Sensitivity of aerosol-borne Influenza A Virus to gaseous nitric acid and carbon dioxide

Sensitivity of aerosol-borne Influenza A Virus to gaseous nitric acid and carbon dioxide | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sensitivity of aerosol-borne Influenza A Virus to gaseous nitric acid and carbon dioxide Ghislain Motos, Celine Terrettaz, Jun Zhang, Beiping Luo, Irina Glas, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9020228/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Transmission via aerosols is an important route of transmission of influenza A virus (IAV) in humans, whose control remains a global challenge. After exhalation, aerosol particles undergo rapid physicochemical changes that lead to substantial aerosol pH variations, which can impair the infectivity of IAV. Here, we investigate the aerostability of IAV, a key step of the transmission chain, using an aerosol chamber that allows assessing the influence of air composition on aerosol pH. At 55% relative humidity, we observe high sensitivity of IAV to nitric acid concentrations as low as 5 ppb, i.e., 400 times lower than the permissible exposure limit, with the time required to achieve a 99% reduction in active viruses being halved. In contrast, even an increase in carbon dioxide concentration to 250,000 ppm has a negligible effect on IAV. Our results underscore the crucial role of trace acids in transmitting aerosolized respiratory viruses, including strong synergies with air humidification. Biological sciences/Microbiology/Environmental microbiology/Air microbiology Health sciences/Diseases/Infectious diseases/Influenza virus Biological sciences/Biological techniques/Mass spectrometry Figures Figure 1 Figure 2 Figure 3 1. Introduction Influenza A virus (IAV) poses a social and environmental burden, affecting humans, birds, and mammals through seasonal epidemics and pandemics. The World Health Organization (WHO) recognizes IAV as a major public health challenge and is actively preparing for a future influenza pandemic 1 . It is widely accepted that IAV transmission also occurs via the air 2 , 3 , 4 . Tools to prevent or mitigate the spread of IAV include vaccination and antiviral treatments, the use of personal protective equipment, ventilation, ultraviolet irradiation, air filtration, and isolation of infected patients. However, these measures are either insufficiently accepted or difficult to implement at the level of the general population. To achieve a better understanding of how influenza spreads, further work is needed across several research areas, such as epidemiology, immunology, building ventilation, transmission modelling, but also studies on the role of environmental factors regarding the ability of infectious respiratory particles to maintain their infectivity and ultimately transmit the disease. The physicochemical evolution of exhaled infectious respiratory particles is an important aspect for understanding the transmission of infectious viruses, as conditions of salinity, acidity, and other factors change greatly after exhalation and influence the inactivation of viruses. Yang and Marr 5 were the first to hypothesize that concentration changes in H + ions during particle evaporation after exhalation could lead to a reduction in pH. Conversely, experiments by Oswin et al. 6,7 using virus transport media concluded that exhaled particles would become alkaline due to CO 2 outgassing from the bicarbonate buffer. Again, conversely, Klein et al. 8 and Luo et al. 9 argued, based on laboratory experiments and biophysical modeling, that exhaled submicron aerosol particles can quickly become acidic due to the condensation of trace acids from the ambient air. Irreversible structural changes in viral surface proteins have recently been shown to be the acid-induced inactivation mechanism for IAV and SARS-CoV-2 10,11 , but it remains unclear which aerosol particle sizes are responsible for transmission and how fast they take up acids from the ambient air. Particles indoors may have lower acidity levels than outdoors, as trace acids and bases concentrations are different 12 . Controlled exposure of virus-laden aerosol particles to different levels of gas phase compounds allows to assess the sensitivity of viruses to pH variations, compared to the effects of other environmental factors, and study potential interactions and synergies between factors. To date, this has been done in two studies, both of which for SARS-CoV-2 in large (50 µm initial diameter) particles 13 , 14 . The results indicated increased aerostability with increased CO 2 concentration, which the authors explained by the reduction in CO 2 outgassing required for the particles to reach equilibrium with the gas phase, thus minimizing pH variations. Further, they found that the addition of nitric acid vapours slightly enhanced virus stability. Here, we investigate the effect of gas phase composition on IAV in submicrometer particles (dry size) whose composition mimics particles exhaled during breathing. Using a