| Duck viral hepatitis (DVH) type A is a kind of highly contagious and lethal infectiousdisease which caused by duck hepatitis A virus (DHAV), and rapidly spreads infection inyoung ducklings aged1to3weeks. DVH type A can result in approximately90%mortality inducklings. DHAV is classed into three serotypes: DHAV-1, DHAV-2and DHAV-3. There areno cross-immunity or antigenic relationships but obvious differences among thethree serotypes. DVH type A which distributes cosmopolitism is considered as the mostharmful disease to the industry. In this study, an infectious full-length cDNA clone of duckhepatitis A virus type1was constructed and part of the bioactivity of the rescued virus wasstudied.1. Construction of an infectious full-length cDNA clone of DHAV-1The full-length cDNA of DHAV-1LY0802strain, isolated from a twelve-days-old duckling,was obtained by the method of RT-PCR using the nine pairs of specific primers which aredesigned in accordance with sequences published on GenBank. The cDNA from the5’ and3’ends of the viral genome were amplified by the Rapid Amplification of cDNA Ends (RACE)method. The homology of those two can approach to93.2%-95.2%compared with thesequences published onGenBank.The full-length cDNA clone of the LY0802strain was divided into five overlappingfragments to assemble. Seven mutations on the recombinant plasmid were found aftercompartment between LY0802and recombinant plasmid. In order to create optimal AUGenvironment and also to make a genetic marker to distinguish between parental virus andrescued virus, the position at13and19were mutated by PCR. The position at5and7ofamino acid sequence changed as a result of the PCR mutation. Other mutations onrecombinant plasmid were silence mutation.The Xho-linearized plasmid DNA was used to transfect BHK-21cells in vitro. As apositive control, BHK-21cells were transfected with viral RNA purified from the parentalLY0802DHV virus isolate. As a negative control, X-tremeGENE HP DNA Transfenction Reagent with PBS was added to BHK-21cells. The identification results by IFA and RT-PCRwere regarded as positive for DHAV. After the qualification experiment on gentetic marker,we are so sure the BHK-21cells were transfected by rescued virus but not wild DHAV.2. Bioactivity analysis of the rescued virusThe TCID50values at24,36,48,60and72h post-infection were analysised to generate thegrowth curve of the rescued virus and the parental virus. The results showed that the amountof virus particle approached to the top at60h post-infection, the growth characteristics weresimilar between the rescued virus and the parental virus.Three groups of101-day-old ducklings susceptible to DHAV-1, were inoculatedintramuscularly with0.25ml of104TCID50rescued virus,0.25ml of104.2TCID50parentalvirus and0.25ml aseptic PBS (pH7.4), respectively. The duckings of the rescued virus groupand rescued virus group exhibited characteristic clinical signs and gross lesions of DVH typeA. The results indicated that the rescued virus of LY0802was same as the wild virus LY0802on the biological characteristics.The success on constructing of the infectious full-length cDNA clone of DHAV-1willcontribute on the researches of infectious mechanism of DHAV-1. |
