新型恩替卡韦耐药突变特征

StephenW

Research Article
Characterization of novel entecavir resistance mutations

    Sanae Hayashi1, Shuko Murakami1, Katsumi Omagari1, Takeshi Matsui1, 2, Etsuko Iio1, Masanori Isogawa1, Tsunamasa Watanabe1, Yoshiyasu Karino3, Yasuhito Tanaka1, ,


Background & Aims

Entecavir (ETV) is approved for the treatment of chronic hepatitis B virus (HBV) infections, but the virus can acquire resistance to the drug. This requires lamivudine resistance mutations (LAMr) and at least one additional mutation. Here, we characterized two novel mutations, rtI163V and rtA186T, associated with viral breakthrough (VBT) in an ETV-refractory patient.
Methods

HBV from an ETV-refractory patient was sequenced, and newly identified mutations were inserted into a replication-competent clone by mutagenesis. Clones were analyzed for replication efficacy and susceptibility to ETV in vitro. Chimeric mice with human hepatocytes were inoculated with the patient’s serum at VBT, and monitored for viral mutation pattern using a next-generation sequencing approach.
Results

RtI163V and rtA186T mutations were detected together with LAMr (rtL180M and rtM204V) at VBT. RtA186T plus LAMr reduced susceptibility to ETV more than 111.1-fold compared with the wild-type clone, while rtI163V plus LAMr resulted in a 20.4-fold reduction. RtA186T significantly reduced viral replication efficacy, while the rtI163V mutation rescued it. Interestingly, the viral mutation pattern in the chimeric mice indicated dominant (or selective) proliferation of a clone containing rtI163V and rtA186T mutations plus LAMr under ETV treatment. Three-dimensional docking simulation indicated that rtA186T reduced the binding affinity of the HBV polymerase to ETV.
Conclusions

VBT in this ETV-refractory patient is attributable to the novel ETV resistance mutations rtI163V and rtA186T. RtA186T was apparently responsible for ETV resistance but the selection of a clone with the double mutation plus LAMr suggests that rtI163V is required to sustain viral fitness.
Abbreviations

    ETV, Entecavir; LAMr, lamivudine resistance mutations; VBT, viral breakthrough; HBV, Hepatitis B virus; HCC, hepatocellular carcinoma; IFN-α-2b, interferon α-2b; PegIFN-α-2a, pegylated interferon; NAs, α-2a nucleos(t)ide analogues; LAM, lamivudine; ADV, adefovir; RT, reverse transcriptase; ETV-TP, ETV-triphosphate; Chimeric Mice Hereafter, chimeric mice with human hepatocytes; HBsAg, Hepatitis B surface antigen; HBcrAg, HBV core-related antigen; SCID, severe combined immunodeficiency; uPA, urokinase-type plasminogen activator gene

Keywords

    Hepatitis B virus; Entecavir; Lamivudine; Antiviral resistance; HBV mutations; Viral breakthrough; Hepatitis B treatment; Combination therapy; rtA186T; rtI163V; Entecavir resistance

    Corresponding author. Address: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Science, Kawasumi 1, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: +81 52 853 8191; fax: +81 52 842 0021.

Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier Ireland Ltd. All rights reserved.

StephenW

研究论文
新型恩替卡韦耐药突变特征

    早苗Hayashi1，修子Murakami1，克己Omagari1，金城武Matsui1，2，悦子Iio1，正德Isogawa1，纲Watanabe1，义康Karino3，保仁Tanaka1，


背景与目的

恩替卡韦（ETV）被批准用于治疗慢性乙型肝炎病毒（HBV）感染的治疗，但病毒可以获得抵抗药物。这需要拉米夫定耐药突变（LAMr）和至少一种另外的突变。在这里，我们的特点两个新的突变，rtI163V和rtA186T，与病毒突破（VBT）相关联的ETV难治病人。
方法

从ETV难治患者的HBV进行测序，和新近鉴定的突变插入由诱变有复制能力的克隆。克隆体外复制功效和易感性ETV进行分析。嵌合体小鼠与人肝细胞接种在VBT患者的血清，并使用下一代测序方法监测病毒突变模式。
结果

与LAMr（rtL180M和rtM204V）在VBT进行检测在一起RtI163V和rtA186T突变。 RtA186T加LAMr敏感性降低ETV超过111.1倍的野生型克隆相比，同时rtI163V加LAMr导致20.4倍的降低。 RtA186T显著降低病毒复制的功效，而rtI163V突变救出。有趣的是，在嵌合体小鼠中的病毒突变图案所表示含有下ETV治疗rtI163V和rtA186T突变加LAMr克隆的显性（或选择性）的增殖。三维对接模拟表明rtA186T减少HBV聚合酶到ETV的结合亲和力。
结论

VBT在此ETV难治患者可归因于新颖ETV耐药突变rtI163V和rtA186T。 RtA186T显然负责ETV耐药但克隆的具有双突变的选择加上LAMr表明rtI163V需要维持病毒健身。
缩写

    ETV，恩替卡韦; LAMr，拉米夫定耐药突变; VBT，病毒性的突破;乙肝病毒，乙型肝炎病毒;肝癌，肝癌; IFN-α-2b中，干扰素α-2b的; PegIFN-α-2A，聚乙二醇化干扰素; NAS，α-2a的核苷（酸）类似物; LAM，拉米夫定; ADV，阿德福韦; RT，逆转录酶; ETV-TP，ETV三磷酸;嵌合体小鼠此后，嵌合体小鼠与人类肝细胞;乙型肝炎表面抗原，B型肝炎表面抗原; HBcrAg，HBV核心相关抗原; SCID，重症联合免疫缺陷;的uPA，尿激酶型纤溶酶原激活物基因

关键词

    乙型肝炎病毒;恩替卡韦;拉米夫定;抗病毒药物耐药性; HBV基因突变;病毒性突破;乙肝治疗;联合治疗; rtA186T; rtI163V;恩替卡韦耐药

    通讯作者。地址：病毒学和肝单位，医学科学的名古屋市立大学研究生院，川澄1，瑞穗区467-8601，日本系。电话：+81 52 853 8191;传真：+81 52 842 0021。

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