恩替卡韦初治耐药的乙型肝炎病毒体外表型分析  

Phenotypic assay of hepatitis B virus isolates from one treatment-nave patient with entecavir resistance

作　　者：于德敏[1] 张东华[1] 姜节洪[1] 余敏杰[1] 张欣欣[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院感染科,上海200025

出　　处：《内科理论与实践》2013年第2期101-105,共5页Journal of Internal Medicine Concepts & Practice

基　　金：国家自然科学基金(项目编号:30872251);上海市自然科学基金(项目编号:09ZR1418800)

摘　　要：目的:探讨恩替卡韦初治患者发生病毒学突破的可能病毒学原因。方法:扩增1例恩替卡韦初治发生病毒学突破的患者在治疗基线和发生病毒学突破时的乙型肝炎病毒(HBV)全长基因组,构建全长基因组克隆并测序,筛选代表性基因组构建1.3倍复制型质粒,转染Huh7细胞后以Southern blot方法观察病毒复制能力,并分别以单克隆病毒和病毒准种群的形式进行恩替卡韦体外药敏试验,Southern blot检测基线及病毒学突破时单个病毒株及准种群的体外药敏情况。结果:在治疗基线和病毒学突破时间点的22个和25个全长克隆中分别筛选出7个和8个代表性克隆,体外药敏结果显示该患者血清中无论是基线还是病毒突破后的病毒株,无论单克隆病毒株还是本研究所构建的病毒准种群在体外情况下均对恩替卡韦敏感。结论:在本例恩替卡韦初治患者未发现与病毒学突破相关的病毒因素,其耐药机制值得进一步研究。Objective To explore the possible viral factors associated with virological breakthrough in one entecavir nave patient.Methods Full length hepatitis B virus（HBV） genome was isolated from one entecavir nave patient who developed virological breakthrough at both baseline and breakthrough time points,and cloned into TOPO-XL-PCR vectors after PCR amplification.Full length HBV clones were constructed and sequenced.Representative plasmids at baseline and breakthrough time points were selected through comparing full length genome sequences.One point three genome recombinant replicative plasmids were constructed by overlapping PCR with corresponding representative plasmids as templates and then transfected into Huh7 cells.Viral replicative ability was determined with Southern blot assay.Recombinant replicative plasmids at each time point were mixed according to ratio of viral replicative abilities to construct HBV quasispecies population plasmids.Each recombinant replicative plasmid and HBV quasispecies population plasmids at each time point were transfected into Huh7 cells and then phenotypic assays were carried out to investigate the in vitro susceptibility of HBV isolates to entecavir.Results Seven representative plasmids at baseline and 8 at breakthrough time points were selected from 20-25 full length colonies.Phenotypic assays suggested both monoclonal virus and HBV quasispecies population remained susceptible to entecavir.Conclusions No viral factor was identified that was associated with virological breakthrough in this entecavir nave patient.Further investigation is needed for mechanisms of entecavir nave drug resistance.

关 键 词：乙型肝炎病毒 病毒学突破 体外表型试验 恩替卡韦 准种 

分 类 号：R512.62[医药卫生—内科学]