HBV反转录酶区rtA181S变异与阿德福韦酯耐药相关性研究

rtA181S mutation in the reverse-transcriptase domain of HBV is associated with adefovir dipivoxil resistance

目的分析HBV反转录酶(RT)区rtA181S变异与阿德福韦酯(ADV)耐药的相关性。方法应用直接测序法筛选分析大样本慢性HBV感染者rtA181S变异的检出频率,并对1例接受ADV单药治疗失败的慢性乙型肝炎患者血清中HBV RT区基因进行克隆测序,分析相关变异形式。用XhoⅠ和SphⅠ双酶切pGEM-Teasy RT及pTriEx-HBV(C)载体后再连接,构建1.1倍HBV野生株和耐药株的重组质粒,转染人肝癌细胞系HepG2细胞,5 h后分别加入不同浓度的ADV(0、0.033、0.100、0.330、1.000、3.300μmol/L)。隔天换药,4 d后收集细胞上清,采用实时荧光定量PCR法检测不同药物浓度作用下细胞培养上清中的HBV DNA载量,并分析其表型耐药特点。结果 9830例慢性HBV感染者的12 000个血清样本中,有46例样本检出rtA181S变异(单独或与其他耐药变异联合出现),在653例中检出经典的rtN236T/A181V变异。其中随访的1例在接受ADV治疗19个月后出现了病毒学和生化学突破,直接测序检出rtA181S+N236T变异,克隆测序分析显示在24个克隆中11个(45.83%)为野生型,6个(25.00%)为rtN236T变异型,5个(20.83%)为rtA181S变异型,1个(4.16%)为rtA181V变异型,1个(4.16%)为rtA181S+N236T变异型。在体外实验中,rtN236T、rtA181S和rtA181S+N236T变异株的相对复制力分别是野生株的91.35%、29.90%和68.53%。表型耐药分析显示rtN236T、rtA181S和rtA181S+N236T变异株对ADV的灵敏性分别为野生株的1/4.41、1/3.05和1/5.43。结论 rtA181S是一种ADV耐药相关变异,可单独或联合其他变异引起患者耐药。但与经典ADV耐药变异相比,rtA181S变异引起的ADV耐药相对较弱,临床检出率较低。

Objective To analyze the association between rtA181S mutation in the reverse-transcriptase （RT） domain of HBV and adefovir dipivoxil （ADV） resistance. Methods Incidence of rtA181S mutation of HBV RT region was screened and analyzed from large samples of chronically HBV-infected patients by direct sequencing. Clonal sequencing was performed and drug-resistance-associated mutations were analyzed from the serum of an ADV-refractory patient with chronic hepatitis B. pGEM-Teasy RT plasmid and pTriEx-HBV （C） plasmid were digested and ligated by the restriction enzyme （ XhoⅠ/SphⅠ）, and the 1.1-ploid genome length HBV recombinant plasmids harboring wild-type and three kinds of mutants in RT region were constructed. Then the replication-competent constructs were transiently transfected into the HepG2 cells. Five hours post-transfection, new medium containing different concentrations of ADV （0, 0.033, 0.100, 0.330, 1.000, 3.300 μmol/L） was supplemented every other day for 4 days. To analyze the phenotypic characteristics of the HBV mutants under the drug pressure, the supernatant was collected and HBV DNA production was quantitated using real-time PCR. Results A total of 12 000 serum samples of 9830 patients with chronic HBV infection were screened. rtA181S mutation was detected in 46 nucleos（t）ide analogues-treated patients. By contrast, signature ADV-resistance mutations rtN236T and A181V were detected in 653 patients. The rtA181S emerged either alone or in concomitance with other drug-resistance mutations. A follow-up patient developed virological and biochemical breakthrough after 19-month ADV monotherapy. At the time of sampling, rtA181S＋N236T was detected by direct sequencing. Clonal sequence analysis of 24 clones showed that there were 11 （45.83%） for wild-type, 6 （25.00%） for rtN236T, 5 （20.83%） for rtA181S, 1 （4.16%） for rtA181V, and 1 （4.16%） for rtA181S＋N236T. The relative viral replication capacity of rtN236T, rtA181S and rtA181S＋N236T mutants was 91 . 35%, 29.90% and 68.53% that of the wild-type strain, respectively. Phenotypic resistance analysis showed that ADV suscep-tibility of mutants harboring rtN236T, rtA181S and rtA181S＋N236T was 1/4.41, 1/3.05, and 1/5.43 that of the wild-type strain. Conclusions rtA181S is an ADV-resistance-associated mutation, which may cause the drug resistance solely or with other mutations in patients with chronic HBV infection. Compared to signature ADV-resistance mutation, rtA181S mutation is less resistant and less frequently detected in ADV-resistance patients.