DEVELOPMENT/STANDARDIZATION/PRODUCT APPLICATION OF RETROVIRUS DETECTION ASSAYS

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Biological products such as vaccines and therapeutics can potentially become contaminated by retroviruses primarily from their 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 cell substrates and biological products. Furthermore, mammalian organs and tissues used in human xenotransplantation must also be demonstrated to be free of infectious agents to minimize potential of cross-species infection. A highly sensitive Alu-PCR assay to was established to detect the integrative potential of retroviral sequences in human DNA. The strategy is based upon amplification of viral-cell junction fragments containing viral LTRs which have integrated in the highly repetitive human Alu repeat regions. The PCR conditions were optimized to reduce the amplification of Alu-Alu fragments and enhance detection of viral-Alu integrants. The assay is being used to investigate the integration potential of endogenous avian retroviral sequences in human DNA . This will address potential safety concerns regarding the presence of defective avian retroviral particles in chicken cell derived vaccines. Simian foamy virus (SFV) is highly prevalent in non-human primates and possess a broad host range including human. In fact, recent reports indicate that human infections can occur due to cross-species transfer from infected animals. Thus sensitive assays were developed to identify SFV infections and to screen biological products. These include general and specific detection assays such as virus isolation using a highly sensitive cell line (M. dunni), reverse transcriptase assay, immunofluorescensce assay, Western blot assay and PCR. These assays can detect prototype strains of SFV serotypes 1, 2, and 3. These assays are being used in multicombinational analysis of monkeys for SFV infection and transmission in an effort to evaluate potential risk of SFV infection in humans. Retroviral induction assay using IdU have been established to investigate the presence of latent, infectious retroviruses in vaccine cell substrates.