Construction And Evaluation Of Immune Efficacy Of Recombinant Chimeric NDV Expressing VP2 Gene Of IBDV

Newcastle disease(ND)is a highly contagious avian infectious disease caused by Newcastle disease virus(NDV).Currently,the ND vaccine widely applied in China is constructed based on the genotype II La Sota strain.However,the molecular epidemiological investigation indicated that the genotype Ⅶ NDV is the predominant strain currently circulating in Asia.NDV fusion(F)gene and hemagglutininneuraminidase(HN)gene are important protective antigens for the ND vaccine.Genetic distance analysis based on the full-length F and HN genes showed that the genotype Ⅶ NDV had high genetic variations compared with the genotype II La Sota vaccine.A genotype-matched vaccine is necessary for providing better protection against genotype Ⅶ NDV infection.Infectious bursal disease(IBD)is a highly contagious,immunosuppressive disease caused by the infectious bursal disease virus(IBDV).IBDV is an important pathogen that seriously endangers the global poultry industry.IBDV mainly infects the bursa of Fabricius of chickens,resulting in disintegration and necrosis of B lymphocytes in the bursa.In recent years,atypical IBD caused by novel IBDV variants has brought serious threats to the Chinese poultry industry.The novel IBDV variants can escape the immune protection of traditional IBDV vaccines,causing serious immunosuppression in chickens.VP2 gene of IBDV is an important antigen gene that induces neutralizing antibodies against IBDV.The prevalence of genotype Ⅶ NDV and IBDV variants has caused considerable economic losses to the poultry industry.Therefore,in this study,the HN and F genes of the La Sota vaccine strain were replaced by the HN and F genes of the genotype Ⅶ NDV virulent strain,respectively.The VP2 gene was optimized and synthesized based on novel IBDV variants was inserted between the P and M genes of the La Sota genome.The recombinant chimeric NDV vaccine r rLaS-VIIF/HN-VP2 expressing the VP2 protein of novel IBDV variants was constructed for the first time.The immunogenicity and efficacy of r La S-ⅦF/HNVP2 were evaluated in SPF chickens.The thesis includes the following aspects:1.Construction and identification of the recombinant chimeric La Sota strain expressing the IBDV VP2 geneIn order to develop a cheap and efficient vaccine for effectively preventing and controlling the infection of novel IBDV variants and Ⅶ NDV,In this study,the recombinant chimeric La Sota vaccine strain containing genotype Ⅶ NDV F and HN genes was used as the vector,and the optimized synthesis sequence based on the VP2 gene of the representative strain SHG19 of novel IBDV variants was inserted between the P and M genes of La Sota vaccine strain genome.The recombinant NDV r La SⅦF/HN-VP2 was successfully rescued.The constructed r rLaS-VIIF/HN-VP2 was identified by sequencing and Western blotting.The results showed that the IBDV VP2 gene was successfully inserted,and the VP2 protein was successfully expressed.The biological characteristics of the 25 th generation of r rLaS-VIIF/HN-VP2 in chicken embryos showed that it retained the biological characteristics of the La Sota parental strain in chicken embryos,such as high-titer replication,low pathogenicity and genetic stability.The peak value of EID50 of r rLaS-VIIF/HN-VP2 reached 10-8.16/0.1 m L,and the mean death time(MDT)of the inoculated chicken embryos was 134 h.The ICPI of one-day-old chickens and IVPI of six-week-old chickens were both 0.The constructed r rLaS-VIIF/HN-VP2 expressing the VP2 gene of IBDV will lay the foundation for the development of efficient and cost-effective polyvalent vaccines that can simultaneously prevent and control the infection of the genotype Ⅶ NDV and novel IBDV variants in China.2.Evaluation of immunogenicity and protection efficacy of recombinant NDV r La SⅦF/HN-VP2 vaccineTo evaluate the immunogenicity and protection efficacy of r rLaS-VIIF/HN-VP2 vaccines.One-week-old SPF chickens were immunized with r rLaS-VIIF/HN-VP2 attenuated live vaccine and oil-emulsion inactivated vaccine.Hemagglutination inhibition test was used to detect the antibody level against the genotype Ⅶ NDV,the IBDV antibody ELISA kit was used to detect the antibody level against IBDV,and the virus neutralization test was used to determine the neutralizing antibody titer against IBDV.The results showed that r rLaS-VIIF/HN-VP2 attenuated live vaccine or inactivated vaccine could provide complete protection against the infection of the genotype Ⅶ NDV and novel IBDV variants in chickens,and the protective efficacy of r rLaS-VIIF/HN-VP2 attenuated live vaccine was better than that of the inactivated vaccine.It is concluded that r rLaS-VIIF/HN-VP2 can be used as a seed virus to prepare a cheap and efficient bivalent vaccine against IBDV variants and Ⅶ NDV strains in China.