Biological products such as vaccines, therapeutics and xenotransplants can potentially be contaminated with infectious retrovirus that may arise from the cell substrate or tissue by induction of an endogenous, latent virus or by recombination between endogenous retroviral sequences to produce a novel virus. Thus development of sensitive retrovirus detection assays and studies to evaluate potential human risk due to an unexpected and inadvertent retrovirus infection are important to public health safety. 1) A low level reverse transcriptase (RT) activity, is produced from chick embryo fibroblasts (CEF) and therefore is present in all chicken cell-derived vaccines, such as measles virus vaccine. The RT activity was detected by a highly sensitive PCR-based RT assay (PERT) and is associated with avian endogenous retroviral sequences related to ALV and EAV families. Infectivity studies using a variety of human cell lines and PBMCs demonstrated the absence of replication-competent retrovirus in the RT activity produced by CEF. Furthermore, there was no evidence of integration by avian retroviral sequences in human DNA using highly sensitive DNA PCR assays, including Alu-PCR, which can specifically detect viral-cell junctions. Similar results were found with a measles vaccine equivalent preparation (MVE). These studies confirmed the absence of an infectious human-tropic 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 were done to investigate virus replication of primary SFV isolates obtained from rhesus and pig- tailed macaques. Infection of 6 different human cell lines identified one for latent SFV infection. This cell line will be used to study SFV latency in humans and regulation of SFV activation in cell substrate used in production of biologics. 3) Retrovirus induction assays may detect the presence of known and novel, latent, infectious retroviruses in vaccine cell substrates. Induction conditions were optimized using 5'- iododeoxyuridine and 5'-azacytidine on K-Balb mouse cells as a model system. PERT analysis indicated early and late production of retrovirus from the induced mouse cells. Retrovirus production correlated with inducer dose. Type C and type A particles were induced from the mouse cells. Conditions used for activation of mouse cells were not successful in activating endogenous retrovirus from non-murine cells. Other strategies are underway to evalute induction of retroviruses in primate cells. These results may help in developing a general strategy for sensitive detection of latent, infectious retroviruses in vaccine cell substrates.
