| BackgroundThe genomes of hepatitis C virus(HCV)are highly level of variability.According to the differences in nucleic acid sequences.HCV can be classed to 8 genotypes and more than 90 subtypes.The HCV genotypes has distinct geographic distribution.The genotypes of 1,2 and 3 distribute worldsidely,shile other genotypes only emerged in certain district.The distribution of HCV genotypes is different in China.The prevalent genotypes were lb and 2a in northern China,while genotype 3a,3b and 6a were significantly increased in southern China.From 2004 to 2011,our team had investigated the epidemic condition of HCV among blood donors in Guangzhou,which was significantly higher than that of other regions in China.At the same time,the survey showed that the proportion of 6a in the residents outside Guandong Province was rising rapidly.The distribution of HCV genotype may be affected by the change of transmission route.Before Chinese government banned the paid blood donation,HCV was mainly transmitted via blood transfusion and plasma derivatives usage,and most HCV infected persons were subtype lb.With the introduction of voluntary blood donation laws and strict blood screening,the risk of HCV transmission through blood transfusion has reduced dramatically.However,the risk of transmission of HCV through lifestyle-related means such as sexual openness and tattooing,especially through intravenous drug abuse,had been greatly increased.Unlike the majority of patients infected through blood transfusions who were subtypes lb,the majority of intravenous drug users were subtypes 6a or 3a.Thus,changes in major transmitted routes may lead to changes in HCV genotype distribution.In addition,global trade and cultural exchanges make the movement of people in different regions more frequent,and the advantages or unique genotypes of different regions may spread locally and affecting the distribution of genotypes due to the people flow.To study the distribution of HCV genotypes in a specific area will help to understand the spread trend of HCV in the area.And it can also help to find possible high-risk infectious sources and routes,so as to further take effective preventive measures.The distribution and trend of HCV genotypes in Guangzhou are still unknown,it is very important to carry out this research.In addition,in order to further understand the sequence characteristics of HCV enveloped protein E1,the amino acid variation of E1 and its substitution patterns were analyzed in this study.Envelope protein El locates on the surface of virus particles,playing an important role on virus invasion,E1 is also one of the main targets of host immune system attack,which induces strong humoral and cellular immunity.E1 protein had been focused for the study of HCV function and vaccine design.However,due to the high mutation of E1 protein and its failure to fold properly when independently expressed,its structure is still not clear.The sequence characteristics of E1 protein in Guangzhou are rarely reported.In this study,we analyzed the variation characteristics of HCV El ectodomain for the main genotypes in Guangzhou.It may be helpful to further understand the structure characteristics of E1 protein and the relationship between variation and host immunity.Objectives1.To explore the distribution characteristics of HCV genotype and its association with age,sex and different regions among blood donors in Guangzhou,and to analyze the trend of HCV genotype in this region.2.To investigate the mutation characteristics of ectodomain amino acid sequence of E1 of HCV dominant genotypes in Guangzhou.Methods1.The positive samples for HCV antibody testing and their demographic information of 1988 cases donated in Guangzhou Blood Center from 2012 to 2019 were collected.HCV RNA testing were performed,and 800 positive samples were randomly selected for genotyping.2.The cDNA was generated using viral RNA extracted from the positive samples through reverse transcription.The E1 gene sequence was amplified by nested PCR,and the positive PCR products were sequenced.The sequencing results and the reference sequences were used to construct a phylogenetic tree to determine the HCV genotype.3.The associations between HCV genotype and demographic characteristics such as different gender,age and region were performed.The trend of HCV genotype distribution in Guangzhou was analyzed since 2004.4.Analyze the ectodomain sequences of E1 of HCV genotypes lb and 6a in Guangzhou,construct the consensus sequences of 1b and 6a,respectively.And gene sequences were translated to amino acid sequences.5.The amino acid mutation and substitution patterns of the acid sequences of E1 proteins of HCV 1b and 6a were analyzed by bioinformatics software.Results1.From 2012 to 2019,a total of 1988 positive samples of HCV antibody were collected for nucleic acid Testing,and 1360 samples were HCV RNA positive.2.Seven HCV subtypes were detected in this study,the numbers of 6a,1b,3a,3b,2a,la and 4a samples were 314,293,49,37,33,11 and 1,respectively.And the proportion of these samples accounted to 42.55%,39.70%,6.64%,5.01%,4.47%,1.49%and 0.14%,respectively.3.In the 18-25 age group,the proportion of subtype lb was higher(74.00%),and the difference was statistically significant compared with other age groups(χ2=91.91,P<0.01).4.In the four periods 2004-2007,2008-2011,2012-2015 and 2016-2019,HCV subtype 6a increased in Guangzhou.According to donors’ region of origin,the proportion of 6a in outside Guangdong Province increased from 10.47%during 2004-2007 to 37.38%during 2016-2019,showing a significant difference(χ2=21.01,P<0.01).5.There were 70(55.00%)and 79(61.24%)amino acid sites mutated in El ectodomain(192-320 aa)of HCV lb and 6a subtypes,respectively,with no significant difference(χ2=1.27,P=0.74).6.Mutations occurred mainly in the region 192-260aa.Highly mutated sites of HCV lb were scattered,while HCV 6a concentrated in the region 231-243 aa.7.All highly mutated sites were in the epitope region,most of them were conservative with BLOSUM score>0.8.No mutation was observed at the glycosylation sites of E1,N196,N209 and N305,and the mutation rates of N234 and N250 were low(1.37-6.69%).Conclusion1.HCV 6a showed an increasing trend among blood donors in Guangzhou,and 6a replaced lb as the most important genotype during 2016-2019.2.The distribution of HCV subtypes varied significantly among different age,gender and region groups.3.There was no significant difference in the mutation rate of the El in HCV lb and 6a subtypes,the mutation mainly occurred in 192-260 aa.4.The glycosylation sites of E1 were highly conserved. |
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