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.

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.

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.

Electrodynamic balance-mass spectrometry reveals impact of oxidant concentration on product composition in the ozonolysis of oleic acid.

The chemical and physical properties of atmospheric aerosol particles change upon oxidative ageing, influencing their interaction with radiation, their propensity to serve as nuclei for cloud condensation and ice formation, and their adverse effects on human health. The investigation of atmospheric aerosol oxidation processes is complicated by low oxidant concentrations and long timescales, which are difficult to represent in laboratory studies. Experimental work often attempts to compensate for short timescales with elevated concentrations of oxidative agents, assuming that the ageing progress depends only on the oxidant exposure, i.e. on the product of oxidant concentration and time, [Ox] × t, and not on [Ox] or t independently. The application of electrodynamic balance-mass spectrometry of single particles allows the validity of this assumption to be investigated, since it provides information on the molecular composition of aerosol particles for a wide range of reaction durations under well-defined oxidation conditions. Here, we demonstrate the capabilities of a new setup on levitated oleic acid droplets reacting with ozone at mixing ratios of 0.2 and 15 ppm, i.e. spanning almost two orders of magnitude in [Ox]. We show that the reactive removal of oleic acid can be accurately expressed as a function of ozone exposure [Ox] × t, whereas the product concentrations depend on [Ox] and t independently. As the underlying reason for the breakdown of the exposure metric, we suggest a competition between evaporation of volatile first-generation products and their accretion reactions with reactive Criegee intermediates, converting them into low-volatility dimers and oligomers. This hypothesis is supported by kinetic model simulations using the aerosol process model KM-SUB, which explicitly resolves the competition between evaporation and secondary chemistry as a function of the experimental timescale and ozone mixing ratio. The model successfully reproduces final product distributions. The findings are further supported by the recorded changes of droplet sizes during oxidation. As a heuristic, the breakdown of the exposure metric in a chemical reaction system is possible, when competition between first- and second-order processes of reactive intermediates determines important system properties.

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.

Visualizing reaction and diffusion in xanthan gum aerosol particles exposed to ozone.

Atmospheric aerosol particles with a high viscosity may become inhomogeneously mixed during chemical processing. Models have predicted gradients in condensed phase reactant concentration throughout particles as the result of diffusion and chemical reaction limitations, termed chemical gradients. However, these have never been directly observed for atmospherically relevant particle diameters. We investigated the reaction between ozone and aerosol particles composed of xanthan gum and FeCl2 and observed the in situ chemical reaction that oxidized Fe2+ to Fe3+ using X-ray spectromicroscopy. Iron oxidation state of particles as small as 0.2 µm in diameter were imaged over time with a spatial resolution of tens of nanometers. We found that the loss off Fe2+ accelerated with increasing ozone concentration and relative humidity, RH. Concentric 2-D column integrated profiles of the Fe2+ fraction, α, out of the total iron were derived and demonstrated that particle surfaces became oxidized while particle cores remained unreacted at RH = 0-20%. At higher RH, chemical gradients evolved over time, extended deeper from the particle surface, and Fe2+ became more homogeneously distributed. We used the kinetic multi-layer model for aerosol surface and bulk chemistry (KM-SUB) to simulate ozone reaction constrained with our observations and inferred key parameters as a function of RH including Henry's Law constant for ozone, HO3, and diffusion coefficients for ozone and iron, DO3 and DFe, respectively. We found that HO3 is higher in our xanthan gum/FeCl2 particles than for water and increases when RH decreased from about 80% to dry conditions. This coincided with a decrease in both DO3 and DFe. In order to reproduce observed chemical gradients, our model predicted that ozone could not be present further than a few nanometers from a particle surface indicating near surface reactions were driving changes in iron oxidation state. However, the observed chemical gradients in α observed over hundreds of nanometers must have been the result of iron transport from the particle interior to the surface where ozone oxidation occurred. In the context of our results, we examine the applicability of the reacto-diffusive framework and discuss diffusion limitations for other reactive gas-aerosol systems of atmospheric importance.

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.

Kinetic Limitation to Inorganic Ion Diffusivity and to Coalescence of Inorganic Inclusions in Viscous Liquid-Liquid Phase-Separated Particles.

