Hepatitis B virus mutation pattern rtL180M+A181C+M204V may contribute to entecavir resistance in clinical practice

<b>Background and Aims:</b> Entecavir (ETV) resistance of hepatitis B virus (HBV) conventionally requires rt184, 202, or 250 mutations plus lamivudine-resistance mutation (rtM204V/I ± L180M). This study aimed to clarify whether rtL180M+A181C+M204V mutations may contribute to HBV ETV resistance. <b>Methods:</b> Serum samples were collected from 22,009 patients who underwent resistance testing in Beijing 302 Hospital from 2007 to 2016. HBV reverse transcriptase (RT) gene was screened by direct sequencing and verified by clonal sequencing. Phenotypic analysis was performed for evaluating replication capacity and drug susceptibility. <b>Results:</b> Classical ETV-resistance mutations of HBV were detected in 1252 patients who were receiving ETV therapy. The rtA181C mutation was detected with rtL180M+M204V mutations in 18 lamivudine-experienced ETV-treated patients, and the emergence of the mutations was associated with virological breakthrough or inadequate virological response to ETV. Patient-derived representative rtA181C-containing mutants, rtL180M+A181C+M204V, rtL180M+A181C+M204V+M250V, and rtL180M+A181C+S202G+M204V, exhibited 45.7%, 25.9%, and 25.0% replication capacity and 85.6-, 356.1-, and 307.1-fold decreased susceptibility to ETV respectively compared to the wild-type strain, while the three mutants remained sensitive to tenofovir (TDF). Artificial elimination of rtA181C largely restored the rtL180M+A181C+M204V mutant’s sensitivity to ETV. Molecular modelling of viral RT binding to ETV showed that the rtL180M+A181C+M204V mutant had a less stable conformation compared to rtL180M+M204V mutant. In clinical practice, undetectable serum HBV DNA was achieved in two of five longitudinally followed rtA181C-positive patients who received switching-to TDF therapy, but not in the other three who received add-on adefovir therapy during observation. <b>Conclusions:</b> Both clinical and experimental data support rtL180M+A181C+M204V as a novel non-classical ETV-resistance mutation pattern.

**背景与目的：** 乙型肝炎病毒（hepatitis B virus, HBV）对恩替卡韦（Entecavir, ETV）的经典耐药模式，通常需要rt184、202或250位点突变联合拉米夫定耐药突变（rtM204V/I ± L180M）。本研究旨在明确rtL180M+A181C+M204V突变是否可导致HBV对ETV产生耐药。
**方法：** 收集2007年至2016年于北京302医院接受耐药检测的22009例患者的血清样本。通过直接测序筛选HBV逆转录酶（reverse transcriptase, RT）基因，并经克隆测序验证其结果。开展表型分析以评估病毒复制能力与药物敏感性。
**结果：** 在接受ETV治疗的患者中，共检出1252例存在HBV经典ETV耐药突变。其中18例既往接受拉米夫定治疗且当前使用ETV的患者，同时检出rtA181C突变与rtL180M+M204V突变，且此类突变的出现与病毒学突破或ETV治疗后病毒学应答不足显著相关。源自患者的代表性rtA181C阳性突变体，即rtL180M+A181C+M204V、rtL180M+A181C+M250V及rtL180M+A181C+S202G+M204V，与野生型毒株相比，复制能力分别为野生型的45.7%、25.9%与25.0%，对ETV的敏感性分别下降85.6倍、356.1倍及307.1倍；而上述三种突变体仍对替诺福韦（tenofovir, TDF）敏感。人工敲除rtA181C位点后，rtL180M+A181C+M204V突变体对ETV的敏感性大幅恢复。病毒RT与ETV结合的分子建模结果显示，rtL180M+A181C+M204V突变体的构象稳定性较rtL180M+M204V突变体更差。临床实践队列中，5例接受纵向随访的rtA181C阳性患者里，2例转为TDF治疗后实现血清HBV DNA转阴，其余3例接受阿德福韦酯联合治疗者未达到病毒学转阴效果。
**结论：** 临床与实验数据均支持rtL180M+A181C+M204V为一种新型非经典ETV耐药突变模式。