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Abstract

Abstract Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), remains one of the most economically significant infectious diseases affecting the global swine industry, resulting in annual losses exceeding US $1.2 billion in the USA alone. PRRSV infects porcine alveolar macrophages (PAMs) and induces profound mitochondrial dysfunction, leading to the release of mitochondrial DNA (mtDNA). However, the mechanisms by which PRRSV-induced mitochondrial damage acts as a damage-associated molecular pattern (DAMP) to trigger inflammatory responses have not been fully elucidated. Here, we report that highly pathogenic PRRSV (HP-PRRSV) infection upregulates interleukin-1β (IL-1β) messenger RNA (mRNA) expression and protein secretion in a dose- and time-dependent manner. Transmission electron microscopy revealed progressive mitochondrial swelling, cristae disorganization, and matrix loss during HP-PRRSV infection. Using RNAscope combined with MitoGreen staining, we discovered that mitochondrial double-stranded RNA (mtdsRNA), generated from bidirectional transcription of mitochondrial DNA, is released into the cytosol during PRRSV infection. In vitro, transcription-driven mtdsRNA or mtdsRNA isolated from PAMs directly activated the NLRP3 inflammasome, inducing caspase-1 cleavage and IL-1β secretion. Mechanistically, the PRRSV-encoded nonstructural protein 11 (nsp11) inhibited NLRP3-dependent inflammatory responses by suppressing the oligomerization of the apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) (ASC), thereby constraining excessive inflammation and establishing a delicate balance between pro-inflammatory signaling and viral immune evasion. Collectively, our findings identify mtdsRNA as a previously unrecognized mitochondrial DAMP that links PRRSV-induced mitochondrial injury to NLRP3 inflammasome activation, while also revealing a counter-regulatory role for nsp11 in modulating inflammation.