Construction Of Ge And Gi Dual Deletion Mutant Of Pseudorabies Virus Variant AH02LA Strain And Primarily Study Of Its Immumogenicity

Pseudorabies(PR),also known as Aujesky's disease,is caused by Pseudorabies virus(PRV).It is an infectious disease of many livestocks and wildlife characterized by fever,itching,encephalomyelitis.The major clinical manifestations of the onset of swine PR include fever,neurological symptoms in newborn piglets,respiratory symptoms in fattening pigs,abortion in sows and growth retardness in adult pigs infected with pseudorabies virus,resulting in great harm to the pig industry.Since 2011,in Northern China,pseudorabies outbreaks happened in pig herds that have been immunized with PRV vaccine like Bartha K61.Afterwards,the epidemics gradually spred to most pig farms in China.Preliminary studies showed that field isolates from died piglets immunized PRV Bartha-K61 pig vaccine were new variant strains of pseudorabies virus which showed highly virulent than strains reported before.The traditional PRV attenuated vaccine like Bartha-K61 could not provide complete protection agaist these pseudorabies virus mutants.Therefore,it is extremely urgent and necessary to develop a new vaccine made of the current pseudo-rabies vaccine mutants.We have previously isolated three PRV strains(AH02LA,LH08 and PRV V+)from the brain tissues of pigs that were immunized with Bartha K61 in farms in Jiansu and Anhui provinces,but were later died of PRV infection.All isolates were identified as PRV wild strains by successful amplification of PRV glycoprotein E(gE)gene fragment with specific primers.The genes of 11 major envelope proteins of AH02LA,LH08 and PRV V+were amplified,sequenced and compared with classic strains(Bartha,Kaplan and Becker)and TJ strain in order to investigate the genetic changes as well as changes in antigenicity and virulence.It was found that the homology between these genes of the three isolates was above 99.9%.However,a large number of mutations were identified in the three isolates when compared with classic strains including non-synonymous substitutions,and insertions and deletions in gB,gC,gD,and gE genes.For example,a 12-bp insertion was observed at position 350-361 in gB gene of AH02LA compared with the classic strain Becker(JF797219),whereas a 9-bp deletion was identified at position 227-234 in gB gene when the isolate was compared with Bartha(JF797217)and Kaplan(JF797218).Although the homology between gC,gD and gE genes of AH02LA and those of TJ strain was 100%,there was a 21-bp deletion in gC gene of the isolate compared with Bartha,Kaplan and Becker strains.A 6-bp insertion was observed at position 825-830 in gD gene of AH02LA when compared with Bartha,Kaplan,whereas a 3-bp insertion was located at position 142-144 and 1481-1483,respectively in gE gene of the isolate compared with Kaplan and Becker.Animal studies showed that the motility rate was 100%in PRV antibody negative pigs that received intranasal inoculation of 2 mL of AH02LA virus(106/0.1 mL)at 60 to 65 days of age.These results have suggested that AH02LA,LH08 and PRV V+ are new virulent PRV strains that might originated from the same wild strain,which provide the basis for the epidemiological surveillance of PRV and the development of novel vaccines.A PRV-AH02LA(?gE/gI)-pHA2 double gene deletion strain was constructed by replacing gE and gI genes of AH02LA strain with a bacterial artificial chromosome(BAC)mini-F vector sequence through homologous recombination,and transformed into E coli competent cells in order to obtain PRV BAC(PRV BAC-11).The deletion of gI and gE genes in the constructed BAC was confirmed by subsequent PCR and restriction fragment length polymorphism(RFLP)analyses.PRV AH02LA BAC genomic DNA was transfected into CEF cells pre-treated with Cre enzyme for loxp-Cre recombination.White spots without green fluorescent were observed under UV light.A double gene deletion recombinant strain without mini-F was rescued and named PRV-AH02LA(?gE/gI).A DNA fragment was obtained by PCR amplification using PRV-AH02LA as the template and primers PRV H1F and PRV H2R,and co-transfected into CEF with PRV BAC-11.White spots without green fluorescent were observed under UV light.A double gene deletion recombinant strain without mini-F was rescued and named PRV-AH02LARec.Growth kinetics study revealed that the growth curves of PRV-AH02LA(?gE/gI)and PRV AH02LARec were similar to that of the parent strain AH02LA.After the infection of cells,the growth rates of PRV-AH02LA(?gE/gI)and PRV AH02LARec were slightly slower compared with the parental strain.Moreover,the viral titers of both strains were also slightly lower than that of the parental strain.In this study,the safety and immunogenicity of the gene deletion virus PRV-AH02LA(?gE/gI)were further evaluated.Pigs were vaccinated with PRV-AH02LA(AgE/gl)in a dose of 105,104 and 103 TCID50,respectively.No symptoms of PR were observed after vaccination.The pigs were challenged with a virulent strain of PRV(107.5 TCID50/mL)at 7 days after immunization.None of the vaccinated pigs displayed any clinical symptoms of PR or viral shedding,indicating full protection of the recombinant vaccine against lethal PRV infection.In contrast,both higher body temperatures and viral shedding were detected in pigs that received Bartha-K61 attenuated vaccine.These results have demonstrated that the gene deletion strain PRV-AH02LA(?gE/gl)is safe and provides effective protection against lethal PRV infection,and therefore is a good vaccine candidate strain for PRV.