medium that mimics lung fluid, we first show evidence of particle acidification due to the action of gaseous nitric acid down to concentrations of a few ppb. By injecting controlled amounts of nitric acid in the aerosol chamber of the LAPI BREATH facility (Laboratory of Atmospheric Processes and their Impacts - Bioaerosol Research & Environmental Airborne Transmission Hub 15 ), we then observe how gas phase concentrations correlate with IAV aerostability. We also carry out experiments with variable levels of carbon dioxide, a gas that accumulates inside occupied buildings and is widely used as an indicator of pathogen transmission risk. 2. Material and Methods 2.1 Experimental setup and procedure. We utilize the LAPI BREATH facility (Fig. 1 ), described in detail by Motos et al. 15 . Briefly, the facility consists of a 1.6 m 3 polytetrafluoroethylene (PTFE) chamber, in which we inject IAV-containing particles using a nebulizer based on bubble-bursting (the sparging liquid aerosol generator, SLAG) which are sampled over the course of an experiment with a condensational growth instrument, the BioSpot-VIVAS, and subsequently analyzed via virus titration and genomic copies quantification. Chamber temperature, relative humidity (RH) and gas phase composition are measured and controlled. The study of Motos et al. 15 provides the aerosol particle size distribution generated by the SLAG, the effect of aerosolization on IAV inactivation, and the rate of particle and virus losses to the chamber walls. PBS was chosen as the aerosol medium for its minimal complexity, allowing isolation of the effect of particle salinity, modulated by RH, on IAV inactivation. The effect of adding organic compounds to the medium was also studied. Building upon these findings, we increase the level of complexity to better mimic human exhalation by moving to a physiologically more relevant medium, synthetic lung fluid (SLF; see Supplementary Methods S1). The H1N1 IAV strains used in this study are A/Puerto Rico/8/34 (PR8) and A/Netherlands/602/2009 (Neth/09). The aerosolization and sampling procedures followed for the aerosol exposure experiments in the present study are detailed in Motos et al. 15 . Briefly, SLF powder is resuspended in the appropriate volume of autoclaved Milli-Q water and kept at 4°C for a maximum of 14 days. SLF solution is diluted 50 times in autoclaved Milli-Q water to generate the medium; 22 ml of which is spiked with IAV to a targeted titer of ~ 10 8 -10 9 PFU/ml for PR8 and ~ 10 7 -10 8 PFU/ml for Neth/09 and placed in a 100-ml screw-capped container. To verify the concentration of the viral inoculum, 25 µl are sampled into 2.5 ml PBSi (PBS for infection; see Supplementary Methods S1) and kept in the fridge during the experiment. We aerosolize IAV for 1 minute with the SLAG at a flow rate of 30 liters per minute. Samples are collected in a 35 x 10 mm Petri dish filled with 2.5 ml PBSi every 5 minutes (4 minutes for sampling and 1 minute for changing the Petri dish in the BioSpot-VIVAS) for the first 35 minutes of the experiment. Three additional samples are collected between 40–45 min, 50–55 min and 60–65 min. Once collected, samples are transferred into 50-ml centrifuge tubes and stored on ice until the end of the experiment. Finally, the samples are aliquoted in 96-well plates and 1.5 ml tubes and stored at -20° C. Samples are enumerated for infectious titer and genome copy concentration within 14 days. Virus and SLF preparation as well as titration of infectious viruses and quantification of viral genomes follow the procedures from Schaub et al. 16 and Luo et al. 9 and are detailed in Supplementary Methods S1. 2.2 Metric for displaying data. Following Motos et al. 15 , we express our infectivity results in terms of 99% IAV inactivation time ( t 99 ), i.e., the time required for a 2-log inactivation, assuming first-order inactivation kinetics. Briefly, we fit infectivity data assuming first-order kinetics, using the midpoint of sampling intervals as time values. The fits are then corrected for physical losses on the chamber walls measured as the decrease in the number of viral genome copies over time by digital polymerase chain reaction (dPCR). The t 99 metric provides infectivity results independent of the time of exposure and related to other timescales of importance for transmission risk, such as particle settling time or the room ventilation/filtration timescale. 