Construction And Immunogenicity Analysis Of An Epidemic Pseudorabies Virus Vaccine Candidate Strain With A Dual Gene-deletion Of GE/gI

Pseudorabies（PR）is a highly contagious infectious disease of swine caused by pseudorabies virus（PRV）.Pigs are an important primary host for PRV,and all ages can be infected.Since 2011,the increased virulence of PRV epidemic strains and the expansion of the epidemic scope of the disease,the ineffective immune protection effect of classical vaccine strains led to the increasing mortality of pigs after the disease infection,which has caused heavy economic losses in the pig industry of China.Therefore,it is of great significance to find out the characteristics of the prevailing strains and to seek vaccine candidate strains with good immunogenicity to prevent and cure the disease effectively.In this study,some immunized pig with suspected pseudorabies symptoms in pig farms of Wuchang,Heihe,Daqing,Suihua and other places in Heilongjiang Province were subjected to virus isolation and identification.A total of 4 PRV strains were isolated from the brain tissues of diseased piglets,which were further verified by isolation and cultivation in PRV permissive cell S,electron microscopy morphology,immunofluorescence,PCR and nucleic acid sequencing,and these viruses were named PRV-WC01,PRV-HH01,PRV-DQ01 and PRV-SH01 respectively.The genetic variation analysis of g C and gE gene sequencing of the four isolated virus showed that these PRV strains were significantly different with the classical strains,but had high homology with the PRV epidemic strains reported in recent years and were in the same branch of phylogenetic tree.The virulence test results showed that the LD₅₀of PRV-WC01 strain to56-day-old piglets is 10^-3.17.PRV-WC01 virus with the titer of 10^8.50TCID₅₀/mL was inactivated by0.1%formaldehyde and added with MONTANIDETM ISA201 VG adjuvant to prepare immunogen.Experimental 21-day-old piglets were inoculated with the inactivated immunogen.On the 21st day after the second immunization,each immunized piglet was challenged with PRV-WC01 strain at a dose of 10 LD₅₀.The results showed that the immune protection rate was 100%,indicating that the strain can be an ideal immunogen with its good immunogenicity and complete immune protective effect on the epidemic strain.Based on the deletion site of PRV Bartha-K61,we induced the gI/gE dul-deletion on PRV-WC01 strain by using the CRISPR/cas9 gene editing system.To obtain sg RNAs with high efficiency gene editing,the designed sg RNAs were screened by viral plaque counting and quantitative PCR.Several sg RNAs were designed respectively to target the gE and gI genes in PRV genome,and were screened by viral plaque counting method.In order to increase the expression of Cas9 in cells to obtain higher gene knockout efficiency,a Vero cell line stably knocking out the PRV gE and gI genes was constructed by using the lentiviral transfection system.First,the lentiviral transfer plasmid Lenti CRISPR V2-sg RNA gE/gI was obtained by restriction enzyme digestion,ligation and homologous recombination.The resulted plasmid was transfected into Vero cells,and the transfected cells were infected with wildtype PRV-WC01.After 18h,the virus solution was collected,followed by the viral genome extraction and sequenceing to identify the gene editing function of the constructed plasmid.Subsequently,293T cells were co-transfected with the three plasmids to package lentivirus.These plasmids were the transfer plasmid Lenti CRISPR V2-sg RNA gE/gI,the packaging plasmid p MD2.G and the auxiliary plasmid ps PAX2 expressing Gag-pol protein.The packaged lentivirus was purified and concentrated using the PEG lentiviral purification reagent,and then infected to Vero cells.Puromycin was used to select the cells that stabely transfected by the letiviral expression system and therefore can stably knocking out the gE and gI genes.Confirmed by PCR amplification and Western blot identifying,the cell line genome was integrated with the CRISPR/Cas9 system and can express Cas9 protein.Therefore,a Vero cell line expressing two highly efficient sg RNAs for gE and gI genes and a Cas9 protein was constructed and was named gE/gI-Cas9-Vero.The gene editing efficiency is improved by 13%by passaging the wildtype PRV-WC01 on this cell line,compared with the traditional transient transfection of plasmids for viral gene Remarkably,the gene edited virus accounted for about 80%of the total virus after successive passages.One PRV strain with the gE/gI gene deletion was finally obtained through multiple rounds of plaque purification on daughter cells and was named PRV-WC01 Del-gE/gI.The replication kinetics showed that the viral titer of PRV-WC01 Del-gE/gI virus on Vero cells was slightly lower than that of the parent strain PRV-WC01 at the early growth period,but there was almost no difference after 24 h.The plaque morphology and size of PRV-WC01Del-gE/gI was similar to the parental strain,which was consistent with the above results.The pathogenicity test of mice indicated that PRV-WC01 Del-gE/gI delayed the death of mice compared with the parental viral strain,but still lethal.The PRV-WC01 Del-gE/gI strain with the titer of 10^8.00TCID₅₀/mL was inactivated by 0.1%formaldehyde,and was added with the adjuvant MONTANIDETM ISA201 VG to prepare an immunogen.Experimaental 21-day-old piglets were inoculated.On the 21st day after the second immunization,each immunized piglet was challenged with the wildtype WC01 strain at a dose of10 LD₅₀.The results showed that both the inactivated PRV-WC01 Del-gE/gI antigen and the live vaccine Bartha-K61 induced specific ELISA antibodies and the neutralizing antibody.Piglets vaccinated with the PRV-WC01 Del-gE/gI immunogen produced 16 times more neutralizing antibodies against the PRV-WC01 strain than the live Bartha-K61 vaccine.Piglets vaccinated with the Bartha-K61 vaccine showed persistent high temperature,and even symptoms of convulsions and ataxia after the PRV-WC01 virus challenge.One piglet was died finally.On the other hand,Piglets immunized with the inactivated antigen showed no other clinical symptoms except transient body temperature increase,and provided fully immune protection against the PRV-WC01challenge.After 14 days of infection,the experimental piglets were sacrified,and the tissue viral lords and organ pathogenicity were texted.The copies of virus genome in PRV-WC01Del-gE/gI vaccinated piglets decreased fastly and significantly and with no pathological changes in all tissues and organs observed by necropsy.The above results further proved that the inactivated PRV-WC01Del-gE/gI virus immunization can provide better immune protection on the epidemic strain than the traditional Bartha-K61 vaccine.Conclusively,in this study,we isolated PRV epidemic strains in vaccinated swine herds.We constructed a stable cell line gE/gI-Cas9-Vero expressing sg RNAs for gE and gI and the Cas9protein by the lentiviral packaging system.This cell line was applied to construct the PRV-WC01Del-gE/gI mutant virus inoculating the wildtype PRV-WC01 PRV virus directly on this cell line.And after multiple circles of cell purification and screening,the dual gene deletion strains were obtained.The growth characteristics,virulence characteristics and immunogenicity of this strain were further determined,which provides a theoretical and material basis for the PRV isolate as a potential inactivated vaccine candidate for pseudorabies.