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.