Construction And Evaluation Of The Recombinant Marek's Disease Virus Vectored Vaccine Expressing VP2 Protein From Infectious Bursal Disease Virus

Infectious bursal disease(IBD)and Marek's disease(MD)are two economically important immunosuppressive disease inn chickens.Vaccination is the main measure for the prevention of these two diseases.The MD prevention is currently conferred by using the attenuated MDV serotype 1 vaccine.IBD is currently prevented by using the traditional inactivated and live vaccines.However,with the high levels of circulating maternal antibodies,the immunity of attenuated live vaccines of IBDV can be easily inhibited.To overcome the maternal antibodies,medium virulent vaccines of IBDV were used,which induced bursal damage to some extent and the failure of immunity of other poultry vaccines.Therefore,it is necessary to develop safer and more efficacious vaccines to prevent vvIBDV infection.We analyzed the genetic,antigenic and pathogenic characteristics of the vvIBDV isolates from China,which suggested the continued evolution of the very virulent IBD Vs.The more recent Chinese vvIBDV showed several genetic changes in both segments and clustered in a distinct subgroup from the typical vvIBDV and the early Chinese vvIBDV.Although having the similar antigenicity with the typical vvIBDV strains,the recent Chinese vvIBDV exhibited higher pathogenicity than the European typical vvIBDV.The vvIBDV HLJ0504 strain is currently the prevalent IBDV strain in China.Marek's disease virus vaccine strain is a preferred vector in the construction of recombinant vaccines.Here we developed a system utilizing overlapping fosmid DNAs transfection that rescues an MDV serotype 1(MDV1)vaccine strain.Using this system,we inserted the eGFP gene into five different insertion sites in MDV genome.The eGFP expression levels from the sites in the unique long(UL)region(within UL41,between UL45 and UL46,and between UL55 and LORF10)were comparable,which were significantly higher than those from the sites in the unique short(US)region(US2 and US 10),and the eGFP expression level from US2 was significantly higher than that from US 10.We next inserted the IBDV VP2 gene at MDV1 genome sites UL41,US 10 and US2.Insertion of the VP2 gene did not affect the replication phenotype of MDV in cell cultures.After challenge with very virulent IBDV(vvIBDV),r814US2VP2 conferred full protection against vvIBDV and vvMDV challenge in specific-pathogen-free(SPF)chickens.Further studies showed that a single dose of 2000 PFU r814US2VP2(namely,rMDV-VP2)was sufficient to fully protect chickens against vvIBDV infection in SPF chickens;this vaccine provided 90%protection against vvIBDV in commercial layer chickens with maternal antibodies,which was higher than the protective efficacy using the B87 medium-virulent live vaccine of IBDV.Additionally,the recombinant MDV1 vaccines provided full protection against vvMDV challenge in chickens.The rMDV-VP2 vaccine exhibited good safety in chickens with low level of transmission ability.To sum up,rMDV-VP2 could be used as a promising bivalent vaccine against both Marek's and infectious bursal diseases in chickens.