Analyses Of HCV Genotypes, NS3 Variation Associated With Drug-resistance And Cytotoxic T Lymphocyte Epitopes In Patients With Chronic HCV Infection

Hpeatitis C virus (HCV) is a highly variable virus. After infected with HCV, most individuals will become chronic infection and some of them will further develop to liver cirrhosis and hepatocelullar carcinoma. HCV genotype may obviously influence antiviral efficacy of interferon-α, e.g., genotype I infection had significantly lower response to the antiviral therapy compared with genotype II HCV infection. HCV NS3 protein has multiple functions important for virus life cycle. Therefore, it has become the major target of novel anti-HBV agents. There is a paucity of data from large-size sample showing prevalence of HBV genotypes in North China in very recent years. Furthermore, NS3 variation profiles of the drug-resistance and CTL epitopes have not been documented yet in Chinese patients. In this study, we investigated HCV genotypes and NS3 protease-encoding region in 150 patients with HCV infection from various districts of North China. The study is to clarify recent prevalence of HCV genotypes and variation profile of NS3 region in relation to the drug-resistance and CTL epitopes in North China.Objective (1) To investigate HCV genotype prevalence in North China for further understanding of recent prevalence of HCV genotypes in the area. (2) To develop a practicable assay for amplifying and sequencing HCV NS3 protease-encoding region. (3) To clarify the profile of the drug-resistance and CTL epitope variations in the NS3 region.Materials and methods (1) Sera from 767 patients with chronic HCV infection were collected, comprised of 518 patients with chronic hepatitis C, 213 with chronic hepatitis C in junction with liver cirrhosis, and 36 patients with chronic hepatitis C in junction with HCC. Gene chip was used for classification of HCV genotypes. (2) An in-house RT-PCR assay with carefully-designed primers was developed and used for amplification of viral NS3 region from sera of 150 patients. Direct PCR sequencing and clonal sequencing if necessary were performed. Phylogenetic tree analysis based on NS3 sequence was used for determination of HCV genotypes and the results were compared with that obtained from gene chip. The variations associated with resistance of teleprovir,a novel inhibitor of NS3-encoeded protease, was analyzed. (3) HLA-A2, A11, A24, and A33 genotyping by PCR was performed for 114 patients'samples. The well-known A2 and A2-restricted CTL epitopes in NS3 region was analyzed.Results (1) The prevalence of HCV genotypes of the 767 patients from different regions of North China was as follows: 1b, 59.71%; 2a, 30.90%; 1b/2a, 3.26%; type 6, 2.49%, 3a, 1.56%; 3b, 0.91%; 1b/3a, 0.52%; 2a/3a, 0.26%; 1a, 0.26%; and 1a/1b, 0.13%. No obvious difference in HCV genotype profile was observed among three different disease statuses. (2) A valid assay for amplification of the NS3 regions from various genotypes was established. Highly consistent with the results from gene chip, the results of NS3 sequence-based analysis of HCV genotypes were as follow: 1b, 66.7% (100/150); 2a, 22.7% (34/150); 3a, 4% (6/150), 3b, 4% (6/150); type 6, 2% (3/150); and 1a, 0.7% (1/150). No obvious regional cluster inclination was observed for NS3 sequences within major genotypes, i.e., 1b and 2a. Telaprvevir-resistant variations V36M/A, T54A, R155K/T, and A/156V/T were not detectable. (3) HLA typing showed that in 114 patients, Forty-three (37.7%) was positive for A2, forty-four (38.6%) was positive for A11, Twenty-seven (23.7%) was positive for A24, sixteen (14.3%) was positive for A33. There were 14 cases (11.4%) negative for all the 4 HLA-A genotypes. variations were frequently detected in NS3-drvied A2-restricted CTL epitopes CINGVCWTV and LLCPSGHAN, and A24-restricted CTL epitope AYSQQTRGL. In most cases, there were more than one substitution within the epitope.Conclusion (1) Genotype 1b is still the predominant HCV followed by 2a at present in North China. (2) The NS3 amplification and sequencing assay that we developed is practical for both analyses of HCV genotypes and resistant variations against HCV protease inhibitors. (3) The primary telaprevir resistance caused by primary resistant variations of the virus stands small chance. (4) High occurrence of CTL epitope variation could be involved in the mechanisms for HCV infection chronicity.