Biological products can potentially be contaminated with an infectious retrovirus, which can arise due to induction of an endogenous, latent virus or by recombination between endogenous retroviral sequences to produce a novel virus. Thus sensitive retrovirus detection assays are necessary to demonstrate the absence of known and novel retroviral contaminants in cell substrates used for production of vaccines and therapeutics and for screening xenotransplants. 1) Reverse transcriptase activity has been found in chicken cell-derived vaccines using the highly sensitive PERT assay. The RT activity is produced from the cell substrate and is associated with endogenous retroviral sequences related to ALV and EAV. Infectivity studies using a variety of human cell lines and PBMCs indicated the absence of replication-competent virus in the RT activity produced from chicken embryo fibroblasts. Furthermore, no replicating retrovirus was detected by the PERT assay in human PBMCs inoculated with measles vaccine material. Highly sensitive PCR assays, including Alu-PCR, which can specifically detect viral-cell integration junctions, were used to investigate entry and integration of EAV and ALV sequences in human PBMC DNA that had been inoculated with measles vaccine material. These studies indicated the absence of infectious retrovirus in the measles vaccine and alleviated public concerns regarding vaccine safety. 2) Simian foamy virus (SFV) is highly prevalent in non-human primates and can infect humans by cross-species transfer from infected animals. Sensitive biological and molecular assays were developed to identify SFV infections and to screen biological products. In an effort to evaluate potential outcome of SFV infection in humans, studies are underway to investigate virus replication of primary SFV isolates by infection of different human cell lines and by functional analysis of their LTRs in CAT assays. A biological detection assay for quantitative assessment of SFV is also being developed. These studies will provide important information regarding mechanism and determinants of virus replication which may be useful in assessing the potential risk of SFV cross-infection in humans. 3) Retrovirus induction assay using IdU has been established to investigate the presence of latent, infectious retroviruses in vaccine cell substrates. PERT analysis of IdU-induced K-Balb mouse cells indicated early and late production of retrovirus from the induced cells. Retrovirus production correlated with inducer dose. Studies are underway to analyze the induction of different retrovirus types from the mouse cells. Additional studies are directed towards optimizing induction conditions for activation of endogenous retroviruses from primate cells. These results may help in developing a general strategy for sensitive detection of latent, infectious retroviruses in vaccine cell substrates.
