ASFV major capsid p72 trimers function as a pH sensor during uncoating process of virus endocytosis and facilitate its application as conformational antigen detection

African swine fever virus (ASFV), a member of the nucleocytoplasmic large DNA virus (NCLDV) family, is the sole representative of the Asfarviridae family. Recent studies have shown that the uncoating process of ASFV occurs through a pH-dependent mechanism within late endosomal compartments. However, the molecular mechanisms underlying pH-mediated capsid destabilization remain poorly understood, and the key viral components responsible for pH sensing during uncoating have not yet been clearly identified. In this study, we identified that the major capsid protein p72 of ASFV functions as a pH-sensitive structural component enriched with ionizable residues. By simulating the acidic environment of endosomes, we observed that the molecular weight and particle size of the acidified p72 protein decreased. Cryo-electron microscopy (Cryo-EM) revealed that the p72 protein underwent trimer depolymerization at acidic pH levels similar to those found in endosomes. This destabilization of the trimer provides a mechanistic basis for proposing a model of the uncoating mechanism of ASFV. Based on these insights, we developed a double-antibody sandwich lateral immunochromatographic test strip targeting p72 trimers. This test strip demonstrated high specificity and sensitivity, making it a valuable tool for detecting ASFV infections and evaluating the efficacy of mild disinfectants used in swine farms against African swine fever virus.