Development and standardization of retrovirus detection

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Summary: Biological products such as vaccines, therapeutics and xenotransplants can potentially be contaminated with infectious retrovirus due to induction of an endogenous, latent virus in the cell substrate or xenotranplant tissue or by recombination between endogenous retroviral sequences to produce a novel virus. Additionally, blood and blood products may contain viruses due to the host donor. Thus, to assure public health safety in biolgics, it is critical to develop sensitive retrovirus detection assays and to evaluate potential human risk of retroviruses, which may be of potential safety concern. 1) Safety evaluation of reverse transcriptase activity that is present in all chicken cell derived vaccines has been completed. The results of infectivity and integration studies demonstrated the absence of an infectious human-tropic retrovirus in the measles vaccine. Thus public concerns regarding vaccine safety were alleviated and childhood vaccinations continued without interruption. 2) SFV infections in humans have been reported due to accidental, occupational exposure to infected non-human primates. Therefore, biological products using materials of simian origin need to be demonstrated to be free of SFV. To evaluate the potential risk of SFV infection and transmission in humans, naturally-occurring SFVs were isolated at low passage from peripheral blood mononuclear cells (PBMCs) of rhesus and pig-tailed macaques. Initial infectivity studies indicated that the naturally-occurring macaque isolates had slower replication kinetics than the laboratory-adapted prototype viruses designated as SFV-1 and SFV-2. Further studies using a variety of human cell types have shown that virus production is strain-specific and cell type dependent. Latent infection was seen with all of the naturally-occurring SFVs in one human tumor cell line. Investigations of the viral determinants of latency were initiated by analysis of LTR function. The results indicated promoter activity using the chloramphenoicoal acetyl transferase gene expression assay (CAT assay). Other viral regions, including the internal promoter, are under investigation to identify determinants of virus latency. To investigate SFV infection due to naturally-occurring viruses in non-human primates, a study has been initiated in which virus negative animals were injected with a virus stock prepared from macaque PBMCs. The animals are being monitored virus infection and clinical changes. 3. Endogenous retroviruses can potentially contaminate biological products by activation of viral sequences in the cell susbtrate. We have investigated quantitatively the induction of type A and type C retroviruses in a well-characterized mouse cell line using a highly sensitive PCR based reverse transcriptase assay (PERT). The results indicated earlier and more sensitive detection of retrovirus activation as compared to previous traditional assays. Analysis of different chemical inducers indicated different kinetics of endogenous retrovirus activation in mouse cells. Further analysis will be done to evaluate the different types of retroviruses induced from the cells. The use of different drugs for virus activation may further provide insight into the mechanism of endogenous retrovirus latency. To evaluate endogenous retrovirus induction in primate cell substrates used in vaccine production, initial studies are underway to investigate virus activation in latently infected primate cells using various chemical inducers that have proven to be successful in the mouse system. The results of these studies will provide strategies to evaluate endogenous retrovirus induction in vaccine cell susbtrates.