| BackgroundCurrently,there are nearly 180 million people globally infected with hepatitis C virus(HCV).China is a country where the presence of hepatitis C is very high with about ten million people being anti-HCV positive.Unfortunately,it still lacks effective preventive vaccines.The studies on HCV genotypes and molecular epidemiology helps us to understand the distribution and dissemination laws of HCV genotypes,which are very important for controlling the spread of HCV.According to the Simmond system,HCV was classified into seven genotypes,85 subtypes,and 20 unknown but equally genetically distanced from the different subtypes.The virus of genotype 6 exhibits the most complicated subtypes and the greatest genetic diversity,and was believed to be the oldest strains among many new variants that are continuing to be discovered and separated from other subtypes.It is very difficult to get the full-length sequence of HCV genotype 6 due to the low virus titer,genetic diversity,and without reference sequence.China's latest study showed that 56.8%(582/997)of HCV genotypes are lb,followed by genotype 2,3,and genotype 6.However,the spread of HCV genotypes are different in the north and south of China.In southern China,6a had replaced 2a,which has caused a second local HCV endemic.Experts predict that genotype 6a from Guangdong will spread quickly to the other parts of the country.Hainan Island is located in the South China Sea.The major population includes Han and Li ethnic.In the modern times,the Han ethnic expectedly moved from the mainland into Hainan Island.Currently,HCV molecular epidemiology in the Han ethnic are still not documented very well.Therefore,further study of HCV in the Han will be helpful for exploring the origin and epidemiology of HCV in the Hainan Island,and at the same time for providing an appropriate prevention approach.The study on the Li ethnic population stated that it had originated in Hainan and show that its five branches have a common origin with that of the Dong Ethnic Population.The Li ethnic population in Hainan are called the ancient living fossil of mankind.So far,each subtype of HCV genotype 6 is mainly prevalent in Southeast Asia.In our previous study,we found that some of the HCV genotype 6 from the Li ethnic population in Hainan is relatively old.Therefore,by studying the distribution of the HCV genotype 6 in the Li ethnic population,its evolution,and its relationship with other parts of Southeast Asia,through bioinformatics methods,it will help to improve the general understanding of the origins of the HCV genotype 6 in Southeast Asia.Objectives1.Make an analysis of the HCV genotype distribution between the Han ethnic populations and the Li ethnic populations in Hainan.2.The analysis of the relationships between the hepatitis C virus(HCV)of the Han population in the Hainan Province and HCV of the Han population in mainland China,will clarify the transmission mode and the patterns of the HCV in the Hainan region.This will help to set down preventative measures against HCV and improve scientific knowledge about it.3.The phylogenetic analysis on the specific virus strains was carried out to find unique isolates of HCV,which were not classified into any known subtypes.Two isolates,HN1316 and HN1350,from the unknown isolates mentioned above,were selected in order to determine and amplify the whole genome of the isolates.Thus,the study of the relationship between specific virus strains of the HCV genotype 6 in the Li ethnic population of Hainan and specific virus strains of the HCV genotype 6 from Southeast Asia will help to improve the understanding of the origins of the ancient HCV genotype 6.Methods1.246 HCV RNA positive serum samples were collected from the Hainan General Hospital,between January 2013 and June 2014(176 cases were from the Han People,70 cases were from the Li People).HCV-RNA was extracted,amplified in the HCV Core-E1 region,and sequenced using the phylogenetic tree to analyze and determine the HCV genotype distribution of the Han and Li People in Hainan.2.Furthermore,BEAST1.6 software and a Bayesian Markov chain Monte Carlo(Bayesian-MCMC)method were used to define HCV sequences of the Han population in Hainan and the Han population in China.The last,extending the number of population infected HCV to 402 cases,all of the above methods were applied to the study on HCV epidemiology and clinical characters in the bigger population.And logistic analysis was used to observe the high risk of chronic HCV infection in Hainan.3.The full genome sequence of the two strains,HN1316 and HN1350,in Baisha County,were amplified with "DNA walking on bridges and islands" strategy.Then,the bioinformatics software,DNAStar,BioEdit,MEGA,SimPlot,BEAST,were used to perform genetic similarity analyses.Then,ML trees based on the HN1316 and HN1350 isolates,were reconstructed with the previously determined sequences of the HCV genotype 6 variant strains(HKP7,HKP16,HKP25,HKP26,HKP38,and HKP43)in Haikou city,as well as the known sequences for HCV genotype 6 strains downloaded from GenBank,to determine their subtype relationship and analyze the origin of HCV genotype 6.Results:1.HCV epidemiology in the Han ethnic in Hainan Province:(1)HCV genotypes found in the Han ethnic population of 163 were:la,lb,2a,3a,3b and 6a,among which 6a accounted for 33.7%(55/163),followed by lb for 33.1%(54/163),3b for 14.7%(24/163),2a for 6.7%(11/163),3a for 8.6%(14/163),and la for 3%(5/163).Compared with other areas in China,6a in Hainan is the most prevalent subtype.