Developing an in vitro system for HCV propagation

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)经常导致人类长期和持久的感染，迫切需要开发一种高效的治疗方法和疫苗。不同地理位置的丙型肝炎病毒存在不同的基因型别和亚型。丙型肝炎病毒1型(a和b亚型)感染在美国很普遍，并且对可用的治疗方法相对耐药。最近，我们和其他研究人员报道了丙型肝炎病毒在细胞培养中的生长。大部分工作是在Huh7细胞或其衍生细胞中用丙型肝炎病毒基因2a(JFH1株)完成的。我们在永生化人肝细胞(IHH)中发现了1a型丙型肝炎病毒(H77株)的生长。然而，野生型丙型肝炎病毒1a的产量较低，并且H77株和JFH1株之间存在显著的序列差异。虽然产生了丙型肝炎病毒1a颗粒，但病毒在肝细胞中连续传代的生长尚未确定。因此，拥有一个强大的细胞培养系统对丙型肝炎病毒1a的生长是极其重要的。此外，感染性病毒颗粒的滴定受到免疫荧光或免疫组织化学检测的限制。在这项开发中的R21拨款申请中，我们计划通过不同方法表征病毒在IHH中的生长来产生高滴度的丙型肝炎病毒1a，并使用报告试验开发一种方便的病毒感染读数。拟议工作的结果将有助于进一步开发新的领域，包括高通量抗病毒筛选试验，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(丙型肝炎病毒)通常会在人类身上造成长期和持久的感染，迫切需要开发高效的治疗方法和疫苗。我们最近在细胞培养中产生了丙型肝炎病毒。我们将研究改善这种病毒的体外生长，并开发一种灵敏的读数，用于未来的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.