The Study Of The Hepatitis C Virus HVR1 Quasi-species

The Hepatitis C virus (HCV) is an RNA virus that replicates with a high rate of mutation, which is particularly evident in the hyper-variable region 1 (HVR1) of the N-amino terminal region of the second envelope domain of the viral genome. Under the influence of environmental factors, continuous viral mutation gives raise to a mixed and changing population of mutants, which is known as quasi-species. Taking into account the quasi-species nature of HCV and the marked heterogeneity of patients with HCV infection, sequence analysis of a large number of quasi-species would be necessary for accurate assessment of the role of viral heterogeneity in the pathogenesis of the disease and its response to therapy, however, a simple and high sensitive SSCP method with rigorous standard and determination of the limit of detection has not been shown.In this study, a rapid method for single-strand conformation polymorphism (SSCP) analysis of PCR products that allows the use of ethidium bromide staining is developed. The HVRl PCR products were evaluated for polymorphisms by non-radioactive SSCP in normal polyacrylamide mini-gels. Several electrophoretic parameters (running temperature, buffers, denaturants, DNA concentration, and gel polyacrylamide concentration, et at) were optimized to reach the max. degree of strand separation. Polymorphisms could be detected when a quasi-species comprised as little as 2-3.3% of the total gene copies in a PCR mixture. Compared to standard radioactive SSCP, this novel non-isotopic method has additional advantages of dramatically increased speed, precise temperature control, reproducibility, and easily and inexpensively obtainable reagents and equipment. Combined with clone sequence, this SSCP method provides an accurate tool to evaluate HCV quasi-species.To study the effects of the HIV-induced-immunosuppression on the composition of HCV populations, three groups with different immunology stage were analyzed: HCV mono-infected patients, HIV/HCV co-infected patients (CD4-cell count≥200), HIV/HCV co-infected patients (CD4-cell count<200). No significant differences in the number of SSCP bands, nucleotide diversity, p-distance, HCV viral load, dN, dS and dN/dS were observed in three groups. The CD4-cell count was not significantly correlated with the number of SSCP bands, p-distance, HCV viral load. These observations suggest that thewidth of the mutant spectra of HCV quasi-species, estimated by SSCP analysis, does not correlate with the severity of immunosuppression.In order to analysis hepatitis C virus genome complexity whether or not be associated with various stages of liver disease. We used PCR-SSCP analysis to estimate the degree of complexity of the hyper-variable region (HVRl) in anti-HCV and serum HCV RNA positive patients. They were divided into five groups: mild chronic hepatitis, moderate chronic hepatitis, severe chronic hepatitis, decompensated hepatitis and liver fibrosis. No significant differences in the number of SSCP bands, nucleotide diversity, p-distance, HCV viral load, dS, dN and dN/dS were observed in five groups with different degrees of liver injury. The ALT values were not significantly correlated with the number of SSCP bands, nucleotide diversity, HCV viral load and dN/dS. These observations suggest that the genomic complexity in the HVRl region of HCV is not related to the clinical severity of liver disease when analyzed by SSCP.