Study On The Molecular Epidemiology Of HCV And Its Genetic Diversity Analysis In Yunnan Province

Hepatitis C virus(HCV),confirmed in 1989,is a major causative agent of chronic liver disease.With a global prevalence of 3%it infects estimated 170-200 million people worldwide.In China,HCV prevalence is 3.2%-4.9%,it could be calculated that infected individuals are up to 50 million.China is considered as moderate and higher HCV epidemic area.HCV is the only species of the genus hepacivirus within the family Flaviviridae.It takes on highly mutational frequency and heterogeneous characters because of its RdRp lacking the proofreading function.On the basis of nucleotide similarity,HCV has been classified into 6 major genotypes and at least 80 subtypes.Moreover there are so many quasispecies existing as a swarm of genetically related variants even in a same HCV infected patient.The distribution of HCV genotypes/subtypes is epidemiologically associated with specific regions and modes of transmission.It has been proved the predominant HCV genotypes/subtypes are different in a specific region according to populations and period of time.And the composition of quasispecies also changes rapidly in the course of disease,affected by the immune status of host and the using of antiviral drugs et al.Yunnan province,known as complex geographic and climate characters,locates in the south western China,bordering with countries of Southeast Asia.Because of neighboring with the biggest drug producing area-Golden triangle,Yunnan has become an important mid-point in illegal drug trafficking route from Golden triangle to China,and then to other regions of the world.The drug using is comparatively popular in Yunnan,the most of user developed into injection drug use rapidly.This behavior provides a convenient mode for the transmission of blood borne pathogens.Yunnan has been considered as the epicenter and the most serious area of HIV epidemic in China.In our previous reports,we found that the co-infection of HIV and HCV is very common among the injecting drug users.Between the inner provinces of China and southeastern Asia countries,there are different HCV epidemiological character and genotype distribution.So it is necessary to conduct the research on HCV epidemiology and its genetic diversity in Yunnan province.In our study,a total of 132 injection drug users(IDUs) were recruited from different prefectures(Kumning,Qujing,Wenshan,Honghe,Dehong and Dali) of Yunnan province. Among them,the positive rates of HIV-1 and HCV were 68.2%and 93.1%,respectively (P＜0.001).PCR amplification and followed sequencing of the HCV 5' NCR-C and NS5B regions were performed on 82 cases IDUs coinfected with HCV and HIV-1.Phylogenetic analyses showed the consistent genotyping in 80 IDUs.Among this population,genotype 3 was the most prevalent,which accounted for 53.8%including 23.8%of 3a and 30%of 3b. The second genotype was genotype 6 accounting for 25%including one novel subtype 6 (tentatively assigned novel 6u subtype,accounting for 8.8%).And,genotype 1b accounted for 19.5%(16 cases),which was reported to be the predominant genotype in general population of China.Notably,only 1 case of 1a(accounting for 1.2%) and no genotype 2 were found.By further comparison of HCV genotype distribution in different prefecture,the proportion of patients with HCV 1b tended to decrease from the north to south and from the east to west in this province.Genotype 3 and 6 strains were more frequent in the southern prefectures.This finding showed a unique pattern of HCV genotype distribution,which was similar to that in the southeastern Asian countries but distinct from that among the general population in China. In two IDUs,genotyping results were discordant,suggesting mixed HCV infections or recombination.With multi-cloning analysis,all the two were proved to be mixed infection. HH075 was infected by three kinds of HCV including genotype 3a,3b and 6a.And,DH102 contained genotype 3a and 6u.But with this preliminary result,the possibility of recombination couldn't be excluded absolutely.Moreover,HCV of 61 cases from three different populations in Kunming prefecture were genotyped based on the HCV NS5B sequence.The results showed that the HCV genotypes distribution was distinctly different in three populations.Among IDUs mono-infected with HCV,genotype 3 was the predominant genotype,up to 93.8%including 31.3%of 3a and 62.5%of 3b.Among HIV and HCV coinfected IDUs,1b and 3b were the most prevalent,accounting for 42.9%and 35.7%respectively.No genotype 2a and only 6n were found in these two populations.In comparison,genotype 1b,2a,3b,6(6k,6n,6v )had been found in general population,and the 1b and 2a were the two predominant genotypes.And,genotype 6 consisted of several subtypes including one case of suspected novel subtype(tentatively assigned as novel 6v subtype).With the basic demographic data analysis,it was found that the average age of general population was older and aging span was dispersed than that of IDUs.In order to trace the changing of epidemiological characters of HCV at different times,in Kaiyuan prefecture from IDUs coinfected with HIV and HCV, 22 and 24 plasma samples were collected in 2000 and 2005 respectively.The described PCR amplification and genotyping were conducted.The results showed that there is no evident change of HCV genotype distribution after evolution of 5 years period.Genotype 3 was always the most predominant,followed by genotype 6.The average age and aging span had no distinct difference at different times.In the former studies two kinds of novel subtype 6u and 6v were discovered and suspected in IDUs and general population respectively.In order to confirm their novel status, strategies for amplifying and sequencing of complete genome were designed.Finally the complete genomic nucleotide sequences of four isolates(DH012/DH014/DH028 and NK46 )were obtained.The three isolates discovered in the IDUs DH012/DH014/DH028 were confirmed as a novel subtype 6u.Isolate NK46 found in the general population also was a novel subtype 6 tentatively assigned as 6v.Now subtype 6u had been validated by the HCV database.To understand the origin and evolution pattern of HCV,all the subtype lb isolates in our study were gathered to construct phylogenetic tree with the reference sequences based on the 5'NCR-C and NS5B regions.The results revealed that the isolates coming from the IDUs clustered into one unique cluster,which only constituted with the strains from IDUs in Yunnan.Comparing,the samples from general population dispersed into three clusters,which indicates there were the different origin or transmiting routes.Using multi-cloning method, HCV quasispecies polymorphisms in different population and different genotypes was analyzed.It was showed that the average genetic distance of E2 was distinctly higher than that of the E1 region.And,the quasispecies polymorphism of genotype 6 was more complex than that of the genotype 1.No evidence was found for the immune deficiency caused by HIV having distinct impact on HCV quasispecies polymorphism.These findings should be further confirmed by enlarged cohort.In conclusion,the genotype distribution and evolution characters of HCV in Yunnan province were systemically studied.HCV genotype distribution was distinctly different in different populations.Two novel subtypes 6(6u and 6v) were discovered firstly and confirmed by complete genome sequencing.And,the quasispecies polymorphism of HCV and its potential affecting factors were investigated and discussed preliminary.All of these results will provide a necessary basis for diagnosis,treatment and prevention of hepatitis C in Yunnan province,or further national wide.