Mixed organic/inorganic aerosols may undergo liquid-liquid phase separation (LLPS) when the relative humidity drops in the atmosphere. Phase-separated particles adopt different morphologies, which will have different consequences for atmospheric chemistry and climate. Recent laboratory studies on submicron particles led to speculation whether LLPS observed for larger drops might actually be suppressed in smaller droplets. Here, we report on micron-sized droplets of a ternary mixture of ammonium sulfate (AS), carminic acid, and water at different temperatures, which were exposed to typical atmospheric drying rates ranging from 0.34 to 5.0% RH min-1. Our results reveal that increasing the drying rate and lowering the temperature results in different morphologies after LLPS and may suppress the growth and coalescence of the inorganic-rich phase inclusions due to kinetic limitations in a viscous matrix. The coalescence time was used to estimate the viscosity of the organic-rich phase within a factor of 20, and based on the Stokes-Einstein relationship, we estimated AS diffusivity. Furthermore, we evaluated the initial growth of inclusions to quantitatively determine the AS diffusivity in the organic-rich phase, which is about 10-8 cm2 s-1 at room temperature. Extrapolation of diffusivity to lower temperatures using estimations for the diffusion activation energy leads us to conclude that the growth of the inorganic phase is not kinetically impeded for tropospheric submicron particles larger than 100 nm.

Tracking Water Sorption in Glassy Aerosol Particles using Morphology-Dependent Resonances.

Morphology-dependent resonances (MDRs) can serve as a sensitive probe of the size and composition of microspheres. While the utilization of MDRs to characterize homogeneous spheres is now routine, analysis of spherical particles with more complicated refractive index profiles can be extremely difficult and time consuming. In ultraviscous and glassy aerosol particles, the concentration profile of water during sorption often contains a sharp front that propagates from the particle surface to the particle center over time. Here we show that the MDR positions associated with this type of concentration profile closely match those of a spherical core-shell profile. Due to the similarities, a core-shell model can be used to simplify the analysis of MDR positions that are observed during water uptake by high-viscosity aerosol particles. We examined the applicability and limitations of this core-shell model in the tracking of water sorption by single particles. Overall, the core-shell model allows for the radial position of a sharp diffusion front to be readily found using MDR positions observed during water sorption, making the analysis of light-scattering measurements much faster and less error prone than previously used fitting schemes. Additionally, methods for calculating MDRs in spherical core-shell particles are also discussed.

Ozone uptake on glassy, semi-solid and liquid organic matter and the role of reactive oxygen intermediates in atmospheric aerosol chemistry.

Heterogeneous and multiphase reactions of ozone are important pathways for chemical ageing of atmospheric organic aerosols. To demonstrate and quantify how moisture-induced phase changes can affect the gas uptake and chemical transformation of organic matter, we apply a kinetic multi-layer model to a comprehensive experimental data set of ozone uptake by shikimic acid. The bulk diffusion coefficients were determined to be 10(-12) cm(2) s(-1) for ozone and 10(-20) cm(2) s(-1) for shikimic acid under dry conditions, increasing by several orders of magnitude with increasing relative humidity (RH) due to phase changes from amorphous solid over semisolid to liquid. Consequently, the reactive uptake of ozone progresses through different kinetic regimes characterised by specific limiting processes and parameters. At high RH, ozone uptake is driven by reaction throughout the particle bulk; at low RH it is restricted to reaction near the particle surface and kinetically limited by slow diffusion and replenishment of unreacted organic molecules. Our results suggest that the chemical reaction mechanism involves long-lived reactive oxygen intermediates, likely primary ozonides or O atoms, which may provide a pathway for self-reaction and catalytic destruction of ozone at the surface. Slow diffusion and ozone destruction can effectively shield reactive organic molecules in the particle bulk from degradation. We discuss the potential non-orthogonality of kinetic parameters, and show how this problem can be solved by using comprehensive experimental data sets to constrain the kinetic model, providing mechanistic insights into the coupling of transport, phase changes, and chemical reactions of multiple species in complex systems.

Shikimic acid ozonolysis kinetics of the transition from liquid aqueous solution to highly viscous glass.

