An In Vivo Study Of The Antiviral Effect On Duck Hepatitis B Virus Of Arsenic Trioxide

Objective: To found a fluorescence quantitative polymerase chain reaction (FQ-PCR) for quantification of duck hepatitis B virus(DHBV) cccDNA (covalently closed circular DNA) in liver based on TaqMan chemistry. The FuZhou duckling model of DHBV was established by acquired infection. Then the inhibitory ability on DHBV DNA and DHBV cccDNA of arsenic trioxide(As2O3) in vivo was investigated and the mechanism of this antiviral effect was also discussed.Methods: The PCR fragments of DHBV cccDNA was cloned into vector PUCm-T respectively. The recombinant plasmid was purified and subsequently quantified as DHBV cccDNA standard.A pair of primer from the two sides of the nick in the minus strand of DHBV DNA and a TaqMan probe, modified with 6-Fam at 5'-end and Tamra at its 3'-end ,between two primers were designed to develop a FQ-PCR for quantification of DHBV cccDNA .The experimental conditions and reagents of amplification were sophisticatedly optimized . FuZhou ducklings of DHBV DNA(-) were selected 1 day after hatching and inoculated intravenously(i.v.) with DHBV DNA (+)serum, those ducklings with a successful inoculation were used as the animal models of acquired infection of DHBV 1 week after inoculation. Then they were classified at random, the ducklings in the experimental group, lamivudine(LAM) control group and blank control group were treated with As2O3, LAM and normal saline respectively for 4 weeks and observed for another week after stopping the treatment. The TaqMan FQ-PCR was used to observe the quantity change of DHBV DNA in ducklings'serum and liver, the DHBV cccDNA in liver is also observed. The ALT and AST level in serum were detected; histological observation on the duckling liver was done simultaneously.Results: The detectable limit of assay was 103 copies/m1 in the TaqMan FQ-PCR for quantification DHBV cccDNA. A linear standard curve was obtained between...