Generation And Efficacy Evaluation Of A TK/gE/gI-gene-deleted Pseudorabies Virus Variant

Porcine Pseudorabies(PR)or Aujeszky’s disease,caused by Pseudorabies Virus(PRV),is a serious disease of cattle,sheep,pigs and other livestock and other wild animals.The disease is characterized by fever,unrelieved itchiness,encephalomyelitis,nervous system and respiratory system illness.At present,there is no effective drug for treatment of PR,and used PRV Bartha-K61 is a very safe and effective vaccine against PR and play a critical role in the control and eradication of PR.However,since 2011,PR has occurred among Bartha-K6-vaccinated pig populations on many farms in China.Many studies showed that the Bartha-K61 vaccine cannot provide full protection against the reemerging PRV variant,so the pig industry in China is still in huge risk,and development of a novel vaccine against the PRV variant is urgently needed.In this study,a variant named PRV NY strain was isolated from the diseased piglets suspected of PR from a Bartha-K61-vaccinated pig farm in Nanyang,Henan Province.In the Phylogenetic tree of g E,g B and g C gene,the NY strain and the other recently emergent PRV variants isolated in China were clustered to a relatively independent clade,and distantly related to the classical virulent of the Europe and America.The g E,g B and g C genes of NY strain contain several typical amino acid mutations,insertions or deletions.The gE/gI and TK genes were PCR amplified from PRV NY strain as homologous arms and cloned into p UC-19 plasmid to obtain the vectors p UC-g E/g ILR and p UC-TKLR Take the enhanced green fluorescent protein(EGFP)as a reporter,and obtain the vectors p UC-g E/g ILRE and p UC-TKLRE.The p UC-g E/g ILRE vector and the total DNA of PRV NY strain were co-transfected into ST cells,and the recombinant virus r PRV NY-g E-/g I--EGFP+ was obtained by plaque purification in ST cells.Then the r PRV NY-g E-/g I--EGFP+ genomic DNA was extracted,and with the p UC-g E/g ILRE vector were co-transfected into ST cells.A g E/g I deletion was selected by plaque purification in ST cells,named r PRV NY-g E-/g I-,and sequencing results showed 2586 bp deletion of g E/g I genes.Finally,The p UC-TKLRE vector and the total DNA of r PRV NY-g E-/g I-were further co-transfected into ST cells,and a g E/g I/TK triple deletion mutant(named r PRV NY-g E-/g I-/TK--EGFP+)was picked up during plaque purification in ST cells.The deletion of 311 bp in TK gene of r PRV NY-g E-/g I-/TK--EGFP+ was confirmed by PCR.The safety and immunogenicity of r PRV NY-g E-/g I-and r PRV NY-g E-/g I-/TK--EGFP+ were also evaluated in pigs.The results showed that the r PRV NY-g E-/g I-is not safety to mice,but the r PRV NY-g E-/g I-/TK--EGFP+ had ideal safety to mice.The indirect ELISA and the neutralization test displayed that the r PRV NY-g E-/g I-/TK--EGFP+ can induced higher PRV-specific antibody,and the recombinant virus elicited significant cellular immune response towards the determination of T lymphocyte subsets.The challenge assay showed that groups of r PRV NY-g E-/g I-/TK--EGFP+ could protect the mice against the virulent PRV NY challenge within limit,and the immunizing dose,route and adjuvant should be taken into account.In conclusion,the r PRV NY-g E-/g I-/TK--EGFP+ might be a promising candidate vaccine for the control of the currently epidemic PR in China.