Luo Ying-ying, Tao Ying, Cai Xue-fei, Zhang Wen-lu, Long Quan-xin, Guo Haitao, Huang Ai-long, Hu Jie-li
Institute for Viral Hepatitis, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, the Second Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Institute for Viral Hepatitis, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, the Second Affiliated Hospital of Chongqing Medical University, Chongqing, China; Department of Infectious Diseases, the People's Liberation Army 161 Hospital, China.
Antiviral Res. 2016 Feb;126:99-107. doi: 10.1016/j.antiviral.2015.12.009. Epub 2015 Dec 29.
Nucleos(t)ide analogues (NUCs) susceptibility assay is important for the study of hepatitis B virus (HBV) drug resistance. The purpose of susceptibility assay is to test the sensitivity of a specific HBV variant to NUCs in vitro, by which assesses if and to what extent the mutant virus is resistant to a specific NUC. Among the existing susceptibility assay methods, stable cell line expressing the specific variant is one of the commonly used assessment systems based on its high repeatability. However, establishment of stable cell lines expressing individual variant is laborious and time-consuming. In the present study, we developed a novel strategy for rapidly establishing HBV replicating stable cell lines. We first established an acceptor cell line stably transfected with a polymerase-null HBV 1.1mer genome DNA, then lentiviruses expressing different mutant HBV polymerases were transduced into the acceptor cell line respectively. Stable cell lines replicating HBV DNA with the trans-complemented HBV polymerases were established by antibiotics selection. Lamivudine and entecavir susceptibility data from these polymerase-complementing cell lines were validated by comparing with other assays. Taken together, this transcomplementation strategy for establishment of stable cell lines replicating HBV DNA with clinically isolated HBV polymerase provides a new tool for NUC susceptibility assay of HBV.
核苷（酸）类似物（NUCs）敏感性测定对于乙型肝炎病毒（HBV）耐药性研究至关重要。敏感性测定的目的是在体外测试特定HBV变异体对NUCs的敏感性，据此评估突变病毒是否以及在何种程度上对特定NUC耐药。在现有的敏感性测定方法中，表达特定变异体的稳定细胞系因其高重复性而成为常用的评估系统之一。然而，建立表达单个变异体的稳定细胞系既费力又耗时。在本研究中，我们开发了一种快速建立HBV复制稳定细胞系的新策略。我们首先建立了一个稳定转染无聚合酶HBV 1.1mer基因组DNA的受体细胞系，然后将表达不同突变HBV聚合酶的慢病毒分别转导到受体细胞系中。通过抗生素筛选建立了用反式互补HBV聚合酶复制HBV DNA的稳定细胞系。通过与其他测定方法比较，验证了这些聚合酶互补细胞系对拉米夫定和恩替卡韦的敏感性数据。综上所述，这种用临床分离的HBV聚合酶建立复制HBV DNA的稳定细胞系的反式互补策略为HBV的NUC敏感性测定提供了一种新工具。
