Hybrid genotype I and II ASFV D250R deletions confer protection against parental and genotype II strains and elicit potent immune response

African Swine Fever Virus (ASFV) continues to mutate uncontrollably, with outbreaks caused by genotype II strains inflicting substantial economic losses on the global swine industry. Currently, a more virulent recombinant strain, combining genotype I and II, is spreading across China, Russia and Vietnam, exacerbating the severity of the epidemic. The development of safe and effective ASFV vaccines is therefore urgent. We constructed two gene-deleted strains – SY18ΔD250R (genotype II) and JX23-02ΔD250R (genotype I/II recombinant) – by knocking out the D250R gene from virulent field strains. Both strains were significantly attenuated and induced robust humoral and cellular immune responses in vaccinated pigs, with no clinical signs or mortality observed post-vaccination. Pigs immunized with SY18ΔD250R showed 80% protection (4/5 survived) against homologous SY18 challenge. Pig #60, which succumbed to infection, exhibited persistently negative humoral responses (p54 antibody) and the lowest cell-mediated immunity (IFN-γ response) within the group. The vaccinated pigs demonstrated cross-protection against an attenuated genotype II strain but failed to resist heterologous challenge with genotype I/II ASFV strain, even after booster immunization. In contrast, JX23-02ΔD250R-immunized pigs achieved 100% survival against both the parental I/II recombinant strain and virulent genotype II strains, with rapid viral clearance and low tissue viral loads. Notably, both SY18ΔD250R and JX23-02ΔD250R exhibited low or undetectable shedding, viremia, and viral loads-offering a significant safety advantage. Thus, JX23-02ΔD250R represents a promising vaccine candidate with potential for application against circulating ASFV strains.