Bernice E. Eddy

Identification of the oncogenic substance in rhesus monkey kidney cell cultures as simian virus 40

Abstract Evidence is presented to show that the oncogenic virus in rhesus monkey kidney cell (RMKC) extracts responsible for the production of tumors in hamsters injected when newborn ( Eddy et al. , 1961 ) is simian virus 40 (SV40). Two viruses were isolated from tumors in hamsters, aged 203 and 304 days, which had been injected when newborn with extracts of RMKC. These viruses on injection into newborn hamsters also caused tumors to develop. Tumors did not develop when one virus strain, A426, was combined with anti-SV40 rabbit antiserum before injection into the hamsters. In cell cultures of Cercopithecus aethiops kidney both viruses caused vacuolization of the cytoplasm of the cells, a characteristic of SV40, and this cytopathic effect was inhibited by SV40 rabbit antiserum. A known culture of SV40 also induced tumors in 11 of 13 hamsters. These tumors were indistinguishable macroscopically and the six examined histologically were indistinguishable from the tumors induced by RMKC extracts. The tumors developed subcutaneously at the site of inoculation, although a few animals manifested tumors in the lungs or kidneys. In general the tumors were undifferentiated sarcomas although portions infrequently resembled fibrosarcomas. Other properties of the oncogenic virus in RMKC extracts corresponded to those described by Sweet and Hilleman (1960) for SV40. Both viruses failed to cause hemagglutination of washed guinea pig erythrocytes or to cause symptoms or death in white mice; both were filterable, resisted treatment with diethyl ether, were stable on storage at −70°C, and exhibited considerable resistance to heating. SV40 was not recovered from an extract of RMKC that failed to induce tumors in hamsters, but was present in three extracts that were oncogenic. These findings point to the fact that the monkey, like the mouse, is host for a virus that has the capacity to stimulate tumor formation in another animal species and to cause cytopathic changes in susceptible cell cultures. The recovery of typical SV40 from two tumor-bearing hamsters, the induction of tumors in hamsters by a known strain of SV40, that were indistinguishable from those elicited by RMKC extracts, and the recovery of SV40 from the RMKC extracts that were oncogenic for hamsters, and failure to recover SV40 from an extract which did not induce tumors are proof that the oncogenic agent in the RMKC extracts is SV40.

The induction of neoplasms with a substance released from mouse tumors by tissue culture

Abstract Evidence suggesting replication of a subcellular tumor-inducing substance in mouse parotid gland neoplasms and mouse leukemias was obtained by culturing preparations from these tumors on monkey kidney and chick chorioallantoic membrane cells in vitro. Unlike the results obtained by Stewart (1953, 1955a, b) with mouse leukemia cell-free extracts, newborn mice injected with cell-free inocula prepared from 20 parotid gland tumors have not developed neoplasms, even after life spans in some instances of more than 2 years. However, when preparations from some of the same tumors were put into tissue cultures and newborn mice were inoculated with the supernatant fluids from these cultures, many developed multiple primary tumors. Mice inoculated with fluids from tissue cultures that had not received the tumor cells failed to develop these neoplasms. All the mice that developed neoplasms had both parotid gland tumors and multiple proliferative renal lesions involving the convoluted tubules. In addition, one or more of the following were observed as primary neoplasms in both sexes: tumors of the submaxillary, sublingual and the exorbital lacrimal glands, thymic epithelioid tumors, tumors of the adrenal medulla, a subcutaneous epithelial tumor in one male, and mammary tumors at an early age in some of the females. Cell-free extracts from a transplanted leukemia and a paraganglioma gave results similar to those obtained with the tissue culture supernatant fluids.

Tumors Induced in Hamsters by Injection of Rhesus Monkey Kidney Cell Extracts

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.

Cytopathogenicity in Tissue Cultures by a Tumor Virus from Mice

Summary Cytopathogenic changes were observed in mouse embryo cell cultures that had been inoculated with tissue culture-grown virus isolated from mouse tumor tissues. Quantitative assays of the virus, referred to as the SE polyoma virus, and serum-virus neutralization tests were correlated with the induction of tumors in hamsters.

Specificity of resistance to tumor challenge of polyoma and SV 40 virus-immune hamsters.

