Molecular Genetics Research Of HCV Genotype6and Genotype2

BackgroundHepatitis C virus (HCV) infection has already become a worldwide health problem, its incidence rate increases year by year, what's more, its pathogenicity is stronge, and easy to become chronic, leading to hepatocytes inflammation, necrosis and fibrosis,finally it develops to hepatic cirrhosis and Hepatocellular Carcinoma(HCC). HCC is one of the malignant tumor that harms humankind health mostly.It is the fifth malignant tumor of humankind, its malignancy is very stronge, five years' survival rate is less than three percentages. At present, incidence rate of HCC is increasing persistently. According to statistical analysis, in2005incident number of HCC was triple of1975, from1.6/100000up to4.9/100000. Every year, there is about half million people dead of it. HCV's dissemination is mainly through hematological system, its infection rate is about three percentages in the whole world, that is about one hundred and seventy million people infecting HCV, each year new case is about thirty five thousands. In America, HCV infection is the most frequent infectious diseases of hematological system, there are about ten thousands people dead of this infection, what's more, the number will increase greatly in the next twenty years. In China, HCV infection rate is between two point five percentages to four point nine percentages, above fifty millions people infecting HCV. Initially, most people who infects HCV ha ve no apparente symptom,70%‐80%of acute infecting persons develop to chronic infection,10%‐20%of chronically infecting persons develop to cirrhosis, and finally5%to HCC. Now, research about concrete pathogenic mechanism and pathological process of HCV is limited, and there is no vaccines aimming directly to it and no effective therapies. In clinic, routine treatment to HCV is combinely using pegylated α interferon and ribavirin, but the sustained virological response to HCV of the patient was only fifty percent, what's more, this kind of treatment's side effect is bad, costly, and time consuming. Because of lack high‐performance, stable cell cultivation system and suitable little animal model, progress of manufacturing specific medicine and protection vaccines is too slow.In1991, Interna‐tionalcommitteeon taxonomy of viruses(lCTV) classified HCV to flaviviridae, hepacivirus. HCV is a single‐strand, positive‐sense RNA virus with envelope, the full length of genome is about9.6kb, including a large opening reading framework(OFR), and two non‐translated region(UTR) flanking5'end and3'end. Now commonly used nomenclature and classification of HCV genotype is the system of Simmonds. This system is aiming to analyze the whole genome or partial fragment of HCV sequence, combining with phylogenetic analysis, classify HCV to seven genotype and several subtype.Among the7genotypes, HCV genotype6has the most subtypes, and it is the most diverse, complex genotype. Now, according to Simmonds nomenclature system, HCV genotype6has twenty three subtype,6a to6w, mainly prevailing in southeast Asia and area around it. Standardization of Simmonds system requires determining at least one HCV full genome, analyzing of nucleotide similarity and phyletic evolution, confirming it not belonging to any genotype or subtype ever known, at last, we can classify it to new genotype or subtype. But,there are some special variations, which nucleotide similarity and genetic distances is greater than those within subtypes, but less than those between subtypes, makes the classification and nomenclature of HCV more complex. It can provide important evidence to revise and improve HCV classification and nomenclature by researching and analyzing the full genome sequences of these variants.Although, multiplicity of HCV genotype2is much less than HCV genotype, but it has unique geographicical origin and genetic diversity, and many HCV subtype2have no formal classification, denomination. Combination with HCV database, we research on its geographicical distribution and genetic diversity, in order to define preventive strategies and development of therapies and a vaccine.Objective1. By analyzing the full genome sequences of HCV genotype6isolates DH027,TV533,L349and QC271, to determine the definite subtype designation of these isolates. It can provide important evidence to revise and improve HCV classification and nomenclature by analyzing and discussing the significance of these isolates in HCV genotyping.2. Base the eleven full genome sequences of HCV genotype2isolates, combination with genetic and geographicical source information in the HCV database, investigate HCV genotype2geographicical origin, genetic diversity.Method1. Using the strategy of DNA walking on bridges and islands, to amplify and determine the full genome sequences of HCV genotype6isolates DH027,TV533,L349and QC271. Together with the full genome sequences for all the subtypes of HCV genotype6retrieved from GenBank, DNAStar, BioEdit, SimPlot, MEGA and other bioinformatical softwares were used to do the nucleotide similarity comparative analysis and phylogenetic analysis. And then a Maximum likelihood(ML) phylogenetic tree was reconstructed to determine the subtype designation of these isolates, discuss the significance in HCV genotyping.2. Determining the full genome sequences of eleven HCV2isolates, using DNAStar, BioEdit, SimPlot, MEGA, reference HCV genotype2sequences in Genebank, making phylogenetic analysis, confirming their designation. Combining the information of HCV genotype2in the database, investigate HCV genotype2geographicical origin, genetic diversity.Results1. The whole genome length of DH027,TV533,L349and QC271is9460nt,9446nt,9454nt and9553nt respectively. Comparison with63referecne sequences of HCV genotype6in Genebank, difference of full genome between DH027and6m is18.7%～19%,15%～16.1%(DH027,6n);19.5%(TV533,6k),16.9%～17.3%(TV533,6l);19%(L349,6k),13.8%～14.5%(L349,6l)； 16.2%～17%(QC271,6j),14.5%～14.8%(QC271,6i).2. The range of11whole genome length is between9508nt～9825nt. Confirm nomenclature of5HCV2subtype. QC114,QC182,QC289,QC297,QC302and QC331represent other6new lineages. In the genome area of E2, P7and NS5A, QC297,QC331,QC182and QC64have some unique Genetic Variation.Conclusion1. According to nucleotide similarity comparison analysis and phylogenetic analysis, DH027,TV533,L349and QC271can be designated into HCV6n,6l and6j subtype reluctantly, but these isolates are also close to6m,6k and6i.Besides, put these4sequences and reference sequences together to analysis, the boundary between6m,6n；6k,6l；6j,6l in the ML tree became ambiguous. The existence of these variants makes the classification and nomenclature of HCV more complex. This research can provide important evidence to revise and improve HCV classification and nomenclature.2. In the11isolates, confirm5HCV2subtype classification and nomenclature; observe6new HCV2subtype candidate; analyses the geographicical distribution of HCV genotype2;find certain unique amino acid variation,showing some genetic specificity of HCV genotype2...