Yamada Norie, Sugiyama Ryuichi, Nitta Sayuri, Murayama Asako, Kobayashi Minoru, Okuse Chiaki, Suzuki Michihiro, Yasuda Kiyomi, Yotsuyanagi Hiroshi, Moriya Kyoji, Koike Kazuhiko, Wakita Takaji, Kato Takanobu
Department of Virology II National Institute of Infectious Diseases Tokyo Japan.
Department of Internal Medicine Center for Liver Diseases, Seizankai Kiyokawa Hospital Tokyo Japan.
Hepatol Commun. 2017 Mar 9;1(2):110-121. doi: 10.1002/hep4.1022. eCollection 2017 Apr.
The emergence of resistance mutations in the reverse transcriptase gene of hepatitis B virus (HBV) is associated with treatment failure. Entecavir (ETV) is one of the most potent anti-HBV reagents; it has a very low resistance rate and is used as the first-line treatment for chronic hepatitis B. In this study, we isolated HBVs in 4 ETV-refractory patients (2 with viral breakthrough, 1 with partial virological response, and 1 with flare-up) and assessed ETV resistance using replication-competent 1.38-fold HBV genome-length molecular clones. The full genome sequences of infected HBVs in ETV-refractory patients were determined. The HBV molecular clones were generated with the patient-derived sequences. After transfection of these molecular clones into HepG2 cells, viral replications and ETV susceptibilities were evaluated by measuring the amount of intracellular core-particle-associated HBV DNA using Southern blotting and real-time polymerase chain reaction. Among these cases, ETV-resistant variants were detected in 2 patients with viral breakthrough and responsible amino acid mutations in reverse transcriptase were successfully identified in these variants. No ETV-resistant mutation was detected in the other cases. The identified ETV-resistant mutations did not confer resistance to tenofovir disoproxil fumarate. : The HBV replication model with patient-derived sequences is useful for assessing replication efficiency, susceptibility to anti-HBV reagents, and responsible resistance mutations and can aid in choosing the appropriate treatment strategy for treatment-failure cases of chronic hepatitis B. ( 2017;1:110-121).
乙型肝炎病毒（HBV）逆转录酶基因中耐药突变的出现与治疗失败相关。恩替卡韦（ETV）是最有效的抗HBV药物之一；其耐药率极低，被用作慢性乙型肝炎的一线治疗药物。在本研究中，我们从4例ETV治疗无效的患者（2例病毒突破、1例部分病毒学应答和1例病情复发）中分离出HBV，并使用具有复制能力的1.38倍HBV基因组长度分子克隆评估ETV耐药性。测定了ETV治疗无效患者中感染的HBV的全基因组序列。用患者来源的序列生成HBV分子克隆。将这些分子克隆转染到HepG2细胞后，通过Southern印迹法和实时聚合酶链反应测量细胞内核颗粒相关HBV DNA的量来评估病毒复制和ETV敏感性。在这些病例中，在2例病毒突破患者中检测到ETV耐药变异体，并在这些变异体中成功鉴定出逆转录酶中相应的氨基酸突变。在其他病例中未检测到ETV耐药突变。所鉴定的ETV耐药突变对富马酸替诺福韦二吡呋酯不产生耐药性。：具有患者来源序列的HBV复制模型可用于评估复制效率、对抗HBV药物的敏感性以及相应耐药突变，有助于为慢性乙型肝炎治疗失败病例选择合适的治疗策略。（2017；1：110 - 121）
