Coxsackie virus, whether the antibiotic was removed or not. Exposure to hyperimmune sera did not so protect the cultures. Since pre-treatment of the cultures was necessary and since the effect could not be removed by extensive washing, it is quite likely that the antibiotic exerted its activity by being incorporated into the cellular metabolism of the host cells, although a surface phenomenon cannot be precluded.

-31Effect on Adsorption and Attachment of Virus Further attempts to characterize the activity of M-8450 and to test the hypothesis of intracellular action of this antibiotic are presented in the following experiment. Three five-day bottle cultures of HeLa cells containing 9.0 ml of maintenance solution were incubated for twenty-four hours at 37~C. One of these bottles contained 1/10 M-8450. A fourth bottle containing normal maintenance solution (no cells) was included to determine the virus thermal inactivation factor in the experiment. Poliomyelitis (Type-l) virus having a titer of 10-6 was diluted to 10 and one ml of this dilution was added to each bottle giving a final dilution of 10-3. The bottles were returned to 370C, and one ml samples were taken from each bottle at sixty minutes, two hours and thirty minutes, and four hours after infection. The samples were then titered and the plaqueforming ability determined in monkey kidney cultures (n.b., Materials and Methods). These results are given in Table 9. It can be seen that there was a one-half log drop in the titer of the incubator control sample after four hours, probably due to thermal inactivation. The results obtained with the remaining samples represent virus that has not been inactivated nor adsorbed. No significant difference could be found between the virus controls and the M-8450 treated sample. In comparing the four hour titer of the incubator control with those of the virus controls and the M-8450 treated sample, it can be seen that approximately one log of virus was adsorbed both in the virus controls and in the M-8450 treated. This adsorption occurred within the first hour. Thereafter, the amount or rate of adsorption appeared

-32to be very low. It can be concluded then, that this lack of effect on attachment and adsorption of virus militates against a surface action by M-8450. Effect of Medium The possibility of finding a metabolite which could reverse the virus inhibitory property of M-8450 was considered. Since the active principle has not been defined chemically, modification of the inhibitory effect might be feasible by altering some component in the medium. The medium employed in this study was composed of Hanks balanced salt solution, sodium bicarbonate, glucose, horse serum, and a complex of amino acids, vitamins, and nucleic acids (n.b., Materials and Methods). The amino acid-vitamin-nucleic acid components were altered by grouping the required compounds into separate stock solutions (A through E) and increasing the volumes of each individual stock solution while correspondingly decreasing the volume of the final maintenance medium. This, in essence, increased the concentration of each component of that particular stock solution. Stock A consisted of glycine, histidine, cystine, and parenamine. Stock B consisted of glycerol, sodium pyruvate, and sodium acetate. Stock C consisted of succinic and malic acids. Stock D contained adenine sulfate, guanine, xanthine, uracil, thymine, and cytosine. Stock E consisted of monobasic potassium phosphate, thiamine, nicotinamide, calcium pantothenate, pyridoxal hydrochloride, pyridoxamine dihydrochloride, ribose, riboflavine, inositol, biotin, folic acid, and

-33 - p-amino benzoic acid. The toxic limit produced by increasing the relative volume of each individual stock solution in the final medium was determined, and the highest non-toxic combinations were then used in attempts to reverse the inhibition by M-8450. The therapeutic index for each combination is given in Table 10. It can be seen that all but stock solution B could be increased to 50, of the final medium without the appearance of toxic effects. Increasing the relative volume of any one stock solution reversed the inhibitory effect of M-8450. Eagle et al (1956) demonstrated that HeLa cells could be maintained in a medium consisting of Hanks balanced salt solution, glucose, and glutamine, and that the virus of poliomyelitis could be grown to expected titers in this medium. Similar experiments were performed and these findings confirmed. It was then of interest to see if the reciprocal relation was true; that is, whether the virus inhibition by M-8450 was greater in a simpler medium. It can be seen in Table 11 that the therapeutic index of M-8450 in Eagle's medium was approximately five times higher than that found when Syverton's maintenance was employed. Attempts to Recover Antigen from Treated Cultures It appeared pertinent to determine whether virus could be recovered from the M-8450-treated poliomyelitis infected cultures where no evidence of infection was observed, and whether virus was produced by the M-8450-treated, Coxsackie-infected cultures which did exhibit a delayed cytopathology.

-34Cultures were treated overnight at 37~C with a 1/10 dilution of M-8450. One group was infected with one hundred tissue culture doses of Type-2 poliomyelitis virus (MEF), and another group with one hundred tissue culture doses of Coxsackie virus. Twenty-four hours after infection, the fluids were removed from the cultures and frozen. Fresh drug-free maintenance medium was added to all the cultures. This process was repeated at twenty-four hour intervals until cytopathology was complete or until the end of the experiment (6th day). Virus recovery was attempted by inoculating 0.5 ml of the samples into replicate HeLa cultures. It can be seen from Table 12 that when the M-8450-treated cultures were challenged with poliomyelitis virus there was no evidence of cytopathology nor could virus be isolated from the fluid medium. However, when the M-8450-treated cultures were challenged with Coxsackie virus, the virus was detectable in the medium as early as twenty-four hours after infection and cytopathology was complete at seventy-two hours. Since an altered (non-cytopathogenic) virus might not be detected with the isolation method employed, attempts to detect incomplete or inactive virus were made. Pools were made from the M-8450-treated poliomyelitisinfected, poliomyelitis virus, and M-8450 control culture fluids. These pools were inoculated into mice by introducing three weekly inoculations (0.5 ml) of each pool intraperitoneally. The mice were bled on the fourth week, and the sera obtained were run in a tissue culture neutralization test at undiluted, 1/4, 1/8, and 1/16 increments.

-35 - The results of the neutralization test are given in Table 13. Fluids from the virus control cultures produced antibody detectable at a dilution of 1/16 when inoculated into mice, whereas the fluids from the M-8450-treated poliomyelitis infected cultures did not induce the formation of detectable antibody. These results indicate that either no detectable antibody-producing virus is produced by the virus-infected drug-treated cultures, or that if a form of incomplete virus is produced, it does not have the ability to stimulate the formation of antibody that would neutralize the original virus. It is possible that the fluids cjntain incomplete virus which does produce antibody, but that this antibody is so altered that it will not neutralize the complete form of virus. In an attempt to further investigate whether incomplete virus was produced, the fluids from the M-8450-treated poliomyelitis-infected cultures were used as antigen in a complement fixation test. The complement fixing ability of the mouse sera, obtained by inoculating these fluids into mice, were also determined. The results of the complement fixation tests were also negative. No antigen could be detected in the culture fluids, nor could complement fixing antibody be detected in the mouse antisera. Differential Inhibition in Monkey Kidney Epithelial Cultures Efiectiveness of HeLa System Virus Inhibitors Since it was necessary to determine whether certain compounds which had shown virus inhibitory activity in the HeLa culture system would act independently of a specific host tissue, they were tested against the same viruses in cultures of monkey kidney epithelial tissue. The study

-36was also expanded by including fourteen other viruses, the Enteric Cytopathogenic Human Orphan (ECHO) viruses (n.b., Materials and Methods). The maximum non-toxic and the minimal inhibitory concentrations fur the four significant HeLa system virus inhibitors are given in Table 14. The pertinent therapeutic indices tor these compounds can be found in Table 15. It can be seen from the indices in Table 15 that the antibiotic M-8450 inhibited the viruses of poliomyelitis and Coxsackie. The antibiotic did not completely protect the cultures against either virus, but merely delayed the cytopathology for twenty-four hours. Some low level activity against most of the ECHO viruses was also obtained (n.b., Table 14). Metkionine sulfoximine, a potent inhibitor of poliomyelitis, Coxsackie, and vaccinia viruses in the HeLa system, did not inhibit any of the viruses when assayed in monkey kidney cultures. Benzaldehyde thiosemicarbazone appeared to be specific in its inhibition of vaccinia virus. Another inhibitor in the HeLa system, 5,6-dichloro-l-D-ribosylbenzimidazole, failed to inhibit any of the viruses tested. Effectiveness of other Experimental Compounds Several benzimidazole derivatives, and a number of water soluble plant extracts which have been shown to have anti-tumor activity (Lucas et al, 1957) were similarly processed in monkey kidney cultures 1. Differential inhibitory activity has been demonstrated against some of the ECHO viruses. Thus, the mushroom extracts M-2 from Calvatia 1 The plant extracts were obtained from Dr. E.H. Lucas, Mich. State Univ.

