Phenotypic assay of a hepatitis B virus strain carrying an rtS246T variant using a new strategy

• Published in: Journal of medical virology Vol. 84; no. 1; pp. 34 - 43
• Main Authors: Hu, Jie-li, Cui, Jing, Guo, Jin-jun, Zhang, Wen-lu, Cai, Xue-fei, Yuan, Zuo-wei, Li, Qing-ling, Deng, Xiao-yan, Zeng, Ai-zhong, Hu, Yuan, Tang, Ni, Huang, Ai-long
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.01.2012; Wiley; Wiley Subscription Services, Inc
• Subjects: Adult; Antiviral Agents - therapeutic use; Base Sequence; Biological and medical sciences; Cloning, Molecular - methods; DNA, Viral - chemistry; DNA, Viral - genetics; Drug Resistance, Viral; entecavir; Fundamental and applied biological sciences. Psychology; Guanine - analogs & derivatives; Guanine - therapeutic use; HBV 1.1 genome; hepatitis B virus; Hepatitis B virus - drug effects; Hepatitis B virus - genetics; Hepatitis B virus - isolation & purification; Hepatitis B, Chronic - drug therapy; Hepatitis B, Chronic - virology; Human viral diseases; Humans; Infectious diseases; Male; Medical sciences; Microbiology; Miscellaneous; Molecular Sequence Data; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation, Missense; Phenotype; Recombination, Genetic; RNA-Directed DNA Polymerase - genetics; RNA-Directed DNA Polymerase - metabolism; Sequence Analysis, DNA; susceptibility assay; Viral diseases; Virology; Virology - methods; Hepatitis A virus; hepatitis Bvirus; Picornaviridae; susceptibility assay; Entecavir; Orthohepadnavirus; Hepatic disease; HBV 1.1 genome; Hepatovirus; Strain; Virus; Hepatitis; Phenotype; Sensitivity; Hepadnaviridae; Digestive diseases; Antiviral; Hepatitis B virus; Genome
• ISSN: 0146-6615; 1096-9071; 1096-9071
• DOI: 10.1002/jmv.22260

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.