Generation and Characterization of Recombinant Pseudorabies Virus Delivering African Swine Fever Virus CD2v and p54

African swine fever (ASF) leads to high mortality in domestic pigs and wild boar, and it is caused by the African swine fever virus (ASFV). Currently, no commercially available vaccine exists for its prevention in China. In this study, we engineered a pseudorabies recombinant virus (PRV) expressing ASFV CD2v and p54 proteins (PRV-triangle TK-(CD2v)-triangle gE-(p54)) using CRISPR/Cas9 and homologous recombination technology. PRV-triangle TK-(CD2v)-triangle gE-(p54) effectively delivers CD2v and p54, and it exhibits reduced virulence. Immunization with PRV-triangle TK-(CD2v)-triangle gE-(p54) neither induces pruritus nor causes systemic infection and inflammation. Furthermore, a double knockout of the TK and gE genes eliminates the depletion of T, B, and monocytes/macrophages in the blood caused by wild-type viral infection, decreases the proliferation of granulocytes to eliminate T-cell immunosuppression from granulocytes, and enhances the ability of the immune system against PRV infection. An overexpression of CD2v and p54 proteins does not alter the characteristics of PRV-triangle TK/triangle gE. Moreover, PRV-triangle TK-(CD2v)-triangle gE-(p54) successfully induces antibody production via intramuscular (IM) vaccination and confers effective protection for vaccinated mice upon challenge. Thus, PRV-triangle TK-(CD2v)-triangle gE-(p54) demonstrates good immunogenicity and safety, providing highly effective protection against PRV and ASFV. It potentially represents a suitable candidate for the development of a bivalent vaccine against both PRV and ASFV infections.