2.3 Particle acidification procedure. To investigate the impact of aerosol particle acidification on the conservation of IAV infectivity, we precondition the chamber air by adding nitric acid prior to injection of infectious respiratory particles. Condensation of acid vapors to the particle phase reduces the pH of the particles, which in turn affects IAV infectivity. The LAPI BREATH is equipped with an acid injection system, based on the evaporation of an acid solution (Fig. 1 ). Once the chamber air has been conditioned to the desired RH, the airflow is directed to an Erlenmeyer flask containing 250 mL of 3% nitric acid through a tight-fitting cap, with a flow rate of 20 liters per minute; the outlet flow is directed to the chamber via the injection port. The choice of nitric acid for the aerosol particle acidification experiments is made after a rigorous selection of the ideal compound, based on 4 criteria: solubility, volatility, acid strength and occupational exposure limit value. High solubility at 20 ° C is crucial because the injection system we use is based on evaporation. The volatility of the compound, i.e., its propensity to remain in the gas phase or to condense to the particle phase, must be neither too low, in order to avoid adsorption on the PTFE lines and walls of the chamber before condensing on the aerosol particles, nor too high, as this would cause the acid to remain in the gas phase without affecting the particles. We estimated the volatility of acids based on their Henry’s law coefficient, following the approach of Liu et al. 17 . This study showed a relationship between this characteristic of volatile organic compounds and small polar compounds and their instrument response time, or in other words, the delay times in measurements caused by the partitioning of these compounds between the gas phase and the surface of the inlet tubing used for measurement. The third criterion, acid strength, indicates how effectively the condensed phase of the acid can reduce particle pH. Finally, the occupational exposure limit value, provided by the Health Institute for Occupational Safety and Health (NIOSH), provides information on the risk of exposure to the compound for human health. Based on this selection, nitric acid, a strong water miscible acid (pKa = -1.38) with intermediate volatility (Henry’s law constant of 89,166 M/atm 18 , corresponding to a response time of approximately 335 s detected by a mass spectrometer) and a safety limit of 2 ppm averaged over a 10-hour workshift 19 , appears to be the most suitable acid for our study. The fact that nitric acid is ubiquitous in the atmosphere, and a widely used, well characterized acid in atmospheric studies is a further argument in favor of its use. 2.4 Gas phase composition measurements. Gas phase nitric acid is detected by a VOCUS chemical ionization mass spectrometer with iodide-adduct ions (hereafter VOCUS, Tofwerk AG, Thun, BE, Switzerland). It is equipped with AIM (adduct ionization mechanism) ion-molecule reactor (IMR), allowing real-time monitoring of volatile compounds in the ppt range 20 with little fragmentation and memory effect. We connect the instrument to the chamber directly through a sealed opening in one of the chamber walls, with PTFE tubing as short as possible to minimize nitric acid losses. To avoid sampling infectious viruses by the VOCUS, we measure the nitric acid concentration before and after each experiment and interpolate the data to estimate the concentration during the experiments. The decay rate of nitric acid concentration with time during an experiment is characterized based on a test experiment with virus-free SLF. Details concerning the calibration of the Vocus can be found in Supplementary Fig. S1 and Methods S2. Carbon dioxide is injected directly into the aerosol chamber from the laboratory gas network supplied via PTFE tubing connected to the chamber injection port. CO 2 concentration is measured using an Aranet4 household sensor (Aranet, Riga, Latvia) attached to the stainless-steel tubes connected to the chamber sampling port. In experiments carried out without injection of nitric acid or carbon dioxide, the gas phase only includes what is hereafter referred to as “filtered air”, namely compressed outside air captured on the building roof, filtered for particles by a high-efficiency particle air (HEPA) filter and for organic substances by an activated carbon filter. In experiments conducted without the addition of nitric acid or carbon dioxide, the chamber is filled with compressed outdoor air collected on the roof of the building, previously HEPA-filtered for particles and activated carbon-filtered for organic substances. The concentration values measured in this filtered air are between 390 and 450 ppm CO 2 and mass spectrometric measurements of the HNO 3 background concentration in the chamber show values between 0.03 and 0.05 ppb. This background is irrelevant for our experiments with ≥ 5 ppb HNO 3 , but is important for our experiments without HNO 3 addition, which we will revisit in the discussion. 2.5 Particle-phase composition measurements. High-resolution time-of-flight aerosol mass spectrometer (HTOF-AMS, thereafter “AMS”, Aerodyne Research, Inc.) with a mass resolution of ∼ 2000 over the range of m/z 100 to m/z 400 is deployed for online, non-refractory aerosol particle characterization. Aerosols are continuously sampled through a PM 2.5 aerodynamic lens. The ionization efficiency of nitrate is calibrated with 300 nm ammonium nitrate particles against simultaneous scanning mode particle sizer (SMPS, DMA model TSI long and CPC model 3772 TSI Inc., Shoreview, MN, USA) measurements to convert ion counts to particle mass concentration. The relative ionization efficiency of ammonium, sulfate, and sodium is calibrated in the same approach. We connect a HEPA filter to the inlet of the AMS and sampled air for 15 minutes to determine the following limits of detection (in µg/m 3 ): 0.013 for SO 4 , 0.0034 for NO 3 , 0.00096 for NH 4 and 0.35 for NaCl + . A detailed description of the AMS can be found in DeCarlo et al. 21 . 