-37maxima, M-4 from Boletus edulis, and a species of Cattleya orchid, M-14, inhibited some oi the ECHO viruses. It can be seen from the indices in Table 15 that the M-2 1fraction appeared to induce significant inhibition of ECHO-4,9, and 11 viruses. The M-4 fraction was slightly active against ECHO-7 and 8 viruses. The M-14 extract appeared to be specific in its action against ECHO-2 virus. It can be seen from Table 16 that these and other plant extracts did not appear to inhibit significantly any other ECHO virus, nor did they demonstrate activity against the viruses of poliomyelitis, Coxsackie, or vaccinia. Studies on the Activity of the Plant Extract M-2 Extent of ECHO-4 and 11 Inhibition - The previous studies showed that the M-2 extract was outstanding in its ability to inhibit ECHO-4 and 11 viruses. Therefore, an experiment was designed to determine 1) how much virus could be inhibited, 2) the necessity of pre-treatment and, 3) whether infection could precede drug treatment and still be influenced by this extract. Cultures were pre-treated with two-fold dilutions of M-2 for twenty-four hours at 37~C, following which 0.1 ml of various dilutions of virus were added. It can be seen from Table 17 that the M-2 fraction inhibited as much as 10,000 tissue culture doses of ECHO-4 and 3,200 tissue culture doses of ECHO-11. Dilutions of 1/120 and 1/240 oi the M-2 ftraction completely protected the cultures from the cytopathology caused by 100 tissue culture doses of' either virus, or from that caused by 1000 tissue culture doses of ECHO-4 virus.

-38To two other groups of cultures, 1/120, 1/240, 1/480, and 1/960 dilutions of M-2 were added at the time of, and one hour following, infection with ECHO-4 virus. As can be seen from Table 18 it was possible to infect the cultures at the time of drug addition or one hour prior to the addition of drug and still demonstrate complete inhibitory activity with the more concentrated dilutions of the drug. The desirability of a twenty-four hour pre-treatment rather than the later addition of M-2 was seen when the therapeutic indices for M-2 were compared (Table 19). The M-2 fraction decreased in effectiveness when 1) a virus inoculum larger than 1000 tissue culture doses was introduced or 2) when the cultures were drug-treated at the time of virus introduction or one hour after infection. IN VITRO Effect Upon ECHO-4 and 11 Viruses In considering the influence of M-2 on the course of virus infection, its action in vitro upon these viruses was studied. Three one ml portions of ECHO-4 infected tissue culture fluid were placed into separate test tubes. To one of these, one ml of M-2 (1/20'inal dilution) was added. One ml of normal maintenance solution was added to the other two tubes. One of the latter was stored at 4~C for twenty-four hours while the remaining two tubes incubated at 37~C for the same time. All three samples were then titrated for virus content (n.b., Materials and Methods). The titers of the control tubes incubated at 4~C and 37~C and of the M-2 treated sample incubated at 57~C were found to be 10-5.0, 10-5.0, and 10-4'5 respectively. The same procedure was followed for ECHO-11 virus, and

-39the titers obtained were 10 4, 10 3, and 10 35 respectively. It is clear that the maximum concentration of M-2 which was used in prior experiments has no pronounced in vitro effect upon the stability of the viruses. Hence, the effect of M-2 directly upon the virus is not a consideration in interpreting its influence on virus mulitplication. Concurrent Inhibition and Growth of Viruses Since the M-2 fraction has been shown to inhibit the cytopathology of considerable amounts of ECHO-4 and 11 viruses, and since no other virus tested is so inhibited (with the possible exception of ECHO-9), evidence of virus inhibition in the absence of any cell damage which would interfere with the multiplication of other viruses within the same tissue should now be demonstrable. Cultures were pre-treated with 1/120, 1/240, 1/480, and 1/960 dilutions of M-2 for twenty-four hours at 37'C. The cultures were then divided into five groups. Three of the groups were exposed to 100 tissue culture doses of ECHO-4 virus and two groups were exposed to 100 tissue culture doses of ECHO-11 virus. Forty-eight hours later and twenty-four hours prior to complete cytopathology of the virus controls, the fluids were removed, the cultures washed once, and fresh drug was added to all the cultures. At this time, large doses of either ECHO-4 (10,000 tissue culture doses), ECHO-11 (3200 tissue culture doses), or ECHO-1 (100,000 tissue culture doses)virus were added to one of the three groups of cultures previously exposed to ECHO-4 or 11 virus. Table 20 presents the experiment in outline form and the results obtained. It can be seen from the

results obtained with the drug control cultures in group 6 that all the dilutions of M-2 tested protected the cultares against the cytopathology of 100 tissue culture doses of either ECHO-4 or 11 viruses for 72 hours after the initial virus exposure. There was also complete protection with the lower concentrations of M-2 against a repeated exposure even when larger doses of ECHO-4 or 11 viruses were employed. However, no protection was afforded against re-exposure to a large dose of ECHO-1 virus, which had been shown in previous experiments not to be inhibited by the M-2 fraction. When smaller quantities of M-2 (1/480 and 1/960) were employed, cytopathology was prevented on the first exposure to virus, but not when larger doses of virus were added on re-exposure. In all these instances the virus which finally broke through the inhibitory mechanisms was recovered and typed, and always corresponded to the second virus. An additional experiment was designed to complement the above findings. A 1/200 dilution of M-2 was used to protect the cultures. The cultures were exposed to 100 tissue culture doses of ECHO-11 virus, and forty-eight hours later were re-exposed to 0.1 ml of a 10 dilution of ECHO-1,2,5,4,5,6,7, and 11 viruses, as well as poliomyelitis, Coxsackie, and vaccinia viruses. These viruses represented challenge doses of 100 to 100,000 tissue culture doses, depending upon the original titer of the virus pools. In this experiment the fluids were not changed prior to the second virus exposure. As can be seen in Table 21, essentially identical results were obtained. Cytopathology was prevented when the cultures were

re-exposed to ECHO-4 or 11 virus, but was complete when the cultures were re-exposed to a virus not affected by the M-2 fraction. These experiments strongly suggest that the influence of M-2 upon cellular reactions is associated with the replication of ECHO-4 and 11 viruses specifically, and does not alter the metabolic processes required for the replication of other viruses not affected by the M-2 fraction. Effect on the Antibody Response of Mice Inoculated with ECHO-S and 11 Viruses Since the ECHO viruses do not produce known characteristic disease signs in laboratory animals (Melnick, 1955a), attempts to demonstrate the efficacy of the M-2 fraction in vivo might be possible if a measurable antibody response to ECHO virus inoculation could be suppressed. In experiments not described herein, it was demonstrated that three intraperitoneal inoculations of ECHO virus were sufficient to produce detectable antibody levels in the sera of white mice. Accordingly, three weekly inoculations (0.5 ml) of undiluted monkey kidney tissue culture antigen were given to two groups of mice. One group received ECHO-4 virus and the other group received ECHO-11 virus. A 1/4 dilution of M-2, given at 0.1 ml/gm of body weight, was inoculated intraperitoneally on the day before antigen was given, at the same time as antigen inoculation, and the day following antigen inoculation. This triad of drug inoculations was repeated each time that virus was to be given, i.e., a total of three drug inoculations per week for three weeks. A group of drug-control mice received the full complement of drug inoculations without receiving antigen.

