title: Phenotypic assay of a hepatitis B virus strain carrying an rtS246T variant using a new strategy.
abstract: 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.
authors: Institute for Viral Hepatitis, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China.
authors: Jie-li
authors: JL
authors: Hu
authors: Jing
authors: J
authors: Cui
authors: Jin-jun
authors: JJ
authors: Guo
authors: Wen-lu
authors: WL
authors: Zhang
authors: Xue-fei
authors: XF
authors: Cai
authors: Zuo-wei
authors: ZW
authors: Yuan
authors: Qing-ling
authors: QL
authors: Li
authors: Xiao-yan
authors: XY
authors: Deng
authors: Ai-zhong
authors: AZ
authors: Zeng
authors: Yuan
authors: Y
authors: Hu
authors: Ni
authors: N
authors: Tang
authors: Ai-long
authors: AL
authors: Huang
mesh_terms: D000328:Adult; D000998:Antiviral Agents / Q000627:therapeutic use*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D024882:Drug Resistance, Viral; D006147:Guanine / Q000031:analogs & derivatives / Q000627:therapeutic use; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010641:Phenotype; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014773:Virology / Q000379:methods*
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D050505:Mutant Proteins; C413685:entecavir; D006147:Guanine; D012194:RNA-Directed DNA Polymerase