DEVELOPMENT/STANDARDIZATION/PRODUCT APPLICATION OF RETROVIRUS DETECTION ASSAYS

逆转录病毒检测方法的开发/标准化/产品应用

• 批准号: 6101185
• 负责人: Arifa S Khan
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6101185
• 关键词: RNA directed DNA polymerase; Retroviridae; animal tissue; diagnosis quality /standard; drug quality /standard; human immunodeficiency virus 1; method development; microorganism culture; polymerase chain reaction; simian immunodeficiency virus; tissue /cell culture; viral vaccines; virus replication

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.

疫苗和治疗剂等生物制品可能 被逆转录病毒主要从其细胞基质污染 由于存在内源性逆转录病毒序列， 宿主基因组的一部分在某些情况下，感染性病毒可能会出现， 诱导感染性潜伏病毒或通过产生新的 通过内源性病毒序列之间的重组获得病毒。因此 需要使用逆转录病毒检测试验来证明 细胞基质中已知的以及新的逆转录病毒污染物， 生物制品。此外，哺乳动物器官和组织用于 人类异种移植还必须证明没有 感染性病原体，以尽量减少跨物种感染的可能性。 建立了一种高灵敏度的PCR检测方法， 逆转录病毒序列在人类DNA中的整合潜力。战略 是基于病毒-细胞连接片段的扩增， 整合在高度重复的人Alu中的病毒LTR 重复区域。优化了PCR反应条件， 扩增病毒-Alu片段和增强病毒-Alu检测 整合者该试验用于研究整合 内源性禽类逆转录病毒序列在人类DNA中的潜力。这 将解决有关存在的潜在安全问题， 鸡细胞衍生疫苗中的缺陷禽逆转录病毒颗粒。 猿猴泡沫病毒（SFV）在非人灵长类动物中高度流行， 具有广泛的宿主范围，包括人类。 事实上，最近的报道 这表明人类感染可能是由于跨物种转移 感染的动物。因此，开发了灵敏的检测方法来鉴定 SFV感染和筛选生物制品。 其中包括一般 和特异性检测测定，例如使用高度特异性的免疫组织化学的病毒分离， 敏感细胞系（M. Dunni），逆转录酶测定， 免疫荧光法、Western blot法和PCR法。 这些测定可 检测SFV血清型1、2和3的原型菌株。这些测定 用于猴SFV感染的多组合分析 和传播，以评估SFV感染的潜在风险 在人类身上。 已建立使用IdU的逆转录病毒诱导试验来研究 疫苗细胞中潜伏感染性逆转录病毒的存在 印刷受体.