2.6 pH retrievals. To observe changes in pH caused by aerosol particle exposure to nitric acid, we perform two chamber experiments, one with and one without gaseous nitric acid. Virus-free SLF is aerosolized in the chamber, while nitric acid in the gas phase and organic and inorganic compounds in the particle phase are continuously measured (together with chamber temperature and RH, respectively 24.5°C and 55% RH with negligible variations). We retrieve the pH value of particles based on the thermodynamic equilibrium of gas- and particle-phase compounds measured simultaneously by VOCUS and AMS. While we used a biophysical model (Respiratory Aerosol Model, ResAM) in our previous work 9 , the partitioning of HNO 3 between the gas and particle phases due to the small size of the aerosol particles quickly leads to thermodynamic equilibrium, which is also confirmed by VOCUS and AMS measurements. This greatly simplifies pH determination. First, we utilize the ISORROPIA-Lite thermodynamic model, which calculates the composition and phase state of NH 4 + -SO 4 2− -NO 3 − -Cl − -Na + -organics-water solutions, allowing for metastable aerosol states and assuming gas-particle equilibrium. The pH is then determined from pH = log 10 ( a H+ ) and a H+ = H·p HNO3 / a NO3− , where a H+ is the H + activity, H is the Henry’s Law constant for HNO 3 (g) ↔ H + (l) + NO 3 − (l) and a NO3− = c NO3− ⋅ γ NO3− is the nitrate activity. Detailed descriptions of this model can be found in Nenes et al. 22 and Fountoukis and Nenes 23 . Second, we apply the S curves method 24 , 25 , 26 , which relies on a theoretical relationship between nitrate partitioning, water content and pH and can be visualized using sigmoid-shaped curves calculated from solubility and dissociation of nitrate in water, including activity coefficients. 3. Results We first describe the dependence of IAV aerostability on air composition and then present evidence of aerosol particle acidification by detailing pH measurements of particles nebulized in the chamber in the presence or absence of nitric acid. 3.1 Infectivity experiments in filtered, nitric acid-enriched and carbon dioxide-enriched air. The inactivation of IAV in aerosolized SLF medium is measured over a RH range from 10% to 95% RH (Fig. 2 A) and compared with previous data obtained using the same experimental setup with PBS and sucrose-supplemented PBS as media (Supplementary Fig. S2). In general, the infectivity decay measured in these three media are very similar (Supplementary Fig. S2) and much less variable than many dependencies reported in the literature 15 . Infectivity decreases monotonically with increasing RH in SLF medium, with a decline of more than one log level in t 99 between 10% and 95% RH. This behavior is probably due to the decreasing viscosity of SLF with increasing humidity. While we found a slight minimum in t 99 as a function of RH in PBS as a medium (“U-shape”), this was hardly present in PBS+sucrose, which we attributed to the protective effect of the organics 15 . With SLF, this minimum disappears completely. In the following, we use SLF as the medium most similar to body fluids to investigate the influence of air composition on IAV infectivity. Starting with filtered air (0.03–0.05 ppb HNO3 and 420–440 ppm CO 2 ), we investigate the influence of increasing the concentration of each compound on the aerostability of an IAV strain PR8 (H1N1), which was also used in Motos et al. 15 . Nitric acid inactivates IAV very effectively, as shown by the cascading anticorrelation between t 99 and HNO 3 concentration (Fig. 2 ). At 55% RH, the sensitivity of IAV to gaseous HNO 3 is high: 5 ppb reduces t 99 by almost a factor of 2, and 30 ppb by almost an order of magnitude. It should be noted that this latter concentration remains 67 times lower than the 8-hour time-weighted average threshold limit value (TWA TLV) for HNO 3 , 2 ppm, as shown by the green vertical bar in Fig. 2 B. However, this sensitivity shows a clear dependence on RH. Under dry conditions (25% RH), 30 ppb HNO 3 only reduces t 99 by 25%, while 80% RH leads to results below the limit of quantification (LOQ) in the PFU titer. We compare the sensitivity of IAV PR8 to acidic conditions with that of the more clinically relevant Neth/09 strain and find that HNO 3 concentrations of 30 ppb at 25% RH and 9 ppb at 55% RH lead to infectivity below the LOQ, although reference measurements (without acid injection) show similar t 99 values as for the PR8 strain (Fig. 2 A). Injecting large concentrations of CO 2 into the chamber (4000 and 6500 ppm, values attainable in poorly ventilated indoor environments but which surround the 8-hour TWA TLV of 5000 ppm; see Fig. 2 B) bears no impact on IAV inactivation at low and high RH, but provides some limited protection at 55% RH, with a near doubling of t 99 values compared to the reference case. We also performed an experiment with an extremely high CO 2 level (about 250000 ppm, i.e., 25% v/v CO 2 in air), resulting in inactivation kinetics comparable to the 4500 and 6500 ppm cases (Fig. 2 B). Overall, our results show that IAV infectivity is very sensitive to the concentration of nitric acid present in the air, moderated by relative humidity, but largely insensitive to carbon dioxide concentration. 