基于CRISPR/Cas9 技术构建PRV UL56基因缺失毒株及其致病性评价OA

To investigate the impact of the pseudorabies virus(PRV)UL56 gene on viral replication and pathogenicity,we constructed a PRV NY-ΔUL56 gene-deleted strain using CRISPR/Cas9 technology.Successful deletion of the UL56 gene was confirmed by Western blot and RT-qPCR.The deletion strain ex-hibited good genetic stability after 15 consecutive passages in PK-15 cells.One-step growth curve and plaque assay results showed that the replication efficiency of the deletion strain was generally con-sistent with that of the parental strain,indicating that UL56 is a non-essential gene for PRV replica-tion.In vivo pathogenicity evaluation using a BALB/c mouse infection model revealed that,compared with the parental strain,the PRV NY-ΔUL56-infected group achieved a survival rate of 60％,and viral loads in various tissues were significantly reduced.Further analysis showed that following infection with the deletion strain,the expression levels of multiple cytokines and chemokines in the spleen,in-cluding IL-2,IL-10,IFN-α/β,CCL5,CXCL10,and TGF-β,were significantly lower than those in the wild-type virus-infected group.These findings suggest that UL56 gene deletion may attenuate viral pathogenicity by downregulating the excessive expression of immune factors in the spleen.In summary,we successfully constructed a genetically stable UL56 gene-deleted strain and,for the first time,systematically re-vealed the role of this deletion in mitigating excessive immune activation,providing a theoretical ba-sis for the development of novel PRV attenuated vaccines targeting UL56.