Stability of influenza A virus in droplets and aerosols is heightened by the presence of commensal respiratory bacteria.

Aerosol transmission remains a major challenge for control of respiratory viruses, particularly those causing recurrent epidemics, like influenza A virus (IAV). These viruses are rarely expelled alone, but instead are embedded in a consortium of microorganisms that populate the respiratory tract. The impact of microbial communities and inter-pathogen interactions upon stability of transmitted viruses is well-characterized for enteric pathogens, but is under-studied in the respiratory niche. Here, we assessed whether the presence of five different species of commensal respiratory bacteria could influence the persistence of IAV within phosphate-buffered saline and artificial saliva droplets deposited on surfaces at typical indoor air humidity, and within airborne aerosol particles. In droplets, presence of individual species or a mixed bacterial community resulted in 10- to 100-fold more infectious IAV remaining after 1 h, due to bacterial-mediated flattening of drying droplets and early efflorescence. Even when no efflorescence occurred at high humidity or the bacteria-induced changes in droplet morphology were abolished by aerosolization instead of deposition on a well plate, the bacteria remained protective. Staphylococcus aureus and Streptococcus pneumoniae were the most stabilizing compared to other commensals at equivalent density, indicating the composition of an individual's respiratory microbiota is a previously unconsidered factor influencing expelled virus persistence.IMPORTANCEIt is known that respiratory infections such as coronavirus disease 2019 and influenza are transmitted by release of virus-containing aerosols and larger droplets by an infected host. The survival time of viruses expelled into the environment can vary depending on temperature, room air humidity, UV exposure, air composition, and suspending fluid. However, few studies consider the fact that respiratory viruses are not alone in the respiratory tract-we are constantly colonized by a plethora of bacteria in our noses, mouth, and lower respiratory system. In the gut, enteric viruses are known to be stabilized against inactivation and environmental decay by gut bacteria. Despite the presence of a similarly complex bacterial microbiota in the respiratory tract, few studies have investigated whether viral stabilization could occur in this niche. Here, we address this question by investigating influenza A virus stabilization by a range of commensal bacteria in systems representing respiratory aerosols and droplets.

The neuraminidase activity of influenza A virus determines the strain-specific sensitivity to neutralization by respiratory mucus.

IMPORTANCE: The respiratory tract of humans is constantly exposed to potentially harmful agents, such as small particles or pathogens, and thus requires protective measures. Respiratory mucus that lines the airway epithelia plays a major role in the prevention of viral infections by limiting the mobility of viruses, allowing subsequent mucociliary clearance. Understanding the interplay between respiratory mucus and viruses can help elucidate host and virus characteristics that enable the initiation of infection. Here, we tested a panel of primary influenza A viruses of avian or human origin for their sensitivity to mucus derived from primary human airway cultures and found that differences between virus strains can be mapped to viral neuraminidase activity. We also show that binding of influenza A viruses to decoy receptors on highly glycosylated mucus components constitutes the major inhibitory function of mucus against influenza A viruses.

Expiratory Aerosol pH: The Overlooked Driver of Airborne Virus Inactivation.

Respiratory viruses, including influenza virus and SARS-CoV-2, are transmitted by the airborne route. Air filtration and ventilation mechanically reduce the concentration of airborne viruses and are necessary tools for disease mitigation. However, they ignore the potential impact of the chemical environment surrounding aerosolized viruses, which determines the aerosol pH. Atmospheric aerosol gravitates toward acidic pH, and enveloped viruses are prone to inactivation at strong acidity levels. Yet, the acidity of expiratory aerosol particles and its effect on airborne virus persistence have not been examined. Here, we combine pH-dependent inactivation rates of influenza A virus (IAV) and SARS-CoV-2 with microphysical properties of respiratory fluids using a biophysical aerosol model. We find that particles exhaled into indoor air (with relative humidity ≥ 50%) become mildly acidic (pH ∼ 4), rapidly inactivating IAV within minutes, whereas SARS-CoV-2 requires days. If indoor air is enriched with nonhazardous levels of nitric acid, aerosol pH drops by up to 2 units, decreasing 99%-inactivation times for both viruses in small aerosol particles to below 30 s. Conversely, unintentional removal of volatile acids from indoor air may elevate pH and prolong airborne virus persistence. The overlooked role of aerosol acidity has profound implications for virus transmission and mitigation strategies.

Inactivation mechanisms of influenza A virus under pH conditions encountered in aerosol particles as revealed by whole-virus HDX-MS.

Multiple respiratory viruses, including influenza A virus (IAV), can be transmitted via expiratory aerosol particles, and aerosol pH was recently identified as a major factor influencing airborne virus infectivity. Indoors, small exhaled aerosols undergo rapid acidification to pH ~4. IAV is known to be sensitive to mildly acidic conditions encountered within host endosomes; however, it is unknown whether the same mechanisms could mediate viral inactivation within the more acidic aerosol micro-environment. Here, we identified that transient exposure to pH 4 caused IAV inactivation by a two-stage process, with an initial sharp decline in infectious titers mainly attributed to premature attainment of the post-fusion conformation of viral protein haemagglutinin (HA). Protein changes were observed by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) as early as 10 s post-exposure to acidic conditions. Our HDX-MS data are in agreement with other more labor-intensive structural analysis techniques, such as X-ray crystallography, highlighting the ease and usefulness of whole-virus HDX-MS for multiplexed protein analyses, even within enveloped viruses such as IAV. Additionally, virion integrity was partially but irreversibly affected by acidic conditions, with a progressive unfolding of the internal matrix protein 1 (M1) that aligned with a more gradual decline in viral infectivity with time. In contrast, no acid-mediated changes to the genome or lipid envelope were detected. Improved understanding of respiratory virus fate within exhaled aerosols constitutes a global public health priority, and information gained here could aid the development of novel strategies to control the airborne persistence of seasonal and/or pandemic influenza in the future. IMPORTANCE It is well established that COVID-19, influenza, and many other respiratory diseases can be transmitted by the inhalation of aerosolized viruses. Many studies have shown that the survival time of these airborne viruses is limited, but it remains an open question as to what drives their infectivity loss. Here, we address this question for influenza A virus by investigating structural protein changes incurred by the virus under conditions relevant to respiratory aerosol particles. From prior work, we know that expelled aerosols can become highly acidic due to equilibration with indoor room air, and our results indicate that two viral proteins are affected by these acidic conditions at multiple sites, leading to virus inactivation. Our findings suggest that the development of air treatments to quicken the speed of aerosol acidification would be a major strategy to control infectious bioburdens in the air.

