An In Vivo Study Of The Antiviral Effect On Duck Hepatitis B Virus Of Thymic Factor D

Objective: To develop a fluorescence quantitative polymerase chain reaction(FQ-PCR) for quantification of duck hepatitis B virus(DHBV) DNA based onTaqMan chemistry. Investigate the natural carrying rate of DHBV in adult Fuzhouducks and etablish the FuZhou duckling model with acquired infection of DHBV.Then the method and the animal model were used to investigate the inhibitory abilityon DHBV of thymic factor D (TFD) in vivo and the mechanisms of this antiviraleffect.Methods: The PCR fragment of DHBV DNA was cloned into vector PUCm-T.Therecombinant plasmid was purified and subsequently quantified as DHBV-DNAstandard.A pair of primers from DHBV S gene and a TaqMan probe, modified with6-Fam at 5'-end and Tamra at its 3'-end ,between these primers were designed todevelop a FQ-PCR for quantification of DHBV DNA , the experimental conditionsand reagents of amplification were sophisticatedly optimized .The detection ofDHBV-DNA by dot hybridization with DIG-labelled probe was also established,boththese two methods were evaluated.DHBV in adult FuzZhou ducks, sera was detecedby PCR; FuZhou ducklings of DHBV DNA(-) were selected 1 day after hatching andinoculated intravenously(i.v.) with DHBV DNA positive serum,those ducklings witha sucsess inoculation were used as the animal models of acquired infection of DHBV2 weeks after inoculation. Then they were classified at random, the ducklings in theexperimental group, IFN control group and blank control group were treated withTFD, IFN-α1b and normal saline respectively for 4 weeks and observed for anotherweek after stopping the treatment. The TaqMan FQ-PCR was used to observe thequantity change of DHBV DNA in ducklings'serum and liver during the treatment.ALT and AST level in serum were also detected at the end of the treatment,histological observation on the duckling liver was done simultaneously.Results: The detectable limit of assay was 1000 copies/m1 in the TaqMan FQ-PCRfor quantification of DHBV DNA .A linear standard curve was obtained betweenl05-109copies/m1(Ct=-2.8361ln(x)+41.45,r=-0.9983) and the coefficient of variationwas l.50%-2.98% and 2.95%-3.24% for intra-and inter-assay,respectively.162 serasamples of adult FuZhou duck were tested by PCR and 89 samples among those werepositive for DHBV-DNA,which told us the natural carrying rate of DHBV in adultFuZhou duck was 55%; 84 FuZhou ducklings of DHBV DNA(-) were inoculated withDHBV DNA positive serum just 1 day after hatching and 80 of them have a sucsessinoculation,the positive rate was 95.24%.The virus inhibitory rate in the TFD treatedgroups was significantly higher than which in the blank control group(8.73% and-5.12% respectively, P<0.05) after 4 weeks treatment. The inhibitory effect wasrelated to the dose and course of the treatment, but the dose have an upper limitbeause the virus inhibitory rate have not significantly difference between the middleand high dose TFD treated groups. After stopping the treatment for 1 week , theDHBV DNA level in serum of the TFD treated groups didn't return and the level inlivers of these groups were significantly lower than which in the blank controlgroup.The level of ALT / AST and the features of liver histopathology in the TFDtreated groups were not different from those in the blank control group.Conclusions: The TaqMan FQ-PCR we etablished for quantification of DHBV DNAis a highly sensitive and specific method. FuZhou ducklings are susceptible to DHBVand they are an idea animal model of HBV infection.Finally,this study confirmed theantiviral effect on DHBV of TFD in vivo and indicated the treatment with TFD havenot toxicity to the duckling's liver.