An E120R Gene–Deleted African Swine Fever Virus Is Fully Attenuated and Induces Partial Protective Immunity in Pigs

African swine fever virus (ASFV) causes a lethal hemorrhagic disease in domestic pigs and represents a major threat to the global swine industry. Until now, 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 biological 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. RNA sequencing and RT-qPCR analyses revealed that ASFV-ΔE120R infection of porcine alveolar macrophages (PAMs) significantly upregulated the expression of chemokines (CCL5, CCL2, CCL4), interferon-related genes (IRF7, IRF8, ISG15, ISG54, ISG56), and the proinflammatory cytokine IL-1β compared with ASFV-WT. In vivo safety evaluation demonstrated that piglets immunized with a single dose of 5 × 10⁶ TCID₅₀ of ASFV-ΔE120R exhibited no clinical signs of ASF and detectable viral nucleic acid in any organ upon necropsy at days 4, 7, 10, and 14 post-immunization. Notably, even after two rounds of blind cell passage using filtered tissue homogenates, no viral genome was detected. Furthermore, two immunizations at the same dose, administered 21 days apart, did not induce clinical signs or viral shedding during a 28-day observation period. Immunogenicity analysis showed that ASFV-ΔE120R elicited both p54-specific antibody production and IFN-γ-secreting peripheral blood mononuclear cell (PBMC) responses. Upon intramuscular challenge with a lethal dose (10 TCID₅₀) of parental ASFV SY18, two out of five immunized pigs (40%) survived. These survivors displayed increased levels of p54-specific antibodies and IFN-γ-secreting PBMCs. No viral nucleic acid or histopathological lesions were detected in their tissues, and tissue immunofluorescence revealed elevated frequencies of CD8⁺ IFN-γ⁺ T cells. Moreover, mRNA levels of antiviral genes (IFN-α, TNF-α, ISG15, ISG54, ISG56, and MX1) in the liver and bone marrow of surviving pigs were significantly upregulated compared with unvaccinated controls. In summary, ASFV-ΔE120R is fully attenuated and safe, and it induces both humoral and cellular immune responses. This study provides further insights into the role of pE120R in viral egress and immune evasion, and highlights its potential as a rational target for the development of live attenuated ASF vaccines.