FIG. 1. Undifferentiated sarcomatous cells, some multinucleated in a section of a subcutaneous tumor in a hamster sacrificed at 223 days. H & E. Mag. 100 X. FIG. 2. Multinucleated giant cells within subcutaneous sarcoma. H & E Mag. 215 X. FIG. 3. A giant cell with basophilic nuclei in subcutaneous tissue in hamster that died at 173 days. H & E Mag. 450 X. FIG. 4. Rounded, vesiculated nuclei of tumor cells surrounding tubules and glomeruli in pole of kidney, in hamster sacrificed at 48 days after subcutaneous transplantation. H & E Mag. 100 X. MONKEY KIDNEY EXTRACT-INDUCED TUMORS TABLE II. Attempts to Culture a Virus from Tumors in Hamsters Induced with Extracts of Monkey Kidney Cells. Hamsters <1 to 3 days of age inj. with the same tumor extract that was added to cell cul tures failed to develop tumors when kept for 200 to 300 days. + The monkey kidney stable cell line originated from rhesus monkey kidney cells, occasionally infiltrated by tumor and the Three hamsters had tumors in the lungs and 2 had similar masses in the kidney. The subcutaneous tumors consisted mainly of cells with pleomorphic, anaplastic nuclei, and variable amounts of cytoplasm (Fig. 1). The nuclei were rounded or ovoid, usually vesiculated, with chromatin arranged as in the spokes of a wheel and or marginated at the perimeter of the nucleus. In some tumors, there were spindle shaped cells and nuclei arranged in interlacing and whorling bundles or in a loose matrix with sparse amounts of cytoplasm. Numerous multinucleated giant cells were present in the sumors (Fig. 2). Many of these were clearly delineated with distinct rounded basophilic nuclei arranged closely within the cell. Others contained fragmented, basophilic nuclear debris within abundant eosinophilic cytoplasm (Fig. 3). The central portions of the subcutaneous tumors frequently were necrotic. There were many congested and occasionally thrombosed blood vessels within the growths. The tumors in the lungs and kidneys had a similar histologic pattern as described above (Fig. 4). The neoplasms appeared to be undifferentiated sarcomas in subcutaneous tissue. kidneys and lungs. Infrequently, there were subcutaneous lobules with histological features resembling fibrosarcomas. Virologic studies. As shown in Table II 3 kinds of tissue culture were incubated with material from the hamster tumors. No de generative changes that could be attributed to a virus were observed and the fluids from the second and fifth tissue culture passage of one tumor did not induce tumors in 2 litters of hamsters that were inoculated and kept for 300 days. Portions of 6 of the tissue extracts that were added to the tissue cultures were injected into litters of newborn hamsters and kept for 200 to 300 days. No tumors were MONKEY KIDNEY EXTRACT-INDUCED TUMORS observed in any of these animals. Tumor No. A 115, was the first tumor transplanted to other hamsters. As mentioned above, tumors developed in 10 of the 12 animals injected. Portions of the liver, kidneys and tumor tissue of one tumor-bearing hamster were ground separately, diluted in 2-fold steps and incubated at 4°C with 0.4% washed guinea pig erythrocytes. No hemagglutination was noted. Eighteen of the tumor-bearing animals were bled and their sera tested for hemagglutination-inhibition antibodies against 2 strains of SE polyoma virus. Inhibition did not occur. Sera were not obtained from the monkeys at the time cell cultures were prepared but sera from 100 normal monkeys from the same colony, tested for hemagglutinationinhibition antibodies against the SE polyoma virus were negative. Discussion. The SE polyoma virus is the only biological material, other than certain of the monkey kidney cell extracts mentioned in this report, that is capable of inducing tumors in all or almost all hamsters in a litter when injected as newborn. Histologically there are similarities between the present tumors and those induced by the polyoma virus. The characteristics of lobulation, giant cell reactions, polygonal anaplastic cells, sarcomatous cells