Tylvalosin tartrate inhibits PRRSV replication by suppressing cellular pyroptosis through the TLR4/NF-κ <scp>B</scp> signaling pathway

, it effectively inhibits the replication of not only L1 PRRSV but also other major lineage strains (L8.1/CH-1a-like, L5/VR2332-like, and L8.3/JXA1-like) in porcine alveolar macrophages (PAMs). Transcriptomic analysis suggested that tylvalosin tartrate suppresses PRRSV replication by inhibiting cellular pyroptosis. We confirmed that L1 PRRSV infection induces pyroptosis in PAMs, and viral replication was hindered upon inhibition of caspase-1 (CASP1) or gasdermin D (GSDMD). Tylvalosin tartrate inhibited GSDMD-mediated pyroptosis induced by either L1 PRRSV or lipopolysaccharide (LPS)/nigericin. Further investigation revealed that tylvalosin tartrate acts upstream by suppressing the activation of the TLR4/NF-κB signaling pathway, a key driver of inflammatory gene expression and pyroptosis. Pharmacological inhibition of TLR4 using resatorvid and genetic knockdown through siRNA replicated the antiviral and anti-pyroptotic effects of tylvalosin tartrate. Our findings elucidate a host-directed mechanism by which tylvalosin tartrate restricts PRRSV replication across diverse lineages, highlighting its potential as a multifaceted therapeutic agent against PRRSV infection.IMPORTANCEPorcine reproductive and respiratory syndrome (PRRS) causes enormous economic losses worldwide. Controlling PRRSV is challenging due to viral genetic diversity, limited vaccine efficacy, and the lack of specific antiviral drugs. Tylvalosin tartrate, a widely used veterinary antibiotic for respiratory and enteric diseases, has been empirically observed to improve outcomes in PRRSV-affected herds. This study provides novel mechanistic evidence that tylvalosin tartrate possesses direct, broad-spectrum antiviral activity against PRRSV by targeting a host pathway. We show that it inhibits viral replication across major epidemic lineages by dampening the TLR4/NF-κB signaling axis and the subsequent gasdermin D (GSDMD)-dependent pyroptosis. This "old drug, new use" strategy repurposes an approved, safe compound to counteract both viral replication and excessive inflammation-a hallmark of severe PRRS. Our work not only clarifies the molecular basis for its clinical utility but also positions tylvalosin tartrate as a promising host-targeted antiviral and immunomodulatory agent for PRRS management, offering a practical and immediate intervention strategy for the swine industry.