Sparfloxacin Reveals a Host–virus Dual-Target Antiviral Scaffold Against Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is a major cause of severe respiratory illness across the lifespan, particularly in infants and older adults. Nonetheless, effective antiviral therapies remain limited. Here, we performed a clinically guided, medium-throughput screening of approved drugs used in pediatric populations and identified sparfloxacin (SPFX), a fluoroquinolone antibiotic, as a mechanistically informative antiviral scaffold. SPFX showed antiviral activity against genetically distinct RSV clinical isolates in vitro, and suppressed RSV infection in a mechanistic mouse model in vivo. Mechanistically, our findings support a host–virus dual-target framework in which SPFX suppresses RSV replication through activity consistent with modulation of viral RNA polymerase function together with a pro-viral HSP70-associated host pathway. Using primary human pediatric airway epithelial cells, we identified SPFX-responsive host factors and observed suppression of HSP70 expression independent of infection. Together, these findings establish SPFX as a mechanistically informative antiviral scaffold and support a host–virus dual-target framework for future RSV therapeutic development. Sparfloxacin inhibits respiratory syncytial virus through dual targeting of the viral RNA polymerase and the host HSP70 pathway, providing a promising antiviral scaffold for future RSV therapeutic development.