title: [Construction of a duck hepatitis B virus YMDD mutant and identification of its resistance phenotype].
abstract: OBJECTIVE
  To construct a lamivudine-resistant plasmid containing 1.2 unit genome of duck hepatitis B virus and identify its replication and drug-resistance in avian LMH hepatica cells.
  
  
  METHODS
  The recombinant plasmid PBS-DHBV1.2 was constructed using the 1.2-genome length DHBV DNA sequence from a dimer DHBV genome with pcDNA3.1 as the template. With site-directed mutagenesis, we obtained PBS-DHBV1.2-M512V plasmids with polymerase gene mutation from PBS-DHBV1.2. Two constructed plasmids were transiently transfected into LMH cells using FuGENETM6 transfection reagent and cultured in the medium containing different concentrations of lamivudine. Southern blot hybridization was performed to detect DHBV replication intermediates.
  
  
  RESULTS
  PCR amplification, restriction digestion and plasmid sequencing all confirmed successful construction of PBS-DHBV1.2-M512V recombinant plasmid. Southern blot analysis identified the presence of all the expected DHBV replication intermediates in LMH cells. The replication capacity of the mutant plasmid was decreased by 2.7 times compared with that of the wild plasmid. The IC(50) of lamivudine was 37.12∓8.81 ng/ml for the mutant, greater than that of the wild plasmid (10.90∓4.80 ng/ml).
  
  
  CONCLUSIONS
  Compared with the wild plasmid, the mutant plasmid has a lower replication capacity and sensitivity to lamivudine in vitro.
authors.affiliation: Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. fuxihua80929@yahoo.com.cn
authors.forename: Xi-Hua
authors.initials: XH
authors.lastname: Fu
authors.forename: Wei-Fang
authors.initials: WF
authors.lastname: Liang
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Wu
authors.forename: Guo-Jun
authors.initials: GJ
authors.lastname: Shen
authors.forename: Hai-Tang
authors.initials: HT
authors.lastname: He
authors.forename: Jin-Jun
authors.initials: JJ
authors.lastname: Chen
authors.forename: Jin-Lin
authors.initials: JL
authors.lastname: Hou
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics; D015611:Hepatitis B Virus, Duck / Q000187:drug effects* / Q000235:genetics*; D019259:Lamivudine / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010957:Plasmids
chemical_list: D000998:Antiviral Agents; D019259:Lamivudine