Zilber and his associates(1) were the first to demonstrate an abnormal antigen in a virus-induced tumor when they worked with Rous sarcoma. More detailed studies of the relationships between the inducing virus and the new cellular antigen in the transformed cells were later made by Klein and his associates(2), and by Habel(3), using polyoma virus-induced tumors in the mouse and the hamster. Since that time there has rapidly appeared published evidence for the existence of new cellular antigens in Gross leukemia cells(4), Shope papilloma virus tumors(5), and Moloney virus leukemia(6). The biological method which most readily demonstrated the new antigen in polyoma tumors, tested the resistance of virus immunized adult animals to challenge with isologous transplantable polyoma tumor. This same technic has been used to demonstrate new cellular antigens in SV 40 virus induced hamster tumors in the experiments reported here. The original work with polyoma tumors had demonstrated the specificity of the new antigen insofar as there was no cross resistance by polyoma immune mice against challenge with tumors induced by other oncogenic agents. SV 40 and polyoma viruses have several physical and chemical characteristics in common(7). They are both DNA viruses of the same size, both have the same number of capsomeres, and both produce sarcomas on inoculation of newborn hamsters. For this reason it was felt that a cross-immunity test, in which animals immunized with each of these 2 viruses were challenged with the corresponding tumors, would be a more severe and direct test of the specificity of the new virus-induced cellular antigens. It will be shown that in such a cross-immunity test each virus appears to induce a resistance to tumor challenge which is quite specific. Materials and methods. Viruses. Polyoma virus was grown in mouse embryo tissue culture from random-bred Swiss mice and a standard pool had a titer of 1.5 × 107 pfu/ml. SV 40 virus was grown in primary green monkey kidney tissue cultures and had a titer of 4 × 107 TCID50 per ml. Antiviral antibody.

Tumor Immunity in Hamsters Infected with Adenovirus Type 12 or Simian Vims 40

Summary The oncogenicity of adenovirus type 12 and SV40 administered to hamsters when newborn could be inhibited or prevented by administration of large doses of the homologous virus at later dates. The protection achieved appeared to vary inversely with the concentration of virus in the initial infecting dose. The administration of greater initial infecting doses of virus resulted in fewer tumor-free animals in the treated groups. The size of the dose of virus used in treatment was a factor, and a large dose (1 ml) gave a little more protection than a 0.25 or 0.5 ml dose. The usual course of treatment was a total of 13 doses of virus administered at twice weekly intervals. Smaller numbers of doses used in treatment were sometimes effective. Treatment with virus heated to 50°C for an hour in the presence of magnesium chloride was not effective in preventing the induction of tumors.

Lymphocytic choriomeningitis virus as related to chemotherapy studies and to tumor induction in mice.

In 1956 Stewart and Haas’ recovered and identified LCM virus in 2 sublines of L 1210 leukemia

Influenza Virus in Sectioned Tissues

. One subline designated AMD, FX, reported as A-Methopterin dependent, was obtained from A. Goldin, and the other, subline C, G 153, an ascites tumor carried with 8-azaguanine to the 98th transfer, was obtained from L. W. Laws. Since optimal growth of both sublines occurred in the presence of drugs, the question arose as to whether drug action was in any way conditioned by the presence of the virus. have reported on the sparing effect of A-Methopterin and, to a lesser degree, of 8-azaguanine on mice inoculated with otherwise uniformly lethal doses of LCM. The drugs did not prevent virus survival and replication, but they did prevent death of a large proportion of the mice inoculated with the virus. Humphreys et aZ.6 reported on a contaminant in the same AMD, FX A-Methopterin-dependent subline of leukemia L 1210 referred to above; this agent retarded growth of the tumor if A-Methopterin was withheld. The contaminant was identified as LCM virus.’ The tumor, ordinarily growing optimally in the presence of A-Methopterin, developed equally well in mice immunized against the contaminating agent, even though A-Methopterin was withheld. The “contaminant-free ” leukemia (tumor passed through immune mice) was relatively resistant to A-Methopterin. These reported findings are of interest to experimental chemotherapy of mouse tumors, for mice are known to harbor latent viruses, one of which is LCM. I t is conceivable that a latent virus in tumor cells, or elsewhere in the host, could be activated by the drug under study. The presence of a n active virus in a tumor could make it difficult to interpret the results of chemotherapy studies. Haas and S t e ~ a r t , ~ and Haas et

The Polyoma Virus Section B

Summary Particles having the known dimensions of the influenza virus are observed in electron micrographs of thin sections cut from infected chorioallantoic membranes and mouse lungs. These particles are in groups and clusters apparently developing from the borders of membrane cells and from the walls of the alveoli.

Influence of Tissue Culture Passage, Storage, Temperature and Drying on Viability of S E polyoma Virus

Publisher Summary This chapter discusses polyoma virus, which has characteristics that are typical of many of the well-known viruses. It can be freed from other viruses by the plaque technique, concentrated, and at least partially purified by methods that have been useful for other viruses—namely, adsorption and elution on erythrocytes, centrifugation, alcohol precipitation in the cold, and by a combination of drying and dialysis. Virus or its specific antibody can be measured by in vitro tests, such as cytopathogenic changes in roller tube tissue cultures, hemagglutination, complement fixation, or mouse antibody production. Virus production in mouse embryo tissue cells with nucleic acids derived from the SE poIyoina virus or nucleic acid treated with the enzyme ribonuclease has been demonstrated. No virus production has been detected in preliminary experiments with viral nucleic acid treated with deoxyribonuclease. The virus is resistant to many physical and chemical agents that are deleterious to other microorganisms. The virus can be recovered from tumor tissue from animals such as mice, hamsters, rats, rabbits, and guinea pigs in mouse embryo tissue cultures.