江西地区儿童感染的乙型肝炎病毒全基因组序列分析

摘要

目的：初步调查江西地区乙肝疫苗免疫儿童感染的乙型肝炎病毒全基因组的分子特征。方法收集江西省儿童医院11445份乙肝疫苗免疫儿童血清标本，采用酶联免疫吸附试验检测 HBV-M，抽提乙肝表面抗原（HB-sAg）阳性血清中的 HBV DNA，分3个相互重叠片段 PCR 扩增 HBV 基因并测序，采用 ContigExpress 软件对所测序列拼接成全基因组序列，分析各读码框核苷酸和氨基酸变化。对 HBV 不同基因型以及所获得的 HBV 全基因组的 S 基因序列进行系统进化树的构建以确定基因型。结果11445份血清样品中，检测出117份 HBsAg 阳性标本，进行分段重叠扩增并测序，最终拼接出58份完整的 HBV 基因组，均为 B 基因型和 adw 血清型。对各开放读码框进行序列比对发现 PreS 区存在散在分布的突变：如 A2990C→ H48P、A2999C→ N51T、C3041T→ P65L、C3097A→L84I 和 A3136G→ T97A；S 区突变集中在120～200 aa，主要突变位点为 C533A→ P127T、A541C→Q129H、G588C→G145A、T753A→F200Y；前 C 区突变位点有 A1814C／T1815C→M1P、G1896A→ W28*，C 区出现1例 C1913A→P5T 变异；P 区出现散在分布的突变位点，未发现与耐药性有关的突变位点，其 RT 区 YMDD 核序也未发生变异；X 区氨基端主要突变点为：G1437T／A→ G22C／S、G1476A→ G35R 和 G1503A／T／C／T1504G→V44I／F／L／C，羧基端突变集中在120～150 aa，出现5例 A1762T／G1764A→K130M／V131I 突变。结论了解分析儿童 HBV 全基因组的分子特征和突变情况，为疫苗免疫儿童感染乙肝的预防和治疗提供病毒学分子信息。%Objective To investigate the molecular characteristics of complete genome of hepa-titis B virus (HBV)in HBV-infected children.Methods A total of 11 445 serum samples from children with HBV immunization were gathered in Jiangxi Children’s Hospital.ELISA analysis was used to determine HBV-M.PCR method with three overlapping primers was used to amplify the gene of HBV DNA extracted from HBsAg positive serum samples and the PCR products were sequenced directly.The sequences of three amplicons were spliced as a complete HBV genome by Contig Express,and the nucleotide and amino acid of complete HBV genome were divided into 4 ORFs to analyze the main and significant mutation sites.The phylogenetic tree of HBV S gene was constructed to determine the genotype of HBV isolates.Results A total of 117 HBV-M posi-tive samples were collected from the 11 445 serum samples,and complete HBV genomes from 58 samples characterized by genotype B and serotype adw were successfully amplified and sequenced. Mutations in the PreS region were found through analyzing the sequence of ORFs,including A2990C→H48P,A2999C→N51T,C3041T→P65L,C3097A→ L84I and A3136G→T97A.Muta-tions in S region mainly occurred in the domain of 120-200 aa and the mutation sites included C533A→P127T,A541C→Q129H,G588C→G145A and T753A→F200Y.Mutation sites in PreC region included A1814C/T1815C→M1P and G1896A→ W28*.Moreover,C1913A→ P5T muta-tion in C region was found in 1 case.Scattered distribution of mutations was observed in P re-gion.Furthermore,no drug resistance associated mutations were found in P region and no YMDD mutations in RT region.Mutation sites in N-terminal of X region included G1437T/A→G22C/S, G1476A→G35R and G1503A/T/C/T1504G→V44I/F/L/C.Mutations in C-terminus mainly oc-curred in the domain of 120-150 aa,including A1762T/G1764A→K130M/V131I mutation in 5 ca-ses.Conclusion In this study,we analyzed the molecular characteristics and mutations of com-plete genome of HBV in HBV-infected children and provided molecular information for the pre-vention and treatment of hepatitis B among children with HBV immunization.