Construction Of Vaccine Strains With Genes Deletion Of PRV Variant And Their Partial Biological Characteristics Research

Pseudorabies（PR）is an acute and febrile infectious disease caused by Pseudorabies virus（PRV）.Pigs are the main hosts of PRV.The clinical characteristics of piglets with PRV are fever,neurological symptoms,etc.,and the mortality rate is high.The breeding pigs mostly show reproductive disorders.At present,there is no effective treatment,vaccine immunization is the core method for the prevention and control of porcine pseudorabies.However,since the end of2011,PRV mutants have appeared and spread in pig farms in various regions of China,and the healthy development of pig industry has been impacted again.In this study,PRV epidemic strain was isolated from suspected disinfected pigs.After virulence identification and genetic evolution analysis,PRV homologous recombination was assisted by CRISPR/Cas9 gene editing system to construct the 11K,g I,g E and 28K gene deletion strain r PRV（FJ）-p EGFP.TK,11K,g I,g E,28K five gene deletions strain r PRV（FJ）-5D and five gene deletions strains r PRV（FJ）-GM-CSF and r PRV（FJ）-CD40L recombinant porcine cytokines,respectively.The safety and immunogenicity of the constructed strains were compared through a series of animal tests,which provided technical support for the research and development and application of new PRV vaccines.The main results are as follows:1.Isolation and identification of epidemic strains of pseudorabies virus and comparison of pathogenicity and immunogenicityOne strain of pseudorabies virus was isolated and identified from 65 clinical samples of sick piglets in Fu Jian province,China,and named PRV FJ strain.When BHK-21 cells were passaged to F10 generation,the TCID₅₀ of F10 cells was determined to be 10^-7.30/0.1m L.The neutralization test was carried out with porcine PRV standard positive serum.the results showed that the isolated PRV FJ strain could be neutralized by PRV positive standard serum,and the neutralization titer was 1:128.The pathogenicity of PRV FJ strain was compared with that of PRV XJ strain and Fa strain isolated and preserved in laboratory.The results showed that the LD₅₀of PRV FJ strain,XJ strain and Fa strain were 10^2.29TCID₅₀,10^3.21TCID₅₀ and 10^3.97TCID₅₀,respectively.Three strains of virus were inoculated into SPF mice at a dose of 1 LD₅₀.In FJ group,clinical symptoms first appeared and 1 mouse died at about 72 h,4 mice died and 2 mice survived at96 h and later,2 mice died at about 84 h in XJ and Fa groups,2 mice died in XJ group and 1mouse in Fa group,4 mice died and survived in 96 h and later in XJ group,and 3 mice died and survived in Fa group after 96 h.Histopathological sections showed that the pathological changes of related tissues in FJ group were relatively severe.The above results showed that PRV FJ strain was the most virulent.The virus was harvested after the three strains were inoculated with BHK-21 cells respectively.After determining the virus content,the virus solution was diluted to 10⁷TCID₅₀,then inactivated with 0.5%formaldehyde for 30 hours and mixed with Freund’s adjuvant phacoemulsification.28 days after immunization,mouse sera were collected to detect neutralizing antibodies.The average neutralizing antibody titers induced by FJ strain,XJ strain and Fa strain were 1:26.7,1:16.3 and 1:21.7,respectively.The results showed that the level of neutralizing antibody induced by PRV FJ strain was the highest and the immunogenicity was good.2.The whole genome sequencing and genetic evolution analysis of of PRV FJ strainThe F10 generation of PRV FJ strain was inoculated into BHK21 cells and the virus solution was harvested.After the DNA was concentrated and extracted by ultrafiltration,the third generation sequencing platform Pac Bio SMRT combined with the second generation sequencing platform MGISEQ-2000 was used to assemble the whole genome sequence.The complete genome sequence is 143703bp,with a GC content of 73.67%,containing 70 coding genes（Genbank number:MW286330）,and functional annotations were carried out one by one.The genetic evolution analysis of the whole genome of 15 PRV reference strains on NCBI showed that all the strains were divided into two branches:European and American gene type I and Asian gene type II,and both FJ strain and domestic epidemic variants belonged to gene typeⅡ.The genetic evolution trees of the main virulence and immune-related genes TK,,g B,g C,g D,g G,g H,g L,g M,g N,g I and g E of FJ strain and other reference strains were constructed respectively.The results showed that all the genes of FJ strain were located in the typeⅡbranch,in which the g G gene was in the same subbranch as the domestic classic strain,the g E gene was in a relatively independent subbranch,and the other genes were in the same subbranch as the domestic mutant strain.The results of homology analysis showed that,PRV FJ strain had the highest homology with the reference strain of PRV epidemic variation published in China after2011,between 99.9%and 100.0%,and the lowest homology with European and American strains,no more than 96.0%.The recombination events of PRV FJ strain were detected by RDP4software.The results showed that the recombination signals were found in the three non-coding regions.The recombinant parent strains were HLJ8 strain and Ea strain.The N-glycosylation sites and potential antigenic sites of PRV Bartha,FJ and Ea strains g B,g C and g D were predicted by software.The results showed that the N-glycosylation sites of g B and g C proteins of three PRV strains were the same,including 7 g B proteins,8 g C proteins and 0 g D proteins.The B cell linear epitope sequences of three glycoproteins of FJ strain were less different from those of Ea strain,g B and g D protein epitopes were less different from those of Bartha strain,and