Influence of Carbon Dioxide and pH on Influenza Virus in Sessile Saliva Droplets

Upon exhalation, virus-laden respiratory droplets experience rapid changes in environmental conditions that lead to chemical and physical alterations that can affect virus infectivity. By manipulating the concentration of gaseous carbon dioxide (CO₂) surrounding sessile saliva droplets, we altered their chemistry and then assessed the impacts of these changes on the infectivity of influenza A virus at relative humidities of 30, 50, and 80%. For virus exposed to low CO₂ (<0.005% CO₂ in N₂) vs high CO₂ (4.3-5% CO₂ in N₂), differences in inactivation were small except at 80% RH, where the virus decayed less (i.e., maintained greater infectivity) in low CO₂ than in high CO₂. The difference exceeded 1log₁₀ at 2 h. For comparison, virus inactivation in ambient air (0.04% CO₂) varied across conditions, sometimes exceeding and sometimes falling below that observed under high- and low-CO₂ atmospheres. Collectively, these results suggest that the driving factors for virus inactivation vary with RH. We measured droplet pH using gold nanoprobes in combination with surface-enhanced Raman spectroscopy and found that pH increased in low CO₂ and decreased in high CO₂ at 80% RH by ∼1 pH unit in both cases. Results were consistent with chemical equilibrium modeling, which indicated that both carbonate and phosphate buffering were important. Changes in pH were smaller or insignificant at 30 and 55% RH. At these low and medium RHs, rapid evaporation of water from the droplets and the resulting increase in viscosity may limit changes in pH. Measured changes in pH did not appear to be sufficient to drive virus inactivation under any tested condition. This finding suggests that pH likely does not impact influenza transmission by fomites.