| Newcastle disease (ND) that caused by Newcastle disease virus (NDV) is one of the most important avian diseases. It is widespread in many country and areas throughout the world, and can cause severe economic losses in the poultry industry. NDV is a paramyxovirus possessing two major glycoprotein components of the virion envelope, one of which is the Fusion (F) protein. It is involved in membrane fusion and virus penetration, and is known to play an important role in immunity to the disease. In order to develop new genetic engineering vaccines which are safe and effective, we constructed four recombinant fowlpox virus (rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4 -SFB, rFPVLp-SFB) expressing F gene of ZJ-1 strain of NDV and evaluated its genetic stability and protective efficacy in this study.1 Selection of recombinant fowlpox viruses (rFPVs) expressing F gene of NDV and their genetic stabilityThe transfer vectors P11SF and PN11SF were transfected by Fugend? Transfection Reagent mediated transfection on CEF infected with parent 282E4 and large plaque strain FPV. The four rFPVs ( rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4-SFB, rFPVLp-SFB) were selected and purified by blue plaques expressing 3 -galactosidase. The insertion and expression of F genes in rFPVs were confirmed by PCR and IFA respectively.Four rFPVs were serially passaged to the 20th on CEF monolayers, the expression of F genes were indentified by IFA. The F genes of passages 0, 10, and 20 were amplified by PCR and sequenced. There were no changes on the nucleotide and amino acid. The results showed that the four rFPVs were genetically stable for at least 20passages.2 Vaccination trials with rFPVs in chickens2.1 Protective efficacy of the rFPVs in SPF chickensSix groups of 5-day-old SPF chickens were vaccinated with Wt-FPVLp, rFPV282E4 -SFA, rFPVtp-SFA, rFPV282E4-SFB, rFPVtp-SFe and inactivated vaccine in oil emulsion by SC route in the nape of neck at the dosage of 104PFU in 0.2 ml, 3 weeks after vaccination, all chickens were challenged with 105ELD5o of F48E8 strain NDV by intranasal. Serum antibodies, morbidity and mortality were used to evaluate the efficacy of the rFPVs. Chickens vaccinated with rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4-SFB, rFPVtp-SFB and inactivated vaccine in oil emulsion all survived, while the protection rate of the group inoculated with Wt-FPV was 0.2.2 Protective efficacy of the rFPVs in commercial chickens26-day-old commercial chickens were immunized with the four rFPVs respectively by SC route at the dosage of 105PFU, and chickens vaccinated with Wt-FPV, inactivated vaccine in oil emulsion and unvaccinated were used as control. 21 days after vaccination, all chickens were challenge with 105ELD50 of F48E8 strain NDV. The protection rates of rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4-SFB, rFPVLp-SFB, and inactivated vaccine in oil emulsion were 64%, 60%, 52%, 88% and 100% respectively, while the chickens vaccinated with Wt-FPV and unvaccinated all died.3 Development of indirect ELISA for detecting antibodies of fowlpox virusThe FPV propagated on CEF was harvested as antigen to develop an indirect ELISA for detecting antibodies of fowlpox virus. The optimal coating concentration of antigen was 2.7ug of FPV protein per well, the serum sample were diluted to 1:100 and the cutoff was determined to be 0.098. The indirect ELISA detected a total of 400 serum sample originated from chickens immunized with FPV or rFPV, and the positive efficacy was 81.25% (325/400). The test also revealed that indirect ELISA was 400-800 tunes more sensitive than AGP. Otherwise, it was simple, convenient, specific and rapid for detecting antibodies of fowlpox virus.The findings showed that the four rFPVs we got not only had good genetic stability, but also provided adequate protection against the challenge of 105ELDso of F48E8 strainNDV in SPF chicken. So the rFPVs vaccines will be a promising substitution of conventional vaccines to prevent and control NDV.
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