Construction And Protective Efficacy Of Recombinant Turkey Herpesvirus Vaccine Strains Expressing Fusion Protein Of Geneotype ? Newcastle Disease Virus

Newcastle disease(ND)is an acute,severe and highly contagious disease of poultry caused by Newcastle disease virus.In recent years,the genotype ? is the mainly prevalent NDV virulent strains in China.Although the widely used genotype II vaccine can provide good clinical protection against genotype VII NDV strains,it cannot effectively prevent the virus shedding of infected chickens.Therefore,it has great significance for prevention and control of ND to develop vaccines that can match the genotype of epidemic NDV strains.Herpesvirus of turkey(HVT)FC126 strain is similar to Marek's disease(MDV)in antigenicity and is usually used to prevent of Marek's disease(MD)of chickens.Since the genome of HVT FC126 strain contains abundant replication unnecessary regions for insertion of exogenous genes.At the same time,it can be freeze-dried and preserved and is always used as a carrier for recombinant virus vaccines for poultry.Fusion(F)protein is the main glycoprotein on the surface of NDV envelope,which is directly related to the pathogenicity of NDV and is also the main protective antigen.In this study,the US2 replication nonessential region of HVT FC126 strain is used as the insertion site of the exogenous gene to construct a recombinant virus vaccine strain that can express NDV F protein genotypes ?,In addition,in order to avoid neutralization of antibodies in vivo,a recombinant virus lacking the transmembrane region of F protein was constructed.The immune efficacy of two recombinant viruses was evaluated.Firstly,the intermediate plasmid pCR2.1-US2 containing the upstream and downstream sequences of exogenous gene sertion site of HVT FC126 strain was constructed.Then,the EGFP gene expression cassette is cloned into the intermediate plasmid to construct the transfer vector pCR2.1-US2-EGFP,the transfer vector pCR2.1-US2-EGFP and HVT FC126 strains genomic DNA are co-transfected into chicken embryo fibroblast(CEF)by using calcium phosphate coprecipitation method.Recombinant virus rHVT-EGFP expressing EGFP is obtained by homologous recombination.Using similar methods and steps,EGFP gene expression cassette of rHVT-EGFP was replaced by F gene expression cassette of NDV strain of genotypes VII to constructe recombinant virusThe genome of genotypes VII NDV DT 2014 strain was used as template to amplified F gene by RT-PCR.The F gene was inserted into the eukaryotic expression vector pEASY(?)-M3,and then the F gene expression cassette including CMV promoter,TK ployA terminator and FLAG-tag was amplified by PCR and cloned into the intermediate plasmid pCR2.1-US2 to construct the transfer vector pCR2.1-US2-F.The analysis of the transmembrane region of F gene showed that F protein contained two transmembrane regions(117-139aa and 502-525aa).Transfer vector pCR2.1-US2-F was used as template,and two transmembrane regions were deleted respectively by using a overlapping PCR to construct a transfer vector pCR2.1-US2-AF.The rHVT-NDV-VII-F and rHVT-NDV-VII-AF were rescued by co-transfected pCR2.1-US2-F or pCR2.1-US2-AF with rHVT-EGFP genomic DNA into CEF respectively.The recombinant virus was purified repeatedly by finite dilution method until the virus plaque without fluorescence under fluorescence microscope.The F gene was correctly inserted into the genome of the recombinant virus by PCR verification.Positive serum of genotypes VII NDV was taken as primary antibody,both recombinant viruses reacted positively by indirect immunofluorescence assay.With western blot,recombinant virus rHVT-NDV-VII-F successfully expressed about 55kDa and 60kDa proteins,and recombinant virus rHVT-NDV-VII-AF successfully expressed about 60kDa protein.The growth characteristics of the two recombinant viruses on CEF cells were similar to that of HVT FC126 vaccine strain.In order to evaluate the immune efficacy of recombinant viruses rHVT-NDV-VII-F and rHVT-NDV-VII-AF,one-day-old SPF chickens were divided into 5 group as rHVT-NDV-VII-F group,rHVT-NDV-VII-AF group,attenuated vaccine group,challenge control group and blank control group.Each group had twenty chickens.First,one-day-old chickens in rHVT-NDV-VII-F group and rHVT-NDV-VII-AF group were immunized the recombinant viruses by subcutaneous injection at a dose of 5000 plaque forming units(PFU);the attenuated vaccine group was vaccinated with La Sota vaccine at seven-day-old.Except the blank control group,the other groups were inoculated NDV DT-2014 strain with 105EID50 dose by dropping nose and dropping eyes at twenty-one-day-old.After challenge,the clinical symptoms and mortality of each group they were observed continuously for fourteen days.On the third day after challenge,swabs samples from oropharynx and cloaca of each group were collected,and the rate of virus shedding was detected by real time PCR.The virus shedding of recombinant virus rHVT-NDV-VII-F group was lowest,followed by rHVT-NDV-?-?F group.In terms of DT-2014 NDV challenge,two recombinant viruses can greatly delay the morbidity and death of chickens.The survival rate of rHVT-NDV-?-F group was forty percent.These results indicated that rHVT-NDV-?-F can provide certain protection for chickens against genotypes ? NDV.