Differentiated primary human respiratory epithelial cells grown at air-liquid interface have become a widely used cell culture model of the human conducting airways. These cultures contain secretory cells such as goblet and club cells, which produce and secrete mucus. Here, we characterize the composition of mucus harvested from airway cultures of nasal and bronchial origin. We find that despite inter-donor variability, the salt, sugar, lipid, and protein content and composition are very similar between nasal and bronchial mucus. However, subtle differences in the abundance of individual components in nasal versus bronchial mucus can influence its antimicrobial properties. The ability of mucus to neutralize influenza A virus varies with the anatomical origin of the airway cultures and correlates with the abundance of triglycerides and specific sialylated glycoproteins and glycolipids.IMPORTANCERespiratory mucus plays an important role during the transmission and infection process of microbes in the human respiratory tract. In the case of influenza A virus, the mucus stabilizes the virions in infectious respiratory particles and droplets but hampers virus particles before they reach the respiratory epithelium through its physicochemical properties and the presence of sialylated decoy receptors. However, it is thus far not well understood which components of mucus mediate protection and inhibition. Our study now provides a comprehensive analysis of bronchial and nasal mucus from primary human airway cultures that can be used as a resource for future experimental designs and interpretations.

We describe a novel biosafety aerosol chamber equipped with state-of-the-art instrumentation for bubble-bursting aerosol generation, size distribution measurement, and condensation-growth collection to minimize sampling artifacts when measuring virus infectivity in aerosol particles. Using this facility, we investigated the effect of relative humidity (RH) in very clean air without trace gases (except ∼400 ppm CO2) on the preservation of influenza A virus (IAV) infectivity in saline aerosol particles. We characterized infectivity in terms of 99%-inactivation time, t99, a metric we consider most relevant to airborne virus transmission. The viruses remained infectious for a long time, namely t99 > 5 h, if RH < 30% and the particles effloresced. Under intermediate conditions of humidity (40% < RH < 70%), the loss of infectivity was the most rapid (t99 ≈ 15–20 min, and up to t99 ≈ 35 min at 95% RH). This is more than an order of magnitude faster than suggested by many previous studies of aerosol-borne IAV, possibly due to the use of matrices containing organic molecules, such as proteins, with protective effects for the virus. We tested this hypothesis by adding sucrose to our aerosolization medium and, indeed, observed protection of IAV at intermediate RH (55%). Interestingly, the t99 of our measurements are also systematically lower than those in 1-μL droplet measurements of organic-free saline solutions, which cannot be explained by particle size effects alone.

ABSTRACT The composition of respiratory fluids influences the stability of viruses in exhaled aerosol particles and droplets, though the role of respiratory organics in modulating virus stability remains poorly understood. This study investigates the effect of organic compounds on the stability of influenza A virus (IAV) in deposited droplets. We compare the infectivity loss of IAV at different relative humidities (RHs) over the course of 1 h in 1-µL droplets consisting of phosphate-buffered saline (without organics), synthetic lung fluid, or nasal mucus (both containing organics). We show that IAV stability increases with increasing organic:salt ratios. Among the various organic species, proteins are identified as the most protective component, with smaller proteins stabilizing IAV more efficiently at the same mass concentration. Organics act by both increasing the efflorescence RH and shortening the drying period until efflorescence at a given RH. This research advances our mechanistic understanding of how organics stabilize exhaled viruses and thus influence their inactivation in respiratory droplets.IMPORTANCEThis study investigates how the composition of respiratory fluids affects the stability of viruses in exhaled droplets. Understanding virus stability in droplets is important as it impacts how viruses spread and how we can combat them. We focus on influenza A virus (IAV) and investigate how different organic compounds found in lung fluid and nasal mucus protect the virus from inactivation. We demonstrate that the ratio of organics to salt in the fluid is an indicator of IAV stability. Among organics, small proteins are particularly effective at protecting IAV. Their effect is in part explained by the proteins’ influence on the crystallization of salts in the droplets, thereby shielding the viruses from prolonged exposure to harmful salt concentrations. Understanding these mechanisms helps us grasp how viruses sustain their infectivity over time in respiratory droplets, contributing to efforts in controlling infectious diseases.

ABSTRACT 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. IMPORTANCE It 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.

