title: Duck HBV DNA copy numbers in isolated hepatocyte nuclei vary dramatically and decline during entecavir therapy.
abstract: BACKGROUND
  We aimed to develop a quantitative assay to measure duck HBV (DHBV) DNA in single hepatocyte nuclei from DHBV-infected animals and to observe intranuclear DHBV DNA kinetics undergoing entecavir (ETV) therapy.
  
  
  METHODS
  DHBV DNA in isolated nuclei was amplified by quantitative real-time PCR. Liver tissues from chronically-infected ducks with or without ETV treatment were assessed. Cell cycle phases were defined with flow cytometry in single nuclei.
  
  
  RESULTS
  We successfully established a quantitative assay to measure intranuclear DHBV DNA in single nuclei with high specificity, sensitivity and acceptable interassay variations. The intranuclear viral DNA copy numbers varied dramatically (2-204 copies/nuclei) in 11 ducks with active viral replication. Average intranuclear DHBV DNA copies from individual animals (7.57-57.67 copies/nuclei) significantly correlated with total intranuclear (rs=0.955, P<0.001) and serum (rs=0.745, P=0.008) viral DNA levels. The median intranuclear DHBV DNA copies in virus-positive nuclei were greater in gap 0/1 than those in gap 2/mitosis and synthesis phases (P<0.001). Median intranuclear viral DNA copies in virus-positive nuclei decreased from 21 to 6 (P<0.001) under 14-19 weeks of ETV therapy. However, subsequently, further reductions were not achieved in four animals after extended 16 week treatment (6 versus 11, P=0.034).
  
  
  CONCLUSIONS
  Intranuclear DHBV DNA levels varied significantly, which could be partially attributed to effects of cell cycle phases, and could be decreased by ETV therapy.
authors.affiliation: Hepatology Unit and Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, PR China.
authors.forename: Guojun
authors.initials: G
authors.lastname: Shen
authors.forename: Xihua
authors.initials: X
authors.lastname: Fu
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Junhua
authors.initials: J
authors.lastname: Yin
authors.forename: Chunxiu
authors.initials: C
authors.lastname: Zhong
authors.forename: Jinjun
authors.initials: J
authors.lastname: Chen
authors.forename: Jinlin
authors.initials: J
authors.lastname: Hou
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use; D001715:Bird Diseases / Q000235:genetics / Q000821:virology; D002467:Cell Nucleus / Q000235:genetics / Q000821:virology; D004279:DNA, Viral / Q000235:genetics*; D004372:Ducks; D005260:Female; D018628:Gene Dosage; D006147:Guanine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D015613:Hepadnaviridae / Q000235:genetics; D018347:Hepadnaviridae Infections / Q000235:genetics / Q000662:veterinary / Q000821:virology; D006509:Hepatitis B / Q000188:drug therapy* / Q000235:genetics / Q000662:veterinary* / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics; D022781:Hepatocytes / Q000821:virology; D008297:Male; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; C413685:entecavir; D006147:Guanine