(1) Regulatory Analysis a) Sensitive PCR-enhanced reverse transcriptase (PERT) assays have detected RT activity in viral vaccines produced in chicken cells. Cells susceptible to infection with various retrovirus types were co-cultivated with chick embryo fibroblasts (CEF) or CEF supernatants and were subsequently examined by EM. So far, 200-300 cells from each of 28 samples have been studied and no adventitious agents have been found. b) As part of studies to define a sensitive and reproducible test for the presence of foamy retrovirus, 4 different cell lines (Mus dunni, CF2TH, HeLa and Vero) were infected with foamy virus and examined by EM at different times post-infection. The rate of viral particle formation was different for each cell type and was inversely correlated with the interval from infection to CPE. (2) Research Using immunoEM we have previously shown that transfection of the MHC Class I H-2Kb or H-2 Kd, but not the H-2Dd or H-2Ld genes, into clones of the B16 melanoma results in the elimination of two actively produced endogenous retroviruses. The absence of the intracisternal A-type particles was found to be due to lack of IAP-specific mRNA, while the loss of ecotropic C-type retroviral particles was associated with rearrangements of the proviral DNA. In recent studies, JB/RH, a carcinogen-induced melanoma of C57BL/6 mice unrelated to B16, was transfected with the H-2Kb gene. This melanoma produces IAP and a C-type retrovirus with envelope antigens similar to those of the B16 C-type retrovirus. Following transfection with H-2Kb production of both IAP and C-type retroviruses was eliminated from the JB/RH cells. In other studies, a normal mouse melanocyte cell line, Melan-A, was infected with the C-type virus from B16 BL6 cells. These cells became productively infected with the C-type virus and also began expressing A-type retroviral particles. Moreover they show evidence of a transformed phenotype (tumor formation in mice, TPA-independence in culture). (3) Research support a) Examination of cell cultures containing plasmids with viral sequences for evidence of viral particle formatiion. b) Immunogold labeling of surface antigens on tuberculosis bacteria. c) Examination of liver biopsies for evidence of infection with Hepatitis viruses.
