In Vitro Virucidal Assays to Evaluate the Antiviral Efficacy of Nitric Oxide Nasal Spray Against Different Sars-Cov-2 Variants

Background: Frequent mutations and geographical variants of the SARS-CoV-2 virus have been rapidly emerging which show varying susceptibility to the existing vaccines and treatments. Nitric oxide nasal spray (NONS) has been found effective in reducing the intranasal viral load and expediting viral clearance of SARS-CoV-2 in clinical trials. The aim of this study was to assess its in vitro effectiveness in neutralizing the Delta and Omicron variants grown on Vero and Caco-2 cell lines, respectively. Materials and Methods:Vero cells were incubated with increasing concentrations of NONS to select the optimum NONS concentration for the infection experiment. The Delta and Omicron SARS-CoV-2 variants were exposed to 10% NONS v/v with media for different durations and subsequently incubated with the Vero cells for Delta variant and Caco-2 cells for the Omicron variant (BA.1) for 48 hours. The cytopathic effects and cell viability of Vero cells were observed and quantified post-incubation, while the viral copy number and cycle threshold (Ct) values from the Caco-2 cell culture supernatant were analyzed to determine the antiviral activity of NONS against Omicron variant. The active comparators were 70% ethanol as a positive control and phosphate buffer solution (PBS) as a negative control. Results: The viability of Vero cells with 10% NONS in the absence of the virus was found to be optimum. Subsequently, incubation of NONS with the Delta variant for 2 min led to a significant reduction in virus mediated cytopathic effects (CPE) and an increase in cell viability (p=0.0021) in comparison to the negative control group. In case of the treatment of the Omicron variant with NONS, Ct value was found to be significantly higher at 2 minutes (p=0.032) and 15 minutes (p=0.0021) of NONS exposure. The viral copy number in infected cells was significantly lower after 15 minutes of NONS treatment compared to the infection control (p=0.0332).​ Conclusion: NONS exhibited significant antiviral activity against the Delta and Omicron variants of SARS-CoV-2, with a quick onset of action. The results of our study support the potential application of NONS as a post-exposure prophylaxis agent to reduce the severity of infection and control secondary transmission for multiple variants.