-42At the end of the fourth week all the mice were bled, and the sera diluted in two-fold increments (1/4 through 1/128) and assayed for antibody content in a monkey kidney tissue culture neutralization test. It can be seen from Table 22 that the M-2 fraction does inhibit the formation of antibody in mice inoculated with ECHO-4 or 11 viruses. Inhibition of Poliomyelitis and Coxsackie Viruses by M-8450 In Monkey Testicular Cultures Inhibition of poliomyelitis virus by M-8450 in plasma-clot roller-tube cultures of monkey testicles has been reported by Hull and Lavelle (1935 and 1954). In view of the inhibitory patterns established by this antibiotic in HeLa and monkey kidney cultures, it was of interest to determine whether the antibiotic would inhibit both poliomyelitis and Coxsackie viruses in cultures of monkey testicle grown without a supporting plasma-clot (n.b., Materials and Methods). The cultures were pre-treated for twenty-four hours at 37~C with a 1/10 and 1/20 dilution of M-8450. Virus inocula containing 1770, 177, and 17 tissue culture doses of poliomyelitis and 1000, 100, and 10 tissue culture doses of Coxsackie virus were added to the cultures, and the cultures were observed for characteristic viral pathology. Pre-treatment of the cultures with 1/10 M-8450 afforded complete protection to the cultures against the maximum quantities of both viruses employed. Complete protection was also obtained against the same quantities of poliomyelitis virus when a 1/20 dilution of the antibiotic was employed.

DISCUSSION Evidence compiled in this investigation indicated that many compounds were capable of inhibiting virus in tissue culture at or near the maximum tolerated dose. When the effectiveness of the compounds was judged by the therapeutic index, i.e., according to the ratio of the maximum tolerated dosage of the compound to the minimal virus suppressive dose, most of these inhibitors had a therapeutic index of two, four, or eight. In view of the high degree of specificity required of a compound in order that it be considered a specific virus inhibitor, it seemed logical to assume that these low indices represented inhibition of virus by virtue of toxicity which was directed against the host cells but which was not detectable morphologically. Exclusion of the compounds demonstrating therapeutic indices of two, four, or eight reduced the number of virus inhibitors to those compounds having therapeutic indices greater than eight. When the HeLa culture system was employed in the virus studies, only four compounds out of ninety-five assayed had an index greater than eight. Methionine sulfoximine appeared to inhibit all three viruses employed in these cultures (poliomyelitis, Coxsackie, and vaccinia). The antibiotic M-8450 inhibited the viruses of poliomyelitis and Coxsackie. Benzaldehyde thiosemicarbazone and 5,6-dichloro-l-ribosylbenzimidazole appeared to be differential in their action and inhibited vaccinia and poliomyelitis viruses respectively. -43 -

-44When monkey kidney cultures were employed to evaluate these four inhibitors, it was found that only M-8450 and benzaldehyde thiosemicarbazone retained their specific inhibitory patterns. Methionine sulfoximine and 5,6-dichloro-l-D-ribosylbenzimidazole did not inhibit any of the viruses employed (ECHO viruses included). The inability of compounds to act independently of a specific host tissue has also been recently reported by Knox, Robbins, and Smith (1957). The only substance tested in monkey testicular cultures, M-8450, again inhibited the poliomyelitis and Coxsackie viruses. Considerable variation was observed in the degree of inhibition when assays were attempted in the various culture systems. Thus, M-8450 completely inhibited both poliomyelitis and Coxsackie viruses in monkey testicular cultures. In the HeLa system, the antibiotic completely inhibited poliomyelitis virus but merely delayed the Coxsackie virus infection fur forty-eight to seventytwo hours. When monkey kidney cultures were employed the cytopathology of both viruses was delayed for twenty-four hours, and neither virus could be -ompletely inhibited. Although there was variation in the degree of inhibition, the significant point, however, is that two substances, M-8450 and benzaldehyde thiosemicarbazone, specifically inhibited virus in the culture systems employed. The specific action of M-8450 against poliomyelitis and Coxsackie viruses supports the in vivo experiments reported by Powell and Culbertson (1953) and by Cochran, Brown, and Francis (1954). The specificity of benzaldehyde thiosemicarbazone for vaccinia virus replication is in agreement with the reports of the effectiveness of the compound in another tissue

-45 - culture system, in chick embryos, and in mice (Hamre, Bernstein, and Donovick, 1950; Hamre, Brownlee, and Donovick, 1951; Thompson, Price, and Minton, 1951). That methionine sulfoximine, a potent inhibitor of all three viruses in the HeLa system, is tissue specific rather than virus specific is demonstrated by the unlimited growth of these same viruses in the presence of the compound in monkey kidney cultures. It is possible that methionine sulfoximine has a toxic affinity for the HeLa cell (the concept of differential tissue toxicity affinity has been experimentally demonstrated by Hsu, Robins, and Cheng in 1956). Another indication that the HeLa culture is more sensitive to the action of methionine sulfoximine is the observation that the compound is at least five times more toxic to the HeLa cultures than it is to the monkey kidney cultures. The inability of methionine sulfoximine to ultimately alter the infection of mice with poliomyelitis virus (Ainslie, 1952) can be cited as additional evidence of the non-specific behavior of methionine sulfoximine as an inhibitor of poliomyelitis virus. Another demonstration of differential inhibition of viruses was seen in monkey kidney culture assays with the ECHO agents. The extracts from the mushrooms Calvatia maxima (M-2) and Boletus edulis (M-4), and from a species of Cattleya orchid (M-14) were found to have therapeutic indices greater than eight against some but not all of the ECHO viruses. The specificity of the ECHO virus inhibition with these extracts, though not confirmed in other tissue cultures systems nor by animal steudies, appeared to be significant in that many other virus entities employed were not so

inhibited. It would be desirable to assay these extracts in at least one other tissue culture system. The specific nature of one of these plant extracts, M-2, has been conclusively demonstrated however, by the concurrent inhibition and growth, respectively, of two distinct viruses in the same monkey kidney culture. The M-2 extract has been shown to inhibit either ECHO-4 or 11 virus while allowing the completion of an entire infection cycle produced by another one of several ECHO viruses (as well as poliomyelitis Coxsackie, and vaccinia) within the same tissue. An in vivo experiment has shown that the M-2 fraction also inhibits the formation ol antibodies in mice inoculated with ECHO-4 or 11 viruses. These experiments strongly suggest that the influence of M-2 upon the cellular reactions is associated with the replication of ECHO-4 and 11 viruses, and does not alter the metabolic processes required four the replication of the other viruses employed within the same tissue. One can deduce, with less certainty however, that ECHO-4 and 11 viruses utilize similar pathways in their replicating processes since both viruses are individually inhibited by the M-2 fraction and since both viruses can be concurrently inhibited within the same culture.

