Fucoidan SPW-05-S2 from Sargassum fusiforme inhibits African swine fever virus replication by blocking PAK2/Talin-1 mediated viral endocytosis and factory formation

African swine fever (ASF), caused by the African swine fever virus (ASFV), is currently a major threat to the global swine industry and food security because no safe and effective vaccines or antiviral drugs are available. Developing a defense strategy that incorporates antiviral agents and other approaches is urgently needed. Here, we found that a fucoidan, SPW-05-S2, from a brown alga, inhibits ASFV infection by over 95% in porcine alveolar macrophages (PAMs) via suppressing viral entry and factory formation. Using an affinity precipitation workflow, we identified 4 viral and 665 cellular proteins as potential binding partners of SPW-05-S2 in ASFV-infected PAMs, of which 3 viral proteins and 2 cellular proteins (PAK2 and Talin-1) were experimentally validated. Mechanistically, our results suggest that SPW-05-S2 inhibits ASFV replication by both suppressing endocytosis via targeting the PAK2/LIMK/Cofilin/F-actin remodeling pathway and blocking viral factory formation through perturbing Talin-1-mediated cytoskeletal remodeling networks. Collectively, our findings show that SPW-05-S2 blocks ASFV infection by modulating host cytoskeletal pathways at two critical stages of the viral life cycle. Given that fucoidans are already widely used in food and pharmaceuticals, SPW-05-S2 represents a promising, low-risk candidate for antiviral intervention against ASFV.