Developing an in vitro system for HCV propagation

描述（由申请人提供）：丙型肝炎病毒（HCV）经常引起人类长期和持续的感染，迫切需要开发高效的治疗和疫苗。HCV的不同基因型和亚型存在于不同的地理位置。HCV基因型1（亚型a和B）感染在美国很普遍，并且对现有治疗相对耐药。最近，我们和其他研究人员报道了HCV在细胞培养中的生长。大部分工作是在Huh7细胞或其衍生物中用HCV基因型2a（JFH 1株）进行的。我们开发了HCV基因型1a（H77株）在永生化人肝细胞（IHH）中的生长。然而，野生型HCV 1a产量低，并且HCV H77和JFH 1株之间存在显著的序列变异。虽然产生了HCV 1a颗粒，但尚未确定肝细胞中连续传代的病毒生长。因此，具有用于HCV 1a生长的稳健的细胞培养系统极其重要。此外，感染性病毒颗粒的滴定受限于基于免疫荧光或免疫组织化学的测定。在这项开发R21资助申请中，我们计划通过以下不同方法表征IHH中的病毒生长来产生高滴度HCV 1a，并使用报告基因测定开发病毒感染的方便读数。拟议工作的结果将有助于进一步开发新的领域，包括用于抗病毒筛选的高通量测定，对药物和疫苗发现计划产生巨大影响。 公共卫生相关性：丙型肝炎病毒（HCV）常引起人类的长期和持续感染，迫切需要开发高效的治疗和疫苗。我们最近在细胞培养中产生了HCV。我们将研究如何改善这种病毒的体外生长，并开发一种灵敏的读数，以供将来用于高通量抗病毒筛选和疫苗发现计划。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) often causes a prolonged and persistent infection in humans, and there is an urgent need for the development of a highly effective therapy and vaccine. Distinct genotypes and subtypes of HCV exist in different geographic locations. Infection with HCV genotype 1 (subtypes a and b) is prevalent in the USA, and is relatively resistant to available therapies. Recently, HCV growth in cell culture has been reported by us and other investigators. Majority of the work was done with HCV genotype 2a (JFH1 strain) in Huh7 cells or its derivatives. We have developed growth of HCV genotype 1a (H77 strain) in immortalized human hepatocytes (IHH). However, wild type HCV 1a yield is low and a significant sequence variation exists between HCV H77 and JFH1 strains. Although, HCV 1a particles are generated, virus growth from serial passages in hepatocytes has not been established. Therefore, it is extremely important to have a robust cell culture system for HCV 1a growth. Furthermore, titration of infectious virus particles is restricted with immunofluorescence or immunohistochemistry based assay. In this Developmental R21 grant application, we plan to generate a high titer HCV 1a by characterizing virus growth in IHH following different approaches, and develop a convenient readout for virus infection using a reporter assay. The results from the proposed work will help in further developing novel areas, including high throughput assay for antiviral screening with tremendous impact for drug and vaccine discovery programs. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) often causes a prolonged and persistent infection in humans, and there is an urgent need for the development of highly effective therapy and vaccine. We have recently generated HCV in cell culture. We will study to improve the in vitro growth of this virus and develop a sensitive readout for future use in high throughput antiviral screening and vaccine discovery programs.