SUMMARY AND CONCLUSIONS One hundred and twenty compounds were assayed either in HeLa, monkey kidney, or monkey testicular cultures for their inhibitory capacities against poliomyelitis, Coxsackie, vaccinia, and fourteen ECHO viruses. In comparing the virus inhibitory abilities of these compounds in the three tissue culture systems, it has been possible to demonstrate the difierential and specific virus inhibitory nature of several of the compounds. The demonstration of such specific action appears to rule out the influence of drug-incited host injury as an explanation for the observed inhibition. The results of the present investigation indicate the following with respect to the compounds employed: 1. The antibiotic M-8450 is a specific inhibitor of poliomyelitis and Coxsackie virus infection. The antibiotic will significantly inhibit both viruses when assayed in HeLa, monkey kidney, or monkey testicular cultures. 2. Benzaldehyde thiosemicarbazone is a specific inhibitor of vaccinia virus in HeLa and monkey kidney cultures. 5. Methionine sulfoximine, a potent inhibitor of poliomyelitis, Coxsackie, and vaccinia in HeLa cells, failed to restrict the growth of these (and also fourteen ECHO viruses) viruses when assayed in the monkey kidney culture system. 4. Although considered a significant inhibitor oi poliomyelitis virus in the HeLa cultures, 5,6-dichloro-l-D-ribosylbenzimidazole also -47

-48failed to inhibit this virus in the monkey kidney cultures. 5. Assays of plant extracts against ECHO virus infection in monkey kidney cultures demonstrated the difrerential specificity of these substances, the fraction M-2 from Calvatia maxima inhibited ECHO-4, and 11 viruses; the fraction M-4 from Boletus edulis inhibited ECHO-7 and 8 viruses; and the fraction M-14 from Cattleya inhibited ECHO-2 virus. 6. The specificity of ECHO-4 and 11 virus inhibition by the M-2 fraction from Calvatia maxima was conclusively demonstrated by the concurrent inhibition and growth, respectively, of two distinct viruses within the same tissue -- growth was accomplished by a virus which was not affected by the M-2 fraction. 7. The in vivo activity of the M-2 fraction was demonstrated by the inhibition of antibody production in mice inoculated with ECHO-4 and 11 viruses.

-49TABLE 1 HYPOTHETICAL RESULTS OF CYTOPATHOLOGY OF VIRUS CONTROL AND DRUG-TREATED CULTURES CYTOPATHOLOGY* CULTURES (Time in Days) 1 2 4 5 6 7 100 Tissue Culture Doses of - - + Virus - - + 100 Tissue Culture Doses of - - - Virus plus 1/10 Drug - - - - 100 Tissue Culture Doses ok - - - - Virus plus 1/20 Drug - --- 100 Tissue Culture Doses of - - Virus plus 1/40 Drug - - 100 Tissue Culture Doses of - Virus plus 1/80 Drug - -- 100 Tissue Culture Doses ol' - - Virus plus 1/160 Drug --- 100 Tissue Culture Doses of - - -P* -p + Virus plus 1/320 Drug - -p + 100 Tissue Culture Doses of - - + Virus plus 1/640 Drug - - + 100 Tissue Culture Doses of - - + Virus plus 1/1280 - - + ** The designation (+) is given when all the cells are off the glass, a (-) designation indicates protection of the cultures. * The designation (-p) indicates an occasional virus plaque.

-50APPENDIX TABLE 2 COMPOUNDS ASSAYED FOR INHIBITION OF ONE HUNDRED TISSUE CULTURE DOSES OF POLIOMYELITIS, COXSACKIE, AND VACCINIA VIRUSES IN HELA CULTURES HIGHEST LOWEST INHIBITORY COMPOUNDS NON-TOXIC CONCENTRATION" CONC. (mg/ml)* Polio. Cox. Vacc. Abbott #1713, — methyl-Ndinethyl indole 0.1 > 0.1 > 0.1 Abbott #3665, p-aminoacetophenone 0.1 0.05 O.05 Abbott #4462*** 0.1 0.05 0.05 Abbott 4271, dimethyl glycine 2.5 1.0 > 2.5 Abbott /6238, 2-thio-4, 6diaminopyrimidine 0.1 0.05 > 0.1 Amycetin 0.025 > 0.025 > 0.025 > 0.025 Anisomycin 0.006 > 0.006 > 0.00 6 > 0.00oo6 8-Azaguanine 0.1 > 0.1 > 0.1 > 0.1 8-Azauracil 0.625 > 0.625 > 0.625 > 0.625 1-P-D-Glucopyranosyl-2ethyl-5-methylbenzimidazole >2.5 > 2.5 > 2.5 5-Chlorobenzimidazole HC1 0.1 0.012 0.1 0.025 1-3-D-Glucopyranosyl- 5methylbenz.imidazole > 2.5 > 2.5 > 2.5 > 2.5 * Highest concentration tested was 2.5mg ml ~ All cultures pre-treated 24 hours at 37 C ~** Identity of the compLound restricted

-51TABLE 2- -Continued HIGHEST LOWEST INHIBITORY COMPOUNDS NON-TOXIC CONCENTRATION CONC. (mig/ml) Polio Cox. Vacc. 5-Methyl-2-isopropyl benzimidazole HC1 0.1 > 0.1 > 0.1 > 0.1 5,6-Dichlorobenzimidazole 0.05 > 0.05 > 0.05 > 0.05 5,6-Dichloro-l-D-ribosyl benzimidazole 0.1 0.0015 0.05 0.05 1- - D-Ribopyranosyl-5-6dimethyl benzimidazole 1.0 0.25 0.5 0.5 1,2-Deoxy-D-ribopyranosyl5,6-dimethyl benzimidazole 1.0 0.25 1.0 > 1.0 1-Ca-D-Arabopyranosyl- 5,6dimethyl benzimidazole > 2.5 1.0 1.0 Benzimidazole(Eastman Kodak) O.1 0.01 > 0.1 Benzimidazole (British Drug Houses, Ltr, London) 0.5 0.01 0.1 5,6-Dimethyl benzimidazole 0.1 > 0.1 > 0.1 > 0.1 2-Ethyl-5-methylbenzimidazoleO.1 > 0.1 > 0.1 > 0.1 2,5-Dimethyl benzimidazole 0.1 > 0.1 > 0.1 > 0.1 2-Benzimidazolethiol 0.1 0.05 0.05 5-Amino-2-benzimidazolethiol 0.1 0.05 0.05 2-Aminobenzimidazole 0.1 > 0.1 > 0.1 > 0.1 6-Methoxy-4 -nitrobenzimidazole 0.031 >0.031 > 0.031 > 0.031 4-Methoxy-6-nitrobenzimidazole 0.024 > 0.024 > 0.024 > 0.024 Benzaldehyde thiosemicarbazone 0.0057 0.0014 0.0028 0.00023 Crotoxin 1.0 > 1.0 > 1.0 > 1.0 Cysteic Acid 1.0 0.1 0.1 2.4-Dinitrophenol 0.05 > 0.05 > 0.05 > 0.05

