RIG-I-dependent sensing of PEDV shapes epithelial antiviral immunity in the intestinal mucosa

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that causes lethal diarrhea in neonatal piglets, yet the viral pathogen-associated molecular patterns (PAMPs) that initiate mucosal antiviral immunity remain poorly defined. Here, integrating intestinal single-cell transcriptomics with in vivo and in vitro infection models, we delineate how PEDV is sensed by host innate immunity. We found that RIG-I, rather than MDA5, functions as the dominant cytosolic sensor that detects PEDV and initiates interferon-mediated antiviral defense in the intestinal mucosa. RIP-seq and RNA structural analyses identified nucleotides 375-760 within the 5' ORF1a region of the PEDV genome as a core RNA PAMP directly engaged by RIG-I. Molecular docking and mutational analyses further revealed that specific nucleotide motifs and cooperative stem-loop assemblies are required for optimal RIG-I activation, establishing higher-order RNA architecture as a key determinant of PAMP potency. Functionally, this PEDV-derived RNA PAMP robustly activated RIG-I signaling, amplified interferon and ISG responses, and restricted replication of PEDV and multiple heterologous RNA viruses, demonstrating broad-spectrum activity against several enteric and respiratory RNA viruses. Moreover, co-administration of this viral RNA with an inactivated influenza vaccine significantly enhanced antigen-specific immunity and protection. Together, these findings define the molecular basis of RIG-I-mediated recognition of PEDV, identify a structurally encoded viral PAMP with both antiviral and adjuvant properties, and provide new insights into how coronaviruses are sensed and controlled at mucosal surfaces.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) causes devastating enteric disease in newborn piglets, yet how the intestinal mucosa detects this coronavirus and mounts antiviral immunity has remained unclear. Our study identifies RIG-I as the dominant epithelial sensor responsible for detecting PEDV and initiating interferon-driven antiviral defense. We further define a structurally encoded RNA element within the 5' ORF1a region of the PEDV genome that functions as a core viral PAMP directly activating RIG-I. This RNA element acts as a molecular alarm that triggers robust innate immune responses, restricts infection by PEDV and other RNA viruses, and enhances the protective efficacy of an inactivated influenza vaccine. These findings illuminate fundamental mechanisms of coronavirus sensing at mucosal surfaces and highlight viral RNA structures as promising natural immunostimulants for antiviral and vaccine strategies.