Abstract

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.