Ageing of particulate organic matter affects the composition and properties of atmospheric aerosol particles. Driven by temperature and humidity, the organic fraction can vary its physical state between liquid and amorphous solid, or rarely even crystalline. These transitions can influence the reaction kinetics due to limitations of mass transport in such (semi-) solid states, which in turn may influence the chemical ageing of particles containing such compounds. We have used coated wall flow tube experiments to investigate the reaction kinetics of the ozonolysis of shikimic acid, which serves as a proxy for oxygenated, water-soluble organic matter and can form a glass at room temperature. Particular attention was paid to how the presence of water influences the reaction, since it acts a plasticiser and thereby induces changes in the physical state. We analysed the results by means of a traditional resistor model, which assumes steady-state conditions. The ozonolysis rate of shikimic acid is strongly increased in the presence of water, a fact we attribute to the increased transport of O3 and shikimic acid through the condensed phase at lower viscosities. The analysis using the resistor model suggests that the system undergoes both surface and bulk reaction. The second-order rate coefficient of the bulk reaction is 3.7 (+1.5/-3.2) × 10(3) L mol(-1) s(-1). At low humidity and long timescales, the resistor model fails to describe the measurements appropriately. The persistent O3 uptake at very low humidity suggests contribution of a self-reaction of O3 on the surface.

Retrieving the translational diffusion coefficient of water from experiments on single levitated aerosol droplets.

The time-dependent growth and shrinkage of aqueous aerosol particles trapped in an electrodynamic balance exposed to changes in relative humidity (RH) depend on the translational diffusion coefficient of water (DH2O). Resonances in the Mie scattering patterns of the illuminated micrometre-sized droplets are used to follow the compositional evolution through stepwise changes in RH. Under conditions where the diffusion of water molecules becomes sufficiently slow, e.g. in the highly viscous or even glassy regime, the concentration and temperature dependent values of DH2O can be determined iteratively by comparing the observed shifts in the Mie resonant wavelengths with predicted shifts from a diffusion model of a multi-layered sphere. It is shown that condensation and evaporation of water vapour from or to highly viscous or glassy droplets follow different kinetic regimes, a result that is consistent with previous studies of adsorption and desorption on glassy surfaces.

Ion depletion near a solution surface: is image-charge repulsion sufficient?

Grazing-incidence Rutherford backscattering and angle-resolved x-ray photoelectron spectrometry are used to determine the ion-concentration profiles near the surface of a solution consisting of a salt (TEABr) in a weakly polar organic liquid (polyethylene glycol) with atomic-layer depth resolution. The predictions of a model, in which ions in solution are repelled from the surface due to a screened Coulomb interaction with their image charge, are in good agreement with measured ion profiles. This contrasts with the behavior of salts in aqueous and highly polar organic solutions.

Morphologies of mixed organic/inorganic/aqueous aerosol droplets.

Despite major progress in the understanding of properties of tropospheric aerosol particles, it remains challenging to understand their physical state and morphology. To obtain more detailed knowledge of the phases, phase transitions and morphologies of internally mixed organic/inorganic aerosol particles, we evaluated liquid-liquid phase separation (LLPS), deliquescence relative humidity (DRH) and efflorescence relative humidity (ERH) of 33 organic/ammonium sulfate (AS)/H2O systems from our own and literature data. The organic fraction consists of single compounds or mixtures with up to ten aliphatic and/or aromatic components with carboxylic acid, hydroxyl, carbonyl, ether, and ester functionalities, covering O : C ratios between 0.29 and 1.33. Thirteen out of these 33 systems did not show LLPS for any of the studied organic-to-inorganic mixing ratios, sixteen underwent LLPS showing core-shell morphology, and four showed both core-shell and partially engulfed configurations depending on the organic-to-inorganic ratio and RH. In all cases the organic fractions of the systems with partially engulfed configurations consisted of dicarboxylic acids. AS in mixed organic/AS/H2O particles deliquesced between 70 and 84% RH. AS effloresced below 58% RH or remained in a one-liquid-phase state. AS in droplets with LLPS always showed efflorescence with ERH between 30 and 50% RH, providing clear evidence that the presence of LLPS facilitates AS efflorescence. Spreading coefficients of the organic-rich phase on the AS-rich phase for systems containing polyethylene glycol 400 (PEG-400) and a mixture of dicarboxylic acids are in agreement with the optically observed morphologies of droplets deposited on the hydrophobic substrate. Analysis of high resolution elastic Mie resonance spectra allowed the detection of LLPS for single levitated droplets consisting of PEG-400/AS/ H2O, whereas LLPS was difficult to detect in (2-methylglutaric acid + 3-methylglutaric acid + 2,2-dimethylsuccinic acid)/AS/H2O. Measured Mie spectra of PEG-400/AS/H2O at 93.5% and at 80.9% RH agreed with computed Mie spectra for a homogeneous and a core-shell configuration, respectively, confirming the results obtained from droplets deposited on a hydrophobic substrate. Based on the presented evidence, we therefore consider the core-shell morphology to be the prevalent configuration of liquid-liquid-phase-separated tropospheric organic/AS/H2O particles.

