A. J. Girardi

Development of Tumors in Hamsters Inoculated in the Neonatal Period with Vacuolating Virus, SV40

Summary Intracerebral and subcutaneous injection into newborn hamsters of vacuolating virus, SV40, grown in renal cell cultures of grivet monkey resulted in single or multiple fibrosarcomas at site of injection which were histologically of varying degree of malignancy. These occurred 3 1/2 to 8 months post-inoculation and in both sexes. Animals injected with appropriate control materials or held uninoculated failed to develop tumors. Tests to exclude mouse polyoma virus as a factor were clearly negative. Evidence for the role of SV40 virus as a primary oncogenic agent was provided by recovery of the virus from the tumor and by demonstration of SV40 antigen in tumors by fluorescent antibody staining. The agent appeared to be localized in the tumors since the virus was not detected in blood or excretions, since antibody response was minimal or lacking, and since gross metastases were lacking. Transplantation and serial passage of SV40-induced tumors were accomplished with ease. The data represent the first definitive evidence implicating a virus of primate origin as a malignant oncogenic agent in experimental animals. The findings relate to observations of oncogenesis in hamsters and do not warrant extrapolation to oncogenesis in other mammalian species.

TESTS IN HAMSTERS FOR ONCOGENIC QUALITY OF ORDINARY VIRUSES INCLUDING ADENOVIRUS TYPE 7.

Summary Studies were conducted to measure the in vivo oncogenic potential of a variety of “ordinary” viruses. Adenovirus 1, 2, 7, 12 and 18, influenza A2, parainfluenza 1, 2 and 3, respiratory syncytial, mumps, measles, rhinovirus 11, 23 and 30, poliovirus 3, salivary gland virus, varicella, pneumonia virus of mice, Rous sarcoma, visceral lym-phomatosis and resistance-inducing factor (avian leucosis) viruses were tested for ability to induce tumors in hamsters inoculated when newborn, A majority of animals given adenovirus types 12 or 18 developed tumors. Additionally, a portion of animals which received type 7 adenovirus developed sarcomas. Evidence is presented for the significance of type 7 adenovirus in tumor development. Two animals given Bryans Rous sarcoma virus also developed sarcomas. None of the animals which received any of the other viruses presented significant evidence for induction of malignancy. Studies of additional viruses are in progress. Addendum: Since the time the present manuscript was submitted, type 7 Pickney virus was found to belong to adenovirus hemagglutinin group 1 in that it agglutinated rhesus but not rat erythrocytes. The strain was identified serologically with antisera to various type 7 adenoviruses and there was no serologic crossing with type 12 or 18 adenovirus. In a repeat experiment. 7 of 60 hamsters inoculated with type 7 Pinckney virus developed tumors to date. A portion of newborn hamsters inoculated with two additional type 7 adenoviruses, Grider and Champagne, have developed palpable masses resembling tumor, suggesting that tumori-genesis may be a generic quality of type 7 adenoviruses. The authors are indebted to W. Raupp, T. Bed-dow, and R. Grady for valuable technical assistance.

Interruption of SV40 Virus Tumorigenesis Using Irradiated Homologous Tumor Antigen.

Summary X-irradiated SV40 transplant tumor or cell culture antigens were highly effective in preventing occurrence of homologous tumor in hamsters inoculated with SV40 virus when newborn. The X-irradiated immunizing tumor antigen was effective when given in a single dose as early as 34 days or as late as 76 days after SV40 virus was administered. The immunizing antigen was retained in tumor transplants in hamster for at least 20 passages and in cell culture of tumor for at least 38 passages. The significance of the findings in relation to protection against the hypothetical virus-induced tumor in man is discussed.

Factors Influencing Tumor Induction in Hamsters by Vacuolating Virus, SV40.∗

Summary Descriptive studies of the pathogenesis of SV40 virus in the hamster model were carried out. Ninety-six per cent of animals given SV40 virus when newborn, 60% of 7 day old animals, 23% of 1 month old animals, and none of 3 month old animals developed tumors. Tumors developed earlier, on the average, in the younger animals. All tumors which did develop were highly malignant, irrespective of host age at time of inoculation. Development of SV40 tumors in hamsters was unrelated to appearance or absence of SV40 antibody although stimulation of such antibody was progressively enhanced with increase in age at time of inoculation. Tumorigenesis was also related to virus dose, 300.000 TCD50, being required to effect near 100% efficiency and 1000 TCD50 being necessary for minimal response. The subcutaneous route was most efficient for tumor induction. A few tumors followed intrapulmonary or intraperitoneal inoculation and these appeared in the subcutaneous tissue. Litter or cage mate contact transmission of tumorigenesis did not occur. The significance of the findings is discussed.

Studies of Oncogenicity of Adenovirus Type 7 Viruses in Hamsters.

Summary All of 3 strains of type 7 ade-novirus isolated in primary human embryonic kidney cell cultures from respiratory secretions of human subjects with acute respiratory illness proved oncogenic for newborn hamsters. The type 7 viruses were isolated and propagated in human embryonic cell cultures and were free of extraneous agents. Detailed information relative to the virology, oncology, pathology and immunology of the type 7 adenovirus-hamster tumor system is presented. The tumors appeared after a long latent period, were highly malignant, were readily transplantable and were free of detectable infectious virus. The cells in most of the tumors were sufficiently differentiated to permit a pathologic diagnosis of malignant lymphoma, or lymphosarcoma when evidence of invasiveness was present. A majority of tumors induced by the virus presented a specific complement-fixing antigen which reacted with sera from hamsters bearing homologous tumor. The authors are indebted to T. Beddow, W. Clark, P. Gosnell, B. Keech and W. Raupp for technical assistance.

