Development and standardization of retrovirus detection

摘要：疫苗、治疗药物和异种移植等生物制品可能会受到传染性逆转录病毒的污染，这是因为在细胞底物或异种移植组织中诱导内源性潜伏病毒，或者通过内源性逆转录病毒序列之间的重组产生新的病毒。此外，由于宿主献血者的原因，血液和血液产品可能含有病毒。因此，为了确保生物制药的公共卫生安全，关键是要发展灵敏的逆转录病毒检测方法，并评估人类感染逆转录病毒的潜在风险，这可能是潜在的安全问题。 1)人类感染SFV的报告是由于意外、职业接触受感染的非人类灵长类动物造成的。因此，使用猿源材料的生物制品需要证明不含SFV。为了评估SFV在人类中感染和传播的潜在风险，从恒河猴(Rh)和猪尾(Pt)猕猴的外周血单核细胞(PBMC)低传代时分离到自然发生的SFV。 答：体外传染性研究表明，自然产生的猕猴分离株的复制速度慢于实验室适应的原型病毒，如SFV-1和SFV-2。事实上，在一个人类肿瘤细胞系中，没有复制任何Rh或铂等价体。对病毒潜伏期决定因素的研究包括LTR和内部启动子等调节区。此外，还对反式激活因子(Taf)的表达和功能进行了分析。 为了研究非人类灵长类动物中自然产生的病毒引起的SFV感染，并分析病毒感染的任何潜在致病效应，将SFV阴性动物注射从猕猴PBMCs中分离的SFV制备的病毒菌种。这些动物目前正在接受病毒感染和临床变化的监测。这些结果将为开展实验提供基础，以解决献血者传播SFV的潜在安全问题。 2)内源性逆转录病毒可通过激活细胞悬液中的病毒序列来潜在地污染生物制品。我们用高灵敏的基于聚合酶链式反应的逆转录酶分析(PERT)定量研究了A型和C型逆转录病毒在一个特征良好的小鼠细胞系中的诱导作用。结果表明，与以前的传统检测方法相比，该方法更早、更灵敏地检测到逆转录病毒的激活。对不同化学诱导剂的分析表明，内源性逆转录病毒在小鼠细胞中的激活动力学不同。比较了不同诱导剂对病毒的诱导效果。结果表明，不同的诱导剂对内源性逆转录病毒的产生有不同的调节作用，由于RT细胞RT产生的增加，在数据解释中应谨慎。 为了评估内源性逆转录病毒在疫苗生产中使用的灵长类细胞底物中的诱导作用，最初将细胞暴露在各种化学诱导剂中，条件已被证明在小鼠系统中是成功的。这些研究结果将为评估内源性逆转录病毒在疫苗细胞悬液中的诱导作用提供策略。

Summary: Biological products such as vaccines, therapeutics and xenotransplants can potentially be contaminated with infectious retrovirus due to induction of an endogenous, latent virus in the cell substrate or xenotranplant tissue or by recombination between endogenous retroviral sequences to produce a novel virus. Additionally, blood and blood products may contain viruses due to the host donor. Thus, to assure public health safety in biolgics, it is critical to develop sensitive retrovirus detection assays and to evaluate potential human risk of retroviruses, which may be of potential safety concern. 1) SFV infections in humans have been reported due to accidental, occupational exposure to infected non-human primates. Therefore, biological products using materials of simian origin need to be demonstrated to be free of SFV. To evaluate the potential risk of SFV infection and transmission in humans, naturally-occurring SFVs were isolated at low passage from peripheral blood mononuclear cells (PBMCs) of rhesus (Rh) and pig-tailed (Pt) macaques. A. In vitro infectivity studies indicated that the naturally-occurring macaque isolates had slower replication kinetics than the laboratory-adapted prototype viruses, such as SFV-1 and SFV-2. In fact, in one human tumor cell line, there was no replication of any of the Rh or Pt isoaltes. Investigations of the viral determinants of latency include the regulatory regions such as the LTR and internal promoter. Additionally, expression and function of the transactivating factor (taf) is also being analyzed. B. To investigate SFV infection due to naturally-occurring viruses in non-human primates and analzye any potential pathogenic effect of virus infection, SFV-negative animals were injected with a virus stock prepared from SFV isolated from a macaque PBMCs. The animals are currently being monitored for virus infection and clinical changes. These results will provide a basis to develop experiments to address potential safety concerns regarding SFV transmission by blood donors. 2) Endogenous retroviruses can potentially contaminate biological products by activation of viral sequences in the cell susbtrate. We have investigated quantitatively the induction of type A and type C retroviruses in a well-characterized mouse cell line using a highly sensitive PCR based reverse transcriptase assay (PERT). The results indicated earlier and more sensitive detection of retrovirus activation as compared to previous traditional assays. Analysis of different chemical inducers indicated different kinetics of endogenous retrovirus activation in mouse cells. Virus induction with different inducers was compared using TEM and RT assays. The results indicated different regulation of endogenous retrovirus production with different inducers and caution in data interpretation due to enhanced RT cellular RT production. To evaluate endogenous retrovirus induction in primate cell substrates used in vaccine production, the cells will be exposed to various chemical inducers, initially, under the conditions that have proven to be successful in the mouse system. The results of these studies will provide strategies to evaluate endogenous retrovirus induction in vaccine cell susbtrates.