g C protein epitopes were significantly different from those of Bartha strain.3.Construction of PRV genes deletion strains and analysis of some biological characteristics.According to the genomic sequence of PRV FJ strain,the homologous arms of g I gene upstream,28K gene downstream,TK gene upstream and downstream,and fusion protein gene sequences T2A-6×His-GM-CSF and T2A-6×His-CD40L,were synthesized and inserted into p EGFP-C1 vector directionally,and the transfer vectors p EGFP-C1-g I28K,p EGFP-C1-TK,p EGFP-g I28K-GM-CSF and p EGFP-g I28K-CD40L were constructed.Using online prediction software,using g E and TK gene sequences as templates,g RNA sequences specifically cleaving g E and TK genes were designed,and psg-g E and psg-TK cutting plasmids were constructed.The transfer vector and g RNA cleavage plasmid were co-transfected into BHK-21 cells and inoculated with PRV FJ strain 12 hours later.The virus solution was harvested after 80%cytopathic effect,diluted and inoculated into BHK-21 cells.Green fluorescent plaque was selected and three rounds of plaque purification were carried out.Four PRV genes deletion strains:r PRV（FJ）-p EGFP,r PRV（FJ）-5D,r PRV（FJ）-GM-CSF and r PRV（FJ）-CD40L were obtained.The one-step growth curve and spot morphology of the four genes deletion strains were determined respectively.the results showed that the growth curve and spot morphology of the four genes deletion strains were basically the same as those of their parents.The deletion genes（TK,11K,g I,g E,28K）and insertion genes（GM-CSF,CD40L）of the four genes deletion strains were detected by PCR amplification,Western Blot and TK gene sequencing.The results showed that the deletion and insertion genes of the four genes deletion strains were inherited stably in the F1-F21 generation.PRV FJ strain and each genes deletion strain were inoculated into BHK-21 and Vero cells with 10⁴TCID₅₀ titer,respectively.The pathological changes of the two kinds of cells at different times and the proliferation characteristics of each genes deletion strain and parent strain were compared.The results showed that the virulence of PRV FJ strain to the two kinds of cells was higher than that of each genes deletion strain,and there was no significant difference in virulence between the two kinds of cells.4.Comparison of safety and immunogenicity of PRV genes deletion strains.SPF mice were inoculated with r PRV（FJ）-p EGFP strain,r PRV（FJ）-5D strain,r PRV（FJ）-GM-CSF strain and r PRV（FJ）-CD40L strain F21 generation at the lowest dose of the single,10and 10³ times(10³,10⁴ and 10⁶TCID₅₀),respectively,six mice in each group.The results showed that all the mice inoculated with 10⁴ and 10⁶ TCID₅₀ in the r PRV（FJ）-p EGFP inoculation group died,only 2 mice survived inoculated with 10³TCID₅₀,and the other groups genes deletion strains survived normally.The mice were immunized with r PRV（FJ）-5D,r PRV（FJ）-CD40L and r PRV（FJ）-GM-CSF strains,and the mice were immunized for 14 days.The titers of g B,g E,neutralizing antibody and the concentration of cytokine IL-2/IL-6/TNF-α/IFN-γwere determined.The results showed that all the g B antibodies were positive and the g E antibodies were negative.There was a negative correlation between the level of neutralizing antibody and the dilution of all genes deletion strains.There was no significant difference in the level of IL-2 between the immunized group and the control group,but the concentrations of IL-6,TNF-αand IFN-γin the immunized group were higher than those in the negative control group.After challenge with 10⁴TCID₅₀ PRV FJ strain,the lowest immune dose of r PRV（FJ）-5D group,r PRV（FJ）-GM-CSF group and r PRV（FJ）-CD40L group was 10⁴TCID₅₀.Histopathological sections showed that the pathological changes of brain,spleen and lung were obvious in the challenge group,but there were no pathological changes in the immune group.PRV genes deletion strains r PRV（FJ）-5D,PRV（FJ）-GM-CSF and r PRV（FJ）-CD40L F21generation with high safety in mice were intranasally inoculated with 3-4-week-old PRV antibody negative weaned piglets at the lowest dose of the single,10 and 100 times(10⁵TCID₅₀,10⁶TCID₅₀ and 10⁷TCID₅₀),respectively.There were 5 piglets in each group.The results showed that during the observation period,all the piglets in the genes deletion virus inoculation group survived normally and the control group began to die on the 5th day after challenge.All died within 7 days.When the genes deletion strain 10⁷TCID₅₀ PRV FJ was inoculated 21 days after inoculation,the lowest immune dose of each genes deletion strain to weaned piglets was10⁵TCID₅₀.The titers of g B,g E and neutralizing antibody were regularly monitored within 21days after immunization with the lowest immune dose.The results showed that the seroconversion rate of g B antibody in:r PRV（FJ）-GM-CSF group and r PRV（FJ）-CD40L group was higher than that in r PRV（FJ）-5D group,and g E antibody was negative.On the 21st day after immunization,the average neutralizing antibody titers of r PRV（FJ）-5D,PRV（FJ）-GM-CSF and r PRV（FJ）-CD40L groups were 1:41.6,1:57.6 and 1:51.2,respectively,while those of the negative control group were less than 1:2.Within 14 days after challenge,the seroconversion rate of g E antibody level,r PRV（FJ）-GM-CSF group and r PRV（FJ）-CD40L group was lower than that in r PRV（FJ）-5D group.All the piglets in the immune group and the negative control group had no clinical symptoms and had good immunogenicity,while all the piglets in the challenge control group died within 7 days.The results of detoxification test showed that,r PRV（FJ）-GM-CSF and r PRV（FJ）-CD40L groups could delay the start time and shorten the duration of detoxification of PRV FJ strain in piglets compared with r PRV（FJ）-5D group.Generally speaking,r PRV（FJ）-GM-CSF and r PRV（FJ）-CD40L strains can be used as vaccine candidates for further study.