Further Tests in Hamsters for Oncogenic Quality of Ordinary Viruses and Mycoplasma, with Correlative Review.

Summary Previous and present findings demonstrated the failure of tumor induction in newborn hamsters by adenovirus 1, 2, 3 (tentative), 4, 5, and 6 (tentative), vaccinia, herpes simplex, varicella, cytomegalo-virus, reovirus 1 (tentative), poliovirus 1 and 3, ECHO 25, Coxsackie A10 and A21, rhino virus 11, 23 and 30, encephalo-myocarditis, chikungunya, Hawaiian dengue, Marituba, Sicilian sandfly, influenza A2, parainfluenza 1, 2 and 3, respiratory syn-cytial, mumps, measles (virulent and attenuated), rubella, rabies, visceral lymphomatosis (including RPL-12 and RIF agent), pneumonia virus of mice and lymphocytic chorio-meningitis viruses and two Mycoplasma species including Eaton agent. Adenovirus 7 was moderately and adenovirus 12 and 18 were highly oncogenic. Tumors occurred in 2 animals which Received Bryan strain Rous sarcoma. The findings are discussed in relation to the familial, biophysical, biochemical and structural properties of the viruses studied and with respect to the evidences which suggest a possible role of virus in human neo-plasia. The authors are indebted to W. Raupp, T. Bed-dow, R. Grady and W. Clark for valuable technical assistance.

Search for Virus in Human Malignancies. 2. In vivo Studies.

Summary Exhaustive investigations were made employing cell culture methods to recover virus from specimens from human cases of leukemia and other malignancies. Altogether, 316 clinical specimens were tested. Primary culture of the human malignant tissue and passage of extracts of clinical specimens to primary cell cultures and to a cell strain were done. The specimens were tested crude and after treatment to remove hypothetical inhibitors and the cultures were observed by a variety of procedures to detect virus by direct and indirect methods as well as before and after treatment to remove inhibitors. No detectable virus or virus-like agent of human malignant tissue origin was recovered. The finding of a simian virus multinucleating agent, MNA, emphasized the need for reliable controls and for caution in interpretation. The negative findings in these experiments were interpreted as examples of methods which failed and were not construed as positive evidence for a non-viral etiology of malignancies in man. W. Raupp contributed significantly to the studies in the conduct of the virus recovery attempts, and E. A. Ridley provided technical assistance. Specimens from cases of human malignancy were kindly supplied by Dr. Irving Wolman, Childrens Hospital of Philadelphia, by Dr. H. T. Tamaki, Montgomery Hospital, Norristown, Pa. and by Dr. J. Gershon-Cohen, Albert Einstein Medical Center, Philadelphia. Collection of specimens by Dr. Wolman was supported in part by grant from U. S. Public Health Service.

ATTEMPTS TO INTERRUPT VIRUS TUMORIGENESIS BY IMMUNIZATION USING HOMOLOGOUS "BJORKLUND-TYPE" ANTIGEN.

Summary Studies were carried out to determine whether “Bjorklund-type” lipoprotein tumor antigen could interrupt the appearance of tumors in animals infected at birth with oncogenic viruses. The polyoma and SV40 hamster systems were employed. “Bjorklund-type” antigen was prepared from corresponding virus-induced tumor and was made into aqueous and mineral oil adjuvant formulations. These vaccines were given during the time period between virus inoculation and first appearance of tumors. Also, adult animals, not previously exposed to virus, were immunized prior to challenge with homologous tumor transplant. None of the adult hamsters given “Bjorklund-type” antigens developed detectable cytolytic or precipitating antibody against homologous tumor. The vaccine was antigenic, however, as proved by development of such antibody in the heterologous guinea pig and rabbit species. The experiments were designed to determine whether protection would be afforded by the vaccine and such was clearly not obtained. Instead, a small but statistically significant enhancement of tumor induction was noted. The implications of these findings are discussed and a word of caution is given, on theoretical bases, for studies of such vaccines in man.

HOST-VIRUS RELATIONSHIPS IN HAMSTERS INOCULATED WITH SV40 VIRUS DURING THE NEONATAL PERIOD.

Summary Findings relative to the pathogenesis of SV40 virus in inducing tumor in hamsters are discussed. Infectious virus disappeared rapidly from the animal and was not demonstrable until its appearance in tumor. The quantity of virus was related to tumor size. Infectious virus was commonly demonstrable in tumor but was not always present and appeared not to be essential to retention of oncogenic quality. Initial neutralizing antibody response in a portion of animals to inoculated SV40 virus appeared unrelated to subsequent development or failure to develop tumor. The technical assistance of W. Raupp and T. Beddow is gratefully acknowledged.

Search for virus in human malignancies. 4. Tests of human neoplastic tissues in sex-segregated mice.

Summary A total of 38 neoplastic tissue preparations from a variety of human cancers were tested for presence of a tumor-inducing principle in newborn ICR/Ha mice. The mice were sex-segregated to prevent pregnancy and to limit occurrence of spontaneous mammary carcinoma. The malignant tissue specimens were tested as crude extracts, after fluorocarbon extraction, or following treatment by the Burton-Friedman method which was reported to make possible tumor induction in mice. There was no evidence for a tumor-inducing principle in any of the specimens tested. Numerous spontaneous neoplasias were observed and mammary carcinoma and malignant lymphoma were most commonly present.