DEVELOPMENT AND STANDARDIZATION OF RETROVIRUS DETECTION ASSAYS AND SAFETY STUDIES

生物制品可能会受到传染性逆转录病毒的污染，这可能是由于诱导内源性潜伏病毒或通过内源性逆转录病毒序列之间的重组产生新的病毒而引起的。因此，有必要进行敏感的逆转录病毒检测分析，以证明用于生产疫苗和治疗药物以及筛选异种移植的细胞底物中没有已知和新的逆转录病毒污染物。1)用高灵敏的PERT法在鸡细胞衍生疫苗中发现了逆转录酶活性。RT活性是由细胞底物产生的，与ALV和EAV相关的内源性逆转录病毒序列有关。使用各种人类细胞系和PBMC进行的感染性研究表明，从鸡胚胎成纤维细胞产生的RT活性中不存在复制能力强的病毒。经PERT检测，接种麻疹疫苗材料的人PBMC中未检测到复制型逆转录病毒。应用高灵敏度的聚合酶链式反应技术，包括Alu-PCR技术，研究了EaV和ALV序列在麻疹疫苗接种的人外周血单核细胞DNA中的进入和整合情况。这些研究表明，麻疹疫苗中没有传染性逆转录病毒，并缓解了公众对疫苗安全性的担忧。2)猴泡沫病毒(SFV)在非人类灵长类动物中高度流行，可通过受感染动物的跨物种传播感染人类。开发了敏感的生物学和分子检测方法来识别SFV感染和筛选生物制品。为了评估在人类中感染SFV的潜在后果，正在进行研究，通过感染不同的人类细胞系来调查SFV初级分离株的病毒复制，并通过CAT检测中对它们的LTRs进行功能分析。还在开发一种用于定量评估SFV的生物检测方法。这些研究将提供有关病毒复制机制和决定因素的重要信息，这可能有助于评估SFV在人类中交叉感染的潜在风险。3)建立了IDU逆转录病毒诱导试验，用于研究疫苗细胞底物中潜伏的、传染性的逆转录病毒的存在。对IDU诱导的K-Balb小鼠细胞的PERT分析表明，诱导细胞产生逆转录病毒的早期和晚期。逆转录病毒的产生与诱导剂剂量相关。目前正在进行研究，以分析从小鼠细胞诱导不同类型逆转录病毒的情况。其他研究针对的是优化从灵长类细胞激活内源性逆转录病毒的诱导条件。这些结果可能有助于开发一种灵敏地检测疫苗细胞底物中潜伏的传染性逆转录病毒的一般策略。

Biological products can potentially be contaminated with an infectious retrovirus, which can arise due to induction of an endogenous, latent virus or by recombination between endogenous retroviral sequences to produce a novel virus. Thus sensitive retrovirus detection assays are necessary to demonstrate the absence of known and novel retroviral contaminants in cell substrates used for production of vaccines and therapeutics and for screening xenotransplants. 1) Reverse transcriptase activity has been found in chicken cell-derived vaccines using the highly sensitive PERT assay. The RT activity is produced from the cell substrate and is associated with endogenous retroviral sequences related to ALV and EAV. Infectivity studies using a variety of human cell lines and PBMCs indicated the absence of replication-competent virus in the RT activity produced from chicken embryo fibroblasts. Furthermore, no replicating retrovirus was detected by the PERT assay in human PBMCs inoculated with measles vaccine material. Highly sensitive PCR assays, including Alu-PCR, which can specifically detect viral-cell integration junctions, were used to investigate entry and integration of EAV and ALV sequences in human PBMC DNA that had been inoculated with measles vaccine material. These studies indicated the absence of infectious 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 are underway to investigate virus replication of primary SFV isolates by infection of different human cell lines and by functional analysis of their LTRs in CAT assays. A biological detection assay for quantitative assessment of SFV is also being developed. These studies will provide important information regarding mechanism and determinants of virus replication which may be useful in assessing the potential risk of SFV cross-infection in humans. 3) Retrovirus induction assay using IdU has been established to investigate the presence of latent, infectious retroviruses in vaccine cell substrates. PERT analysis of IdU-induced K-Balb mouse cells indicated early and late production of retrovirus from the induced cells. Retrovirus production correlated with inducer dose. Studies are underway to analyze the induction of different retrovirus types from the mouse cells. Additional studies are directed towards optimizing induction conditions for activation of endogenous retroviruses from primate cells. These results may help in developing a general strategy for sensitive detection of latent, infectious retroviruses in vaccine cell substrates.