Construction And Preliminary Study On Immune Efficacy Of Recombinant PRV Strains With Partial Epitope Regions Of TGEV And PEDV S Gene

Transmissible gastroenteritis(TGE)and porcine epidemic diarrhea(PED)are caused by porcine transmissible gastroenteritis virus(TGEV)and porcine epidemic diarrhea virus(Porcine epidemic diarrhoea,PEDV),respectively,and theirclinical symptoms are similar,including piglet diarrhea,vomiting,anorexia,dehydration,and weight loss.In 2010,the virulence of PEDV mutated was enhanced,and the traditional PEDV vaccine strain(CV777)could not provide complete protection.The PEDV mutated strain could still circulate in pig farms immune to the CV777 strain,which brought great trouble to the prevention of PEDV in production.Porcine pseudorabies virus(PRV)infection can lead to neurological disorders,respiratory distress,weight loss,death of young pigs and abortion of pregnant sows.Since the end of 2011,a large numberof PRV variant strains have emerged in many areas of China,which are more pathogenic than the PRV classic strains.Furthermore,the classic Bartha-K61 vaccine does not fully protect piglets from these new variants.Clinically,there are often mixed infections of TGEV,PEDV and PRV.Vaccine immunization is still one of the effective means to prevent TGEV,PEDV mutant strains and PRV mutant strains.Therefore,the development of safe and effective dual ortriple vaccines is of great significance forthe prevention of TGEV,PEDV and PRV infection.Recombinant viruses represent a particularly promising avenue of vaccine research both forimproving existing vaccines and fordeveloping new ones.The existence of numerous nonessential genes in the large PRV genome permits the simultaneous insertion of multiple foreign genes in the hope of vaccinating against several diseases at the same time.In this study,the rPRV NY-g E-/g I-/TK-strain preserved in the laboratory was used as the parental strain to construct two recombinant viruses(rPRV-AD and rPRV-AD-CS)forexpression of TGEV S gene AD epitope region and co-expression of TGEV S gene AD epitope region and PEDV S gene CS neutralizing epitope region.The three deletion strains(rPRV NY-g E-/g I-/TK-)were constructed from the PRNY mutant strain isolated in ourlaboratory at the end of 2012 as the parent.At the same time,theirbiological properties and immunogenicity are explored in orderto obtain an ideal candidate strain fora safe and effective double ortriple vaccine.In this study,the main antigenic epitope region containing A and D antigenic sites in the TGEV S gene was inserted into the pG transferplasmid through the Bam H I restriction site.The transferplasmid pG-AD-EGFP was successfully constructed.The transferplasmid pG-AD-EGFP contains the same sequence of the PRV g G gene as the homology arm..The parental strain(rPRV NY-g E-/g I-/TK-)was first inoculated into ST cells,and then the transferplasmid pG-AD-EGFP were transfected into ST cells.The parental strain undergoes homologous recombination with the transferplasmid in ST cells.The lesions with the green fluorescence were picked undera fluorescence microscope,and the recombinant virus rPRV-AD-EGFP was obtained by plaque purification.EGFP was knocked out by CRISPR/Cas9 technology,and the nonfluorescent lesions were selected and purified by plaque to obtain the recombinant virus rPRV-AD.The recombinant virus rPRV-AD was identified at the transcriptional and expression levels,and the results showed that rPRV-AD successfully expressed AD epitope on ST cells.The neutralizing epitope region of PEDV CS was inserted into eukaryotic expression vectorpBApo-EF1α_Pur_DNA through Bam H I and Hind III restriction sites,and Koza K sequence(GCCACC)was introduced before CS gene to obtain eukaryotic expression plasmid pBA-CS.The Bstz17 I restriction site cuts the transferplasmid pG-AD-EGFP into a linearvector.About 20 bpat each end of the linearvectorwas used as the homology arm to introduce the primers foramplifying the CS expression cassette,and the CS gene expression cassette was amplified with the eukaryotic expression plasmid pBA-CS as the template.The linearvectorand CS expression cassette were recombined into the transferplasmid pG-AD-CS-EGFP by seamless cloning technology.The parental strain(rPRV NY-g E-/g I-/TK-)was first inoculated into ST cells,and then the transferplasmid(pG-AD-CS-EGFP)was transfected into ST.The recombinant virus with green fluorescence was selected and purified by plaque to obtain the recombinant virus pG-AD-EGFP.EGFP was knocked out by CRISPR/Cas9 technology,and the recombinant virus rPRV-AD-CS was obtained by plaque purification.The recombinant virus rPRV-AD-CS successfully expressed the CS neutralizing epitope region by RT-PCR,Western blot and indirect immunofluorescence assay.Recombinant viruses(rPRV-AD and rPRV-AD-CS)and parental strains(rPRV NY-g E-/g I-/TK-)had similarculture properties on ST cells,virus titers on fourdifferent cells resemblance.Theirone-stepgrowth curve growth trends are basically the same.They have the same physicochemical properties and are genetically stable.Afterimmunizing 2-week-old piglets with the two recombinant virus strains,the piglets’ body temperature,body weight gain,feed intake and mental state were all normal,indicating that they are safe forpiglets.The ELISA and neutralization tests showed that the recombinant virus could stimulate piglets to produce immune responses against PRV,TGEV and PEDV,respectively.The immune effect against TGEV and PEDV was lowerthan that of commercial attenuated dual vaccine,while the immune effect against PRV was basically the same as that of commercial vaccine.The results of the challenge test showed that the recombinant viruses(rPRV-AD and rPRV-AD-CS)had a certain protective effect on the virulent TGEV strain,and the recombinant virus(rPRV-AD-CS)had a certain protective effect on the virulent PEDV strain.However,the protective effect was lowerthan that of commercial attenuated duplex vaccines.In this experiment,the numberof animals in each groupis relatively small,and the immune effect of the recombinant strain can only be shown initially.Therefore,the recombinant viruses(rPRV-AD and rPRV-AD-CS)still need furtheroptimization if they are to become ideal double ortriple vaccines.In conclusion,two recombinant viruses(rPRV-AD and rPRV-AD-CS)were successfully constructed in this study,and theirbiological properties and immune efficacy were explored.It will lay a certain theoretical and technical foundation forthe research and development of the recombinant virus live vectorvaccine using PRV as the vectorin the future.