An Experimental Study Of The Antiviral Effect On Duck Hepatitis B Virus Of Artesunate

Objective: With the fluorescence quantitative polymerase chain reaction (FQ-PCR) for quantification of duck hepatitis B virus(DHBV) DNA and cccDNA (covalently closed circular DNA) based on TaqMan chemistry, the model of FuZhou duckling DHBV infected and the culture model of the primary duck hepatocytes were established to investigate the inhibitory ability on DHBV DNA and DHBV cccDNA of artesunate(ART) in vivo and in vitro. And the mechanism of this antiviral effect was also discussed.Methods: (1). FuZhou ducklings of DHBV DNA(-) were selected 1 day after hatched 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. Ducks with congenitally infection of DHBV were randomly divided into five groups: untreated group, high,medium,low-dose artesunate treated group,lamivudine-treated group. The ducks in the lamivudine-treated group were fed lamivudine with a dose of 50mg/kg once. Ducks in the three-dose artesunate-treated groups were treated with different doses of decoction of this medicine for 28 days respectively. The serum content of DHBV-DNA was determined by FQ-PCR method before and 7 days after the treatment, and on the 7th, 14th, 21st and 28th day of the treatment. The histopathological changes of liver were evaluated before and after the treatment. (2). The primary duck hepatocytes were cultured in the entironment of 37℃,5%CO2, arfter 24 hours the live duck hepatocytes were divided into five groups: untreated group, high,medium,low-dose artesunate treated group,IFNα-2b group. The conformation and livability of duck hepatocytes were observed, and the DNA of the primary duck were collected by the UNIQ-10 method and were taken to detect the inhibition of ART on DHBV.Results: (1)Lamivudine showed a rapid inhibiting effect on DHBV-DNA, but this effect didn't last long, and the serum level of DHBV-DNA increased again after treatment. The serum level of DHBV-DNA dropped markedly in the high-dose artesunate-treated group on the 21st and 28th day. Low-dose and medium-dose artesunate also showed certain inhibiting effect on DHBV-DNA. The features of liver histopathological changes in the ART treated groups showed no statistical significance as compared with that in the untreated group. (2)The livabilities of duck hepatocytes treated with low-dose,medium-dose and high-dose artesunate were all over 95%.The DHBV DNA levels in cytoplasm of duck hepatocytes treated with ART(15,30,60μg/ml) were significantly lower than those of control cells(P < 0. 05).Conclusions: The TaqMan FQ-PCR established for quantification of DNA is a highly sensitive and specific method. ART has the antiviral effect of DHBV DNA replication in vivo and in vitro, and has some inhibitory effect on DHBV cccDNA in liver. Otherwise the study indicated the treatment with suitable dosage of ART have no toxicity to the duckling's liver. Thus this herbal medicine is an effective,safe and economical drug for hepatitis B. Future studies are needed to explore the active mechanism of the antiviral effect on duck hepatitis B virus of artesunate.