-52TABLE 2 —Continued HIGHEST LOWEST INHIBITORY C OMPOUNDS NON-TOXIC CONCENTRATION CONC. (mng/ml) Polio. Cox. Vacc. 2,2-Diphenyl-3-hydroxy-,ropyl dicarbamate 0.1 0.05 0.05 2-n-Butyl-2-ethyl-1-3propanediol dicarbamatt 0.01 > 0.01 > 0.01 > 0.01 2,2-Diethyl-1-3-propanediol dicarbamnate 0.5 0.5. 5 2,6-Diaminopurine sulfate 0.5 O.1 0.1 Ethionine 0.5 > 0.5 > 0.5 Erythromycin 0.1 > 0.1 > 0.1 Florida(B17-FSC) 0.625 > 0.625 > 0.625 > 0.625 Florida(Al4-FSC) 0.625 > 0.078 0.078 0.156 Gallic acid 0.25 0.12 0.12 0.12 Helenine 10.0* 10.0* 80.0* 10.0* Helenine** 10.0* 80.0* 80.0* 10.0* Hydroxylamine hydrochloride 0.1 > 0.1 0.01 Hydrazine dihydrochloride 0.1 0.01 0.01 Isopropionine 1.0 0.5 > 1.0 Malononitrile 0.156 > 0.156 > 0.156 > 0.156 Me )henesin 0.1 0.01 0.01 Methionine sulfoximine 0.5 0.0017 0.0075 0.0025 Meprobamate 0.5 > 0.5 > 0.5 > 0.5 M-+850 10.0* 495.0* 422.0* 42.0* *Reciprocal of the dilution **48-hour pre-incubation

-53 - TABLE 2 —Continued HIGHEST LOWEST INHIBITORY COMPOUNDS NON-TOXIC CONCENTRATION CONC. (mg/ml) Polio Cox. Vacc. M5-11496 0.125 0.015 0.06 0.06 Neomycin 1.0 > 1.0 > 1.0 >1.0 Nepara #1245, quinolinic acid in water > 2.5 > 2.5 > 2.5 > 2.5 Nepara #1618, commercial amine fraction 0.5 > 0.5 0.25 > 0.5 Nepara #1716, butyl isonicotinate 1.0 > 1.0 > d.O 0.5 Nepara #1766, N-(n-dodecylN-methyl-N' -N' -dimethyl)ethylene diamine-monoundecylenate 0.05 > 0.05 > 0605 > 0.05 Nepara #1779, 2-nitro-4pyridinecarboxylic acid > 2.5 > 2.5 > 2.5 > 2.5 Nepara #1928, hydroquinone glucose >2.5 >2.5 >2.5 >2.5 Nepara #1931, diazouracil 0.1 0.1 0.05 0.1 Nepara #2336, bioflavonoid complex (from lemon) 1.0 > 1.0 > 1.0 > 1.0 Nepara #2416, Naringin 0.5 > 0.5 0.1 > 0.5 p-Aminophenol 0.01 >0.01 > 0.01 propylene glycol 1% 1% 1% 1i 5- Hydroxy- 1-methyl tetrazolopyrimidine > 2.5 > 2.5 > 2.5 Puromycin 0.0002 > 0.0002 > 0.0002> 0.0002 Pyridacil derivative 0.039 > 0.039 > 0.039 > 0.039 2 -Amino- 4- hydroxy- 5-methyl 6-phenyl pyrimidine 0.05 > 0.05 > 0.05 > 0.05

_54TABLE 2 —Continued ~HIGHEST LOWEST INHIBITORY COMPOUNDS NON-TOXIC CONCENTRATION CONC. (mg/ml) Polio Cox. Vacc. 2-Amino- 4-hydroxy- 5-propyl6-methyl pyrimidine 0.5 > 0.5 > 0.5 > 0.5 2-Amino-4-hydroxy-5- (c-methyl isobutane)-6-methyl pyrimidine 0.01 > 0.01 > 0.01 > 0.01 2, 4-Amino-5-phenyl-6-methyl pyrimidine 0.1 0.1 O.1 > 0.1 2-Amino-4-methoxy-5-methyl-6phenyl pyrimidine 0.05 > 0.05 > 0.05 > 0.05 2-Amina-4-chloro-5-methyl-6phenyl pyrimidine 0.01 > 0.01 0.01 > 0.01 2, 4-Amino-5-methyl-6phenyl pyrimidine 0.1 > 0.1 > 0.1 2-Thio-4-hydroxy-5-methyl-6phenyl pyrimidine 0.1 > 0.1 > 0.1 > 0.1 2, 4- Hydroxy-5-methyl-6phenyl pyrimidine 0.5 > 0.5 > 0.5 > 0.5 2-Amino-4-hydroxy-5-propyl-6phenyl pyrimidine 0.05 > 0.05 > 0.05 > 0.05 2-Amino-4-hydroxy-5-n-butane6-phenyl pyrimidine 0.05 > 0.05 > 0.05 > 0.05 2- Hydroxy-4-amino-5-methyl-6phenyl pyrimidine 0.5 0.1 0.1 2-Amino-4-hydroxy-5,6dimethyl pyrimidine 0.5 0.1 > 0.5 2-Methyl-4-hydroxy-5-methyl6-phenyl pyrimidine 0.1 0.1 O.1 2 -Amino-4-hydroxy-6-furan pyrimidine 0.1 > 0.1 > 0.1 > 0.1 2 -Amino- 4 - hydroxy- 5 -methyl6-thiophene pyrimidine 0.05 > 0.05 > 0.05 > 0.05

-55TABLE 2 —Continued HIGHEST LOWEST INHIBITORY COMPOUNDS NON-TOXIC CONCENTRATION CONC. (mg/ ml) Polio. Cox. Vacc. 2-Amino-4-hydroxy-5methyl-6-furan pyrirnidine 0.1 > 0.1 > 0.1 > 0.1 2-Amino-4-hydroxy-5-methyl6-pyrrole pyrimidine 0.5 > 0.5 > 0.5 > 0.5 2-Amino-4-hydroxy-5-methyl6-pyridine pyrimidine 0.5 > 0.5 > 0.5 > 0.5 Semicarbazide hydrochloride 2.5 > 2.5 > 2.5 Thiosemicarbazide 1.0 > 1.0 > 1.0 0.5 Cz-Amino-phenyl-ethanesulfonic acid 0.1 > 0.1 > 0.1 > 0.1 a-Amino- Shenyl-methane sulfonic acid 0.5 0.1 > 0.5 Thiouracil 1.0 0.5 0.5 6-n-propyl-2-thioracil 0.5 O.1 > 0.5 Thorozine 0.0005 > 0.0005 > 0.0005> 0.0005 Tolseran 0.1 > 0.1 > 0.1 > 0.1 6-Uracil-n-methyl sulfone 0.0097 > 0.0097 > 0.0097> 0.0097

-56TABLE 3 COMPARATIVE TOXICITY OF COMPOUNDS IN TISSUE CULTURE HIGHEST NON-TOXIC CONC. (MG/Mi) COMPOUNDS* MONKEY MONKEY HELA'ESTICLE KIDNEY CULTURES CULTURES CULTURES Abbott #1713, 1-MethylN-dimethyl indole 0.1 0.5 Abbott #4462x** 0.1 > 2.5 Abbott #3665, 3-amino acetophenone 0.1 O.1 Abbott #4271, dimethyl glycine > 2.5 > 2.5 Abbott #6238, 2-thio-4,6- 0.1 0.1 diamino pyrymidine 8-azaguanine 0.1 0.5 l-5-D-Glucopyranosyl-2ethyl-5-methyl benzimidazole > 2.5 > 2.5 5-Chlorobenzimidazole HC1 0.1 > 0.1 1- -D-Glucopyranosyl-5methyl benzimidazole > 2.5 > 2.5 5-Methyl-2- isopropyl benzimidazole HC1 O.1 > 0.1 5,6-Dichlorobenzimidazole 0.1 0.01 5,6-Dichloro-l-D-ribosyl benzimidazole 0.1 0.1 1-p-D-ribopyranosyl-5, 6dimethyl benzimidazole 1.0 1.0, 2 - Deoxy-D-ribopyranosyl5,6-dimethyl benzimidazole 1.0 1.0 l-ac-D-arabopyranosyl-5,6dimethvl berzimidazole > 2.5 S *Underlined compounds indicate those for which different toxic levels have been found. **Plasma-clot roller tubes. -***Identity of compound restricted.

