Gammaretroviruses in Biologics

摘要：使用猪组织或细胞的临床试验正在进行中。由于所有的猪在其基因组中携带猪内源性逆转录病毒（PERV），这些异种移植程序具有暴露于PERV和传播PERV的风险。今年，我们分析了来自几个不同物种的非人灵长类动物（恒河猴、狒狒和非洲绿色猴）的原代细胞和细胞系是否允许PERV复制。 我们进行该分析以确定非人灵长类动物是否是用于评估PERV的体内感染性和致病性潜力的合适模型。 在所有情况下，我们发现病毒可以进入细胞，整合并表达病毒RNA，但病毒感染在NHP培养物中没有扩散。 进一步的分析表明，对允许复制的阻断可能发生在几个不同的阶段，包括无效的病毒进入和病毒组装（A. Ritzhaupt等人，J. Virol.，In Press）. 分析表明，NHP不太可能作为PERV体内复制的允许模型，因此，应谨慎解释此类研究的数据。 作为该项目的组成部分，我们开发了用于检测和定量PERV RNA和DNA的灵敏测定法，其可用于分析来自细胞培养实验、动物组织和人临床样品的样品（Argaw等人，J. Virol.方法，出版中）。 我们完成的第二个项目涉及对源自猪细胞的PERV的分析，所述猪细胞被工程化以表达人补体调节蛋白之一CD59（Takefman等人，J. Virol. 75：4551）。 本研究的目的是重现该分子转基因猪可能发生的情况。 已经提出，从人CRP转基因猪表达的PERV可能对补体介导的保护的病毒溶解和中和活性具有抗性。 我们发现，人CD59蛋白被纳入病毒体来源于猪细胞表达hCD59，这种分子的存在抑制补体介导的裂解，但不中和PERV的感染性。 因此，针对PERV感染的补体介导的保护的一个组分仍然保留。

Summary: Clinical trials using porcine tissues or cells are underway. Since all pigs carry in their genome porcine endogenous retroviruses (PERV), these xenotransplantation procedures carry the risk of exposure to and transmission of PERV. This year, we analyzed whether primary cells and cell lines derived from non-human primates of several different species (rhesus macaque, baboon, and African green monkey) were permissive to PERV replication. We performed this analysis to determine whether non-human primates are a suitable model for assessing in vivo infectivity and potential for pathogenicity of PERV. In all cases, we found that the virus could enter the cells, become integrated, and express viral RNA, but the viral infection did not spread in the NHP cultures. Further analyses demonstrated that the block to permissive replication may occur at several different stages, including inefficient viral entry and viral assembly (A. Ritzhaupt, et al, J. Virol., In Press). The analysis demonstrated that NHP do not likely serve as a permissive model for PERV replication in vivo, and therefore, data from such studies should be interpreted with caution. As a component of this project, we developed a sensitive assay for detection and quantitation of PERV RNA and DNA that can be used to analyze samples from cell culture experiments, animal tissues, and human clinical samples (Argaw, et al, J. Virol. Methods, In Press). The second project we completed involved an analysis of PERV derived from pig cells that were engineered to express one of the human complement regulatory proteins, CD59 (Takefman, et al, J. Virol. 75:4551). The aim of this study was to reproduce what would likely happen in pigs transgenic for this molecule. It has been proposed that PERV expressed from pigs that are transgenic for human CRPs may be resistant to the virolysis and neutralizing activity of complement-mediated protection. We found that the human CD59 protein is incorporated into virions derived from pig cells expressing hCD59, and that the presence of this molecule inhibited complement-mediated lysis, but not neutralization of infectivity of PERV. Therefore, one component of complement-mediated protection against PERV infection is still retained.