Temporally integrated multiomics analysis elucidates intricate regulatory mechanisms of ASFV in a wild boar lung-derived clonal cell line.

African swine fever virus (ASFV) is a large double-stranded DNA virus that poses a significant threat to the global pig industry. Currently, our understanding of ASFV biology remains limited, as there is a lack of suitable cell lines to support its propagation and analysis. Here, we optimized a wild boar lung cell line to increase its susceptibility to ASFV, followed by temporally integrated transcriptomic and proteomic analyses of ASFV infection. Multiomics analysis revealed more than 17,000 genes and 5100 proteins, with 1594 differentially expressed genes (DEGs) and 923 differentially expressed proteins (DEPs) identified. Temporal dynamics revealed stage-specific host modulation: early-phase DEPs orchestrated metabolic reprogramming and transmembrane transport via the solute carrier superfamily (e.g., SLC25A, SLC30A, and SLC44A2), whereas the late infection phase featured concurrent upregulation of innate immune effectors (e.g., Mx1, OAS1, ISG15, and TRIM21) and suppression of apoptosis inhibitors (e.g., TMEM192, PDCD4, and DPP9), suggesting synchronized antiviral activation and apoptotic regulation. Unexpectedly, siRNA-mediated knockdown of key DEGs or DEPs involved in antiviral immunity, interferon signalling, DNA repair, genome stability, immune regulation, the inflammatory response and vesicular transport did not significantly affect viral replication. Only knockdown of the genes STX17 and ZNF512 significantly impaired ASFV replication. This systematic investigation provides a comprehensive framework for further studies of ASFV-host interactions, identifies candidate host dependency/support factors and establishes critical groundwork for the development of targeted antiviral interventions and next-generation vaccine platforms.