-57TAI3LE 3 —Cont inued HIGHEST NON-TOXIC CON. (MG/Mi) COMPOUNDS MONKEY MONKEY HELA TESTICLE KIDNEY CULTURES CULTURES CULTURES Benzimidazole (Eastman Kodak') (.1 0.1 Benzimidazole (British Drug Houses, Ltr, London) 0.5 5,6-Dimethyl benzimidazole 0.1 0.1 0.1 2-Ethyl-5-methyl benzimidazole 0.1 0.1 2,5-Dimethyl benzimidazole 0.1 0.1 2-Benzimidazolethiol 0.1 0.1 5-Amino-2-benzimidazolethiol 0.1 0.1 2-Amino benzimidazole 0.1 0.1 4-Methoxy-6-nitrobenzimidazole 0.024 0.039 6-Methoxy-4-nitrobenzimidazole 0.031 0.031 Benzaldehyde thiosemicarbazone 0.005 0.039 Cysteic acid 1.0 > 2.5 Helenine 10* 10* Hydroxylamine HC1 0.1 0.05 > 0.1 Hydrazine dihydrochloride 0.1 0.1 > O.1 Mephenesin 0.1 0.1 Methionine sulfoximine 0.5 > 2.5 Meprobamate 0.5 0.1 M-8450 10* 10** 10* Semicarbazide hydrochloride >2.5 > 2.5 > 2.5 *Reciprocal of the dilution. **Reciprocal of the dilution; cultures grown without jlasma-clot.

-58TABLE 4 EFFECT OF M-8450 ON THE MULTIPLICATION OF HELA CELLS IN CONTINUOUS CULTURE Dilution of Fold Increase Average Growth M-8450 per Weekly Passage Fold in Growth Increase (%) Medium 1st 2nd 3rd 4th 5th * Control** 3.68 6.0 5.2 4.9 5.5 5.05 100 1/10 1.88 2.1 1.9-2.1 1.86 1.96 40 1/40 3.51 5.1 4.9** 4.3 6 1/80 4.1 4.0 4.05 80 1/160 5.4 5.4 5.4 108 *Arithmetic average **M-8450 withheld from the medium

-59TABIZ 5 COMPOUNDS DEMONSTRATING INHIBITORY ACTIVITY AGAINST THE VIRUSES OF POLIOMYELITIS AND COXSACKIE GROWN IN HELA CULTURES m VIRUSES *** 0OOTCID50 50TCID50 10TCID50 COMPOUNDS (MS/M1) Polio Cox Polio Cox Polio Cox Abbott #3665, P-amino - - - - - aceto henone *Abbott #462, 0.1 - - - - - Abbott #6238, 2-Thio-4,6diamino pyrimidine O.1 - + - + - + Abbott #4271, dimethyl glycine, 2.5 - - - - + Benzimidazole, 0.1 - - - - - 2-Benzimidazolethio, 0.1 -- 5-Amino-2-benzimidazolethiol 0.1 - - - - - l-C-D-arabooyranosyl-5,6dimethyl benzimidazole 0.1 - l-p-D-ribo yranosyl-5,6dimethyl benzimidazole, 0.1 + + + + + + 1-2-lDeaxy-D-ribooyranosyl-5,6-dimethyl benzimidazole, 10 - - - 5-Chlorobenzimidazole Hcl, 01 - -- 5,6-Dichloro-l-D-ribosylbenzimidazole, 0.1 - - b Cysteic acid, 1.0 - - Erythromycin, ). + + + + + + Hydroxylamine B1, 0.1 + +? + - 4? Hydrazine dihydrochlod ride, 0.1 - - - M-8450, 10** - - Methionine sulfoximine,0.1 - Mephenesin, 0.1 Milltown, 0.1 + + + + + + Diazouracil, 0.1 2,2-Diethyl-1, 3-propanediol dicarbamate, 0.5 2,2-Diphenyl-3-hydroxy propyl dicarbamate 2-Amino-4-chloro-5-methyl 6-rhenyl yrimidine 0.01 + + - + 2, 4-Amino-5-methyl-6henyl yrimidine, 0.1 2-Methyl-4-hydroxy-5methyl-6- )henyl pyrimidine, 0.1 2-Hydroxy-4-amino-5methyl-6-phenyl pyrimidine, 0.1 2-Amino-4-hydroxy-5,6 dimethyl pyrimidine 0.1 - + + - + Semicarbazide HN1, 2.5 +? - - +? *Identity of the compount restricted. **Reciprocal of the dilution. ***The (-) (+) designation represents the protection and degeneration of the cultures

/^^ r 4 _//^" io ^~ / ^^ /~~~~~~~~~~~~ -3/ 10 / Cl) -2 cr10 10 0 2 4 6 8 1012141618 2022242628303234363840424446 TIME FROM' INOCULATION, HOURS. Figure 1. Effect of Benzimidazole (0.1 mg/nl) and Cyteic Acid (1.0 mg/ml) on Poliomyelitis (Mahoney) virus Grown in HeLa Cultures.

-5 10 10" / " -4 4 I0~~~~~~~~~~~~~~0 -3/ U) -2 15z 10 cc Ig w~f H 9-~~~~~~~r 10 0 2 4 6 8 10 12 14 16 18 20 22 2426283032 34363840424446 TIME FROM INOCULATION, HOURS. Figure 2. Effect of Benzimidazole (0.1 mg/ml) and Cysteic Acid (1.0 rmg/mi) on Coxsackie (Conn. 5) Virus Grown in HeLa Cultures.

-62TABLE 6 THERAPEUTIC INDICES* OF SELECTED VIRUS INHIBITORS IN HELA CULTURES VIRUSES COMPOUNDS Poliomyelitis Coxsackie Vaccinia M-8450 49.5 42.2 4.2 Helenine 1.0 8.0 1.0 Helenine ** 8.0 8.0 1.0 Methionine sulfoximine 294.0 66.5 200.0 M5-11496 8.3 2.0 2.0 Benzaldehyde thiosemicarbazone 4.0 2.0 24.0 Diazouracil 1.0 2.0 1.0 A14-(FSC) 8.0 8.0 4.0 5,6-Dichloro-l-Dribosyl benzimidazole 66.5 2.0 2.0 *Therapeutic Index = Maximum tolerated dose/minimal suppresive dose. **48-Hour pre-treatment of cultures at 37~C. TABLE 7 THERAPEUTIC INDICES* OF FRACTIONS OF ULTRACENTRIFUGED M-8450 TESTED AGAINST ONE HUNDRED TISSUE CULTURE DOSES OF POLIOMYELITIS VIRUS IN HELA CULTURES SAMPLE THERAPEUTIC INDEX 4~C Control 44.7** Room Temperature Control 11.3 Supernatant 1.4 Pellet Wash 1.4 Reconstituted Pellet 32.0 *Therapeutic index = Maximum tolerated dose/minimal suppressive dose. **This figure represents the mode of the results obtained in other similar tissue culture assays.

-63 - TABLE 8 PROTECTION OF M-8450 TREATED AND WASHED HELA CULTURES FROM THE CYTOPATHOLOGY OF POLIOMYELITIS AND COXSACKIE VIRUSES TREATMENT CYTOPATHOLOGY* Poliomyelitis virus + Coxsackie virus + M-8450 plus poliomyelitis virus M-8450 plus Coxsackie virus M-8450, washed, plus poliomyelitis virus M-8450, washed, plus Coxsackie virus Polio.antiserum, washed, plus polio. virus + Cox. antiserum, washed, plus Cox. virus + *The (-) (+) designations represent the protection and degeneration of the cultures.

