Jacobs, A. I. | bioRxiv Science

bioRxiv2026

Antiviral drug synergy and mutational signatures in different epithelial cell models of RSV and hPIV infection

Despite the huge global health burden presented by respiratory viruses, effective broad-spectrum antiviral therapeutic options remain limited. Here we evaluated the antiviral activity of four RNA-dependent RNA polymerase (RdRp) inhibitors, remdesivir, ribavirin, favipiravir, and molnupiravir, against respiratory syncytial virus (RSV) and human parainfluenza (hPIV) using monotherapy or dual-drug combinations with epithelial cell lines and primary human airway culture models. Remdesivir showed the greatest potency across both viruses, while ribavirin and favipiravir also demonstrated inhibition. Molnupiravir was active against RSVA but not hPIV3. Several dual-drug combinations, including remdesivir-favipiravir, remdesivir-molnupiravir and favipiravir-molnupiravir, produced marked synergy against RSVA, and more limited synergy for hPIV3. Antiviral efficacy was validated in primary airway epithelial cultures, where effective concentrations preserved epithelial integrity and attenuated viral disruption of ciliary function. Across both viruses, increasing antiviral exposure was associated with dose-dependent signature mutagenesis. Importantly, antivirals induced significantly higher RSVA mutation burden in the primary airway model. These findings highlight the therapeutic potential of RdRp inhibitor combinations for RSVA and hPIV3, provide mechanistic insight through antiviral-related mutational signatures and demonstrate advantages of the primary human airway culture model for development of effective multi-drug regimens and broad-spectrum antiviral preparedness. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/699296v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1eb00dforg.highwire.dtl.DTLVardef@186358forg.highwire.dtl.DTLVardef@265181org.highwire.dtl.DTLVardef@1b10d0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Neutrophil myeloperoxidase as a functional biomarker for RSV severity: implications for in vitro therapeutic screening.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, yet effective therapeutics are lacking. The aim of this study is to develop an in vitro model that recapitulates key clinical outcomes in infants with RSV bronchiolitis to help accelerate the discovery of effective therapeutics. Neutrophil activation and influx into the airways are hallmarks of severe RSV infection, but these responses are difficult to quantify in clinical trials. Here we profile peripheral blood-derived neutrophils from infants with RSV admitted to the Paediatric Intensive Care Unit (PICU) and identify myeloperoxidase (MPO) as a key indicator of disease severity when compared to age matched controls. To mechanistically model this response, we established a paediatric airway epithelial air-liquid interface (ALI) system incorporating an endothelial layer and primary neutrophils to recapitulate the tissue microenvironment at the blood-airway barrier. Following RSV infection, neutrophil migration and activation were assessed using flow cytometry. The inclusion of an endothelial layer enhanced physiological relevance and more accurately replicated in vivo MPO responses. We then evaluated two antiviral candidates (remdesivir (RDV) and RSV604) to assess their ability to modulate neutrophil activation. While both compounds reduced viral load at 24 hours post-infection, only RSV604 attenuated MPO expression. These findings establish MPO as both a biomarker of RSV disease severity and a functional readout of therapeutic efficacy and demonstrate that targeting neutrophildriven inflammatory pathways may be critical for reducing pathology in infant RSV infection. Take home messageAntiviral drug discovery should include neutrophil MPO reduction as a readout of therapeutic efficacy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/682115v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@170058corg.highwire.dtl.DTLVardef@17c344corg.highwire.dtl.DTLVardef@14d79baorg.highwire.dtl.DTLVardef@172ac3c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