An attenuated African swine fever virus with deletions of the CD2v and A137R genes offers complete protection against homologous challenge in pigs

African swine fever (ASF) has caused a devastating pandemic among domestic and wild swine, leading to significant economic losses in the global swine industry. Recombinant live-attenuated vaccines are a potential option for the control of ASF. However, safe and effective vaccines against the ASF virus (ASFV) are not yet commercially available, and thus, additional vaccine candidates still need to be developed. In this study, we demonstrate that the simultaneous deletion of the ASFV genes CD2v and A137R from the highly virulent isolate ASFV HuB/HH/2019 substantially attenuated the virulence in swine. All pigs (4/4) infected with 10 5 TCID 50 doses of the double gene deletion virus HuB/HH/2019Δ CD2v Δ A137R (HuBΔ CD2v Δ A137R ) remained healthy during a 27-day observation period, with no fevers (>40.5°C) observed. Cases of viremia were mild and cleared by 24 days post-inoculation (dpi). Antibodies against ASFV p30 were induced, and the inoculated pigs were well-protected against homologous challenges. All inoculated pigs survived after being challenged with 10 2 HAD 50 ASFV HuB/HH/2019, and no fever or clinical symptoms developed during the 22-day monitoring period. In contrast, all control group pigs died before 11 dpi (2/2). Cytokine assays and RNA sequencing revealed mild responses elicited by HuBΔ CD2v Δ A137R . Successful innate and passive immune responses were also triggered. The findings suggest that deletion of the CD2v and A137R genes can attenuate ASFV, and the deletion mutant virus HuBΔ CD2v Δ A137R is a promising vaccine candidate. IMPORTANCE The emergence of ASF has caused substantial economic losses in the global pig industry. In light of this, the development of a safe and effective vaccine is crucial to control the spread of ASF. In this study, a live-attenuated ASFV strain was developed by simultaneously knocking out the viral genes CD2v and A137R . The mutant virus exhibited weakened virulence in pigs, elicited robust humoral and cellular immunity, and conferred protection upon challenge with high doses of the parental virus. Notably, no fever or clinical symptoms were observed during a 27-day monitoring period. Consequently, our research presents a highly promising candidate strain for a live-attenuated vaccine, offering a significant step forward in the quest for effective prevention measures. Additionally, the identification of these specific gene targets opens up avenues for further exploration and refinement of strategies aimed at combating ASF outbreaks.