-64 - TABLE 9 EFFECT OF 1/10 M-8450 ON POLIOMYELITIS VIRUS ADSORPTION AND ATTACHMENT ONTO HELA CULTURES POLIOMYELITIS INFECTED CULTURES TIME VIRUS TITER (NEG. LOG.) IN NUMBER OF PLAQUES PER MONKEY KIDNEY TUBE CULTURES MONKEY KIDNEY BOTTLE AFTER Virus M-8450 370C* Virus M-8450 INFECTION Control Treated Control Control Treated 60 minutes 1.66,2.25 1.83 - 22,40 29 2 hours 30 min. 1.66,1.66 1.66 - 21,19 19 4 hours 1.5, 1.5 1.5 2.5 20,28 19 *In-ubator control without cells.

TABLE 10 EFFECT OF MEDIUM UPON THE INHIBITION BY M-8450 OF ONE HUNDRED TISSUE CULTURE DOSES OF POLIOMYELITIS VIRUS IN HELA CELLS STOCK HIGHEST NON-TOXIC THERAPEUTIC SOLUTIONS CONCENTRATION OF INDEX * STOCK SOLUTIONS ** A 50% 12.0 B 15% 11.0 C 50% 8.0 D 50% 4.0 E 50% 4.0 Control Medium-*** 100o 44.7x x* *-Composition of stock solutions given in text. **Therapeutic index = maximum tolerated dose/ minimal suppressive dose. ***Complete Syverton maintenance medium. ****This figure re resents the mode of the results obtained in other similar tissue culture assays. TABLE 11 EFFECT OF EAGLES'S MEDIUM UPON THE INHIBITION BY M-8450 OF POLIOMYELITIS VIRUS IN HELA CULTURES MAINTENANCE TISSUE CULTURE THERAPEUTIC MEDIUM DOSE OF POLIO- INDEX* MYLITIS VIRUS Eagle's 320 330.5 Syverton's 178 64.o *Therapieutic index = maximum tolerated dose/ minimal su pressive dose.

-66 - TABLE 12 CYTOPATHOLOGY AND VIRUS ISOLATIONS FROM M-8450-TREATED VIRUS- INFECTED HELA CULTURES HOURS POLIOMYELITIS INFECTED COXSACKIE INFECTED AFTER CULTURES CULTURES INFECTION Controls M-8450 Controls M-3450 Treated Treated I* C** I C I C I C 24 nd +?*** -- nd +? + 38 nd + - - nd + + +? 72 - - nd + 96 - - 120 - - 144- - *Virus isolation attempts in Hela cultures **Cyt opathology ***The designation (+) represents degeneration of the cultures or a positive virus isolation. The designation (-) represents no evidence of degeneration or a failure to isolate virus nd = not done. TABLE 13 ANTIGENICITY OF M-8450-TREATED, POLIOMYELITIS-INFECTED CULTURE FLUIDS IN MICE SERUM MOUSE ANTISERA PRODUCED AGAINST- DILUTIONS Virus Control Drug-Treated Drug-Treated, Culture Fluids Culture Fluids Virus-Infected Culture Fluids Undiluted -* + + 1/4 - + + 1/8 - + + 1/16 - + + *The designations (-) and (+) respectively represent protection and degeneration of cultures in a tissue culture neutralization test.

TABLE 14 LOWEST INHIBITORY CONCENTRATION (MG/M1) OF COMPOUNDS ASSAYED FOR INHIBITION OF ONE HUNDRED TISSUE CULTURE DOSES OF VIRUS IN MONKEY KIDNEY CULTURES Highest COMPOUNDS** Toxic VIRUSES EMPLOYED Conc. Echo Echo Echo Echo Echo Echo Echo Echo Echo Echo Echo Echo Echo Polio Cox Vacc. MG M1 1 2 3 4 5 6 7 8 9 11 12 13 14 Type 1 B-l M-8450* 10** <10 60 60 60 30 60 60 80 80 60 <10 30 20 320* 120* <10 Methionine Sulfoximine 2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >25 >2.5 > >.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 >2.5 Benzaldehyde Thiosemicarbazone*.039 >039 >.039 >039 >039 >039 >.039 >.039 >.039 >039 >039 > 039 > 039 >.039.039 >.039>0017* 5, 6-Dichloro1-D-Ribosyl benzimidazole O.1.025 >O.1 >0.1 >O.1.0125 0.05 >0.1 4-Methoxy-6- nitrobenzi- - midazole.039 >.039 >039 > 039.0065 >.039.0097 >.039 >.039 >.039.019 >.039 >039.0095>.039 >.039.019 6-Methoxy-4nitrobenzimazole.031 >.031.0075.0037 >0.31 >.031 0013.015 >.031 >.031 >031 >.031 1,2-Deoxy-DRibopyranosyl 5,6-dimethyl benzimidazole 1.0.125.187 0.5 0.5.125 l-f-D-RibopyranosyL 5,6dimethyl benzimidazole 1.0 >1.0.125 >1.0.375.125 1-a-D-Arabopyranosyl 5,6dimethyl benzimidazole 1.0 >1.0.125 >1.0 >1.0.125 *Compounds considered significantly inhibitory by virtue of large therapeutic index. **Cultures pre-treated 24 hours at 37~C. ***Reciprocal of the dilution.

-68TABLE 15 THERAPEUTIC INDICES* OF SELECTED VIRUS INHIBITORS IN MONKEY KIDNEY CULTURES COMPOUND VIRUSES Polio. Cox. Vacc. Echo-2 Echo-4 Echo-7 Echo-8 Echo-9 Echo-ll M-8450 32.0 12.0 <1.0 6.o. 6.. 6. 8. 8.0 6. Methionine sul- <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 foximine Benzaldehyde thiosemicarbazone <1.0 <1.0 22.0 <1.0 <1.0 <1.0 <1.0 <1.< <1.0 5,6-Dichloro-1-Dribosyl benzimidazole 2.0 - - - <1.0 <1.0 8.0 - Plant extract, M-2<1.0 <1.0 <1.0 <1.0 28.8 4.0 <1.0 12.0 28.0 Plant extract, M-4 2.0 - <1.0 4.0 4.0 8.0 16.0 3.0 3.0 Plant extract, M-14<1.0 <1.0 <1.0 48.0 <1.0 <1.0 4.0 <1.0 <1.0 *Therapeutic index = Maximum tolerated dose/minimal suppressive dose.

