Immunogenicity evaluation of a recombinant pseudorabies virus co-expressing PCV2 and PCV3 capsid proteins in mice and piglets.

Porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), and pseudorabies virus (PRV) are major pathogens posing significant threats to the swine industry. Viral evolution and mutations have limited the efficacy of current commercial vaccines, necessitating the development of more effective prophylactic strategies. In this study, a novel recombinant virus strain, designated as rPRV-ΔTK-PCV3(Cap)/ΔgIgE-PCV2(Cap), was generated using PRV SX-10 variant as the backbone. CRISPR/Cas9-mediated deletion of TK and gE/gI genes was performed, followed by insertion of PCV3 and PCV2 capsid protein genes into the respective loci. The engineered recombinant strain demonstrated stable proliferation in BHK-21 cells, efficiently expressed heterologous PCV3 and PCV2 capsid proteins, while maintaining biological properties comparable to its parental strain. The rPRV-ΔTK-PCV3(Cap)/ΔgIgE-PCV2(Cap) demonstrated favorable safety and immunogenicity profiles in mice and piglets, eliciting robust immune responses characterized by high titers of specific antibodies against PRV, PCV3, and PCV2, along with significantly elevated levels of cytokines (IFN-γ, IL-2, and IL-4). Histopathological analysis and viral load quantification demonstrated that rPRV-ΔTK-PCV3(Cap)/ΔgIgE-PCV2(Cap) immunization significantly attenuated tissue lesions and decreased viral copies of PRV and PCV in mice and piglets. Collectively, these findings suggest that rPRV-ΔTK-PCV3(Cap)/ΔgIgE-PCV2(Cap) serves as a promising candidate vaccine against PRV and PCV infections.