(2)Phylogeographic distribution was found:HCV isolates from Hainan merged into the isolates from the Chinese mainland.They distributed alternately in each cluster.Then,we continued to detect the genotype of 402 patients with chronic HCV infection in the Han Ethnic,and found that genotype distribution and epidemiological characteristics were as follows:(3)6a was still the most prevalent subtype in Han Ethnic of Hainan Island followed by 1b,3b,2a,3a,and 1a.(4)Based on the results of logistic analysis,the high risk of chronic HCV infection were used the medical apparatus with pollution(P<0.05)and IDUs(P<0.01),blood transfusion(P<0.05).6a was more frequent in the patients through IDU with Chi-Square test.2.There were 58 HCV positive serum samples from the Li ethnic population in Hainan.Genotype 6 accounted for 94.8%(55/58),lb for 1.7%(1/58),2a for 1.7%(1/58),3b for 1.7%(1/58).Furthermore,55 of HCV genotypes 6 found in the Han ethnic population were:6a for 3.5%(2/58),6xa for 3.5%(2/58),6w for 5.2%(3/58),6e for 1.7%(1/58),6v for 1.7%(1/58)and 6r 1.7%(1/58),and the others were unique strains for 77.6%(45/58).This result indicated that HCV genotype 6 was a specific viral strain found in the Hainan Li ethnic population that was distributed with high genetic diversity.In addition,most of the 45 viruses,isolate sequences in the phylogenetic tree were clustered independently from the HCV sequence in Southeast Asia and other parts of the agglomeration,as a separate cluster evolution.3.Two samples of HN1316 and HN1350 were successfully amplified along their whole genome.They had similarities with other full genome sequences from 70-80%.Phylogenetic analysis of the whole genome sequences suggests that HN1350,the six new variants(HKP7,HKP16,HKP25,HKP26,HKP38 and HKP43),6g and 6w have a common ancestor.HN1316,6a,6b,6xd,and 6_ZS202,6_L373 all shared a common ancestor.The full genome of HN1350,HN1314 and HN1411 shared 7.6%-14%genetic distance.Conclusion1.The distribution pattern of HCV genotypes among the Han and Li ethnic population exhibits significant differences.In the Han ethnic population,HCV isolates were defined as:la,lb,2a,3a,3b,and 6a.They were consistent with the distribution in the Han Chinese mainland,but the proportion of each subtype was not the same as the distribution.Among them,6a was the major HCV in the Han ethnic population,followed by to lb,3b,2a,3a,and la.The HCV genotypes 6,along with many new unclassified subtypes,have a very high diversity in the Li ethnic population,in Hainan.2.Two ethnic populations of people,the Han and Li in Hainan,were shown to have different modes of transmission routes.Geographical phylogenetic analysis showed that HCV in the Han population displayed 1a,1b,2a,3a,3b,and 6a,which mainly originated from the Chinese mainland.It suggests that the transmission routes of the HCV in the Han population are part of the whole transmission cycle of HCV in the mainland of China.The HCV 6a genotype in the Han ethnic population is the most important genotype,suggesting that 6a might have started growing in China after the rapid spread of HCV in the Guangdong region began.In the Li population,HCV genotype 6 has a high genetic diversity with unique patterns.The system shows that the phylogenetic analysis of genotype 6 in the Li population is specifically different from genotype 6 in other parts of Southeast Asia.The HCV in the Li population clustered together and expanded independently along evolution history,which suggests that the HCV genotype 6 in the Li ethnic population lived in a closed-off type of life style.The virus had to evolve over a very long period of history.The genotype 6 HCV in the Li ethnic population might be involved from the origination of the HCV virus in Southeast Asia.3.For the first time,we identified a new subtype of HCV genotype 6 from the chronic hepatitis C patients in the Li ethnic population living in Hainan.HN1350,HN1411,and HN1304 clustered together showing the same epidemiology spread.They met the new Subtype assignment conditions,meaning they could be classified into a new subtype in the HCV genotype 6.HN1316 had a complete genome sequence,but it lacked three other independent isolates.It will remain as a single viral string,and it will not be determined as new subtype unless more isolates are found. |
PDF Full Text Request