African swine fever (ASF), a devastating infectious disease in swine, severely threatens the global pig farming industry. Disease control has been hampered by the unavailability of vaccines. Here, we report that deletion of the QP509L and QP383R genes (ASFV-DQP509L/QP383R) from the highly virulent ASF virus (ASFV) CN/GS/2018 strain results in complete viral attenuation in swine. Animals inoculated with ASFV-DQP509L/QP383R at a 104 50% hemadsorbing dose (HAD50) remained clinically normal during the 17-day observational period. All ASFV-DQP509L/QP383R-infected animals had low viremia titers and developed a low-level p30-specific antibody response. However, ASFV-DQP509L/QP383R did not induce protection against challenge with the virulent parental ASFV CN/GS/2018 isolate. RNA-sequencing analysis revealed that innate immune-related genes (Ifnb, Traf2, Cxcl10, Isg15, Rantes, and Mx1) were significantly lower in ASFV-DQP509L/QP383R-infected than in ASFV-infected porcine alveolar macrophages. In addition, ASFV-DQP509L/QP383R-infected pigs had low levels of interferon-β (IFN-β) based on enzyme-linked immunosorbent assay (ELISA). These data suggest that deletion of ASFV QP509L/383R reduces virulence but does not induce protection against lethal ASFV challenge. [ABSTRACT FROM AUTHOR]