| Hepatitis C virus (HCV), a member of the Flavivirdate family is the major cause of chronic liver disease worldwide. HCV is a positive-sense single-strand RNA virus with a genome that encodes one large polyprotein in which putative structural proteins are located at the N-terminal end, and the putative nonstructural (NS) proteins are located at the C-terminal end. One of the important characteristics of HCV is that its genome exhibits significant genetic heterogeneity as a result of the accumulation of mutations during viral replication. The genetic sequences of HCV variants are very heterogeneous, varying by more than 30% across the entire genome among the six major genotypes, 20% among subtypes, and up to 10% within a subtype. Analogous to other RNA viruses, HCV circulates in an infected individual as a population of closed related, yet heterogeneous, sequences: the quasispecies. The quasispecies distribution of HCV might have important biological consequences. It has been proposed that this genetic heterogeneity allows HCV to escape immune pressure and to establish chronic infection. In addition, the existence of a heterogeneous population of HCV may influence the outcome of antiviral therapy; resistance to treatment might result from selection of minor viral populations during this therapy. Therefore, it is important to define accurately quasispecies populations of HCV.Many analyses of viral quasispecies of HCV have been published. The majority of these studies have focused on the most variable part of the HCVingenome, hypervariable region 1 (HVR1) of glycoprotein E2. Mutation of this region of the genome is believed to be associated with viral persistence via immune escape mechanisms. However, it is well known that genetic heterogeneity of HCV extends throughout the entire genome. It is our hypothesis that significant mutation occurred in other regions of the viral envelope genes during the chronic infection. In addition, most studies assessing the diversity of HCV quasispecies are conducted by amplifying selected portions of the genome by PCR, isolating individual subgenomic fragments by a cloning produce, and then characterizing the nucleotide sequence of each clone. Evaluating the diversity of HCV quasispecies in clinical samples often requires the sequencing of a large number of clones, but because of the effort and expense, published studies obtain sequence information from a small number of colonies per subject.In order to investigate genetic variation of HCV quasispecies and its relationship with the outcome of acute hepatitis C, HCV quasispecies were characterized in specimens collected every two to six months from a cohort of acutely HCV-infected individuals (the duration of specimen collection, 34 months after seroconversion). We evaluated 2 individuals who spontaneously cleared viremia and 3 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned El and the 5' half of E2, including HVR1. To assess the quasispecies complexity and to detect variants for sequencing and the expression of their functional proteins , Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined a single-stranded conformational polymorphism (SSCP) method and heteroduplex analysis (HD) analysis.1. The mixed oligonucleotides primed amplification of the HCV structural region and Cloning of cDNAThe fragment spanning the 3' half of C region, El and 5' half of E2IVincluding HVR1 of Chinese viral strain with HCV type la> Ib or 3b, which was determined by RT-PCR and restriction fragment length polymorphism (RFLP) analysis of the 5' untranslated region (UTR), was amplified by using the mixed oligonucleotides primers so as to increase detective sensitivity toward variants in the plasma of an HCV infected patient. The 1 -kb amplicons were ligated into the vector pT-adv and used to transform E. coli TOP 10'F competent cells, in which cloning efficiency was > 90%.2. Optimal screening of viral quasispeces in the structural region of HCVFor e...
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