基于长期研究队列的广西隆安县乙型肝炎病毒准种研究  

Study on the characterization of hepatitis B virus quasi-species based on the long-term cohort study in Long′an County,Guangxi

作　　者：贾蕙华 陈钦艳[1] 蒋智华[1] 王学燕[1] 张文嘉 TIM J Harrison J BROOKS Jackson LI Wu 方钟燎[1,2] JIA Huihua;CHEN Qinyan;JIANG Zhihua;WANG Xueyan;ZHANG Wenjia;TIM J Harrison;J BROOKS Jackson;LI Wu;FANG Zhongliao(Guangxi Zhuang Autonomous Region Center for Disease Control and Prevention,Guangxi Key Laboratory for the Prevention and Control of Viral Hepatitis,Nanning,Guangxi 530021,China;School of Preclinical Medicine,Guangxi Medical University Nanning,Guangxi 530021,China;Division of Medicine,University College London Medical School,London,UK;Carver College of Medicine,University of Iowa,Iowa City,USA)

机构地区：[1]广西壮族自治区疾病预防控制中心,广西病毒性肝炎防制研究重点实验室,广西南宁530021 [2]广西医科大学基础医学院,广西南宁530021 [3]Division of Medicine,University College London Medical School,London,UK [4]Carver College of Medicine,University of Iowa,Iowa City,USA

出　　处：《中国热带医学》2023年第8期822-827,共6页China Tropical Medicine

基　　金：国家自然科学基金项目(No.81860595)。

摘　　要：目的明确广西隆安县队列HBsAg无症状携带者感染的乙型肝炎病毒(hepatitis B virus,HBV)准种序列长期演化特征。方法收集9名广西隆安县队列HBsAg无症状携带者2004年、2007年、2013年、2019年或2020年4个不同时间点血清样本。酶联免疫吸附测定法检测HBV血清学标志物;聚合酶链式反应(polymerase chain reaction,PCR)荧光探针法检测HBV病毒载量;试剂盒提取HBV DNA;PCR法扩增HBV全基因组,进行二代测序;利用Mega等软件对所获得的序列进行生物信息学分析。结果共获得23份血清样本,309条全长基因准种序列,每份标本平均获得(0.18±0.07)G测序数据量。55.55%(5/9)的研究对象携带的HBV毒株基因型在长期进化过程中发生了基因型转换,PreS/S区系统进化树基因分型结果与全基因组分析结果完全一致;发现B/C、I/C重组体;各研究标本的Sn值范围为0~0.37,D值范围为0~0.11;共检出21种特殊单核苷酸/氨基酸突变位点(S区7种,X区2种,PreC区3种,BCP区9种)和6种缺失突变,发现多联突变组合形式,未发现任何耐药突变,77.8%(7/9)研究对象在2004年携带的HBV毒株发生了BCP区nt1762(A→T)/1764(G→A)双突变和PreC区1896位(G→A)点突变,HBV基因可在无抗病毒药物压力下由突变型可恢复为野生型和(或)野生型转变为突变型;HBV基因组进化率为2.03×10^(-5)~3.50×10^(-3)。结论在HBV自然感染进程中,HBV基因型和重组体、准种复杂性和多样性可随时间发生改变;HBV基因突变型和野生型可相互转换,一定程度上降低了利用基因型和相关突变来预测临床结局的价值;隆安县乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)无症状携带者HBV基因组进化率很高。Objective To clarify the long-term evolution of hepatitis B virus(HBV)quasi-species in HBsAg asymptomatic carriers in Long'an county,Guangxi.Methods ELISA was used to detect serological markers of HBV.Viral loads were measured by real time PCR.HBV DNA was extracted from serum by kits.The whole HBV genome was amplified using nested PCR and amplicons were sequenced by next-generation sequencing(NGS).These sequences from NGS were analyzed by the software like Mega.Results Serum samples were collected from 9 HBsAg asymptomatic carriers in Longan County,Guangxi at 4 different time points in 2004,2007,2013,2019 or 2020.A total of 23 serum samples and 309 full-length gene quasi-species sequences were obtained,with an average amount of(0.18±0.07)G sequencing data for each sample.Genotype of 55.54%(5/9)the studied subjects underwent genotype conversion during the long-term evolution process of HBV quasi-species,and the genotyping results of the phylogenetic tree in the PreS/S region are in perfect agreement with the results of the whole genome analysis;recombinant B/C,I/C were found;the Sn ranged from 0 to 0.37 and the genetic diversity ranged from 0 to 0.11,respectively.A total of 21 special single nucleotide/amino acid mutations(7 in the S region,2 in the X region,3 in the PreC region and 9 in the BCP region)and 6 deletion mutations were detected,multiple mutations were found and no drug resistant mutations were found;77.8%(7/9)of the HBV strains carried by the subjects in 2004 had double mutations at nt1762(A→T)and 1764(G→A)and a stop mutation at nt1896(G→A);HBV mutations can be restored from the mutant type to the wild type and(or)vice versa without antiviral drug pressure,and The evolution rate of HBV genome was 2.03×10^(-5)~3.50×10^(-3).Conclusion HBV genotype,recombinants,genetic complexity and diversity of HBV quasi-species can change over time during in natural infection.The transformation between HBV mutation type and wild type reduces the value of predicting clinical outcomes by genetic types and relat

关 键 词：乙型肝炎病毒 基因型 二代测序 准种 基因突变 

分 类 号：R512.62[医药卫生—内科学]