Positive and negative controls were included at the extraction step and in both rounds of amplification. genotype. These studies indicate that the anti-HCV antibody immune response to HCV peptides varied across regions and among races. The distribution of HCV genotypes among Tibetans in Tibet and Uighurs in Sinkiang was different from that in the inner areas of China. In addition, a master genotype, type II, was found to exist in HCV infection with multiple HCV genotypes. Keywords: Hepatitis C virus, anti-HCV, ELISA, neutralization test, genotype Introduction The incidence of Hepatitis C virus (HCV) infection has been steadily increasing in the last few decades in China and is expected to intensify in the coming years worldwide 1. Peptide-based vaccines have been generated and tested in pre-clinical and clinical trials 2. However, the development of effective peptide-based vaccines has been significantly hampered by the high genetic variability of HCV. Anti-viral humoral immune response plays a fundamental role during HCV infection. Viral clearance is associated with the production of anti-envelope antibodies, and high serum levels of anti-viral envelope antibodies can prevent HCV infection in chimpanzees 3. Nevertheless, the correlations between antibody production and its potential neutralizing immune response are still largely unknown. HCV belongs to the hepacivirus genus in the Flaviviridae family 4. There are 6 different genotypes of HCV and more than 70 sub-types based on the nucleic acid sequences 5. HCV is therefore characterized by high levels of genetic heterogeneity, which accounts for the difficulties in vaccine development and for the lack of therapeutic efficacies. In China, the prevalence of HCV genotypes I Rabbit polyclonal to AMPK2 and II has been previously reported 6. However, little is known about the immune response to HCV peptides, and the region-specific distribution of HCV genotypes in China has not been fully evaluated. In this report, we conducted serological and molecular studies on a large cohort of HCV carriers in 6 regions of China to determine the HCV antibody immune responses to HCV peptides and the distribution model of HCV genotypes in different areas and races of China. Materials and Methods Study population Human HCV-positive serum samples from 363 Rosuvastatin HCV-infected patients were collected from hospitals in different areas of China: 35 from Shanghai, 20 from Shaanxi, 19 from Tianjin, 62 Tibetans, 23 Hans from Tibet, 146 Uighurs, 42 Hans from Sinkiang, and 16 from Hebei (Table ?(Table1).1). The selection of the study population was based on the following criteria: presence of HCV RNA in plasma confirmed by nested reverse transcription polymerase chain reaction Rosuvastatin (nRT-PCR); absence of other concomitant liver diseases; negative HIV test; no prior interferon and/or ribavirine treatment, and neither habitual alcohol nor active intravenous drug users. Control serum samples (n=3) were obtained from subjects who Rosuvastatin were negative for Hepatitis A, B, C, CMV (cytomegalovirus) and HIV. The peptides CP1, CP2 and NS4 used in this study were prepared in our laboratory: Table 1 Characters of 363 HCV-infected patients in different areas of China XL1-Blue and cultured in LB plate at 37C overnight. Recombinant clones were selected randomly and positive clones were detected and genotyped by PCR as described above. Partial HCV-C genes from the positive clones were amplified by nest-PCR with external primers 5′-ATGAGCACGAACATTCCTAAAACC-3′ and 5′-AGCGGAAGCTGGGAGTGGT-3′ and internal primers: 5′ -CACTCTCGAGCACCCTATCAGGCAGT-3′ and 5′-TTCACGCAGAAAGCGTCTAG-3′. Positive and negative controls were included at the extraction step and in both rounds of amplification. PCR products were sequenced using the dideoxy-mediated chain-termination method with a 373A Automatic DNA Sequence Analysis Machine (Applied Biosystems, Weiterstadt, Germany). Individual sequences were analyzed with MegAlign software (DNAStar Inc., Madison, WI). Statistics Data are expressed as means SD. Statistical analysis was conducted using StatView. Significant differences between groups were determined by ANOVA, and p<0.05 was considered significant. In order to detect the differences among categorical variables of neutralization rate (%), chi-square (2) test was applied. In order to detect differences among categorical variables of neutralization ODSD, test (stands for test) The data in Table ?Table22 also show. that pre-neutralization, the OD value of samples from Tianjin was the highest (1.540.64), while that of samples from Tibet was the lowest (0.660.26). Post-neutralization, the average OD values of samples from Shanghai, Shaanxi, Tianjin and Hebei appreciably declined (by 50% or nearly 50%), indicating that those samples.