Measurements of thermodynamic and optical properties of selected aqueous organic and organic-inorganic mixtures of atmospheric relevance.

Atmospheric aerosol particles can exhibit liquid solution concentrations supersaturated with respect to the dissolved organic and inorganic species and supercooled with respect to ice. In this study, thermodynamic and optical properties of sub- and supersaturated aqueous solutions of atmospheric interest are presented. The density, refractive index, water activity, ice melting temperatures, and homogeneous ice freezing temperatures of binary aqueous solutions containing L(+)-tartaric acid, tannic acid, and levoglucosan and ternary aqueous solutions containing levoglucosan and one of the salts NH(4)HSO(4), (NH(4))(2)SO(4), and NH(4)NO(3) have been measured in the supersaturated concentration range for the first time. In addition, the density and refractive index of binary aqueous citric acid and raffinose solutions and the glass transition temperatures of binary aqueous L(+)-tartaric acid and levoglucosan solutions have been measured. The data presented here are derived from experiments on single levitated microdroplets and bulk solutions and should find application in thermodynamic and atmospheric aerosol models as well as in food science applications.

Comparing the mechanism of water condensation and evaporation in glassy aerosol.

Atmospheric models generally assume that aerosol particles are in equilibrium with the surrounding gas phase. However, recent observations that secondary organic aerosols can exist in a glassy state have highlighted the need to more fully understand the kinetic limitations that may control water partitioning in ambient particles. Here, we explore the influence of slow water diffusion in the condensed aerosol phase on the rates of both condensation and evaporation, demonstrating that significant inhibition in mass transfer occurs for ultraviscous aerosol, not just for glassy aerosol. Using coarse mode (3-4 um radius) ternary sucrose/sodium chloride/aqueous droplets as a proxy for multicomponent ambient aerosol, we demonstrate that the timescale for particle equilibration correlates with bulk viscosity and can be ≫10(3) s. Extrapolation of these timescales to particle sizes in the accumulation mode (e.g., approximately 100 nm) by applying the Stokes-Einstein equation suggests that the kinetic limitations imposed on mass transfer of water by slow bulk phase diffusion must be more fully investigated for atmospheric aerosol. Measurements have been made on particles covering a range in dynamic viscosity from < 0.1 to > 10(13) Pa s. We also retrieve the radial inhomogeneities apparent in particle composition during condensation and evaporation and contrast the dynamics of slow dissolution of a viscous core into a labile shell during condensation with the slow percolation of water during evaporation through a more homogeneous viscous particle bulk.

Exploring the complexity of aerosol particle properties and processes using single particle techniques.

The complex interplay of processes that govern the size, composition, phase and morphology of aerosol particles in the atmosphere is challenging to understand and model. Measurements on single aerosol particles (2 to 100 µm in diameter) held in electrodynamic, optical and acoustic traps or deposited on a surface can allow the individual processes to be studied in isolation under controlled laboratory conditions. In particular, measurements can now be made of particle size with unprecedented accuracy (sub-nanometre) and over a wide range of timescales (spanning from milliseconds to many days). The physical state of a particle can be unambiguously identified and its composition and phase can be resolved with a high degree of spatial resolution. In this review, we describe the advances made in our understanding of aerosol properties and processes from measurements made of phase behaviour, hygroscopic growth, morphology, vapour pressure and the kinetics of water transport for single particles. We also show that studies of the oxidative aging of single particles, although limited in number, can allow the interplay of these properties to be investigated. We conclude by considering the contributions that single particle measurements can continue to make to our understanding of the properties and processes occurring in atmospheric aerosol.