Analysis Of Part Sequences Of HCV Genome Before And After Chronic Hepatitis C Patients Treated By Interferon

Hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide, infecting an estimated 170 million people. More than one million new cases of infection are reported annually throughout the world. Epidemiological studies have revealed that more than 80% of acutely HCV-infected patients fail to eradicate the virus and they subsequently develop chronic hepatitis, cirrhosis, and hepatocellular carcinoma.The HCV genome is a positive-sense, single-stranded RNAgenome approximately 9.4-9.6 kb long, with a high degree ofheterogeneity. It is thought that genetic heterogeneity of HCV mayaccount for some of the differences in disease outcome and responseto treatment observed in HCV-infected persons. Analysis of thenucleotide sequence homology of different viral genomic regions suchas the 5' untranslating region (5'UTR) and regions coding for theenvelopel (El), core, and nonstructural 5B (NS5B) proteins led to theidentification of six major genotypes and numerous subtypes withinthe genotypes. The gold standard for determination of HCV genotypeis sequence analysis of phylogenetically informative coding regions ofthe HCV genome and comparison to consensus sequences of knowngenotypes. The genotypes and subtypes of HCV differ in geographicdistribution. In China, the common genotypes is HCV 1b and 2a, HCV1b is the most common. Natural viable intra genotypic and inter genotypic recombinants have been reported. Diagnostically recombinants represent an intriguing challenge.Recently, therapy combining IFN and ribavirin, has been used to treat patients with chronic hepatitis C worldwide to increase the effectiveness of IFN-based therapy. The therapy is not highly effective, as there still remains resistance or relapse of effective cases after the withdrawal. The HCV genotype has emerged as an important factor both in predicting a sustained response to and in determining the duration of antiviral therapy. The sustained virologic response rates are dependent on several key clinical and virologic factors.In this study, part regions of four CHC patients' (Y- a patient before treatment of IFN+Rib, R-during treatment, L and D- after treatment) HCVRNA were amplified by RT-PCR, and then cloned into T vectors. After being sequenced, these genes were analyzed. We intend to discover the genome sequence and its variation, amino acid variations of HVR about HCV Changchun-strain, using of cross-sectional study and future longitudinal study, in order to provide clues to researches on the mechanisms of resistance to interferon, the exploitation of hepatitis C vaccine and the immune mechanism of eliminating hepatitis C virus.The results are as follows: Fragments 39-2601bp (1b type)of the patient R and Y were amplified successfully. Fragments 7234-8583bp (1a type) of all the four patients were amplified successfully. 5' NCR of patient R and Y have homology of 100% to some known sequences (including some CN strain), C and E1 regions have better homology to some known sequences too. As to the HVR1 (hypervariable region 1) and HVR2, both of patient R and Y have obvious heterogeneities . Fragments 7234-8583bp (1a) of all the four patients are highly homologous to AM strains. The four patients are consistent with each other at nucleotide sites 7942,8490, and amino acid sites 114,180,297 of NS5b, but different from many other strains. Fragments 7975-8196 bp( 222bp )of all the four patients are highly homologous (>99%) to M62321,M32084,E66593.Conclusion: (1) Furtherly, it proved that 5'NC is highlyconserved, and C and El regions are relative conserved. All of these reqions can be the potential candidates for gene diagnosis and treatment. (2) Conserved amino residues of threonine (T) at position 2, glycine (G) at position 23 and glutamic acid (Q) at position 26 in HVR1 were further confirmed . (3) Sequences of the four patients' NS5b (HCV la) have good homology to AM strains, and seemed to have relation to the effects of interferon.. (4) (6) Nucleotide sites 7942,8490 and amino acid sites 114,180,297 of NS5b may have regional differences. (5) Fragments 7975-8196bp (222bp) of all the four patients are highly homologous (>99%) to M62321,M32084,E66593, they may in the same branch of phylogenetic tree analysis. (6) Patients R and Y may be infected with la type and 1b type simultaneously, or infected with recombinants.(7) It is confirmed that there is infection of HCV gene type la in Changchun.