Use Of Adeno-associated Virus Carring The CRISPR/Cas9 Gene Editing System For Defense The Porcine Pseudorabies Virus

Pseudorabies virus is pathogen of porcine pseudorabies,it's genome is prone to mutation which will lead to the vaccine defenses ineffective therefore will be hard to heal once infected,and it can also lead a high mortality rate in piglets.PRV infection can cause reproductive problems in sows.Infected piglets have symptoms such as vomiting,diarrhea,lethargy.The other animals can also be infected and the symptoms often include itching,fever,etc.The pseudorabies outbreak can bring about serious economic losses.In order to inhibit PRV infection,we packed the CRISPR/Cas9 systems in adeno-associated virus by targeting TK,gE and VP 16 genes of PRV.The goal is to ameliorate the symptoms caused by PRV toxicity via partly removing PRV.The experimental details are described in the following:(1)We constructed CRISPR/Cas9 recombinant vectors(pXTK,pXgE,pXVP16),in which the corresponding sgRNA that target TK,gE and VP 16 genes,are incorparated in the pX601 plasmid that expresses the Cas9 gene.The successful construction these recombinant vectors are verfied by sequencing.(2)Next,we packed each of above recombinant plasmid in adeno-associated virus vectors(AAV)by triple plasmid transfection method,which includes pHelper and pAAV2/5 in addition to the recombinant plasmid.The expression of the corresponding sgRNA and Cas9 in the packed AAV are verified by real-time PCR using the corresponding primers and the yield of AAV isolated from the infected cells is also calculated.(3)Furthermore,we examined the amount of PRV virus as well as the mRNA expression of BCL2,BCL-XL,IRF3 and IFNp in the cells infected by PRV with or without the a'bove modified AAVs by real-time PCR.(4)Moreover,we test the effects of these modified AAVs in treating PRV-infected mice.The death rate is reduced and the time of death is prolonged by treating all three modified AAVs,with the better results by targeting TK and VP 16.The amount of PRV virus is reduced in the spleen tissues of mice treated with the modified AAVs carrying pXgE,pXVP16.The expression of several apoptotic and immunity associated regulators are also affected by treating with the modified AAVs as indicated by real-time RT-PCR analyses.However,the effects vary among the three different modified AAVs.In summary,(1)The CRISPR/Cas9 recombinant vectors are successfully constructed;(2)The CRISPR/Cas9 recombinant vectors packing in adeno-associated viruses are successful;(3)The amount of PRV virus is reduced in the cells infected by PRV with above modified AAVs,while the expression of BCL2,BCL-XL,IRF3 and IFN?are changed in the PRV-infected cells treated with above modified AAVs;(4)The results of experiments in mice show that these modified AAVs are effective in inhibiting PRV-infected mice.Our results will provide a novel strategy for inhibiting PRV infection.