Premium
Phenotypic assay of a hepatitis B virus strain carrying an rtS246T variant using a new strategy
Author(s) -
Hu Jieli,
Cui Jing,
Guo Jinjun,
Zhang Wenlu,
Cai Xuefei,
Yuan Zuowei,
Li Qingling,
Deng Xiaoyan,
Zeng Aizhong,
Hu Yuan,
Tang Ni,
Huang Ailong
Publication year - 2012
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.22260
Subject(s) - virology , hepatitis b virus , recombinant dna , entecavir , biology , cloning (programming) , hepatitis b virus pre beta , genome , orthohepadnavirus , hepatitis b , hepadnaviridae , polymerase chain reaction , virus , microbiology and biotechnology , hepatitis b virus dna polymerase , genetics , gene , lamivudine , computer science , programming language
Phenotypic assays of hepatitis B virus (HBV) play an important role in research related to the problem of drug resistance that emerges during long‐term nucleot(s)ide therapy in patients with chronic hepatitis B. Most of the phenotypic assay systems that are available currently rely on the transfection of recombinant replication‐competent HBV DNA into hepatoma cell lines. Cloning clinical HBV isolates using conventional digestion‐and‐ligation techniques to generate replication‐competent recombinants can be very difficult because of the sequence heterogeneity and unique structure of the HBV genome. In this study, a new strategy for constructing an HBV 1.1× recombinant was developed. The core of this strategy is the “fragment substitution reaction” (FSR). FSR allows PCR fragments to be cloned without digestion or ligation, providing a new tool for cloning fragments or genomes amplified from serum HBV DNA, and therefore making the assay of HBV phenotypes more convenient. Using this strategy, a phenotypic assay was performed on an HBV strain carrying an rtS246T variant isolated from a patient with chronic hepatitis B that was only responsive partially to entecavir therapy. The results indicated that this strain is sensitive to entecavir in vitro. J. Med. Virol. 84:34–43, 2011. © 2011 Wiley Periodicals, Inc.