摘要
目的鉴定1例阿德福韦酯(ADV)治疗失败的慢性乙型肝炎患者HBV反转录酶(RT)区的新变异rtN236V,并分析其表型耐药特点。方法从1例ADV治疗失败患者的血清中提取HBV DNA,采用巢式PCR扩增HBV RT区,将PCR产物克隆到pGEM-Teasy载体,转化JM109细胞。挑选34个阳性克隆进行DNA测序并分析耐药相关突变。构建1.1倍HBV野生株和耐药株重组载体,转染人肝癌细胞系HepG2细胞。转染4h后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),转染后第4天收集细胞培养液,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HBV DNA的产量,并分析其表型耐药特点。结果该例患者在接受ADV治疗15个月后出现病毒学突破和生化学突破,测序结果显示34个克隆中,20个(58.8%)为rtN236V变异株,11个(32.4%)为rtN236T变异株,2个(5.9%)为野生株,1个(2.9%)为rtA181V+N236V变异株。rtN236T、rtN236V和rtA181V+N236V变异株的相对病毒复制力分别为野生株的89.43%、83.60%和75.44%。表型耐药分析显示:rtN236T株、rtN236V株和rtA181V+N236V株对ADV的敏感性分别为野生株的1/4.75、1/3.10和1/5.10,但对LAM、ETV和TDF均敏感。结论在ADV长期治疗失败的慢性乙型肝炎患者血清病毒池中鉴定了新的变异形式rtN236V,并与rtN236T和rtA181V株伴随存在,该变异降低了病毒的复制力及对ADV的敏感性,可能是一个新的ADV耐药相关变异。
Objective To identify a novel mutation rtN236V in the hepatitis B virus (HBV) reverse-transcriptase (RT) region of an adefovir dipivoxil (ADV)-refractory patient with chronic hepatitis B, and analyze its phenotypic resistant characteristics. Methods HBV DNA was extracted from an ADV-refractory patient with chronic hepatitis B, and the full-length RT region was amplified by nested PCR. The PCR product was cloned into the pGEM-Teasy vector, and then transfected into JM109 cells. Thirty- four clones were randomly selected for DNA sequencing, and drug-resistance-associated mutations were analyzed. After restriction double enzyme digestion and ligation procedure, the 1.1-ploid genome length HBV recombinant plasmids harboring wild type and three different mutants in RT region were constructed. The replication-competent constructs were then transiently transfected into the HepG2 cells. Four hours post-transfection, new medium containing different concentrations of lamivudine, ADV, entecavir and tenofovir were supplemented every other day for 4 days. The phenotypic characteristics of the HBV mutants were analyzed under the drug pressure, the supernatant was collected and HBV DNA production was quantitatively detected using real-time PCR. Results Virological and biochemical breakthrough appeared in this patient after ADV treatment for 15 months. Sequence analysis of 34 clones showed that there were 20 strains (58.8%) for rtN236V mutant, 11 (32.4%) for rtN236T, 2 (5.9%) for wild type and 1 (2.9%) for rtA181V+N236V. The relative viral replication capacity of rtN236T, rtN236V and rtA181V+N236V mutants was 89.43%, 83.60% and 75.44% of the wild-type strain, respectively. Phenotypic resistance analysis showed that the susceptibility of mutant harboring rtN236T, rtN236V and rtA181V+N236V was 1/4.75, 1/3.10 and 1/5.10 of the wild-type virus. The three mutants were still susceptible to lamivudine, entecavir and tenofovir. Conclusion A novel mutation rtN236V concomitant with rtN236T and rtA181V mutations has been first identified in viral pool of an ADV-refractory patient, rtN236V may decrease the viral replication capacity and the susceptibility to ADV, which might be a novel ADV resistant mutation.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2013年第4期256-259,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家“十二五”传染病重大专项子课题(2012ZX10004503)~~
关键词
肝炎病毒
乙型
变异
抗药性
病毒
hepatitis B virus
variation
drug resistance, viral