Evaluation of An I226R and A137R deletion mutant from a genotype I/II recombinant African swine fever virus strain as a live attenuated vaccine in pigs

The emergence of highly lethal genotype I/II recombinant African swine fever virus (ASFV) strains in China has rendered existing genotype II-based live attenuated vaccines ineffective, underscoring the urgent need for novel vaccine candidates. Although single-gene deletions of A137R or I226R have shown promise against genotype II strains, their efficacy and safety in the context of recombinant strains remain unexplored. Using homologous recombination, we constructed a single gene-deleted mutant (JX23-02ΔI226R) and a double gene-deleted mutant (JX23-02ΔI226RΔA137R) from the genotype I/II recombinant ASFV strain JX23-02. The replication kinetics, pathogenicity, immunogenicity, and protective efficacy of these mutants were evaluated in vitro and in pigs. Both deletion mutants exhibited significantly reduced replication in porcine alveolar macrophages. Immunization with JX23-02ΔI226R resulted in 40% survival, and surviving pigs were protected against lethal challenge. In contrast, JX23-02ΔI226RΔA137R was fully attenuated: all immunized pigs survived challenge without any clinical signs (maximum rectal temperature 40.2 °C) and developed detectable p54-specific antibody responses. Moreover, viral shedding and tissue viral loads were markedly lower in the double-deletion group than in the single-deletion group. The double gene-deleted mutant JX23-02ΔI226RΔA137R represents a promising live attenuated vaccine candidate against genotype I/II recombinant ASFV strains, supporting the use of multigene deletion strategies for ASF vaccine development.