Influenza A virus (IAV) spreads through exhaled aerosol particles and larger droplets. Estimating the stability of IAV is challenging and depends on factors such as the respiratory matrix and drying kinetics. Here, we combine kinetic experiments on millimeter-sized saline droplets with a biophysical aerosol model to quantify the impact of NaCl on IAV stability. We show that IAV inactivation is determined by NaCl concentration, which increases during water evaporation and then decreases again when efflorescence occurs. When drying in air with relative humidity RH = 30%, inactivation follows an inverted sigmoidal curve, with inactivation occurring most rapidly when the NaCl concentration exceeds 20 mol/(kg H2O) immediately prior to efflorescence. Efflorescence reduces the NaCl molality to saturated conditions, resulting in a significantly reduced inactivation rate. We demonstrate that the inactivation rate k depends exponentially on NaCl molality, and after the solution reaches equilibrium, the inactivation proceeds at a first-order rate. Introducing sucrose, an organic cosolute, attenuates IAV inactivation via two mechanisms: first by decreasing the NaCl molality during the drying phase and second by a protective effect against the NaCl-induced inactivation. For both pure saline and sucrose-containing droplets, our biophysical model ResAM accurately simulates the inactivation when NaCl molality is used as the only inactivating factor. This study highlights the role of NaCl molality in IAV inactivation and provides a mechanistic basis for the observed inactivation rates.

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.

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.

ABSTRACT Respiratory mucus lining the airway epithelium constitutes an important first line of defense against infection with influenza A virus (IAV). Previous studies suggested that the inhibitory potential of mucus relies not only on the presence of decoy receptors but also on the dense meshwork blocking virus access to the epithelium independent of sialic acid. Here, we report for a panel of human and avian IAV isolates that susceptibility to neutralization by respiratory mucus varies in a strain-dependent manner. Our data reveal that viral neuraminidase (NA) activity inversely correlates with the mucus sensitivity of a given strain. As NA cleaves sialic acid from glycan chains and thus releases IAV from decoy receptor binding, our results imply that virus inhibition by mucus is mainly driven by decoy receptor binding of IAV. Other inhibitory properties of mucus, such as size exclusion, non-covalent interactions with mucus components, or the presence of immunomodulatory molecules, likely play minor roles in neutralizing IAV. Inhibition of NA activity using oseltamivir was sufficient to increase the sensitivity of a seasonal IAV strain to mucus during infection in a cell-based neutralization assay and in differentiated primary human airway epithelial cultures. Overall, our data highlight the importance of NA for the initiation of virus infection besides its major function in release, especially in overcoming the host defense mechanism of mucociliary clearance in the airway epithelium. Furthermore, strain-specific differences in the ability to penetrate respiratory mucus may be the result of host adaptation and can have consequences for the transmissibility and infectivity of circulating viruses. 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.

Enveloped viruses are prone to inactivation when exposed to strong acidity levels characteristic of atmospheric aerosol. Yet, the acidity of expiratory aerosol particles and its effect on airborne virus persistence has not been examined. Here, we combine pH-dependent inactivation rates of influenza A virus and SARS-CoV-2 with microphysical properties of respiratory fluids under indoor conditions using a biophysical aerosol model. We find that particles exhaled into indoor air become mildly acidic (pH ≈ 4), rapidly inactivating influenza A virus within minutes, whereas SARS-CoV-2 requires days. If indoor air is enriched with non-hazardous 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 seconds.Conversely, unintentional removal of volatile acids from indoor air by filtration may elevate pH and prolong airborne virus persistence. The overlooked role of aerosol pH has profound implications for virus transmission and mitigation strategies.One Sentence SummaryRespiratory viruses are sensitive to aerosol pH, an unidentified factor in the mitigation of airborne virus transmission

Microbial communities are found throughout the biosphere, from human guts to glaciers, from soil to activated sludge. Understanding the statistical properties of such diverse communities can pave the way to elucidate the common mechanisms ...Multiple ecological forces act together to shape the composition of microbial communities. Phyloecology approaches—which combine phylogenetic relationships between species with community ecology—have the potential to disentangle such forces but are often ...

Light-absorbing organic atmospheric particles, termed brown carbon, undergo chemical and photochemical aging processes during their lifetime in the atmosphere. The role these particles play in the global radiative balance and in the climate system is still uncertain. To better quantify their radiative forcing due to aerosol–radiation interactions, we need to improve process-level understanding of aging processes, which lead to either “browning” or “bleaching” of organic aerosols. Currently available laboratory techniques aim to simulate atmospheric aerosol aging and measure the evolving light absorption, but they suffer from low sensitivity and precision. This study describes the use of electrodynamic balance photophoretic spectroscopy (EDB-PPS) for high-sensitivity and high-precision measurements of light absorption by a single particle. We demonstrate the retrieval of the time-evolving imaginary part of the refractive index for a single levitated particle in the range of 10−4 to 10−5 with uncertainties of less than 25 % and 60 %, respectively. The experimental system is housed within an environmental chamber, in which aging processes can be simulated in realistic atmospheric conditions and lifetimes of days to weeks. This high level of sensitivity enables future studies to explore the major processes responsible for formation and degradation of brown carbon aerosols.

