| Duck viral hepatitis(DVH) is a highly mortal disease, caused by duck hepatitis virus(DHV), and spreads rapidly in three-week-old ducklings characterized by high mortality and liver lesions with hemorrhagic foci. DHAV has been caused large economic loss for duckery. DHAV-1 and DHAV-3 are the main types of serum in the domestic epidemic, and there has been no report of DHAV-2 in mainland China. Duck virus hepatitis yolk antibody and duck hepatitis A vaccine are the main measures for the prevention and treatment of DHV. Currently, control of the disease is based upon the use of classic vaccine. Thus, to develop safer and more effective and newer vaccine to protect and control this disease is a promising direction in the future. Newcastle disease(ND), caused by infection with virulent Newcastle disease virus(NDV), is one of the most serious infectious diseases in poultry. Vaccination combined with strict biosecurity practices has been the recommended strategy for controlling NDV outbreaks. With the development of the reverse genetic manipulation technology, the live vector vaccine of Newcastle disease is widely studied and applied.In order to develop a safe and effective bivalent vaccine, LaSota strain recombinant vaccine virus expressing VP1 gene of the duck hepatitis A viruses type 1 and 3 was generated as a polyvalent vaccine against DHAV and NDV diseases in breeder ducks using a reverse genetics approach, not only can fill gaps in the current DHAV-3 vaccine, but also can achieve the effect of stitch anti sick. This study mainly includes the following four parts:1. Construction of recombinant Newcastle disease virus vector expressing duck hepatitis type 1 and type 3 VP1 geneThe DHAV-1 and DHAV-3 virus RNA were extracted by Trizol reagent, and the DHAV-1VP1-2A and DHAV-3VP1 genes were amplified by RT-PCR, successfully synthesized by SOE-PCR. Amplified product DHAV 1VP-2A-3VP1 was detected by 1% agarose gel electrophoresis. The target fragment was about 2368 bp, which was in agreement with the expected result. The recombinant LaSota cDNA clone containing the VP1 gene of the duck hepatitis A viruses type 1 and 3 was constructed by swapping the RFP ORF in the p LS-RFP vector with that of DHAV 1VP-2A-3VP1 ORF.2. Virus rescue and propagationRescue of the recombinant viruses was performed by transfection of the full-length cDNA clone, p LS-1VP1-2A-3VP1, and supporting plasmids that express the NDV NP, P, and L proteins into HEp-2 cells. The rescued virus, rLS-1VP1-2A-3VP1, was harvested by freeze-thawing the infected cells and was amplified by inoculating into the allantoic cavity of 10-day-old SPF chicken embryos. We further performed HA,RT-PCR,IFA tests to identify recombinant viruses.3. The biological characteristics of recombinant virusAnalyses of the virus stock titers were performed by conducting the standard HA, the 50% tissue infectious dose(TCID50) and the 50% egg infective dose(EID50). Pathogenicity of the recombinant viruses was assessed by performing the standard mean death time(MDT), intracerebral pathogenicity index(ICPI) and intravenous pathogenicity(IVPI) tests. The results showed that the recombinant rLS-1VP1-2A-3VP1 kept the high-titer growth property and low pathogenicity in embryonated chicken eggs similar with recombinant vaccine strain r LS-RFP.4. Duck immunity testAfter the initial vaccination, the serum neutralizing antibody level of immune ducks was determined by neutralization test. The average titers of DHAV-1 and DHAV-3 antibodies in the sera of immunized ducks were 1:18.17 and 1:16.60, respectively.5. Western blotWestern blot was used to further verify the reactivity of neutralizing antibody generated by recombinant virus and VP1 protein of the duck hepatitis A viruses type 1 and type 3.These results demonstrated that the rLS-1VP1-2A-3VP1 had the potential of being used as a novel live viral vector vaccine against both duck viral hepatitis and Newcastle diseases in breeder ducks. |
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