Galectin-9 is a restrictor of infection with multiple enteroviruses.

Enteroviruses (EVs), encompassing over 200 sero/genotypes, cause diseases ranging from mild illness to severe systemic infections. Galectin-9 (Gal-9) is an immunomodulatory lectin implicated in various viral infections, yet its role in EVs remains unexplored. Here, we identify Gal-9 as a novel host restriction factor against a broad spectrum of EVs, including EV-A71, EV-D68, and Coxsackievirus B2. LGALS9 deficiency increased viral replication in cells and pathogenicity in a mouse model of EV-A71 infection, while its overexpression inhibited viral replication. Mechanistically, Gal-9 specifically interacts with the viral VP2 protein, and mapping identified residues 179-194 of Gal-9 as critical for this interaction. A Gal-9 Δ179-194 mutant lost VP2-binding capacity and antiviral activity. These findings reveal that Gal-9 is a key component of the host antiviral response against EVs, functioning through a direct interaction with the viral VP2 protein.IMPORTANCEEnteroviruses (EVs) cause a wide spectrum of disease and present a persistent global health challenge, highlighted by the cyclical resurgence of strains like EV-D68 and EV-A71. However, due to a lack of sufficient understanding of their pathogenesis, no EV-specific antiviral drugs are available. This study demonstrates that galectin-9 (Gal-9) is a key host factor that broadly antagonizes EVs, including EV-A71, EV-D68, and Coxsackievirus B2. We show that Gal-9 deficiency enhances viral replication both in cell culture and in an animal model. By identifying Gal-9 as a crucial restriction factor against EVs, our study provides foundational insight for the future development of antiviral strategies.