A High throughput Reverse Genetics System for Hepatitis C Virus

 描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是一种重要的医学核糖核酸病毒，可引起多种肝脏疾病，也是美国肝移植的主要适应症。鉴于其在公共卫生中的高度重要性，无论是基础研究还是翻译研究都迫切需要建立一个描述丙型肝炎病毒生命周期的体外模型，然而，目前的体外模型只能模拟病毒生命周期的某些步骤。到目前为止，只有一种特殊的丙型肝炎病毒2a株，命名为JFH1，能够在体外繁殖。基于JFH1的细胞培养是通过从克隆的病毒基因组中拯救传染性病毒而建立的，即所谓的反向遗传学方法。这种方法的一个众所周知的限制是在准备全长病毒克隆期间内部或外部引入的病毒突变的不可预测性。这些突变中的一些可能对成功抢救感染性病毒是致命的或有害的，特别是在丙型肝炎病毒的背景下，由于其协调 基因组网络和显著的遗传多样性。当被测试的菌株/克隆的数量足够大，足以克服来自异质病毒群体或实验引入的突变的障碍时，可以用JFH1以外的菌株建立丙型肝炎病毒细胞培养。然而，以繁琐的过程著称的传统反向遗传学很难实现这一目标。因此，在前期工作的基础上，我们建议建立一个能够高通量筛选临床分离的丙型肝炎病毒的快速反向遗传学平台。我们假设，通过整合我们的长RT-PCR技术(美国专利号：7,786,294 B2)和无缝组装方法，建立这样一个平台是可行的。该平台是一种以质粒为基础的反向遗传学方法，利用Gibson组装方法将长的RT-PCR和丙型肝炎病毒的3‘非编码区扩增片段融合到目标质粒中，然后直接转染Huh-7细胞，随后检测潜在的感染性病毒。我们将筛选总共61个患者来源的丙型肝炎病毒1a毒株，这些毒株已经在全基因组水平上被表征为病毒突变。该方法对丙型肝炎病毒以外的RNA病毒具有普遍的适用性。

 DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is one of medically important RNA viruses and causes a wide spectrum of liver diseases, which are also a leading indication for liver transplantation in the United States. Given its high priority in public health, an in vito model for HCV life cycle is highly demanded for both basic and translational HCV research, however, current in vitro HCV models could only mimic some steps of viral life cycle. There is so far only one particular HCV genotype 2a strain, named JFH1, is able to be propagated in vitro. The JFH1-based cell culture has been established through the rescue of infectious viruses from cloned viral genomes, so called reverse genetics approach. A well-known constraint in such an approach is the unpredictability of viral mutations introduced internally or externally during the preparation of full-length viral clones. Some of these mutations may be lethal or detrimental for successful rescue of infectious viruses, in particular in the setting of HCV due to its coordinated genome network and remarkable genetic diversity. When the number of strains/clones tested is large enough to overcome the barrier of mutations from either heterogeneous viral population or experimental introduction, HCV cell culture could be established with strains other than JFH1. However, this goal is hard to be achieved by conventional reverse genetics that is notable for its tedious process. Based on our previous work, we thus propose to develop a rapid reverse genetics platform capable of screening clinical HCV isolates in a high-throughput manner. We hypothesize that the establishment of such a platform is feasible by the integration of our long RT-PCR technique (US patent No: 7,786,294 B2) and a seamless assembly method. The proposed platform is a plasmid-based reverse genetics in which long RT-PCR and 3'UTR amplicons of HCV will be fused into the target plasmid using the Gibson assembly method, followed by direct transfection into Huh-7 cells and subsequent measurement of potential infectious viruses. We will screen a total of 61 patient-derived HCV 1a strains that have already been characterized for viral mutations at the full-length genome level. The method developed with the proposed project has general applicability in RNA viruses other than HCV.