Liu Y, Li X, Xin S, Xu Z, Chen R, Yang J, Liu L, Wong V W-S, Yang D, Chan H L-Y, Xu D
Institute of Infectious Diseases/Liver Failure Medical Center, Beijing 302 Hospital of PLA, Beijing, China.
J Viral Hepat. 2015 Mar;22(3):328-34. doi: 10.1111/jvh.12298. Epub 2014 Aug 14.
The study aimed to clarify clinical significance of hepatitis B virus (HBV) rtA181S mutation in Chinese HBV-infected patients. A total of 18 419 patients with chronic HBV infection from Beijing 302 Hospital were investigated. HBV complete reverse transcriptase region of polymerase was screened by direct sequencing, and the results were verified by clonal sequencing. Replication-competent mutant and wild-type HBV genomic amplicons were constructed and transfected into the HepG2 cells and cultured in the presence or absence of serially diluted nucleos(t)ide analogues. Intracellular HBV replicative intermediates were quantitated for calculating the 50% effective concentration of the drug (EC(50)). The rtA181S was detected in 98 patients with 12 kinds of mutational patterns. Genotype C and genotype B HBV infection occupied 91.8% and 8.2% in rtA181S-positive patients, in contrast to 84.6% and 15.4% in rtA181S-negative patients (P < 0.01). All rtA181S-positive patients had received nucleos(t)ide analogues. rtA181S was detected in multiple patients with virologic breakthrough. Phenotypic analysis of patient-derived viral strains showed that rtA181S, rtA181S+N236T, rtN236T and rtA181V strains had 68.5%, 49.9%, 71.4% and 66.2% of natural replication capacity of wild-type strain, and 3.7-fold, 9.8-fold, 7.9-fold and 5.6-fold increased EC(50) to adefovir dipivoxil (ADV). The rtA181S strain remained susceptible to lamivudine, entecavir and tenofovir, and ADV susceptibility was restored after the mutation was eliminated through site-directed mutagenesis. Rescue therapy with entecavir or combination therapy was effective in rtA181S-related ADV-refractory patients. The rtA181S mutation confers moderate resistance to ADV. It could be induced by either lamivudine or ADV and contribute ADV treatment failure.
本研究旨在阐明乙肝病毒（HBV）rtA181S突变在中国HBV感染患者中的临床意义。对北京302医院的18419例慢性HBV感染患者进行了调查。通过直接测序筛选聚合酶的HBV完整逆转录酶区域，并通过克隆测序验证结果。构建具有复制能力的突变型和野生型HBV基因组扩增子，转染至HepG2细胞中，并在存在或不存在系列稀释的核苷（酸）类似物的情况下进行培养。对细胞内HBV复制中间体进行定量，以计算药物的50%有效浓度（EC50）。在98例患者中检测到rtA181S，有12种突变模式。rtA181S阳性患者中C基因型和B基因型HBV感染分别占91.8%和8.2%，而rtA181S阴性患者中分别为84.6%和15.4%（P<0.01）。所有rtA181S阳性患者均接受过核苷（酸）类似物治疗。在多名病毒学突破患者中检测到rtA181S。对患者来源的病毒株进行表型分析显示，rtA181S、rtA181S+N236T、rtN236T和rtA181V株分别具有野生型株天然复制能力的68.5%、49.9%、71.4%和66.2%，对阿德福韦酯（ADV）的EC50分别增加了3.7倍、9.8倍、7.9倍和5.6倍。rtA181S株对拉米夫定、恩替卡韦和替诺福韦仍敏感，通过定点诱变消除突变后，ADV敏感性得以恢复。恩替卡韦挽救治疗或联合治疗对rtA181S相关的ADV耐药患者有效。rtA181S突变赋予对ADV的中度耐药性。它可由拉米夫定或ADV诱导产生，并导致ADV治疗失败。
