Preliminary Studies On Recombinant Pseudorabies Vaccine Expressing African Swine Fever Virus CD2v Protein

African swine fever(ASF)is a highly lethal infectious disease caused by African swine fever virus(ASFV)infection.The death rate of domestic pigs and wild boars is close to100%.The epidemic outbreak was first reported in Kenya in 1921,then gradually spread to countries in Europe and Asia,and currently to commercial vaccine is avaible for prevention.In this study,we constructed a recombinant Pseudorabies Virus(PRV)(PRV-△gE/△gI/△TK-(CD2v))that expresses ASFV CD2 v protein by the CRISPR / Cas9 technology.The proliferation ability of PRV-△gE/△gI/△TK-(CD2v)in Vero cells is not significantly different from that of the attenuated strain(PRV-△gE / △gI / △ TK),but PRV-△gE / △gI / △ TK-(CD2v)and PRV-△gE/△gI/△TK-were significantly less proliferative in mouse peripheral blood mononuclear cell(PBMC)than the virulent strain(PRV-Fa).The CDD2 v protein in the PRV-△gE/△gI/△TK-(CD2v)strain maintained the high level of expression in Vero cells up to 20 consecutive passages.Mouse infection data show that PRV-Fa can cause severe pruritus and the viral nucleic acids can be detected in the brain,lung,heart,liver,spleen,and kidney.PRV-△gE/△gI/△TK-(CD2v)and PRV-△gE/△gI/△TK did not cause itching and no viral nucleic acid was detected.Only PRV-Fa but not the other two PRV strain can induce IL6 transcription level in mouse tissuses and the serum IL6 level was also increased,None of above three PRV strains caused changes in IL1β,TNFα,IFNβ transcription levels,and PRV-△gE/ △gI/△TK-(CD2v)and PRV-△gE/△gI/△TK can also lead to T cell immunity.The recombinant PRV-△gE/△gI/ △TK-(CD2v)strain when immunizes mice can produce high titer specific antibodies against ASFV CD2 v protein.The control group challenged with the strong toxic strain PRV-Fa died on the fourth day,while the protection rate of the recombinant strains was 100%.After the challenge,the peak of detoxification in the immune group appeared earlier than the control group,but the amount of detoxification was much lower than that in the control group.These results indicate that PRV recombinant attenuated strain PRV-△gE/△gI/△TK-(CD2v)protein is safe and effective,and may be used as candidate vaccine strain to fight African swine fever and Pseudorabies.