3.2 Evidence of particle acidification due to exposure to gaseous nitric acid. To confirm that the inactivation of IAV described in Section 3.1 is indeed caused by a low pH of the aerosol particles resulting from the uptake of nitric acid, we performed two experiments, which differ solely by the injection of nitric acid as chamber preconditioning before the aerosolization of virus-free SLF. The nitric acid concentration (~ 30 ppb) and the RH (55%) are chosen to reproduce the conditions under which IAV inactivation occurs rapidly ( t 99 < 4 min; Fig. 2 A). Figure 3 indicates that the presence of nitric acid vapour results in very low levels of nitrate in the particles (the particle-phase NO 3 mass fraction drops from ~ 0.6 to ~ 0.002 based on gas- and particle-phase nitrate measurements; see panels C and G). Since the particles emitted by the SLAG are in the submicron range after drying 15 , they quickly reach equilibrium with the gaseous nitric acid, leading to a rapid reduction in particle pH from approximately 5 to 2. Note that direct pH measurements of our diluted SLF solution using a pH-meter provided a value of approximately 6.4, slightly higher than the particle pH retrieved in the absence of nitric acid (after aerosolization in the chamber; Fig. 3 D). 4. Discussion 4.1 IAV inactivation in SLF particles. In addition to a bubble-bursting nebulizer (SLAG) simulating aerosol generation in the alveolar region, a chamber reproducing an indoor environment with sufficient dimensions to account for aerosol deposition, and a virus sampler reproducing the liquid-to-liquid deposition of aerosol particles in the respiratory tract of a host, our experimental setup now also includes a medium reproducing the essential properties of the respiratory tract fluid. The main limitations to a perfect imitation of the natural situation concern the absence of very large particles produced by the upper respiratory tract (larynx, tongue and lips) that the SLAG cannot generate, and the relatively small amount of bicarbonates present in our SLF compared to body fluids (which helps avoiding carbonate precipitation, but in small particles equilibrium is attained readily irrespective of the initial carbonate concentration by fast outgassing of CO 2 ). The results obtained with LAPI BREATH using SLF as medium indicate that the aerostability of IAV decreases with increasing RH. This clearly contradicts the WHO rating that high humidity prolongs airborne virus infectivity, without however formulating guidelines on RH management due to the lack of relevant data 27 . While RH levels above ~ 80% should be avoided indoors, as they promote the growth of mold, the risk of IAV transmission is maximized in dry environments. These results confirm previous findings 28 , 29 , 30 but disagree with recent studies that found IAV infectivity to be independent of RH 31,32 . The different media, air compositions, and virus strains used in these studies, which may contribute to different experimental outcomes, could explain this discrepancy. 4.2 Effect of air composition on IAV infectivity. While the dependence of virus aerostability on RH and medium composition has been studied for decades, often with contradicting results 15 , its dependence on gas phase compounds has only recently attracted scientific interest 6 , 8 , 9 , 13 , 14 , 33 . In particular, acids and bases are known to affect indoor air quality 12 . They are often semi-volatile, partition to aerosol particles exhaled by people, and affect the pH and potentially the infectivity of viruses contained in these particles. Luo et al. 9 calculated the evolution of particle pH and virus inactivation after exhalation using ResAM, which they constrained by the thermodynamic and diffusion kinetic properties of SLF determined from levitated SLF droplets and by pH-dependent IAV inactivation rate from plaque measurements. They showed that the condensation of trace acids typically present in indoor air is sufficient to lower the pH of fine aerosol particles to pH 4 within minutes, leading to rapid inactivation of IAV but not SARS-CoV-2. They found that enriching the air with non-hazardous levels of HNO 3 could effectively reduce the aerostability of both viruses. When 50 ppb of HNO 3 was added to the air, they calculated that t 99 fell to a few seconds. Our