Construction And Potential Efficacy Of GE/gI/TK Genes Deleted Mutant Of A Pseudorabies Virus Variant

Porcine pseudorabies(PR)is a widespread and acute infectious disease caused by pseudorabies virus(PRV)in a variety of domestic animals and wild animals.It was characterized by high fever,extreme itching(except porcine)and encephalomyelitis.The disease mostly outbreaks in the herd,causes huge economic losses in pig indudtry.At present,there is no effective drug for treatment of PR,so vaccination became the main measures to control the occurrence and prevalence of this disease.However,since 2011,serious pseudorabies disease have occurred in vaccinated pig farms in provinces of China,which means that existing vaccines can not provide completly effective protection against the epidemic PR strains,and pig industry in China is still in huge risk.It is believed that the reemerging PR in China since 2011 is caused by a variant PRV(vPRV).In this study,a gE/gI double-deletion strain vPRV and a gE/gI/TK triple-deletion vPRV strain without reporter gene were construct from a vPRV-XJ strain through Cre-loxP system.The preliminary study on the growth characteristics and potential efficacy of knockout vPRV prototype vaccine provides a technical platform for the development of new vaccines against the reemerging PR in China.1.Construction of gE/gl double deletion strain and gE/gl/TK triple deletion mutant of variant PRV(vPRV)The gE/gI and TK genes were PCR amplified from vPRV-XJ strain as homologous arms and cloned into pBKS plasmid,taking the enhanced green fluorescent protein(EGFP)as a reporter,to obtain pBKSgILgER-GFP and pBKSTKLR-GFP respectively,then transfer the vectors pBKSgILgER-GFP and pBKSTKLR-GFP with loxP sites were constructed by insertion of the EGFP gene into gE/gI gene and TK gene cloned in pBKS.Total DNA of vPRV-XJ infected Vero cells was extracted and the pBKSgELgIR-GFP vector and total viral DNA were co-transfected into Vero cells by calcium phosphate precipitation method,harveste the recombinant virus rPRV-gE’/gl’-GFP with the cytopathic effect(CPE)more than 80%.The recombinant virus rPRV-gE-/gI-GFP genomic DNA was mixed with Cre recombinase and extracted,then transferered into Vero cells by phosphate precipitation method.Subsequently,a gE/gI deletion vPRV mutant was obtained by plaque purification in Vero cells.Sequencing results showed a 2287 bp deletion of gE/gI existed in the genome of the vPRV-gE/gI deletion mutant.The genomic DNA of rPRV-gE-/gI-was further co-transfected with plasmid pBKSTKLR-GFP,then rPRV-gE-/gI-/TK--GFP was also selected by plaque purification.Treated the DNA of rPRV-gE-/gI-/TK-with Cre recombinase,transfered into Vero cells,and a gE/gI/TK triple deletion mutant was picked up during the plaque purification.The deletion of 498 bp in TK gene of rPRV-gE-/gI-/TK-was confiemed by PCR.2.Virus replication and potential efficacy of the deletion mutantsThe titers of PRV-XJ,rPRV-gE-/gI-and rPRV-gE-/gI-/TK-in Vero cells were 10-7 500 50%tissue culture infective dose(TCID50)/mL,10"6 667TCID50/mL and 10-6.571TCIDso/mL,respectively,indicating that the replication rate of the two deletion mutants was similar,but both of them had a low replication titers compared with their parental strain.The 50%lethal dose(LD50)of the wild type strain,the gE’/gI’ mutant and the gE-/gI-/TK-mutant for mice were 1.3×103TCID50,7.9×104TCID50 and more than 1×106TCID50,respectively.Compared with the paretal strain,the virulence of gE/gI mutant in mice was decreased,while gE-/gI-/TK-mutant showed a significant attenuation in mice.Mice immunized with 104.7TCID50 deletion mutants showed no clinical symptom and growth retardation.The antibody level of rPRV-gE-/gI-/TK-group was significantly higher than that of Bartha K61 group evaluated in an indirect immunofluorescenceassay,after challenged with the vPRV-XJ,the efficacy of rPRV-gE-/gI-/TK-was 87.5%and the efficacy of Bartha K61 was about 12.5%,respectively.Pigs immunized with 106.3TCID50 of rPRV-gE-/gI-/TK-did not exhibit any clinical signs,the PRV-specific antibody level increased stabily and quickly.After challenged with vPRV-XJ,the rPRV-gE-/gI-/TK-immunized pigs did not display clinical signs and pathological changes,implying that this prototype vaccine can protect commercial pigs against vPRV-XJ challenge.In conclusion,the rPRV-gE-/gI-/TK-strain was safe and had ideal immunogenicity to pigs and might be a promising candidate vaccine for the control of the currently epidemic PR in China.