中文摘要：

目的分析乙型肝炎病毒（HBV）反转录酶区rtI233V变异的演变规律及其与阿德福韦酯（ADV）耐药的相关性。方法分析9830例慢性HBV感染者血清样本中HBV rtI233V变异的检出率。选择其中2例代表性患者,动态收集血清样本,扩增HBV RT基因并克隆测序（〉20个克隆/样本）,观察rtI233V变异的演变过程。构建pTriEx-HBV（C）1.1倍野生株和3种变异株（rtI233V、rtN236T、rtI233V＋rtN236T）复制子,瞬时转染HepG2肝癌细胞,分别加入不同浓度的拉米夫定（LAM）、ADV、恩替卡韦（ETV）和替诺福韦（TDF）。采用实时荧光定量PCR法检测不同浓度药物作用后细胞培养上清中HBV DNA的水平,分析变异病毒复制力与表型耐药的变化特点。结果9830例慢乙肝患者血清样本中共检出rtI233V位点变异28例,检出率0.28%,其中rtI233V单独变异19例,与rtN236T等经典耐药变异联合出现9例。该变异的检出患者均有ADV治疗史,其中16例（57.1%）接受ADV单药治疗6个月以上,12例（42.9%）接受包括ADV的多药序贯联合治疗1年以上。病毒复制力与表型耐药分析显示,rtI233V变异株的复制力与野生株接近,rtN236T变异株的复制力较野生株显著降低;rtI233V变异株对LAM、ADV、ETV和TDF均敏感,rtI233V＋rtN236T联合变异对耐药性的影响不大,但rtI233V变异可明显恢复rtN236T变异株受损的复制力。结论rtI233V位点变异与ADV应答不佳相关,虽不直接降低病毒对ADV的敏感程度,但可增加经典ADV耐药病毒株的复制力,是一种复制力补偿变异。

英文摘要：

Objective To analyze the evolution of rtlZ33V mutation in the reverse transcriptase domain of hepatitis B virus （HBV） and its association with adefovir dipivoxil （ADV） resistance. Methods The rate of detection of rtI233V mutation in 9830 patients with chronic HBV infection was analyzed. HBV reverse transcriptase genes isolated from serial serum samples of two patients were amplified by nested PCR, and clonal sequencing （〉20 clones/sample） was performed to analyze the evolution of rtI233V mutations. The replica of pTriEx-HBVI.1 vectors harboring wild-type and mutant strains （rtI233V, rtN236T, rtI233V＋rtN236T） were respectively constructed and transiently transfected into HepG2 cells. Media containing serial concentrations of lamivudine （LAM）, ADV, entecavir （ETV）, or tenofovir （TDF） were used to treat the cells. Then HBV DNA in the supernatants was quantitatively determined by real-time PCR in order to analyze HBV mutants＇ replication competence and phenotypic characteristics under the drug pressures. Results The detection rate of rtI233V mutation in 9830 nucleos（t）ide analogues-treated patients was 0.28% （28/9830）, including 0.19% （19 patients） with rtI233V individual mutation and 0.09% （9 patients） with rtI233V mutation combining with rtN236T or other mutations. All of the patients had rtI233V had ADV exposure history： 16 （57.1%） of them received ADV monotherapy for over six months, and 12（42.9%） of them received ADV combined sequential therapy for over 12 months. Replication competence and phenotypic resistance analysis showed rtI233V and wild-type strains had similar viral replication competence, while rtN236T exhibited significantly lower replication competence compared with wild-type strains, rtI233V strains remained sensitive to LAM, ADV, ETV, and TDF and showed little influence on drug resistance when combined with rtN236T, but it showed ability to restore the defected replication capacity ofrtN236T strains. Conclusions The rtI233V mutation is ass