results here provide experimental confirmation of the general trend of these model results for IAV inactivation and indicate that IAV infectivity is indeed anticorrelated to the HNO 3 concentration and that this anticorrelation is modulated by RH. At 55% RH, our pH retrieval (pH = log 10 ( H·p HNO3 /( c NO3− ⋅ γ NO3− )), see Section 2.6) from the measured p HNO3 and c NO3− shows that SLF particles become slightly acidic even when no nitric acid is injected into the chamber (~ pH 5, Fig. 3 D), which is due to the background level of HNO 3 of ~ 0.03 ppb (Fig. 3 B). When HNO 3 is increased to 30 ppb (Fig. 3 F), c NO3− increases by about a factor of 3 (Fig. 3 A,E), pH decreases to about 2 (Fig. 3 H), and the corresponding inactivation time t 99 decreases from 50 to about 6 minutes (brown and green circles in Fig. 2 A). At low RH of 25, the SLF becomes much more viscous than at higher RH and the HNO 3 uptake is limited by liquid phase diffusion (see Fig. S9D of Luo et al. 9 ). This may explain why t 99 at 25% RH is by 1–2 orders of magnitude longer than at higher RH. The intermediate levels with 5 ppb and 17 ppb HNO 3 and the concomitant monotonous reduction in t 99 at 55% RH indicate the increasing impact of acidity on IAV. In contrast to Haddrell et al. 13 , we find no evidence of alkaline states to play a role. They found an enhancement of aerostability of SARS-CoV-2 after the addition of nitric acid to their moisture injection system, which they explained as a transition from alkaline to neutral pH. However, they used aerosol particles with much larger size (~ 14-micron equilibrated diameter compared to 250 nm diameter in the peak of the equilibrated volume distribution in the present study; see Motos et al. 15 ). The characteristic time for uptake of ambient trace gases by freshly emitted or exhaled particles scales roughly with the square of the diameter. Therefore, for our small particles the HNO 3 uptake and acidification take only seconds and alkalinity due to the loss of bicarbonate from the particles cannot prevail 8 . Finally, we note that the agreement of our experiments with Luo et al. 9 is only qualitative. They reported t 99 < 3 s for SLF droplets smaller than 0.5 µm at 50% RH and 50 ppb HNO 3 . In the present experiments, the inactivation time is much longer, namely t 99 ~ 360 s for 55% RH and 35 ppb HNO 3 . This can be due to a small fraction of large particles (radius > 2 µm), for which the uptake of HNO 3 takes more time than for smaller particles, also due to slow liquid phase diffusion of nitrate ions in SLF 9 . Precise measurements of the entire size distribution is a topic of further investigation. 4.3 Perspectives and final remarks. Dry environments concentrate the risk of indoor IAV transmission by increasing the stability of the virus in airborne particles. Assuming that the LAPI BREATH is able to reproduce the conditions that lead to the transmission of IAV, mainly in terms of aerosol particle size and composition, this translates in our measurements to t 99 values that are a factor 3 to 5 higher at 25% RH compared to 55%. Furthermore, t 99 shows a systematic dependence on the HNO 3 concentration in indoor air. Could this contribute to future epidemic risk prevention? A variety of pharmaceutical and non-pharmaceutical risk reduction strategies have been investigated 34 , 35 . Ventilation with outdoor air appears to be the simplest and most effective of all measures, but its application becomes problematic at low outdoor temperatures. Next, air filtration and ultraviolet disinfection (especially far-UVC) have been shown effective, but they respectively influence aerosol pH 9 and increase ozone and ultra-fine particle levels 36 , so that advantages and disadvantages must also be evaluated 37 . Finally, there have been recent advances in research into the inactivation of IAV through exposure to gaseous species via the oxidative pathway, e.g., through the use of solution sprays containing hypochlorous acid 38 , 39 . In the same way that chlorine, a pollutant that is toxic to human health at high aqueous concentrations, is added to drinking water to combat infectious viruses and bacteria, one could consider disinfecting the air with nitric acid, under the strict supervision of experts, during acute transmission risk events such as transport and large indoor gatherings. Although we do not currently recommend injecting nitric acid into buildings, we advocate research into air acidification for disinfection in order to combat airborne pathogens by means of very low acid concentrations considered safe for human health. To this end, studies such as the one presented here should be extended to other virus strains and other environmental conditions, with particular attention paid to the cross-sensitivity of various factors influencing the stability of IAV, as shown here for the interaction between RH and acidification. A combination of laboratory experiments and process-oriented modeling can show whether airborne acids could contribute to future epidemic prevention strategies. Declarations Competing interests The authors declare no competing interests. Author contribution GM wrote the original draft. GM, CT, JZ, KV, FC and AB performed formal analysis. GM, CT, BL, IG, MP, AB, UK, SS, TP, TK and AN discussed the methodology. SS, TP, TK and AN conceptualized the study and acquired funding. Acknowledgments We acknowledge the SNSF through the support of the IVEA (CRSII5_189939) and AirTRAC (IC00I0-228106) projects, and the support by an SNSF R’Equip grant for the purchase of the AMS and VOCUS. We thank Prof. Spyros Pandis for his support. 