Chronic hepatitis C virus (HCV) infection afflicts about 3 million people in the United States and is the leading indication for liver transplantation (CDC, NHANES III). Currently treatments are effective in only about 50% of patients. Improved treatments and improved methods for studying HCV are a research priority of NIDDK. This project addresses a significant research need by introducing novel cell cultures system for HCV and by introducing methods that are needed to define the structure of the HCV replication intermediate. The cells to be used are mononuclear cells derived from both peripheral blood (PBMCs) and from ascitic fluid (AMCs).
In Specific Aim I, we will monitor changes in HCV RNA over time and measure the inhibitory effects of interferon-? and 2'-methyl adenosine (Merck), an inhibitor of the HCV RNA-dependent RNA polymerase, using Real time PCR. Using specialized equipment we developed for this purpose, we will heat RNA extracts to temperatures over 100 C and thereby release genomic (+) and antigenomic (-) HCV RNAs from replication intermediates. Strand-specific PCR assays will be used to detect positive (+) and negative (-) strands.
In Specific Aim II, we will investigate the changes in HCV RNA replication that are produced by exposure of cells to cytokines and we will identify HCV mutations associated with high-level replication. The experiments in this aim will define culture conditions for optimal HCV RNA replication. HCV sequence analysis will be performed on the HCV RNA derived from PBMCs and AMCs.
In Specific Aim III, we will carry out in-depth analysis of the structure of the HCV replication intermediate and determine the ratio of genomic and antigenomic strands. We postulate that the antigenomic strands within the replication intermediate are almost entirely involved in RNA-RNA duplex structure, while the genomic strands contain both single- and double-stranded regions. Strand-specific PCR amplification will be performed on several HCV gene regions to identify domains that are accessible to PCR primers.
In Specific Aim I V, we will be used to investigate the infectivity of cell culture supernatants from the system optimized in Aim II by exposing HCV-naive mononuclear cells to supernatant from the optimized HCV-culture system. Public Health Relevance: The hepatitis C virus (HCV) is a major public health treat;chronic infection causes about 10,000 deaths in the U.S. each year and the number is rising. The development of improved treatments is a research priority. The goal of this project is to develop a model system to study HCV. Our project will contribute to the development of more effective interventions.
|Klepper, Arielle; Eng, Francis J; Doyle, Erin H et al. (2017) Hepatitis C virus double-stranded RNA is the predominant form in human liver and in interferon-treated cells. Hepatology 66:357-370|
|Klepper, Arielle; Branch, Andrea D (2015) Macrophages and the Viral Dissemination Super Highway. EC Microbiol 2:328-336|
|Gutierrez, Julio A; Klepper, Arielle L; Garber, John et al. (2011) Cross-genotypic polyclonal anti-HCV antibodies from human ascitic fluid. J Virol Methods 171:169-75|