Duck HBV Dna Copy Numbers in Isolated Hepatocyte Nuclei Vary Dramatically and Decline during Entecavir Therapy

Abstract

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. 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. 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). Intranuclear DHBV DNA levels varied significantly, which could be partially attributed to effects of cell cycle phases, and could be decreased by ETV therapy.