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PLoS Pathog 3:e151 Noti JD et al (2013) High Humidity Leads to Loss of Infectious Influenza Virus from Simulated Coughs. PLoS ONE 8:e57485 Kormuth KA et al (2018) Influenza Virus Infectivity Is Retained in Aerosols and Droplets Independent of Relative Humidity. J Infect Dis 218:739–747 Dubuis M-E et al (2021) Ozone inactivation of airborne influenza and lack of resistance of respiratory syncytial virus to aerosolization and sampling processes. PLoS ONE 16:e0253022 Longest AK, Rockey NC, Lakdawala SS, Marr LC (2024) Review of factors affecting virus inactivation in aerosols and droplets. J Royal Soc Interface 21:20240018 Morawska L et al (2021) A paradigm shift to combat indoor respiratory infection. Science 372:689–691 Le Sage V, Lowen AC, Lakdawala SS (2023) Block the Spread: Barriers to Transmission of Influenza Viruses. Annual Rev Virol 10:347–370 Sørensen SB, Dalby FR, Olsen SK, Kristensen K (2024) Influence of Germicidal UV (222 nm) Lamps on Ozone, Ultrafine Particles, and Volatile Organic Compounds in Indoor Office Spaces. Environ Sci Technol 58:20073–20080 Marr LC, Samet JM (2024) Reducing Transmission of Airborne Respiratory Pathogens: A New Beginning as the COVID-19 Emergency Ends. Environ Health Perspect 132:055001 Narihata K et al (2025) Rapid inactivation of aerosolised influenza virus using low-concentration gaseous hypochlorous acid. Sci Rep 15:33610 Hew YL, Isoda N, Miura T, Hiono T, Sakoda Y (2026) Evaluation of the Efficacy of Low-Concentration Gaseous Chlorine Dioxide in Inactivating Airborne H5 High Pathogenicity Avian Influenza Virus in Vivo Model. Food Environ Virol 18:4 Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9020228","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":600620250,"identity":"dacdd51f-3763-4b77-97fc-9132c7f455dc","order_by":0,"name":"Ghislain Motos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDACCcYGhgcMEjCuDQMD8+EGBqAgfi0JEC0gdWkMDGyJhLQAcQKECVJ3mLAW+ejm5g8JNRZ5DGKHjz/48Oe8nDkbUP3PHbi1GN452CaRcEyimEE6LbFxZtttY8s2xgbG3jN4tMxIbGNIYJNIbJDOMWzmbbiduOF+YwMzYxteLUCH/YNq4flzrn7DMUb8WuSBiiUS22Ba2A4kGBDSYgBW3wckgX6ZObMt2XAn0C8He/HZMiP98YcP3+oS+6WTD3z48MdO3pyN+eCDn/hsOQBlsMFFgPgANqVwWxowDMGnfBSMglEwCkYkAADGCVaSHSM3UQAAAABJRU5ErkJggg==","orcid":"","institution":"EPFL","correspondingAuthor":true,"prefix":"","firstName":"Ghislain","middleName":"","lastName":"Motos","suffix":""},{"id":600620251,"identity":"9f739304-2879-48a2-9a8c-eee978d0c082","order_by":1,"name":"Celine Terrettaz","email":"","orcid":"","institution":"EPFL","correspondingAuthor":false,"prefix":"","firstName":"Celine","middleName":"","lastName":"Terrettaz","suffix":""},{"id":600620252,"identity":"abdb2562-3ef9-4a63-bc89-7d017c16d761","order_by":2,"name":"Jun Zhang","email":"","orcid":"","institution":"EPFL","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Zhang","suffix":""},{"id":600620253,"identity":"dbaf8b01-4c00-4532-8d9d-18108e280d62","order_by":3,"name":"Beiping Luo","email":"","orcid":"","institution":"Institute for Atmospheric and Climate Science, Swiss Federal Institute of Technology Zurich","correspondingAuthor":false,"prefix":"","firstName":"Beiping","middleName":"","lastName":"Luo","suffix":""},{"id":600620254,"identity":"b67ecb54-f1ae-4167-9072-166f23fa4247","order_by":4,"name":"Irina Glas","email":"","orcid":"https://orcid.org/0000-0001-6976-6360","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Irina","middleName":"","lastName":"Glas","suffix":""},{"id":600620255,"identity":"9d32bea8-53f1-43de-b0f4-5567c788b43f","order_by":5,"name":"Kalliopi Violaki","email":"","orcid":"https://orcid.org/0000-0003-4612-3973","institution":"EPFL, LAPI","correspondingAuthor":false,"prefix":"","firstName":"Kalliopi","middleName":"","lastName":"Violaki","suffix":""},{"id":600620256,"identity":"78a92c2d-e614-4da6-9d60-48e134646aec","order_by":6,"name":"Marie Pohl","email":"","orcid":"","institution":"University of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"","lastName":"Pohl","suffix":""},{"id":600620257,"identity":"a3f5d619-5a42-42f1-8ac2-a81eeada808d","order_by":7,"name":"Frank Charlton","email":"","orcid":"","institution":"EPFL","correspondingAuthor":false,"prefix":"","firstName":"Frank","middleName":"","lastName":"Charlton","suffix":""},{"id":600620258,"identity":"1687ff25-b4bd-4640-a9bc-af690728934a","order_by":8,"name":"Andrea