Abstract Background The nasal epithelium is the primary site of replication and spread for respiratory viruses, including SARS-CoV-2 variants, influenza (A, B, and highly pathogenic avian H5N1), RSV, hMPV, HPIV-3, and rhinovirus. Nitric oxide (NO) is an antimicrobial with broad, pathogen-independent antiviral activity through nitrosylation of viral proteins and disruption of host-virus interactions. During SARS-CoV-2 pandemic, nitric oxide nasal spray was shown to rapidly reduce viral load, symptoms and transmission in individuals with Delta and Omicron variants (Tandon, Wu et al., 2022). This study evaluated the virucidal efficacy of a nitric oxide nasal spray (NONS) against a wider panel of post-pandemic clinically relevant respiratory pathogens, including the avian influenza A (H5N1) strain of current global concern. Methods In vitro assays evaluated NONS against SARS-CoV-2 (wild type; Alpha, Beta, Gamma, Delta, Epsilon, Omicron including XBB), influenza A (H1N1, H3N2, H5N1) and B, RSV A2, hMPV, HPIV-3, and HRV-14. Virus stocks (3.3-4.7 log10 CCID50/0.1 mL) were exposed to 100% NONS for 30 s-2 min, neutralized, and titrated on respective cell lines. Controls included water (virus-only), 70% ethanol (Oh, Shin et al., 2025), toxicity, and neutralized controls. Cytotoxicity was assessed by WST-1 assay in Vero cells (0.5-30% NONS, 24 h). Functional protection was measured by cytopathic-effect assays after pre-exposing SARS-CoV-2 Delta to 10% NONS for 20 s-15 min. Results NONS caused rapid inactivation across all viral families. For SARS-CoV-2 variants, 2-min exposure reduced titers below detection (≥3.5-4.0 LRV, 99.97-99.99%). Influenza A (H1N1, H3N2, H5N1 avian strain) and B were completely inactivated within 30 s (3.8 LRV, 99.98%). RSV, hMPV, and HPIV-3 were undetectable within 2 min (≥2.8-3.3 LRV, ≥99.8-99.95%). NONS also fully inactivated the non-enveloped HRV-14 (3.8 LRV), while 70% ethanol produced only partial reduction (∼0.5 LRV). Pre-exposure of SARS-CoV-2 Delta to 10% NONS prevented cytopathic effects, confirming functional protection. Vero-cell viability remained ≈80% at 10% NONS. Conclusions NONS exhibits broad, rapid virucidal activity against major and clinically significant respiratory viruses, including multiple SARS-CoV-2 variants and the highly pathogenic avian influenza H5N1. Its fast action (30 s-2 min), pathogen-independent mechanism, and favorable tolerability support its potential as an intranasal antiviral for early infection control and reduced transmission. Together with clinical data showing accelerated viral clearance and symptom reduction, these findings highlight intranasal nitric oxide as a promising frontline antiviral for pandemic preparedness and prevention. This abstract is funded by: Sanotize Research & Development Corp.
Miller et al. (Fri,) studied this question.