Formation of organic aerosol by oxidation of gas phase compounds has been intensely studied, and is much better understood than the aerosol ageing transformations during the lifetime of organic aerosol. Aerosol ageing influences how those aerosol particles affect climate and human health and is still not well constrained in current models. Photochemistry in the condensed phase is an important mechanism responsible for ageing of organic aerosol. In the lower troposphere, where UV light intensity with sufficiently low wavelength to directly photolyze aerosol components is low, indirect photochemistry (catalyzing redox processes of non-absorbing molecules) is especially relevant. Recently we studied transition metal complex photochemistry in single particles levitated in an electrodynamic balance. In particular, we investigated the aqueous iron(III)-citrate/citric acid system and found that irradiation at 473 nm led to rapid and significant degradation of the citric acid. Up to 80% of the initial particle mass was partitioned to the gas phase with the degradation rate depending on kinetic transport limitations of oxygen. These kinetic limitations arise are influenced strongly by the relative humidity dependence of particle viscosity where water acts as a plasticizer. Here we will report on photochemical degradation experiments adding various salts in different (ammonium sulfate, ammonium bisulfate, etc.) to the reference system iron(III)-citrate/citric acid. Preliminary experiments suggest that pH of the aerosol particle influences the degradation rate in this system significantly.

The possible use of organic particle emissions as indicators of smoldering fires at low temperatures (early stages, <150 degrees C) is explored in laboratory experiments. Samples consisting of wood pellets were heated under controlled atmosphere. The sample temperature was slowly ramped, mimicking the spontaneous, slow temperature increase during self-heating in organic materials. As the sample temperature reached 90-100 degrees C, a two-orders-of-magnitude increase in the number concentration of sub-micrometer particulate matter was measured under both air and nitrogen atmosphere. A detailed analysis of their chemical composition indicated that the particles formed through evaporation of low-volatile organic compounds followed by condensation downstream of the heated volume. The increase in aerosol concentration precedes any significant increase in measured CO and CO2 concentration in both time and temperature. Our results suggest that the submicron particle concentration could be considered as an indication of the stages that lead to smoldering fires.

The contribution of light absorption by brown carbon aerosols to the Earth’s energy balance still poses a significant uncertainty in our understanding of climate forcing. As a result, one of the main open questions regarding organic aerosols in atmospheric chemistry is related to the formation and degradation of light-absorbing compounds during aging processes. Towards this goal, we explore the use of photophoresis for high sensitivity measurements of light absorption by a single levitated particle in an experimental setup that facilitates realistic atmospheric gas concentrations and aging time.Photophoresis occurs when the surface of an illuminated, light-absorbing particle is unevenly heated relative to its surroundings. The temperature difference between the illuminated and the ‘dark’ side of the particle results in an uneven momentum transfer from colliding gas-phase molecules. This leads to a net photophoretic force, acting on the particle in the direction of the momentum transfer gradient. The photophoretic force is related to the complex refractive index of the particle and to its size parameter through the distribution of internal electric fields. As such, it may lead to a net force away from (positive) or towards (negative) the light source.Using this phenomenon, we were able to retrieve the imaginary part of the complex refractive index (k) of a single particle levitated in an electrodynamic balance (EDB) at 473 nm wavelength. Extremely low values of k from 10-4 to 10-5 were successfully retrieved with uncertainty of less than 35% during a photo-bleaching experiment of a slightly absorbing organic particle used as a model for atmospheric brown carbon.An advantage of the EDB is that heterogeneous chemistry and photochemistry experiments are performed on a single particle that is levitated for days and weeks allowing for realistic atmospheric gas concentrations and aging times. In such experiments, measurements of the particle’s light absorption properties using photophoresis would add valuable information on the evolution of light absorption by the aging of organic aerosols.A future study will implement this approach in an EDB-MS system where the EDB will be coupled with a soft ionization mass spectrometer. This will allow the identification of the molecular species responsible for light absorption as it evolves.

The wavelength-dependence of the complex refractive indices (RI) in the visible spectral range of secondary organic aerosols (SOA) are rarely studied, and the evolution of the RI with atmospheric aging is largely unknown. In this study, we applied a novel white light-broadband cavity enhanced spectroscopy to measure the changes in the RI (400-650 nm) of β-pinene and p-xylene SOA produced and aged in an oxidation flow reactor, simulating daytime aging under NO x-free conditions. It was found that these SOA are not absorbing in the visible range, and that the real part of the RI, n, shows a slight spectral dependence in the visible range. With increased OH exposure, n first increased and then decreased, possibly due to an increase in aerosol density and chemical mean polarizability for SOA produced at low OH exposures, and a decrease in chemical mean polarizability for SOA produced at high OH exposures, respectively. A simple radiative forcing calculation suggests that atmospheric aging can introduce more than 40% uncertainty due to the changes in the RI for aged SOA.

