DEVELOPMENT/STANDARDIZATION/PRODUCT APPLICATION OF RETROVIRUS DETECTION ASSAYS

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We have directed efforts towards development of strategies and assays for the detection of low levels of retrovirus in cell substrates and biological materials used in product development or in xenotransplants. We have developed and standardized an improved reverse transcriptase (RT) assay for the detection of Mg++-requiring retroviruses including HIV, SIV, and type D retroviruses. The improved RT assay can be useful for the detection of known as well as novel retroviruses in biological products using a primate cell substrate. We are also optimizing and standardizing RT assays for the detection of other retroviruses which may contaminate biologics. In addition, we have developed sensitive detection assays for simian foamy virus (SFV) which is a potential contaminant of simian-derived biologics including vaccines and xenotransplants. These include identification of the Mus dunni cell line as highly susceptible for virus isolation; development of PCR primers for direct analysis of infected cells and tissues and Western Blot and IFA for detection of SFV antibodies in simian and human infections. Evaluation of cell substrates and biological materials and products for viral contamination is critical for product safety. Retroviral sequences are present in all species; the majority are defective but in some cases infectious viruses occur which may persist as an integrat part of the host genome and perhaps cause disease. Therefore, it is critical to demonstrate absence of retroviruses in biological materials intended for human use. These include cell substrates, raw materials, final products as well as tissues/organs used for xenotransplants. Thus, assays for detection of low level retrovirus contamination as well as for detection of known as well as novel retroviruses need to be developed/optimized and standardized for evaluation of product safety. Furthermore, experience with the assays will also enable us to provide expert guidance and review of submissions for evaluation at the FDA.