Hydroxychloroquine suppresses replication of PRRSV via dual regulation of autophagic flux and NF-κB-driven inflammatory signaling

Porcine reproductive and respiratory syndrome virus (PRRSV) induces excessive autophagy and aberrant nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) hyperactivation, triggering severe respiratory and reproductive disorders in pigs and causing massive economic losses to the global swine industry. Hydroxychloroquine sulfate (HCQ), a classic autophagy inhibitor, restricts PRRSV proliferation in vitro; however, its in vivo protective efficacy and dual-targeted molecular mechanisms remain poorly characterized. In this study, intramuscular HCQ administration conferred robust protection against lethal PRRSV infection in piglets: it completely eliminated virus-induced mortality, relieved fever and interstitial pulmonary lesions, and restored normal growth performance. In vitro assays confirmed that HCQ with negligible cytotoxicity within the concentration range of 1-100 μM interferes with multiple stages of PRRSV infection. Combined network pharmacology and cellular functional experiments revealed a dual regulatory mechanism of HCQ against PRRSV: HCQ blocks autophagosome-lysosome fusion to disrupt complete autophagic flux, and suppresses the IκBα-p65 signaling cascade to reduce the secretion of proinflammatory mediators. Dose-response analyses demonstrated that HCQ exerts potent antiviral activity against divergent field PRRSV isolates, porcine epidemic diarrhea virus (PEDV) and swine acute diarrhea syndrome coronavirus (SADS-CoV) within a wide safe therapeutic window, with all broad-spectrum in vitro antiviral activity against diverse swine RNA viruses validated in cell culture models. Collectively, HCQ suppresses PRRSV replication by simultaneously regulating autophagic flux and NF-κB-dependent inflammatory signaling, supporting its repurposing as a promising therapeutic candidate for porcine reproductive and respiratory syndrome (PRRS) control. Although HCQ exhibits broad-spectrum inhibitory activity against divergent PRRSV strains, PEDV, and SADS-CoV in cell culture, all anti-PEDV and anti-SADS-CoV effects are only validated in vitro without in vivo animal verification.