Studies on the pathogenesis of hepatitis C virus

Work was carried out to generate molecular clones spanning the polyprotein coding region of the HCV genome. A fragment cloning technique was employed to amplify short regions of the genome that could be sequentially pieced together for the construction of a molecular clone of HCV.;Studies on the pathogenesis of HCV continued, investigating part of the immune response mounted against HCV within infected cells. Studies on the response of HCV patients to interferon-alpha therapy have proven that the majority of patients who undergo treatment, which is both expensive and associated with many severe side effects, do not respond. The search for patient and virus factors that may be used to predict the response of an HCV infected patient to treatment would be great benefit in allowing patients and doctors to make informed choices about undertaking interferon-alpha treatment.;The double-stranded RNA-induced protein kinase (PKR) is a major downstream effector of the interferon-alpha immune response. Expression levels of PKR may therefore play some role in determining the effectiveness of interferon-alpha treatment. Studies were therefore undertaken to investigate the role of PKR as a predictive factor for HCV patient response to interferon-alpha treatment. Assays were developed to measure expression levels of PKR mRNA and PKR protein extracted from liver biopsy tissues of patients infected with HCV. The aim was to analyse and correlate pre-treatment expression levels of PKR with patient response to interferon-alpha treatment. A quantitative competitive RT-PCR assay was successfully developed to measure PKR mRNA in cellular RNA extracts. A quantitative western blot assay was also developed for the quantitation of PKR protein in cellular protein extracts. The work carried out here forms the basis for future experiments in which the analysis and quantitation of specific proteins from cell culture or tissue extracts can be achieved.