title: A novel method for nucleos(t)ide analogues susceptibility assay of hepatitis B virus by viral polymerase transcomplementation.
abstract: 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.
authors.affiliation: 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.
authors.forename: Ying-ying
authors.initials: YY
authors.lastname: Luo
authors.affiliation: 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.
authors.forename: Ying
authors.initials: Y
authors.lastname: Tao
authors.affiliation: 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.
authors.forename: Xue-fei
authors.initials: XF
authors.lastname: Cai
authors.affiliation: 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.
authors.forename: Wen-lu
authors.initials: WL
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Quan-xin
authors.initials: QX
authors.lastname: Long
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, USA.
authors.forename: Haitao
authors.initials: H
authors.lastname: Guo
authors.affiliation: 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; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases (CCID), Hangzhou, China. Electronic address: ahuang1964@163.com.
authors.forename: Ai-long
authors.initials: AL
authors.lastname: Huang
authors.affiliation: 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; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases (CCID), Hangzhou, China. Electronic address: hujieli1977@163.com.
authors.forename: Jie-li
authors.initials: JL
authors.lastname: Hu
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral; D015684:Gene Products, pol / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test / Q000379:methods*; D020022:Genetic Predisposition to Disease; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D057809:HEK293 Cells; D006509:Hepatitis B / Q000188:drug therapy / Q000821:virology*; D006515:Hepatitis B virus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D019694:Hepatitis B, Chronic / Q000188:drug therapy; D006801:Humans; D020128:Inhibitory Concentration 50; D019259:Lamivudine / Q000494:pharmacology; D016086:Lentivirus / Q000235:genetics; D009154:Mutation; D009711:Nucleotides / Q000494:pharmacology*; D012328:RNA Viruses; D060888:Real-Time Polymerase Chain Reaction; D014161:Transduction, Genetic; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D015684:Gene Products, pol; D009711:Nucleotides; C066027:P protein, Hepatitis B virus; D019259:Lamivudine; C413685:entecavir; D006147:Guanine
keywords: Hepatitis B virus; Susceptibility assay