HCV Sequence Adaptation To CD8~+T Cell Immune Pressure

Objective1. To detect the HCV genotyping of chronic HCV patients in Wuhan population, toanalyze the distribution of HCV genotyping and its clinic significance.2. To amplificate HCV NS3region (HCV genotype1b) and finish its sequencing, andanalyze the amino acid mutation of HCV NS3function region and CD8~+T cell epitope.3. Compare the HLA phenotypic frequence distribution between Wuhan and German.To detect HLA typing of HCV1b isolates, and compare the HLA phenotypic frequencedistribution between Wuhan HCV patient population and German healthy population.4. To analyze the sequence variation of peripheral circulating HCV isolates and itscorrelation with the HLA-I alleles, and predict HLA-I restricted CD8~+T cell epitope.Methods1. We collected the serum and whole blood from227patients. The genotyping afterextracting the HCV RNA from the serum is detected for analyzing the distribution of HCVgenotyping in Wuhan patients and the clinical significance of HCV genotype1b.2. HCV NS3(HCV genotype1b) will be amplified by a nested RT-PCR assay withtype specific primers and sequenced. Software for sequence analysis is used for analyzing thesequence of the function region and identified the mutation according to their location insideor outside previously described CD8~+T cell epitope.3. We compared HLA phenotypic frequence distribution which was obtained from HLA phenotypic frequence web between Wuhan population and German population. HLAphenotypic frequence of HLA-class I alleles (A-and HLA-B locus) in patients with HCVgenotype1b was performed for comparing with the HLA-Alleles of Wuhan healthy patients.4. Analyzing HCV NS3(HCV genotype1b) CD8~+T cell epitope mutation withrespect to the host’s HLA-type with ALIGN software. We also predicted the HLA-Irestricting CD8~+T cell epitope with HCV genotype1b by web software.Results1. There were126cases of HCV genotype1b in227Wuhan HCV patients whose ratiowas63.6%, whereas the other HCV genotype had72cases whose ratio was36.4%, whichindicated the major genotype in Chinses HCV patients is genotype1b. Comparing the liverfunction of39cases with HCV genotype1b and19cases with other HCV genotype, we foundthat the liver disfunction of HCV genotype1b happened more easily than other HCVgenotype, that is to say the disease with HCV genotype1b is sicker than HCV other genotype.2. After30HCV NS3sequence were analyzed, it is indicated that the amino acidsequence of HCV NS3function region was difficult to mutate with relatively conservative. Inaddition, the amino acid mutations frequency of inside previously described CD8~+T cellepitope was significantly higher compared with the outside region, which had statisticsignificance (P<0.05). It is indicated that the immune pressure of CD8~+T cell is one of theimportant reason for HCV NS3mutation.3. It is indicated that the HLA-A02/11/24and HLA-B13/15/46frequence was higher inWuhan population than in German population. The HLA phenotypic frequence distributionbetween Wuhan population and German population was different obviously. HLA-A11orHLA-B46frequence which was high in Wuhan population was lower in Germany population,whereas the frequence of HLA-A01/03or HLA-B07/08which is high in German populationis lower in Wuhan population. HLA genotypes of HCV patients population being analyzedand compared with Wuhan healthy population, it is suggested that the HLA phenotypicfrequency of HCV patients population and healthy population is basically the same. 4. HLA-I restricted NS3CD8~+T epitope mutation frequence analyzed in a populationlevel (HCV1b) indicated two HLA-A02positive associated NS3CD8~+T cell epitopemutation frequence was higher than HLA-A02negative, which had statistical significance(P<0.05). In another words, in two previously described CD8~+T cell epitope mutations werehighly reproducible in patients sharing the relevant HLA-Alleles in a population level. HLA-Irestriced NS3region CD8~+T cell epitope (HCV1b) were predicted and analyzed with30NS3sequence with HLA data, which suggested predicted four HLA-I positive NS3region CD8~+Tcell epitope mutation frequence have higher tendency than HLA-I negative.Conclusion1. The major genotype in Chinses HCV patients is genotype1b, and the disease ofwhich is worese than HCV other genotype.2. The amino acid sequence of HCV NS3function region was difficult to mutate orrelatively conservative. The amino acid mutations frequency of inside previously describedCD8~+T cell epitope was significantly higher compared to the outside region, which suggestedthe immune pressure of CD8~+T cell is one of the important reasons for HCV NS3mutation.3. The HLA phenotypic frequence between Wuhan healthy population and Germanhealthy population was different obviously. The frequence of HLA-A11or HLA-B46whichwas high in Wuhan healthy population was lower in German healthy population. The HCVCD8~+T cell epitope mutation frequence which is restricted by HLA with higher phenotypicfrequence is relatively higher in wuhan population.4. Two HCV NS3region (HCV genotype1b) CD8~+T cell epitope mutation wasrestricted with HLA-A02, which indicated HLA-I associated CD8~+T cell pressure was themajor driving force for viral evolution of. HLA-I restriced HCV NS3region CD8~+T cellepitope (HCV genotype1b) were predicted and predicted four HLA-I positive HCV NS3region CD8~+T cell epitope mutation frequence have higher tendency than HLA-I negative.