Immune Status Of Hcv Infection By Virus Genetic Variation And Disease Outcome Study

Viral hepatitis C is a serious, public health problem worldwide, and it is the major cause of hepatitis got by transfusion. The detail information about the genetic structure of HCV is the basis to research its pathogens, pathogenesis, diagnostic agent and design vaccine. HCV takes on highly genetic heterogenetity. its mutation can be found in the whole genomic, but the frequency is different in the different region. 5'untranslated region(5'UTR) is mostly conservative, so many commercial exploitation agents are based this region, such as the qualitative, quantitative and genotype diagnoses kit. Envlope region 2(E2) is the mostly diversity in the full genomic, the N-end first 27 amino acids and followed 91-97 amino acids is highly variable, be called highly variable region 1 and 2(HVRl,HVR2).Their amino acids diversity are 80% and 100% respectively, always be used to analyse quasispecies.In our research work, the genotype analysis was conducted by sequencing PCR products. We amplifyied the 5'UTR of prevalent HCV strains in part regions of China Mainland with nested hot start reverse transcription poiymerase chain reaction (RT-nested-hot start-PCR), purified the products and sequenced directly, confirmed the genotype by analyzing the 7 information region according to the methods described by Stuyver L .Then compared these sequences with standard strains published before, performing phylogenetic tree analysis, in order to get the prevalent trend and distributing characteristic in these region. There are four genotype(1a,1b,2a,1e) in Zhejiang province, Jiangsu province and Xingjiang province, but 1b is the major genotype. There are distinct sex difference in patients, male is absolutely predominance; as for age, there are no difference in different genotypes; the frequency of Liver function abnormity is significantly high in lb patients. This is accordant with the former reports.We selected 9 patients from different regions which belong to lb,2a genotype, amplified theregion including HVR1 with RT-nested-hot start-PCR, purified the products, ligated to pGEM-T easy Vector, the resulting recombinant plasmids were used to transform to TOP10F' cells. After being confirmed as positive clones, sequenced on an automated sequencer Megabase 500. We randomly select 15 positive clones from each sample, compared their nucleotide and amino acid sequence of HVR1, analyzed quasispecies diversity, complexity and phylogenetic distance, discuss their relation with the state of illness, predicted the possible immune epitope. We found that, on the amino acids level, the mutation rate is 80% in HVR1, the homology with Chinese consistent sequence) CCS is 42%-60%, moreover, the nonsynonymous substitutions is high than synonymous substitutions. The amino acids located at 385 T(threonine), 390 G(glycine), 403 F(phenylalanine), 406 G. and 409 Q (glutamine) are conserved, maybe associated with dimensional structure. The diversity and complexity of quasispecies is associated with high virus load and infection time. The quasispecies heterogeneity is not consisted with the liver damage. The evolvement of quasispecies is not only the result of positive immune pressure in the host, but also the accumulation of random mutation. There are CTL and CD4+T cell antigen epitope in the HVR1 gene, there are difference between different genotype and different dominant sequences.