with a tendency to differentiate into fibrous tissue were all similar to those in tumors produced by subcutaneous injection of the SE polyoma virus in newborn guinea pigs (17) and hamsters. The tumors of the present hamsters differed from the polyoma-virus-induced neoplasms in their distribution and cellular detail. No tumors were found in heart. stomach, intestines or liver as occur usually in hamsters inoculated with high titer polyoma virus. The larger number of bizarre giant cells and lack of frequent cellular differentiation of the tumor cells also helped to distinguish these tumors from those produced in response to the polyoma virus. The SE polyoma virus can be propagated in mouse embryo cell cultures inoculated with virus-in fected hamster tissue, particularly after one or 2 passages (18) and characteristic cytopathogenic changes occur in the cells (14). Thus far, there has been no evidence that a virus multiplied in the tissue culture cells inoculated with the hamster neoplasms induced with monkey kidney extracts. If a virus in the monkey kidney ceil extracts is responsible for the hamster tumors then it is not detectable in the induced hamster tumors. Newborn hamsters did not develop tumors after injection with hamster-tumor extracts. Extracts of polyoma-virus-induced tumors injected directly into other hamsters do not induce tumors (1). To demonstrate polyoma virus in hamster tumors it is necessary to increase the concentration of virus and to free it from antibody by again cultivating the virus in a suitable cell culture (18). Failure to propagate a virus in certain tissue culture lines suggests that at present the tumor-inducing material must be labeled a "substance." Extracts prepared either from human or feline tumor tissue or human tissue cultures failed to induce tumors when inoculated into newborn hamsters. Summary. Injection of newborn hamsters with extracts of certain lots of rhesus monkey kidney cell cultures has been followed some months later by occurrence of neoplasms at site of injection in 109 of 154 hamsters. Three of the neoplasms were transplanted to other hamsters and the recipient hamsters developed tumors in 13 to 48 days. Transplants from the second tumor were passed from hamster to hamster 5 times and tumors developed in all surviving animals in 6 to 24 days. Tumors could not be induced in newborn hamsters by inoculation of extracts of the hamster tumors. No changes indicative of a virus were observed when extracts of a tumor or a tumor mince were incubated with primary mouse embryo, primary vervet monkey kidney cells. or a continuous line of rhesus monkey kidney cell cultures. The tumors appeared to be somewhat different from those induced by the SE polyoma virus. 1. Eddy, B. E., Stewart, S. E., Young, R., Mider, G. B.. J. Nat. Cancer Inst., 1958, v20, 747. 2. Rustigian, R., Johnston, P., Reihart, H., PROC. MONKEY KIDNEY EXTRACT-INDUCED TUMORS Soc. EXP. BIOL. AND MED., 1955, v88, 8. 3. Hull, R. N., Minner, J. R., Am. J. Hyg., 1956, v63, 204. 4. Ann. N. Y. Acad. Sci., 1957, v67, 413. 5. Mahlerbe, H., Harwin, R., Brit. J. Exp. Path., 1957, v68, 539. 6. Hull, R. N., Minner, J. R., Mascoli, C. C., Am. J. Hyg. 1958, v68, 31. 7. Sweet, B. H., Hilleman, M. R., Internat. Conference on Live Poliovirus Vaccines. Pan American Health Organization, and World Health Organization, 1960, v2, 79. 8. Morgan, J. F., Morton, J. H., Parker, R. C., PROC. SOC. EXP. BIOL. AND MED., 1950, v73, 1. 9. Lillie, R. D., Histopathologic Technic and Practical Histochemistry, 2nd ed. The Blakiston Co., New York, 1954, p41. 10. Mallory, F. B., Pathologica! Technique, W. B. Saunders Co., Philadelphia 1942, p156. 11., ibid., 161. 12. Earle, W. R., J. Nat. Cancer Inst., 1943, v4, 165. 13. Evans, V. J., Fioramonti, M. C., Sanford, K. K., Earle, W. R., Westfall, B. B., Am. J. Hyg., 1958, v66, 66. 14. Eddy, B. E., Stewart, S. E., Berkeley, W., PROC. SOC. EXP. BIOL. AND MED., 1958, v98, 848. 15. Youngner, J. S., ibid., 1954, v85, 202. 16. Eddy, B. E., Rowe, W. P., Hartley, J. W., Stewart, S. E., Huebner, R. J., Virology, 1958, v6, 290. 