Isolation And Identification Of Pseudorabies Virus Variant And Construction Of Its GE Gene Deletion Mutant

Porcine pseudorabies(PR)is caused by the pseudorabies virus(PRV),which characterized by pruritus neurologic symptoms,and reproductive disorder,resulting in huge economic loss for pig industry.The classical vaccine strain plays an important role in the prevention and control of PR,however,in the past few years PRV appeared in immunized pigs,and cause epidemic in acertain range,indicating that PRV may have variation.Therefore,it has important practical significance to develop a new pseudorabies vaccine based on PRV variant strain.In this paper,PRV variant strain that was isolated from infected pigs with suspected pseudorabies in a pig farm in Jiangsu was confirmed that the virus had the sequence characteristics of PRV variant strain.The isolated PRV variant strain was taken as parent virus,and a gE gene deletion mutant without reporter gene was constructed.Further assessments for the growth characteristic and immunological efficacy of this deletion mutant were carried out.This study will lay a basis for research and development genetic engineering vaccine of pseudorabies.1.The isolation,identification,and purification of the PRV variant strain.The submitted samples from a pig farm with suspected pseudorabies in Jiangsu were detected and diagnosed as mixed infection of PRV and classical swine fever virus by PCR detection.The sample was filtrated by 0.22 μm filter membrane and inoculated into PK15 cells.The cells were observed a typical cytopathic effect with shrink and round after 28 hours post-infection,the PCR detecting result showed that the cells contained PRV and classical swine fever virus at same time.Total DNA of virus was extracted by using proteinase digestion and phenol extraction method,and the virus was further purified by transfection of DNA into PK15 cells using calcium phosphate precipitation.The further result of PCR confirmed that the cells only contained PRV.The purified virus can form typical cytopathic effect in PK15 cells and was shown PRV particle structure under the electron microscope.When PRV gE gene was amplified and sequenced,the amino acid sequence of gE gene showed that there were two aspartic acids inserted into gE genes,which is the characteristic of PRV variant strain that were already reported.So the isolated virus was PRV variant strain,which was named PRV-TX.2.Construction of gE gene deletion mutant of PRV variant strainpSKgERL-GFP transferring plasmid vector with green fluorescent protein marker(established by our lab)and genomic DNA of PRV-TX was co-transfected into PK15 cells in a proportion of 10 μg:1 μg by using calcium phosphate precipitation,when 80%lesions were appeared in the cells,the viruses were collected.After doubling dilution,virus was inoculated into single-layer PK15 cells and the recombinant virus with reported gene was purified by plaque purification method.After reported gene knockout by using recombinant Cre,the rPRV-TX-gE-without reporter gene was obtained after plaque purification.3.Growth characteristic and immune efficacy of rPRV-TX-gE-mutantThe growth curve of recombinant virus was tested in PK15 cells,and the result showed that the growth curve of rPRV-TX-gE-mutant was consistent with parental strain PRV-TX.After 84 hours post infection,the titers of rPRV-TX-gE-mutant and parental strain PRV-TX were 106.14 TCID50/0.1 mL and 106 34 TCID50/0.1 mL,respectively.There was no significant difference between the two strains,indicating that gE gene deletion of mutant had no influence on virus replication.LDsos of the rPRV-TX-gE-mutant and parental strain PRV-TX were determind in mice,in comparison with classic strain PRV-SZ and its gE mutant rPRV-SZ-gE-.The results showed that LD50s of PRV-SZ,rPRV-SZ-gE-,PRV-TX and rPRV-TX-gE-in BALB/c mice were 103 67 TCID50/0.1 mL,104 TCID50/0.1 mL,103.5TCID50/0.1 mLand 104.17 TCID50/0.1 mL,respectively.The results suggested that pathogenicity of classical strains was similar to variant strains.The virulence of gE mutant in mice was decreased when compared with that of parental strains.Mice were immunized with rPRV-SZ-gE-or rPRV-TX-gE-strains at the dose of 103TCID50 by injecting hindlimb muscles.After 21 day post immunization,the mice were challenged by PRV-SZ or PRV-TX at the dose of 104TCID50.The results showed that rPRV-TX-gE-deletion mutant vaccine provided 83.3%protection rate against two virulent strains in mice.The protection rate of rPRV-SZ-gE-deletion mutant vaccine was 66.7%for PRV-SZ challenge and 33.3%for PRV-TX challenge.These results suggested that rPRV-TX-gE-can provide good protection against both classic and variant strains challenge.However,the protection rate of rPRV-SZ-gE-against variant strain challenge was lower.