Comprehensive Search for Channels in African Swine Fever Virus

African swine fever virus (ASFV) is arguably the most critical threat to global food security. With a mortality rate approaching 100%, it threatens pork production, a cornerstone of human nutrition that accounts for more than 35% of all meat consumed worldwide. ASFV is also one of the most structurally complex and largest pathogenic viruses, harboring a genome of 170–190 kbp. Consequently, to better understand this pathogen, we exhaustively screened the genome of the HK_NT_202103 strain for encoded ion channels using three complementary bacteria-based assays. Out of 196 genes screened, 24 candidates exhibited transmembrane potential, and their successful expression and integration were verified using maltose-binding protein fusions and Western blotting. Fourteen of these candidates scored positively across all three assays, confirming their status as potential viroporins. This functionally redundant set of 14 viroporins enables the virus to modulate host potassium levels and proton flux across membranes. Beyond identifying general channels, we mapped specific proton-selective pores. By applying kinetic modeling and ternary clustering, we moved beyond qualitative screening to categorize these viroporins into distinct functional zones based on their specific ion-movement dynamics. Genomic comparisons across eleven diverse global genotypes revealed that these key channels have remained virtually unchanged for over a century. The combination of structural stability and functional critical mass establishes these channels as premier vulnerabilities for therapeutic exploitation. Ultimately, this study provides the first validated functional atlas of ASFV ion channels, offering a detailed roadmap for designing broad-spectrum antivirals targeting the viral metabolic core.