Establishment of in vitro persistent infection of the foot-and-mouth disease virus SAT 2 serotype and comparative viral genome analysis during acute and persistent infections

Abstract

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen affecting global livestock production. This study aimed to establish persistent FMDV SAT 2 infection using ammonium chloride (NH4Cl) in vitro, a lysosomotropic agent that inhibits viral replication by increasing endosomal pH. Cytotoxicity assays determined NH4Cl’s effects on virus-free BHK-21 cells at concentrations of 10 mM, 15 mM, and 20 mM. Persistent infections were established by infecting BHK-21 cells at a multiplicity of infection (MOI = 0.001) and treating with NH4Cl, while acute infections were achieved without NH4Cl treatment. Acute infections exhibited extensive cytopathic effects (CPE), whereas persistent infections displayed delayed CPE. Real-time PCR confirmed reduced viral replication in NH4Cl-treated cultures, as indicated by higher Cq values (25–34) compared to acute infections (15–25). RNA sequencing revealed greater mutation accumulation in acute infections, with deletions and insertions predominantly occurring in non-structural protein (NSP) and 3’ UTR regions. Mutation rates increased over time in both infection types. This study demonstrates that NH4Cl effectively induces persistent SAT 2 FMDV infections in vitro by inhibiting replication and modulating viral evolution. These findings provide a valuable model for studying FMDV persistence and highlight key differences in viral adaptation between acute and persistent infections