DEVELOPMENT/STANDARDIZATION/PRODUCT APPLICATION OF RETROVIRUS DETECTION ASSAYS

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Biological products including vaccines and therapeutics can potentially become contaminated by retroviruses primarily from the cell substrate due to the presence of endogenous retroviral sequences which reside as a part of the host genome. In some cases infectious virus may arise due to induction of an infectious, latent virus or by generation of a novel virus via recombination between endogenous viral sequences. Thus retrovirus detection assays need to be used for demonstrating the absence of known as well as novel retroviral contaminants in the cell substrate and products. Furthermore, mammalian organs/tissues which can be used in xenotransplantation protocols must also be demonstrated free of infectious agents. We have directed our initial efforts towards standardization and utilization of the reverse transcriptase (RT) assay which is a relatively simple, quick and inexpensive assay for the general detection of retroviruses. Although not highly sensitive this assay is useful for the detection of known and novel retroviruses. We assessed the sensitivity and reproducibility of a new RT assay for the detection of HIV-1 and SIV. Our data indicates that the new RT assay is 10-fold better than a published RT assay (Willey, et al., 1988) in a 2-hour incubation period. The new RT assay can be useful for direct detection of retroviruses in virus isolation studies as well as for screening biological products for viral contaminants, subsequent to amplification thru susceptible cells. We are currently optimizing RT assays for the detection of other retroviruses including simian retroviruses which can potentially contaminate vaccines grown in monkey cell substrates. Current efforts are also directed towards developing and optimizing infectivity assays for the detection of potential retroviruses which may arise in primate cell substrates or in simian tissues used in xenotranplantation. Our present focus is simian foamy virus (SFV) which is widespread in monkeys and has a broad tropism for mamalian and avian cells. We have discovered that SFV can replicate very efficiently in Mus dunni cells. We have compared the replication kinetics of SFV in Mus dunni cells with its growth in other currently used cell lines for SFV replication, including dog, monkey and human cell lines. Our data based upon CPE and RT analysis indicate that SFV replicates most efficiently in Mus dunni cells. Thus these cells may be useful for the detection of SFV contamination in biological materials. We are also developing sensitive PCR assays for the direct detection of SFV in tissues. This approach will be used to evaluate the potential risk of SFV infection in man by studying SFV infection and interaction with other retroviruses using a monkey model.