STUDIES OF ADENOVIRUS-SV 40 HYBRID VIRUSES , IV . AN ADENOVIRUS TYPE 2 STRAIN CARRYING THE INFECTIOUS SV 40 GENOME

Recent studies'-5 have demonstrated that a number of human adenoviruses are capable of forming intergeneric "hybrids," or transcapsidants,5 with SV40 genetic material when propagated in monkey kidney cells naturally or deliberately contaminated with SV40. Such hybrids were recognized by their ability to induce SV40 T antigen detectable by complement fixation (CF) and immunofluorescent (FA) techniques, and in some cases by induction of tumors having the cytologic and antigenic characteristics of those induced by SV40 virus. The infectious SV40 was eliminated from one of these adenovirus-SV40 (Ad.-SV40) hybrids, the LL (E46) strain, by two serial passages with SV40 antiserum; this strain appears to carry only a portion of the SV40 genome. However, a number of the other hybrid strains contained infectious SV40 after 4-10 serial passages with SV40 antiserum4' at 7; and heat inactivation, serologic, and virus cloning studies7 I on the hybrid type 4 strain R.N.4 suggested that the persistence of SV40 was due to incorporation of the complete SV40 genome into adenovirus capsids. This report presents further evidence for this phenomenon, using the monkey kidney-adapted adenovirus type 2 (Ad. 2) vaccine strain Ind. 2.4 9 Materials and Methods.-Virus: The passage history of the Ind. 2 strain of Ad. 2 has been described.4 Briefly, this strain was isolated from a human adenoid culture and adapted to rhesus monkey kidney cells naturally infected with SV40 virus. It was subsequently passed four times in African green monkey kidney (AGMK) cultures with SV40 rabbit antiserum in an unsuccessful attempt to remove the SV40 contaminant. Previous studies4 showed that this strain was carrying SV40 genetic material in adenovirus capsids, as judged by induction of SV40 T antigen. The preparations used in the present studies were from the first three subsequent AGMK passages beyond the antiserum passages. Stocks were prepared by inoculating 32-oz flask cultures of AGMK with 1 ml of undiluted virus, harvesting at complete cytopathogenicity (CPE), freezethawing three times, clarifying by low-speed centrifugation, and storing at -70'C. Some experiments were done with the adenovirus band (density 1.33) obtained by two cycles of isopycnic density gradient centrifugation in RbCl. Tissue cultures: Human embryonic kidney (HEK) and AGMK cultures were obtained from Flow Laboratories, Rockville, Md., and Microbiological Associates, Inc., Bethesda, Md. All cultures were maintained in Eagle's basal medium no. 2 (BME) with 2% agammaglobulinic calf serum, glutamine, and antibiotics. Virus titrations: Extinction dilution titrations were done by the inoculation of serial tenfold dilutions into HEK or AGMK tube cultures, 0.1 ml per tube. Four tubes were inoculated per dilution and cultures were observed biweekly for periods of 21-30 days. AGMK cultures were scored for both adenovirus and SV40 CPE, and when the latter was seen near the endpoint of an adenovirus titration the presence of SV40 was confirmed by passage with and without SV40 antiserum. Plaque titrations were done in secondary cultures of AGMK cells by a previously described technique.10 Antisera: Adenovirus and SV40 rabbit antisera were prepared against prototype strains by standard techniques.11 The Ad. 2 sera had neutralizing antibody titers of 1:320, and the SV40 sera, 1:2560 and 1:5120. Fluorescent antibody assays: Procedures for infection, fixation, and immunofluorescent staining of coverslip cultures have been described.'2' 13 Pooled serum from hamsters with SV40 tumors