African swine fever virus (ASFV) is a highly pathogenic large DNA virus that causes African swine fever (ASF) in domestic pigs and European wild boars with mortality rate up to 100%. The DP96R gene of ASFV encodes one of the viral virulence factors, yet its action mechanism remains unknown. In this study, we report that DP96R of ASFV China 2018/1 strain subverts type I IFN production in cGAS sensing pathway. DP96R inhibited the cGAS/STING, and TBK1 but not IRF3-5D mediated IFN-beta and ISRE promoters activation. Furthermore, DP96R selectively blocked the activation of NF-kappa B promoter induced by cGAS/ STING, TBK1, and IKK beta, but not by overexpression of p65. Moreover, DP96R inhibited phosphorylation of TBK1 stimulated by cGAS/STING activation, and TBK1-induced antiviral response. Finally, truncated mutation analysis demonstrated that the region spanning amino acids 30 to 96 of DP96R was responsible for the inhibitory activity. To our knowledge, this is for the first time that DP96R of ASFV China 2018/1 is reported to negatively regulate type I IFN expression and NF-kappa B signaling by inhibiting both TBK1 and IKK beta, which plays an important role in virus immune evasion. (C) 2018 Elsevier Inc. All rights reserved.