大理地区流行3a丙型肝炎病毒E基因序列分析

Sequencing of the E gene of hepatitis C virus genotype 3a in the Dali area

目的了解云南省大理地区主要流行基因型3a丙型肝炎病毒(Hepatitis C virus,HCV)E基因分子特征和遗传进化特点。方法2014年1-5月,在大理州人民医院采集临床诊断为丙型肝炎患者血清,提取病毒RNA,采用3a HCV E基因特异引物进行RT-PCR扩增并测序,采用Clustal X2.1、DNASTAR、Mega 7.1、NetNGlyc 1.0和SYFPEITHI 1.0等生物信息学软件进行序列分析。结果共收集到丙型肝炎患者血清48份,其中4份(标本号为3、4、9、12)3a HCV E基因扩增阳性,测序后分别获得4株病毒1327 nt序列。遗传进化分析显示4株HCV病毒与国内、外流行3a型HCV位于同一进化分支内,核苷酸和氨基酸同源性分别为83.2%~91.5%和82.4%~92.3%,与基因3型其它基因亚型HCV同源性分别为69.6%~73.1%和73.6%~77.7%,与其它基因型HCV病毒同源性均低于66%和70.3%。4、9、12号病毒E基因均存在10个潜在糖基化位点,3号病毒缺少1个224位潜在糖基化位点,其余9个糖基化位点位置与另3株病毒均相同;与其它病毒相比,4株病毒仅有5个T细胞抗原表位预测分值发生变化,其余抗原表位预测分值变化不大。结论大理地区主要流行基因型是HCV3a,其致病性、免疫特性等生物学特征未发生明显改变。

Objective To ascertain the genetic evolution and molecular characteristics of hepatitis C virus(HCV)genotype 3a,the predominant genotype currently circulating in Dali,Yunnan Province.Methods Sera samples were collected from patients infected with HCV in the First People’s Hospital of Dali from January to May 2014.RNA samples were extracted from the serum samples and were then analyzed using RT-PCR with E primers.The PCR products were sequenced,and sequencing was performed using bioinformatic software.Results Of 48 serum specimens from patients with a suspected HCV infection,4 tested positive for HCV according to RT-PCR with HCV 3a genotype E gene specific primers.Four 1327-nt sequences were obtained via sequencing using RT-PCR.Homology analysis of the nucleotide sequences and amino acid sequences of HCV E genes from 4 HCV-positive serum samples collected in Dali indicated that the 4 HCV strains from Dali are genotype 3a.The E gene had a nucleotide sequence similarity of 83.2-91.5%and an amino acid sequence similarity of 82.4-92.3%to the genotype 3a from China and overseas.A phylogenetic tree based on the nucleotide sequence of the E gene indicated that HCV from the 4 serum samples collected in Dali fell within the HCV genotype 3a evolutionary clade.There are 10 potential glycosylation sites on the E gene of HCV from samples 4,9,and 12,but HCV from sample 3 has 9 potential glycosylation sites.The predicted value of 5 T-cell epitopes of the 4 HCV strains from Dali increased or decreased because of amino acid mutations.The predicted value of other T-cell epitopes did not change.Conclusion HCV genotype 3(3a)is the predominant genotype(subtype)currently circulating in Dali,Yunnan Province.The virulence and immunological characteristics of HCV 3a from Dali have not changed significantly.