Genetic Stability And Protective Efficacy Of The Recombinant Fowlpox Virus With Highly Expressed Glycoprotein B Gene From Serotype 1 Marek's Disease Virus

In order to evaluate the genetic stability of the recombinant fowlpox virus highly expressing glycoprotein B gene (rFPV-gB/R) from CVI988/Rispens strains of Marek's disease virus. rFPV-gB/R was passaged in chicken embryo fibroblast (CEF) for 30 times and specific pathogen-free (SPF) chickens for 5 times. The inserted gB genes of different passages of rFPV-gB/R were amplified by PCR. Sequence analysis showed that the inserted gB genes of rFPV-gB/R from different passages were very stable without a single nucleotide change when compared with the original one. A series of animal expriments in SPF chickens were carried out to meet the requirements for applying the certificate of the rFPV-gB/R based vaccine from the Chinese Ministry of Agriculture. Combinational lyophilized vaccine containing rFPV-gB/R, HVT and rFPV-IFN- II was evaluated in commercial Wolfhill chickens for its protective efficacy against Marek's disease. The results showed that the effective dose of rFPV-IFN-II was one tenth of that of rFPV-gB/R.A purity assay was performed with the original recombiant fowlpox virus and the pilot product of the combinational lyophilized rFPV-gB/R vaccine. The results showed that rFPV-gB/R was free from exogenous bacterial and viral contaminations. The experiment for determining the valid storage time of the vaccine showed that the vaccine product remained effective after 12 months preservation at -20℃Experiments for evaluating the biological safety of rFPV-gB/R were completed toevaluate the stability of recombinant virus passaged in SPF chickens and its ability of horizontal transmission. The chickens were challenged with FPV strain of 102E6 30 days after inoculation. The results showed that chickens immunized with rFPV-gB/R were protected from challenge, indicating that immunogenicity of the recombinant virus is stable. The in vitro virus isolation showed that the recombinant virus could survive only at the inoculation site for 7 days. It confirmed that rFPV-gB/R was safe to use.In laboratory trials, the protective efficacy of combinational lyophilized vaccine (rFPV-gB/R+HVT+rFPV-IFN- II) was higher than either monovalent lyophilized HVT or bivalent lyophilized vaccine(rFPV-gB/R+HVT) in Wolfhill chickens that are susceptible to MD. The protection efficacy of the combinational vaccine composed of rFPV-gB/R, HVT and rFPV-IFN-II approached the protection level afforded by conventional serotype 1 CVI988 vaccine when challenged with very virulent Marek's disease virus strain(wMDV) Md5 and RBIB. The effective dose of rFPV-IFN-II as an immune adjuvant was one tenth of the dose of rFPV-gB/R.The results of this study recomfirmed that the new combinational lyophilized vaccine (rFPV-gB/R-HVT+rFPV-IFN- II) was safe, stable and efficacious.