| Hepatitis C virus (HCV) is a globally distributed human pathogen that currently infects estimated170million people, the high chronic infection rate significantly increases the risk of liver cirrhosis and hepatocellular carcinoma. As a single-stranded RNA virus with genome length of about9.6kb, HCV is classified into hepacivirus genus offlaviviridae family. In the process of HCV replication, proofreading function of RNA dependent RNA polymerase is lacking, which result in the high mutation rate of HCV genome. Based on its genetic diversity. HCV can be classified into at least six genotypes and more than80subtypes with phylogenetic analysis. The various HCV genotypes and subtypes have been associated with different epidemiological and geographical spread patterns. Genotypes1and2are globally distributed; genotype3is predominant in Asia, North America and parts of Europe; Endemic prevalence patterns was observed in other genotypes, such as genotype4in Europe, Middle east Asia and Central Africa; genotype5in parts of Africa and Europe, genotype6in Southeast Asia and North America. Regarding to the transmission route. Genotype1,3,6are mainly through intravenous drug use, blood and other rout; Genotype2is mainly through the mode of slave trade; genotype4and5are diffusing mainly due to dirty needles and medical accident. Up to date, four HCV genotypes, including genotype1,2,3and6were prevalent in China. Genotype1b and2a were mainly found in Northern China. The fast spreading of genotype3and6was recently reported in Southern and South-Western China.With the development of evolution theory and related analysis methods, such as coalescent theory and evolutionary molecular, it is possible to understand the characters of HCV evolution and migration. The further understanding on the prevalence and evolution characters of various genotypes is helpful for deep description of HCV epidemic and making of prevention strategy. Here, we selected a total of150HCV antibody positive samples collected from Yunnan in2005,2008and2010, respectively. With HCV sequencing and genotyping analysis, we samples could be classified into4genotypes (1/2/3/6) and9subtypes (1b/2a/3a/3b/6a/6m/6n/6v/6k). In2005, there were6subtypes including1b(14cases,41.3%),2a(6,17.6%),3b(9,26.3%),6n(2,5.9%),6v(1,3.0%), and6k(2,5.9%). In2008, there were6subtypes including1b(8,20.0%),2a(9,22.5%),3a (3,7.5%),3b(18,45.0%),6a(1,2.5%),6n(1,2.5%). In2010, there were5subtypes, which were1b(13,32.5%),3a(8,20.0%),3b(16,40.0%),6m(1,2.5%),6n(2,5.0%). We found that subtypes lb(total35,30.7%) and3b(total43,37.7%) was mainly prevalent in Yunnan area through observation, and part HCV genotypes in Yunnan province have significant change as follow:the3a subtype changed from0(0%) case in2005to8(20.0%) cases in2010; the2a subtype decreased from6(17.6%) cases in2005to0(0%) case in2010. We also found that in three years subtype3b had a high infection rate, subtypes2a,3a had a significant variation through chi-square analysis. This result suggested the development trend of HCV genotype, and the special distribution that was different from the domestic and Southeast Asia was formed in Yunnan province. After that we have taken major subtypes3b as an example and used the BEAST software to estimate the original time and evolutionary rate. We found that Chinese3b subtype derived from Southeast Asian countries, and the3b subtype initial source may be the Pakistan through analysis. And the results showed that the infected populations of subtype3b in Yunnan area were divided into two clusters that were IDUs and non-IDUs. BSP was constructed to evalute the evolution of two clusters, and we found that there may be HCV cross communication behavior between two kinds of people. Subtypes1b (total35,30.7%) and3b (total43,37.7%) was definitely predominant in Yunnan. The other HCV genotypes were shown to be changed,:3a subtype was increased from0case (0%) in2005to8cases (20.0%) in2010; and the2a subtype decreased from6cases (17.6%) in2005to0(0%) case in2010. Based on the diversity of subtypes3b strains, BEAST software was used to deduce its original time and evolutionary rate, the original time was about61years before and the evolutionaty rate was0.55E-03. According to the comparing phylogenetic analysis with reference sequences,3b subtype strains in Yunnan was deduced to originate from Southeast Asian countries, and its initial source seems the Pakistan. The distribution of3b subtype is also connected with transmission routs, its strains could be divided into two clusters from Injecting Drug Users (IDUs) and non-IDUs respectively. BSP analysis was conducted to evaluate the spread of two clusters, it was found the cross transmission between this two population occurring frequently.HCV6u subtype was distinguished in2003, and fast spreading recently. The virulence of6u in culture system was a suit contrast to explain its molecular mechanism. The6u specific primers were designed to amplify the fragment of Core-p7-NS2(～3100bp). We divided PCR template into4parts (named as1-4) to amplify respectively, then get the long fragment with overlap PCR technology. This fragment was digested with double restriction endonucleases (Agel and Spel), and then inserted into plasmid J6/JFH-1to construct the new6u/JFHl recombinant plasmid. Chimeric6u/JFH-1virus RNA was obtained with reverse transcription taken linearized plasmid as template. Subsequently, HCV RNA was transfected into Huh7.5.1cell to construct culture system. The HCV RNA was detected in collected supernatant and infected cells. The results showed that virus RNA has replicated in cultured cells. The viral load in supernatant was up to lO3copies/ml. It means the6u chimeric virus culture system has been preliminary constructed.In this study, HCV genotypes distribution was serially described from2005-2010. The origin and evolutionary characteristics of3b subtype were analyzed in this region. We also speculated on the HCV genotype distribution characteristics of Yunnan province in future. In addition, the chimera virus6u/JFH-l culture system was preliminary established, it do provides a technical platform for the research of pathogenesis, vaccine development and other aspects. |
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