Photolytic radical persistence due to anoxia in viscous aerosol particles.

In viscous, organic-rich aerosol particles containing iron, sunlight may induce anoxic conditions that stabilize reactive oxygen species (ROS) and carbon-centered radicals (CCRs). In laboratory experiments, we show mass loss, iron oxidation and radical formation and release from photoactive organic particles containing iron. Our results reveal a range of temperature and relative humidity, including ambient conditions, that control ROS build up and CCR persistence in photochemically active, viscous organic particles. We find that radicals can attain high concentrations, altering aerosol chemistry and exacerbating health hazards of aerosol exposure. Our physicochemical kinetic model confirmed these results, implying that oxygen does not penetrate such particles due to the combined effects of fast reaction and slow diffusion near the particle surface, allowing photochemically-produced radicals to be effectively trapped in an anoxic organic matrix.

Retrieval of Aerosol Size Distributions From In Situ Particle Counter Measurements: Instrument Counting Efficiency and Comparisons With Satellite Measurements.

The method to derive aerosol size distributions from in situ stratospheric measurements from the University of Wyoming is modified to include an explicit counting efficiency function (CEF) to describe the channel-dependent instrument counting efficiency. This is motivated by Kovilakam and Deshler's (2015, https://doi.org/10.1002/2015JD023303) discovery of an error in the calibration method applied to the optical particle counter (OPC40) developed in the late 1980s and used from 1991 to 2012. The method can be applied to other optical aerosol instruments for which counting efficiencies have been measured. The CEF employed is the integral of the Gaussian distribution representing the instrument response at any one aerosol channel, the aerosol counting efficiency. Results using the CEF are compared to previous derivations of aerosol size distributions (Deshler et al., 2003, https://doi.org/10.1029/2002JD002514) applied to the measurements before and after Kovilakam and Deshler's correction of number concentration for the OPC40 calibration error. The CEF method is found, without any tuning parameter, to reproduce or improve upon the Kovilakam and Deshler's results, thus accounting for the calibration error without any external comparisons other than the laboratory determined counting efficiency at each aerosol channel. Moments of the new aerosol size distributions compare well with aerosol extinctions measured by Stratospheric Aerosol and Gas Experiment II and Halogen Occultation Experiment in the volcanic period 1991-1996, generally within ±40%, the precision of OPC40 moments, and in the nonvolcanic period after 1996, generally within ±20%. Stratospheric Aerosol and Gas Experiment II and Halogen Occultation Experiment estimates of aerosol surface area are generally in agreement with those derived using the new CEF method.

Carbon Dioxide Diffusivity in Single, Levitated Organic Aerosol Particles.

The diffusivity of molecules relevant to condensed-phase chemistry within viscous secondary organic aerosol (SOA) remains highly uncertain. Whereas there has been an effort to characterize water diffusivity as well as the diffusivity of larger compounds, data are lacking almost entirely for small molecules, such as carbon dioxide (CO2). Here we use photochemically generated CO2 in single particles of aqueous citric acid as a SOA proxy, levitated in an electrodynamic balance, to deduce CO2 diffusivity in the particle with unprecedented accuracy. For medium viscosities at intermediate relative humidities (∼25-40% RH), we find CO2 diffusivities DCO2 ≈ 10-14 m2 s-1, agreeing with the Stokes-Einstein relationship based on current viscosity data but 10 times lower than that for water. Conversely, under dry high-viscosity conditions, we find that DCO2 ≈ 10-16 m2 s-1, which is 10 times higher than for water. We infer that the chemical degradation of atmospheric SOA particles will likely not be limited by CO2 diffusivity.

Determination of polar stratospheric cloud particle refractive indices by use of in situ optical measurements and T-matrix calculations.

A new algorithm to infer structural parameters such as refractive index and asphericity of cloud particles has been developed by use of in situ observations taken by a laser backscattersonde and an optical particle counter during balloon stratospheric flights. All three main particles, liquid, ice, and a no-ice solid (NAT, nitric acid trihydrate) of polar stratospheric clouds, were observed during two winter flights performed from Kiruna, Sweden. The technique is based on use of the T-matrix code developed for aspherical particles to calculate the backscattering coefficient and particle depolarizing properties on the basis of size distribution and concentration measurements. The results of the calculations are compared with observations to estimated refractive indices and particle asphericity. The method has also been used in cases when the liquid and solid phases coexist with comparable influence on the optical behavior of the cloud to estimate refractive indices. The main results prove that the index of refraction for NAT particles is in the range of 1.37-1.45 at 532 nm. Such particles would be slightly prolate spheroids. The calculated refractive indices for liquid and ice particles are 1.51-1.55 and 1.31-1.33, respectively. The results for solid particles confirm previous measurements taken in Antarctica during 1992 and obtained by a comparison of lidar and optical particle counter data.