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: 6161250
• 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/6161250
• 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

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.

我们一直在努力制定战略和分析方法，以 细胞底物中低水平逆转录病毒的检测 用于产品开发或异种移植的生物材料。我们 已经开发并标准化了一种改进的逆转录酶(RT) 检测需要镁离子的逆转录病毒包括HIV，SIV， D型逆转录病毒。改进的RT检测方法可用于检测 生物制品中已知和新型逆转录病毒的检测 使用灵长类细胞底物。我们还在优化和标准化 RT检测可能污染的其他逆转录病毒 生物制品。此外，我们还开发了灵敏的检测方法 猴泡沫病毒(SFV)是一种潜在的污染物 猿源生物制剂，包括疫苗和异种移植。这些 包括将小鼠邓尼氏细胞系鉴定为高度敏感 病毒分离；直接分析病毒的聚合酶链式反应引物的开发 感染细胞和组织与Western Blot和IFA检测SFV 猿猴和人类感染中的抗体。 细胞底物、生物材料和产品的评价 病毒污染对产品安全至关重要。逆转录病毒序列 在所有物种中都存在；大多数是有缺陷的，但在某些情况下 传染性病毒可能作为宿主的一部分持续存在。 基因组，也许会导致疾病。因此，至关重要的是 证明生物材料中不存在逆转录病毒 为人类所用。这些包括电池衬底、原材料、最终产品 以及用于异种移植的组织/器官。因此，对 检测低水平逆转录病毒污染以及检测 的已知和新的逆转录病毒需要开发/优化和 产品安全评价的标准化。此外，体验 还将使我们能够提供专家指导和审查 提交给FDA进行评估。