TABLE 16 RECIPROCAL OF THE LOWEST INHIBITORY DILUTION OF PLANT EXTRACTS ASSAYED FOR VIRUS INHIBITORY PROPERTIES IN MONKEY KIDNEY CULTURES PLANT LOWEST VIRUSES EMPLOYED EXTRACT NONFRACTIONS TOXIC Echo Echo Echo Echo Echo Echo Echo Echo Echo Echo Echo Echo Echo Polio Cox. Vacc. ** DILUTION 1 2 3 4 5 6 7 8 9 11 12 13 14 Type 1 B-l M 1 80 < 80 < 80 < 80 < 80 < 80 < 80 < 80 < 80 < < 80 80 320 < < 80 - < 0 < < 80 M 2* 100 <100 <100 <100 2880* <100 <100 400 <100 1200* 2U80* <100 600 <100 <100 <100 <100 M 3 120 <120 <120 <120 <120 <120 <120 240 <120 480 <120 <120 480 <120 240 <120 <120 M 4* 120 480 480 720 480 240 <120 960* 1920* 360 360 240 240 - 240 - <120 M5 10 < 10 20 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 - < 10 - < 10 Ua M 6 120 <120 <120 360 <120 <120 <120 720 720 <120 <120 <120 720 - <120 <120 <120 \O M 7 100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 - 200 <100 <100 M 8 100 200 <100 <100 <100 <100 <100 <100 <100 <100 <100 600 <100 - <100 <100 <100 M 9 200 <200 <200 <200 <200 <200 600 <200 <20 <200 <200 600 <200 -<200 <200 400 MlO 100 400 <100 <100 <100 <100 <100 <100 <100 <100 600 400 <100 - - - Mll 100 400 <100 <100 <100 <100 200 <100 <100 300 <100 <100 <100 - <100 <100 <100 M12 100 <100 <100 <100 <100 <100 <100 <100 <100 300 <100 <100 200 - <100 <100 <100 M13 100 <100 <100 <100 - <100 <100 <100 - - 300 - - - <100 <100 <100 M14* 10 < 10 480* < 10 < 10 < 10 < 10 < 10 40 < 10 < 10 < 10 < 10 - < 10 < 10 < 10 M15 160 <i6o <160 <160 <160 <160 <60 o <60 o <60 o <160 <160 <6 <16 - <160 <160 <160 M16 160 320 <160 <160 320 <160 320 320 320 <160 <16o 320 640 - 320 320 320 M18 200 - 600 - 200 - - - -00 1200 200 800 800 - 200 200 800 M19 100 - - - - - -- - - M20-25 160 - *Compounds considered significantly inhibitory by virtue of large therapeutic index. **All cultures -re-treated 24 hours at 37~C.

TABLE 17 INHIBITION OF CYTOPATHOLOGY* BF M-2-TREATED MONKEY KIDNEY CULTURES INFECTED WITH ECHO-4 AND 11 VIRUSES TIME VIRUS AFTER CONTROLS 24-HOUR PRETREATMENT WITH M-2 VIRUS TCIDo50 INFECTION (NORMAL _____________________________________________________ INOCULATED (HOURS) MEDIUM) 1/120 1/240 1/480 1/960o 1/1920 1/3840 48 ++++ - - - - - - + + + 10,000 72 - - + - + + + + 96 + + + 48 ++++ - - - - - - - - - - + + Echo-4 1,000 72 - --- + + + + 96 - - - - + + 48 - - - - - 100 72 ++++- - - - -- - 96 --- + + + + + 48 ++ - - - - - - - + + + + + 3,200 72 - + + + + 96 + + - + 48 +++ - - - - - - - - - - + + Echo-11 1,000 72 - - -- + + + + + 96 + + + + + 48 ---- - - - - - - - - + + 100 72 +++ - - - - - - - 96 --- + + + + + *The designations (+) (-) represent the degree of involvement of the cultures. When all the cells are off the glass the (+) designation is givenr.

-71TABLE 18 INHIBITION OF CYTOPATHOLOGY* OF MONKEY KIDNEY CULTURES TREATED WITH M-2 AT TIME OF ECHO-4 INFECTION AND ONE HOUR LATER VIRUS TIME VIRUS (100 AFTER CONTROLS DILUTIONS OF M-2 DRUG TCID50 INFECTION (NORMAL (M-2) (HOURS) MEDIM) 1/120 1/240 1/480 1/960 48 - With virus Echo-4 72 + + + + — 96 - + + + + + 1 hour 48.- - - - - after Echo-4 72 + + + + virus 96 - - + + *The designations (+) (-) represent the degree of involvement of the cultures When all the cells are off the glass the (+) designation is given. TABLE 19 THERAPEUTIC INDICES* FOR M-2 TREATED MONKEY KIDNEY CULTURES Drug 1 hr. PRETREATMENT 24 HOURS with after Virus TITER BEFORE VIRUS virus virus Controls ECHO (Neg. 10,000 3200 1000 100 100 100 100 VIRUS Log.) TCD50 TCD50 TCD50 TCD50 TCD50 TCD50 TCD50 #4 5.0 14.4 - 28.8 28.8 14.4 3.6 < 1.0 #11 4.5 - 9.6 28.8 28.8 - - < 1.0 *Therapeutic index = maximum tolerated dose/minimal suppressive dose.

-72TABLE 20 INHIBITION OF CYTOPATHOLOGYX** OF M-2 TREATED MONKEY KIDNEY CULTURES AFTER DOUBLE VIRUS EXPOSURES CULTURES FIRST CYTOPATHOLOGY IN TYPE OF PRETREATED EXPOSURE CULTURES HRS AFTER FIRST ECHO VIRUS GROUP 24 HOURS VIRUS RE-EXPOSED VIRUS EXPOSURE RECOVERED # WITH M-2 (100 TO VIRUS TCID)r) 72 96 120 1/120 Echo-4 Echo-4 - - - nd** 1 1/240 (10,000 - + nd 1/480 " TCID50) - + 4 1/960 + 4 1/120 Echo-4 Echo-11 - - - nd 2 1/240 " (3200 - nd 1/430 TCIDn0) + 11 1/960 " + 11 1/120 Echo-4 Echo- 1 + 1 3 1/240 " (100,000 + 1 1/480 TCID0 ) + 1 1/960 + 1 1/120 Echo-ll Echo-4- - nd 4 1/240 " (o,0ooo - + 4 1/480 " TCID50) - + 4 1/960 " + 4 1/120 Echo 11 Echo-1 - - - nd 5 1/240 " (3200 - - - nd 1/480 TCID50) - + nd 1/9 60 " + 11 6 1/120 Echo 4 - - nd Drug 1/240 or 11 - + nd Control 1/480 - + nd Cultures 1/960 - + nd Virus Controls Echo 4 + nd or 11 *Cultures washed once and fresh M-2 added. **Not done. ***The designations (-) (+) represent protection and degeneration of the cultures.

-73 - TABLE 21 CYTOPATHOLOGY** OF M-2 TREATED MONKEY KIDNEY CULTURES AFTER DOUBLE VIRUS EXPOSURES FIRST SECOND CYTOPATHOLOGY IN EXPOSURE EXPOSURE HOURS AFTER FIRST 24- HOUR VIRUS VIRUS VIRUS EXPOSURE PRETREATMENT (100 (O 1 ml of GROUP WITH M-2 TCIDpo) 10-1 dil.) 72 96 120 1 1/200 Echo-11 Echo 1 + 2 1/200 " Echo 2 + 3 1/200 " Echo 3 + 4 1/200 I Echo 4 - 5 1/200 " Echo 5 + 6 1/200 " Echo 6 + 7 1/200 " Echo 7 + 8 1/200 " Echo 11 - - - 9 1/200 " Polio + 10 1/200 " Coxsackie + 11 1/200 " Vaccinia + Untreated controls " + *EvidCene of occasional virus plaques **The designations (-) (+) represent the protection and degeneration of the cultures

-74 TABLE 22 RECIPROCAL OF THE ANTIBODY TITERS* OF SERA OBTAINED FROM MICE INOCULATED WITH ECHO-4 AND 11 VIRUSES AND TREATED WITH M-2 FRACTION TYPE OF VIRUS M-2 TREATED M-2 TREATED VIRUS CONTROL CONTROL VIRUS-INOCULATED INOCULATED MICE MICE MICE Echo-4 16 6 E-ho-11 64 4 12 *Obtained in monkey kidney tissue culture neutralization test.

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