Luteolin restricts ASFV replication by regulating the NF-κB/STAT3/ATF6 signaling pathway

African swine fever (ASF) is a devastating infectious disease that causes significant economic losses to the pig industry worldwide. Luteolin is abundant in onion leaves, carrots, broccoli, and apple skin and exerts various biological activities, including anti-cancer and anti-virus effects. Our aim was to demonstrate the mechanism of action and potent antiviral activity of luteolin against ASF virus (ASFV) in porcine alveolar macrophages. We performed cell viability, hemadsorption, indirect immunofluorescence, western blotting, and quantitative real-time polymerase chain reaction assays to investigate the effect of luteolin on ASFV. Notably, luteolin restricted ASFV replication in a dose-dependent manner. The anti-ASFV activity of luteolin was maintained for 24-72 h. Subsequent experiments revealed that luteolin could block multiple stages of the ASFV replication cycle, including those at 6-9 h and 12-15 h after infection, instead of directly interacting with ASFV. Moreover, ASFV infection stimulated the expression of phosphorylated nuclear factor (NF)-κB, interleukin (IL)- 6, and phosphorylated signal transducer and activator of transcription 3 (STAT3). However, luteolin downregulated ASFV-induced NF-κB, IL-6, and STAT3 expression. Importantly, NF-κB agonist CU-T12-9 weakened the inhibitory effects of luteolin on NF-κB and STAT3. Moreover, CU-T12-9 partially restored the inhibitory effect of luteolin on ASFV. Similarly, luteolin reduced ASFV-induced activating transcription factor 6 (ATF6) expression, and CU-T12-9 weakened the inhibitory effect of luteolin on ATF6. Our findings suggested that luteolin inhibited ASFV replication by regulating the NF-κB/STAT3/ATF6 signaling pathway and might provide a rationale for anti-ASFV drug development.