乙型肝炎病毒反转录酶区rtI233V变异的演变及病毒学特点分析

发布时间：2018-06-15 20:11

本文选题：肝炎病毒 + 乙型　；参考：《解放军医学杂志》2015年03期

【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。
[Abstract]:Objective to analyze the evolution of rtI233V mutation in the reverse transcriptase region of hepatitis B virus (HBV) and its correlation with adefovir dipivoxil (ADV) resistance. Methods the detection rate of HBV rtI233V mutation in serum samples of 9830 patients with chronic HBV infection was analyzed. The HBV RT gene was amplified and sequenced (20 clones / samples) to observe the evolution of rtI233V mutation. The replicators of pTriEx-HBVC1.1-fold wild strain and three variant strains, rtI233V, rtN236Tn233V rtI233V rtN236T, were transiently transfected into HepG2 hepatoma cells. Different concentrations of lamivudine, entekavir ETV) and tenofovir (TDFV) were added to HepG2 hepatoma cells. The levels of HBV DNA in the supernatant of cell culture after different concentrations of drugs were detected by real-time fluorescence quantitative PCR, and the characteristics of replicability and phenotypic resistance of mutant virus were analyzed. Results there were 28 cases of rtI233V mutation detected in serum samples of 9830 patients with chronic hepatitis B, the detection rate was 0.28%. Among them, 19 cases were rtI233V mutation alone, 9 cases were combined with rtN236T classic drug resistance mutation. All of the patients with ADV had a history of ADV therapy, of which 16 patients (57.1) received ADV alone for more than 6 months and 12 patients (42.9%) received multi-drug sequential combination therapy including ADV for more than one year. The replicability and phenotypic resistance of rtI233V mutant was similar to that of wild strain. Compared with wild strain, the replicative power of rtI233V variant was significantly lower than that of wild strain. The combination of rtI233V variant and TDF sensitive rtI233V rN236T had little effect on drug resistance. However, rtI233V mutation could significantly restore the damaged replicability of rtN236T mutant. Conclusion rtI233V mutation is associated with poor ADV response, although it does not directly reduce the sensitivity of the virus to ADV, it can increase the replicability of classical ADV resistant strains and is a kind of replicative force compensating variation.
【作者单位】：桂林医学院研究生学院;解放军302医院全军传染病研究所、肝衰竭诊疗与研究中心病毒性肝炎研究室;桂林市第三人民医院肝病科;
【基金】：国家自然科学基金(81371852) 北京市自然科学基金(7132224)~~
【分类号】：R512.62

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