17. Eddy, B. E., Borman, G. S., Kirschstein, R. L., . Touchette, R. H., J. Infect. Dis., 1960, v107, 361. 18. Eddy, B. E., Stewart, S. E., Canadian Cancer Conference, Academic Press, New York, 1959, v3, 307. Received March 3, 1961. P.S.E.B.M., 1961, v107. 420 The Vacuolating Virus, S.V40 (26128) B. H. SWEET AND M. R. HILLEMAN Viruses are commonly carried by monkeys and may appear as contaminants in cell cultures of their tissues, especially the kidney. Included among these are B virus, foamy agent, measles-like virus, hemadsorption viruses, LCM virus, arborviruses, and a wide variety of miscellaneous viral agents(1-7). Hull et al.(2,3) have studied extensively viruses encountered in "normal" monkey renal cell cultures. Hull called them simian or SV viruses and classified them into 4 groups. based on the kind of cytopathic change induced in monkey kidney cell cultures infected with the agents. Twenty-eight of these viruses were precisely separated serologically into types and, additionally, 24 unidentified viruses were recorded. Malherbe and Harwin (4) distinguished 7 distinct types among the simian agents or SA viruses they recovered from vervet kidney materials. During the past 2 years, our virus research group* has repeatedly encountered a new simian virus of rhesus and cynomolgus monkey kidney origin. This agent was unique among simian viruses studied hitherto since it grew but did not cause a cytopathic effect in the rhesus or cynomolgus kidney cell cultures from which it was derived. Instead, it grew and caused marked cytopathic changes in cell cultures of a heterologous species, i.e., the green monkey, Cercopithecus aethiops obtained from Equatorial East Africa [grivet, according to Sanderson (8)]. This agent has been called the "vacuolating virus" by us because of the prominent cytoplasmic vacuolation seen in infected cell cultures. Dr. Hullt suggested that this agent be given the "official" designation of S.V-40 in C.P.E. group 4. The discovery of this new virus, the vacuolating agent, represents the detection for the The first such agent was recognized by Dr. L. MeClelland in the course of safety testing of vaceines. + Personal communication. first time of a hitherto "non-detectable" simian virus of monkey renal cultures and raises the important question of the existence of other such viruses. The present report summarizes the current information available concerning the vacuolating agent. Certain of these data have been presented at the Second International Conference on Live Poliovirus Vaccines (9). Materials and methods. Tissue cultures. Primary cell cultures were prepared from trypsinized cell suspensions and were grown in Melnick's lactalbumin-yeast extract medium (10) containing 2% heat-inactivated calf serum. All line cell cultures were grown in Eagle's basal medium (10) containing 10% heat-inactivated horse serum. During virus propagation, all cell cultures were maintained using medium 199 (10) containing 2% heatinactivated chicken serum and at initial pH 7.6. All culture fluids contained 100 μg of penicillin and 100 units of streptomycin per ml. Propagation of vacuolating agent. The virus grew readily in cell cultures of grivet monkey kidney with production of cytopathic change. Such cultures were used routinely for the work described in this report. For ordinary purpose, the virus was passed in an initial dilution of 101 of infected tissue culture fluid using 0.2 ml of inoculum per 2.0 ml of maintenance medium. After 4 to 7 days incubation, at a time when at least 50% of the cell sheet showed cytopathic change typical of that of the vacuolating agent, the cultures were frozen and thawed once and the fluid was harvested. The virus was stored frozen in the dry ice refrigerator. Virus titrations and serum neutralization tests. All virus titrations were performed using serial 10-fold dilutions of virus. The inoculum dose was 0.2 ml per roller tube culture containing 2.0 ml of maintenance medium. The end point was the highest initial |