Deletion of the D345L gene attenuates ASFV and induces protection against homologous and heterologous challenge by enhancing host innate immunity.

African swine fever (ASF) is a highly contagious and lethal disease caused by African swine fever virus (ASFV), with a mortality rate approaching up to 100%. At present, the functions of multiple genes of ASFV remain to be elucidated. In this study, we demonstrate that pD345L is a late-expressed viral protein and localizes to the viral factory. The recombinant strain JX23-02ΔD345L was obtained from the genotype I/II strain JX23-02. In vitro assays demonstrated that the deletion of D345L remarkably impairs viral replication. Further analyses revealed that JX23-02ΔD345L significantly up-regulated pro-inflammatory cytokines (IL-1β, TNF-α) and IFN pathway components in PAMs, induced pyroptosis, apoptosis, and interferon responses. In vivo animal experiments demonstrated that the JX23-02ΔD345L strain was completely attenuated in pigs, failing to induce any typical clinical signs of ASF even with a high-dose inoculation. Moreover, a single immunization with JX23-02ΔD345L can elicit specific humoral and cellular immune responses against ASFV, conferring 80% protection against intramuscular challenge with the homologous strain JX23-02 and 60% protection against the heterologous strain SY18. These results reveal D345L as a crucial virulence-related gene for ASFV pathogenesis, which is highly correlated with viral replication and the host antiviral response. Furthermore, JX23-02ΔD345L represented a candidate for the development of effective and safe ASFV vaccines. This finding provides a foundation for the research on ASFV and offers an important target and evidence for vaccine development.