A High throughput Reverse Genetics System for Hepatitis C Virus

丙型肝炎病毒的高通量反向遗传学系统

基本信息

批准号：
8891839
负责人：
XIAOFENG FAN
金额：
$ 7.58万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-10 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8891839
关键词：
3&apos Untranslated Regions Antiviral Agents Antiviral Therapy Cell Culture System Cell Culture Techniques Cells Characteristics Chronic Hepatitis C Clinical Consensus Sequence Drug resistance Frustration Generations Genetic Genetic Variation Genome Genomics Genotype Goals Hepatitis C Hepatitis C virus In Vitro Legal patent Length Life Cycle Stages Liver diseases Measurement Methods Modeling Mutation Names Nature Patients Phenotype Plasmids Population Preparation Preventive Primary carcinoma of the liver cells Process Public Health RNA Polymerase I RNA Viruses Research Reverse Transcriptase Polymerase Chain Reaction System Techniques Testing Transfection United States Untranslated Regions Vaccines Viral Viral Genome Virus Work base combat genetic approach in vitro Model in vivo liver transplantation particle public health relevance reverse genetics screening tool

项目摘要

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.

描述（由申请人提供）：丙型肝炎病毒（HCV）是医学上重要的RNA病毒之一，可引起多种肝脏疾病，鉴于其在公共卫生中的高度优先性，这也是美国肝移植的主要适应症。 HCV 生命周期的体外模型对于 HCV 基础研究和转化研究都非常有需求，然而，目前的体外 HCV 模型只能模拟病毒生命周期的某些步骤，迄今为止只有一种特定的 HCV 基因型 2a 毒株。名为 JFH1 的细胞培养物是通过从克隆病毒基因组中拯救感染性病毒而建立的，这种方法的一个众所周知的限制是其不可预测性。在制备全长病毒克隆期间从内部或外部引入的病毒突变，其中一些突变可能是致命的或不利于成功拯救感染性病毒，特别是在 HCV 环境中，因为其协调性。当测试的毒株/克隆数量足够大以克服来自异质病毒群体或实验引入的突变障碍时，可以使用 JFH1 以外的毒株建立 HCV 细胞培养物。基于我们之前的工作，传统的反向遗传学很难实现这一点，因此我们建议开发一个能够以高通量方式筛选临床 HCV 分离株的快速反向遗传学平台。这样的通过整合我们的长 RT-PCR 技术（美国专利号：7,786,294 B2）和无缝组装方法，该平台是可行的。所提出的平台是基于质粒的反向遗传学，其中长 RT-PCR 和 3'UTR 扩增子。使用吉布森组装方法将HCV融合到目标质粒中，然后直接转染到Huh-7细胞中，并随后测量潜在的感染性病毒。我们将筛选总共61种患者来源的病毒。 HCV 1a 毒株已在全长基因组水平上进行了病毒突变表征。该项目开发的方法对 HCV 以外的 RNA 病毒具有普遍适用性。