The Relationship Between Hapititis C Virus F Protein Or Genetic Variants In HLA-DP And The Hepatitis C Virus Persistence

Chronic infection with hepatitis C virus (HCV) is a serious infections liver disease in worldwide that frequently results in cirrhosis, or leading to hepatocellular carcinoma (HCC) within two to three decades of infection. It is estimated that more than180million people have been infected with HCV over worldwide, only10-20%patients with acute infection clear the virus spontaneously, among whom75%develop chronic hepatitis and are at risk for cirrhosis and HCC. Currently, HCV infection has become a serious social and public health problem.The mechanisms of chronic HCV infection and factors influencing the disease progression are still not fully understood, but several epidemiological factors such as biological characteristics of HCV, host immune system, host genetic variation and environmental are suspected to affect the clinical outcomes. It is well established that HCV core protein (Core) is involved in virion assembly and the chronic hepatitis C or HCC progression. Its can unregulated many of cytokines, such as c-myc, c-fos and Bcl-2, to active NF-κB and inhibit the cell apoptosis induced by Fas or TNF. Recently, a "new Core" was synthesized by genome open reading frame (ORF) shift and named alternative reading frame protein (F protein). Previous studies have not been able to exclude the influence of F protein on the specific characteristics of the host cellular immune response or the disease development; therefore, the discovery of F protein challenges various activities attributed to HCV Core. In order to evaluate the mechanisms of chronic HCV infection, this study conducted three section experiments to analysis the biological function of F protein:firstly, cytokines secretion and functional properties of F protein and Core were analyzed in PBMCs and PDCs derived from human peripheral blood; secondly, the relationship between HCV F region quasispecies and the viral replication, liver damage and Th2dominant response were analyzed in chronic hepatitis C patients; thirdly, the relationship between genetic variants in human leukocyte antigen-DP and hepatitis C virus persistence and F protein generation were analyzed in the Chinese Han population. The study in this area might explain a possible new vision of the development of chronic HCV.Section I The cytokine response to HCV F protein in peripheral blood mononuclear cells (PBMCs) from patients with chronic HCV in vitroObjective:To investigate the specific humoral immunity against HCV F protein and cytokine response to HCV F protein in peripheral blood mononuclear cells (PBMCs) and plasmacytoid dendritic cells (PDCs) from patients with chronic HCV and healthy donors in vitro.Methods:Blood samples were collected from chronic hepatitis C and healthy controls. F protein was used to detect F antibodies in the serum to divide samples into three subject groups including F-Ab(+)CHP, F-Ab(-)CHP and healthy control. PBMCs and PDCs were isolated from these three groups and then treated with F protein. After72hours, enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of IFN-a and IL-10. The apoptosis of PDCs was detected by flow cytometry.Results:The results demonstrated that the levels of interferon (IFN)-a secreted by PBMCs in patients’positive for the anti-F protein antibody, were lower than those of patients negative for the anti-F protein antibody. Furthermore, HCV F protein and Core not only inhibited specific unmethylated CpG oligonucleotide sequences of type A (CpG-A) induced IFN-a production by PBMCs and PDCs, but also upregulated the production of interleukin (IL)-10by PBMCs in patients with chronic HCV and healthy controls. Notably, following neutralization of IL-10in the media and in vitro Core or F protein stimulation, levels of IFN-a were increased. Moreover, the results revealed that the roles of F protein and Core were similar with regard to the induction of apoptosis of PDCs in patients with chronic HCV.Conclusions:These findings suggest that F protein may inhibit PBMC IFN-a secretion by regulating the production of IL-10, and may contribute to an increase in the rates of apoptosis in PDCs. In conclusion, the results have revealed a potential involvement of F protein in the mechanisms of chronic hepatitis C. Section II Study on diversity and significance of HCV F region quasispecies in ClinicObjective:To explore the relationship between the HCV F region quasispecies and the clinical significance.Methods:HCV quasispecies from78un-treated patients were detected by the single-strand conformation polymorphism (SSCP) analysis. The HCV RNA was quantitative analysed by the real-time fluorescence quantitative polymerase chain reaction. The antibody of HCV F protein and Thl (IL-12, IFN-y) or Th2(IL-5, IL-10) cytokines were detected by the enzyme-linked immunosorbent assay.Results:The positive rate of anti-F antibody in chronic HCV patients was60%and the number of SSCP bands of patients with HCV lb subtype was significantly higher than that of patients with non-HCV lb subtype (P=0.002). Moreover, the number of quasispecies associated with the loads of HCV RNA or levels of serum ALT/AST in chronic patients (all P<0.05). In addition, the levels of Thl (IL-12, IFN-y) cytokines secreted in the patients with the number of F quasispecies>2were lower than those of patients with F quasispecies≤2(P=0.023;P=0.010), and the Th2(IL-5, IL-10) cytokines in the patients with the number of F quasispecies>2were higher than those of the group of patients with F quasispecies≤2(P=0.024; P=0.012).Conclusion:The number of HCV F region quasispecies correlate with the viral replication, liver damage and Th2dominant response. Section Ⅲ Genetic variants in HLA-DP influence both hepatitis C virus persistence and hepatitis C virus F protein generation in the Chinese Han populationObjective:To assess the association of human leukocyte antigen-DP variants with risk of chronic HCV or anti-F antibody generation.Methods:We selected two single nucleotide polymorphisms (SNPs) in a region including HLA-DPA1(rs3077) and HLA-DPB1(rs9277534) and genotyped SNPs in702cases and342healthy controls from Chinese population using TaqMan SNP genotyping assay. Moreover, the exon2of the HLA-DPA1and HLA-DPB1genes were amplified and determined by sequencing-based typing (SBT).Results:The results showed that rs3077significantly increased the risk of chronic HCV infection in additive models and dominant models (OR=1.32and1.53). The rs3077was also contributed to decrease the risk of anti-F antibody generation in additive models and dominant models (OR=0.46and0.56). Subsequent analyses revealed the risk haplotypes (DPA1*0103-DPB1*0501and DPA1*0103-DPB1*0201) and protective haplotypes (DPA1*0202-DPB1*0501and DPA1*0202-DPB1*0202) to chronic HCV infection. Moreover, we also found that the haplotype of DPA1*0103-DPB1*0201and DPA1*0202-DPB1*0202were associated with the anti-F antibody generation.Conclusions:Our findings show that genetic variants in HLA-DP gene are associated with chronic HCV infection and anti-F antibody generation.