Theaflavin inhibits African swine fever virus replication by disrupting lipid metabolism through activation of the AMPK signaling pathway in virto

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), which is one of the most harmful swine diseases in the pig industry because of its nearly 100% mortality rate in domestic pigs and results in incalculable economic loss. Ever since ASF was initially reported, scientists have worked to develop anti-ASF vaccines; however, currently no clinically effective vaccine for ASF is available. Therefore, the development of novel measures to prevent ASFV infection and transmission is essential. In this study, we aimed to investigate the anti-ASF activity of theaflavin (TF), a natural compound mainly isolated from black tea. We found that TF potently inhibited ASFV replication at non-cytotoxic concentrations ex vivo in primary porcine alveolar macrophages (PAMs). Mechanistically, we found that TF inhibited ASFV replication by acting on cells rather than interacting directly with ASFV to inhibit viral replication. Further, we found that TF upregulated the AMPK (5′-AMP-activated protein kinase) signaling pathway in ASFV-infected and uninfected cells, and treatment with the AMPK agonist MK8722 upregulated the AMPK signaling pathway and inhibited ASFV proliferation in a dose-dependent manner. Notably, the effects of TF on AMPK activation and ASFV inhibition were partially reversed by the AMPK inhibitor dorsomorphin. In addition, we found that TF down-regulated the expression of genes related to lipid synthesis and decreased the intracellular accumulation of total cholesterol and total triglycerides in ASFV-infected cells, suggesting that TF may inhibit ASFV replication by disrupting lipid metabolism. In summary, our results demonstrated that TF is an ASFV infection inhibitor and revealed the mechanism by which ASFV replication is inhibited, providing a novel mechanism and potential lead compound for the development of anti-ASFV drugs.