| Hepatitis C virus (HCV) is a single-stranded RNA virus with genome length of about 9.6 kb. It is classified into hepacivirus genus of flaviviridae family. In the process of HCV replication, proofreading function of RNA dependent RNA polymerase is lacking. It results in the high variation of HCV genome. Despite of genotype and subtypes, the HCV sub-mutation in host is displayed. The variants within homological rate of 95% were called as quasispecies. Existence of HCV quasispecies reflectes the results of competitive selection based on the viral replication in certain circumstances. The complexity and composition of the quasispecies were closely related with the progression of disease. HCV quasispecies with mutation in CTL epitopes were the the main reason of virus immune evasion from the host immune monitoring and treatment no-response.According to HCV viral load and other clinical parameters,16 hepatitis C patients were screened from 50 HCV infected individuals, and were recruited in this study. With amplification of HCV 5'NCR and NS5B fragments and subsequently nucleotides sequencing and analyzing, their HCV genotypes/subtypes were determined. It was shown that HCV genotype/subtypes lb,3a,3b and 6n were co-prevelent in this throng. And, lb (41.25%) and 3b (36.75%) were predominant genotype. All the recruits were treated with different therapy, their disease progression was monitored for 1.5 years. According to the detected clinival parameters, the disease was recovered in 8 recruits with undetected viral RNA. And, chronic HCV infection was maintained in other 8 recruits. Moreover, the method of multiple cloning and sequencing to detect NS5A and E2 quasispecies was established. Neighbor-joining evolutionary tree, standardization entropy, average genetic distance and the dN/dS value were used to evaluate HCV quasispecies complexity. At the monitor start point, the range of genetic distance of E2 quasispecies were 0.0625-0.0097 and 0.0037-0.0269 for the chronic infectors and the treatment responders, respectively. The range of entropy were 0.9063～0.9786 and 0.5769～0.8849. For NS5A quasispecies, the range of genetic distance of were 0.0050～0.0113, and 0.0022～0.0095 respectively, and entropy ranged in 0.8847～0.9734 and 0.5187～0.8007, respectively. The values of genetic distances and entropy for these two genes in chronic infector were bigger than that in the treatment responders. It suggested that hepatitis C patients with less quasiapecies heterogeneity were easily lead to treatment response. At monitor start, dN/dS ratio of NS5A and E2 quasispecies were less than 1 for all the chronic infectiors and 6 treated reponders. It was mainly cused by by genetic drift. It was noted that the dN/dS ratio in 2 treated reponders were more than 1. It was considered as the consequence of Darwinian evolution pressure.Furthermore, the disease progression of recruits was monitored, and blood samples were collected at interview times. Using the collected samples, the origin time and evolutionary rate of quasipecies within host was estimated in BEAST software package. And, Bayesian Skyline Plot was constructed to evalute the evolution of quasispecies population. The correlation between the quasispecies variety index (diversity and complexity) and biochemical index (ALT value and AST value) of liver function was further analyzed. For 8 recruits with chronic infection, the quasispecies of E2 region and NS5A region displayed two evolution modes during their disease progression. The evolution modes of quasispecies for these two genes fragments were showed to be in coordination. It means NS5A and E2 quasispecies displayed the same evolution mode for one patient. The evolutionary rate (5.84×10-3 sub/site/month) in treatment responders was significant higher than that of chronic infectors (0.61×10-3～1.31×10-3 sub/site/month). No significant correlation between complexity and diversity of HCV E2 quasispecies with serum ALT and AST levels (p>0.05) was found here.Finally, Elispot was used to detect the changing of active CTL in PBMC during of chronic disease progression. According the nucleotide sequence of major HCV quasispecies, amino acide sequence of CTL epitope at monitor start time was LLFTPGSKQNV. However, this CTL epitope was changed as SFFTSGPKQNI at 1 years monitor (T4). Therefore, antigen peptide (NH2-SLLFTPGSKQNVQ-COOH) was designed and synthetized. It was used to stimulate cultured PBMC from T1 and T4 collection times, respectively. According to the chromogenic spots, the number of active CTL at T1 time was 307. This number was declined to 21 with the disease progression to T4 phage. It was proved that CTL epidtopes coding genes' mutation could lead to viral evasion from immune recognization.In this study, the disease progression of mono HCV infected individuals was monitored. The characters of quacispecies variation and evolution were analyzed with sequence analysis of multiple cloning. The research on mechanism of HCV immune evasion was performed with Elispot method. Although it was still preliminarily, the function of CTL antigen variation on immune evasion could be concluded. These results will be the necessary data to clarify the mechanism of hepatitis C chronicity, and will provide the basis for the development of vaccine. |
PDF Full Text Request