Application and development of molecular biological methods to the issues of va

这个项目的目标是应用和发展分子生物学方法，以确保疫苗和细胞底物不受病毒，特别是逆转录病毒的污染。已经发表了三种检测逆转录酶（RT）的灵敏方法，它们的灵敏度至少是传统RT测定法的一百万倍。虽然这些检测方法被称为PERT（用于产品增强RT）或Amp-RT，但我们选择用PBRT的通用名称来称呼它们，用于基于pcr的RT。这三种检测方法依赖于相同的原理：在RT反应中使用已知序列的RNA作为模板和寡脱氧核苷酸引物；由于RNA序列已知，cDNA产物可通过聚合酶链反应（PCR）扩增；PCR产物可以通过多种方法检测。我们在CBER建立了三种PBRT检测方法，以比较它们的敏感性、特异性和可重复性。对测定法进行了修改，以消除测定法的一个问题，即它们的高背景信号。我们还修改了实验，使细胞DNA聚合酶的RT活性大大降低。最近，我们已经调整了PBRT分析与实时定量系统TaqMan系统一起使用，用于Perkin-Elmer 7700系统。这种改进的分析，TM-PERT分析，在至少6个数量级上是线性的，并且与原始分析一样敏感。有了这种敏感的TM-PERT分析，我们已经与CBER的几个小组进行了合作研究，将该分析应用于几个监管问题。在与Kurt broson及其同事（OTRR/DMA/LMDI）的一项研究中，我们正在评估TM-PERT测定是否可用于监测单克隆抗体制备过程中的病毒清除。在与Carolyn Wilson的另一项研究中，我们确定了猪因子VIII的RT水平，当人因子VIII不适合时，猪因子VIII用于治疗血友病患者。由于该产品来源于猪，并且所有猪细胞都含有并可能表达一种内源性逆转录病毒（PERV），因此存在这种因子VIII制剂含有PERV的可能性。通过我们的TM-PERT分析和Wilson博士的RT-PCR评估，所有测试的因子VIII批次都有低水平的PERV。威尔逊博士证明这些制剂不含传染性病毒。作为我们对鸡RT活性调查的一部分，我们开始了一项研究，以确定伪型是否可以在副粘病毒或正粘病毒的逆转录病毒核心和包膜糖蛋白（Env）之间形成。如果是这样，那么这可能为小鸡RT粒子进入人类细胞提供了一种途径。作为一个模型系统，我们研究了带有HIV核心颗粒的麻疹、腮腺炎和流感病毒Envs。我们的研究结果表明，假型可以在体外形成，因此理论上有这种假型在体内形成的可能性。这种伪型形成的后果尚不清楚。

The goal of this project is to apply and develop molecular biological methods that can ensure that vaccines and cell substrates are free from viral, and particularly retroviral, contamination. Three sensitive methods for the detection of reverse transcriptase (RT) that are at least a million-fold more sensitive than conventional RT assays have been published. Although the assays were variously termed PERT (for product- enhanced RT) or Amp-RT, we have chosen to call them by the generic name of PBRT for PCR-based RT. The three assays depend on the same principle: an RNA of known sequence is used as a template with an oligodeoxynucleotide primer in an RT reaction; because the sequence of the RNA is known, the cDNA product can be amplified by the polymerase chain reaction (PCR); the PCR product can be detected by a number of methods. We set up the three PBRT assays at CBER in order to compare their sensitivities, specificities, and reproducibilities. Modifications were made to the assays to eliminate one problem with the assays, viz., their high background signals. We also modified the assay such that RT activities of cellular DNA polymerases were substrantially reduced. Recently, we have adapted the PBRT assay for use with the real time quantitative system, the TaqMan system, for use with the Perkin-Elmer 7700 system. This modified assay, the TM-PERT assay, is linear over at least 6 orders magnitude and is as sensitive as the original assays. With this sensitive TM-PERT assay, we have undertaken collaborative studies with several groups at CBER to apply the assay to several regulatory issues. In a study with Kurt Brorson and colleagues (OTRR/DMA/LMDI), we are assessing whether the TM-PERT assay can be used to monitor viral clearance during the preparation of monoclonal antibodies. In a separate study with Carolyn Wilson, we determined the RT level in porcine factor VIII, a therapeutic used in those hemophiliacs when human factor VIII is unsuitable. Because the product is pig derived and because all pig cells have and may express an endogenous retrovirus (PERV), the potential exists that such factor VIII preparations have PERV. All factor VIII lots tested had low levels of PERV as assessed by the TM-PERT assay by us and by RT-PCR by Dr Wilson. Dr Wilson showed that these preparations do not contain infectious virus. As part of our investigation into the chick RT activity, we began a study to determine whether pseudotypes can form between a retrovirus core and an envelope glycoprotein (Env) from paramyxoviruses or orthomyxoviruses. If so, then this may provide a means by which the chick RT particle could enter human cells. As a model system, we investigated measles, mumps and influenza virus Envs with HIV core particles. Our results show that pseudotypes can form in vitro and thus there is a theoretical possibility of such pseudotypes forming in vivo. The consequences of such pseudotype formation remains unknown.