The radiative effects of biomass-burning aerosols on regional and global scales can be substantial. Accurate modeling of the radiative effects of smoke aerosols requires wavelength-dependent measurements and parameterizations of their optical properties in the UV and visible spectral ranges along with improved description of their chemical composition. To address this issue, we used a recently developed approach to retrieve the time-and spectral-dependent optical properties of ambient biomass-burning aerosols from 300 to 650 nm wavelengths during a regional nighttime bonfire festival in Israel. During the biomass burning event, the overall absorption at 400 nm increased by about 2 orders of magnitude, changing the single scattering albedo from a background level of 0.95 to 0.7. Based on the new retrieval method, we provide parameterizations of the wavelength-dependent effective complex refractive index from 350 to 650 nm for freshly emitted and slightly aged biomass-burning aerosols. In addition, PM2.5 filter samples were collected for detailed offline chemical analysis of the water-soluble organics that contribute to light absorption. Nitroaromatics were identified as major organic species responsible for the increased absorption at 400 to 500 nm. Typical chromophores include 4-nitrocatechol, 4-nitrophenol, nitrosyringol, and nitroguaiacol; oxidation-nitration products of methoxyphenols; and known products of lignin pyrolysis. Our findings emphasize the importance of both primary and secondary organic aerosols from biomass burning in absorption of solar radiation and in effective radiative forcing.Plain Language Summary The radiative effects of biomass-burning aerosols on regional and global scales are substantial. Accurate modeling of the radiative effects of smoke aerosols requires wavelength-dependent measurements and parameterizations of their optical properties in the UV and visible spectral ranges along with improved description of their chemical composition. To address this issue we used a recently developed approach to retrieve the time-and spectral-dependent optical properties of the ambient aerosol from 300 to 650 nm wavelengths and a high-resolution mass spectrometry analysis of fine particulate matter. We found a significant increase in aerosol light absorption in the UV-Vis spectral range which is correlated to high levels of nitroaromatic compounds identified in the water-soluble extracts of the filter samples. Additionally, for further applications of our results in radiative transfer models, we provide parameterizations of the wavelength-dependent effective complex refractive index from 350 to 650 nm for freshly emitted and aged biomass-burning aerosols.

Lag Ba'Omer, a nationwide bonfire festival in Israel, was chosen as a case study to investigate the influence of a major biomass burning event on the light absorption properties of atmospheric brown carbon (BrC). The chemical composition and optical properties of BrC chromophores were investigated using a high performance liquid chromatography (HPLC) platform coupled to photo diode array (PDA) and high resolution mass spectrometry (HRMS) detectors. Substantial increase of BrC light absorption coefficient was observed during the night-long biomass burning event. Most chromophores observed during the event were attributed to nitroaromatic compounds (NAC), comprising 28 elemental formulas of at least 63 structural isomers. The NAC, in combination, accounted for 50-80% of the total visible light absorption (>400 nm) by solvent extractable BrC. The results highlight that NAC, in particular nitrophenols, are important light absorption contributors of biomass burning organic aerosol (BBOA), suggesting that night time chemistry of •NO3 and N2O5 with particles may play a significant role in atmospheric transformations of BrC. Nitrophenols and related compounds were especially important chromophores of BBOA. The absorption spectra of the BrC chromophores are influenced by the extraction solvent and solution pH, implying that the aerosol acidity is an important factor controlling the light absorption properties of BrC.

The multi-pass photoacoustic spectrometer (PAS) is an important tool for the direct measurement of light absorption by atmospheric aerosol. Accurate PAS measurements heavily rely on accurate calibration of their signal. Ozone is often used for calibrating PAS instruments by relating the photoacoustic signal to the absorption coefficient measured by an independent method such as cavity ring down spectroscopy (CRD-S), cavity-enhanced spectroscopy (CES) or an ozone monitor. We report here a calibration method that uses measured absorption coefficients of aerosolized, light-absorbing organic materials and offer an alternative approach to calibrate photoacoustic aerosol spectrometers at 404 nm. To implement this method, we first determined the complex refractive index of nigrosin, an organic dye, using spectroscopic ellipsometry and then used this well-characterized material as a standard material for PAS calibration.

To reduce uncertainty associated with aerosol impacts on atmospheric radiative transfer and heating, high accuracy measurements of the fundamental absorption properties of atmospheric particles are needed. Photoacoustic spectroscopy (PAS) is increasingly being applied to this problem on account of its potential to offer significantly higher accuracy than traditional techniques. However, the accuracy of the PAS technique depends strongly on the efficacy of its calibration. A common approach used to calibrate aerosol PAS instruments is to use absorption by gaseous ozone. The study of Bluvshtein compares this calibration approach to a new particle-based method. For C1