Deletion of the African swine fever virus E120R gene completely attenuates its virulence by enhancing host innate immunity and impairing virus release

African swine fever virus (ASFV) causes a lethal haemorrhagic disease in domestic pigs and poses a major threat to the global swine industry. Currently, no effective commercial vaccines or antiviral drugs are available for ASF control. In this study, we constructed a recombinant E120R gene-deleted virus, ASFV-ΔE120R, based on the highly virulent genotype II strain SY18, to investigate the role of the E120R gene. ASFV-ΔE120R exhibited impaired virion release and formed aberrant tubular structures, rendering viral particles more susceptible to neutralization by convalescent pig sera. ASFV-ΔE120R induced higher levels of transcription of Cytokines, chemokines, and interferon-regulated genes in porcine alveolar macrophages compared with ASFV-WT. In vivo safety evaluation demonstrated that piglets immunized with 5 × 10⁶ TCID₅₀ of ASFV-ΔE120R exhibited no clinical signs or viral nucleic acid in tissues at necropsy on days 4, 7, 10, and 14 post-immunization. Two immunizations at the same dose, 21 days apart, also induced no clinical signs or viral shedding during a 28-day observation. Immunogenicity analysis showed that ASFV-ΔE120R elicited p54-specific antibodies and IFN-γ-secreting PBMC responses. Upon challenge with parental ASFV SY18, two of five pigs (40%) survived, showing elevated antibody levels, IFN-γ-secreting PBMCs, and increased CD8⁺ IFN-γ⁺ T cells. Moreover, Cytokines and interferon-stimulated genes were significantly upregulated in survivors. In summary, ASFV-ΔE120R is fully attenuated, safe, and induces both humoral and cellular immune responses, highlighting pE120R as a rational target for ASF vaccine development.