Baccarini","email":"","orcid":"https://orcid.org/0000-0003-4614-247X","institution":"EPFL","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Baccarini","suffix":""},{"id":600620259,"identity":"585ea29c-b946-4cea-b7d2-4b57dd67c247","order_by":9,"name":"Ulrich Krieger","email":"","orcid":"https://orcid.org/0000-0003-4958-2657","institution":"ETH Zurich","correspondingAuthor":false,"prefix":"","firstName":"Ulrich","middleName":"","lastName":"Krieger","suffix":""},{"id":600620260,"identity":"83f0c8af-dfdd-43ed-b13f-ed284645427c","order_by":10,"name":"Silke Stertz","email":"","orcid":"https://orcid.org/0000-0001-9491-2892","institution":"University of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Silke","middleName":"","lastName":"Stertz","suffix":""},{"id":600620261,"identity":"49f54947-8e9d-4232-b287-0cd48c84b054","order_by":11,"name":"Thomas Peter","email":"","orcid":"","institution":"Institute for Atmospheric and Climate Science","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Peter","suffix":""},{"id":600620262,"identity":"8c039242-9ccf-4e96-b029-283d587ae5bc","order_by":12,"name":"Tamar Kohn","email":"","orcid":"https://orcid.org/0000-0003-0395-6561","institution":"EPFL","correspondingAuthor":false,"prefix":"","firstName":"Tamar","middleName":"","lastName":"Kohn","suffix":""},{"id":600620263,"identity":"4b017c5d-d95e-4b2b-97df-4bc90d76224e","order_by":13,"name":"Athanasios Nenes","email":"","orcid":"https://orcid.org/0000-0003-3873-9970","institution":"Ecole Polytechnique Fédérale de Lausanne (EPFL)","correspondingAuthor":false,"prefix":"","firstName":"Athanasios","middleName":"","lastName":"Nenes","suffix":""}],"badges":[],"createdAt":"2026-03-03 12:26:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9020228/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9020228/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104404887,"identity":"641f09f9-49a8-41ce-b10e-9dd91624e53c","added_by":"auto","created_at":"2026-03-11 12:21:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":160802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of the LAPI BREATH. \u003c/strong\u003eThe experimental setup, already detailed in the study of Motos et al.\u003csup\u003e15\u003c/sup\u003e, is shown and complemented by the newly used instrumentation in the present work (in red). The Erlenmeyer contains a nitric acid solution (3.0% v/v in water). AMS, aerosol mass spectrometer; HEPA filter, high-efficiency particulate air filter; MFC, mass flow controller; SLAG, sparging liquid aerosol generator; BioSpot-VIVAS, viable virus aerosol sampler; SMPS, scanning mobility particle sizer; RH/T, relative humidity/air temperature sensor. The crossed circles represent manual three-way valves.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9020228/v1/a48b31b394764384467fc859.png"},{"id":104289576,"identity":"97505cf0-b60f-4ea8-b4a8-f32a495a4843","added_by":"auto","created_at":"2026-03-10 06:27:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":352641,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of nitric acid and carbon dioxide enrichment on the conservation of IAV infectivity in SLF. (A)\u003c/strong\u003e \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e vs. RH in SLF medium under filtered air (brown; 0.03 - 0.05 ppb HNO\u003csub\u003e3\u003c/sub\u003e and 410 - 440 ppm CO\u003csub\u003e2\u003c/sub\u003e), nitric acid-enriched (green) and carbon dioxide-enriched air (grey and black) conditions. Circles indicate PR8, a laboratory-adapted strain while squares represent the clinical strain Neth/09. \u003cstrong\u003e(B)\u003c/strong\u003e Same data as (A) for nitric acid- and carbon dioxide-enriched air at 25% and 55% RH, presented as \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e vs. concentration of nitric acid and carbon dioxide. Vertical lines indicate the 8-hour time-weighted average threshold limit values (TWA TLV) of both gases. 95% confidence intervals are indicated as error bars in (A) and (B).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9020228/v1/87093e3f794869be9b1d3ae4.png"},{"id":104405524,"identity":"e64fe334-3763-44bd-8cdc-2e4d2e5f047a","added_by":"auto","created_at":"2026-03-11 12:23:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":325447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epH retrievals of SLF particles. \u003c/strong\u003e(A, E) Gas phase, (B, F) particle phase, (C, G) calculated nitrate fraction and (D, H) retrieved pH performed in the absence (left panels) and presence (right panels) of nitric acid supplementation. Chamber temperature and RH are respectively 24.5°C and 55% with little variation (not shown). The ISORROPIA-Lite model requires measurement of all particle-phase compounds, while the S curves method is only based on nitrate. Gas phase nitric acid, temperature and RH are needed for both methods. The x-axes indicate the time elapsed compared to virus aerosolization. Due to the small mass of aerosol present in the chamber before aerosol injection (indicated by dotted vertical bars), the pH retrievals depicted during this period in panels D and H are very uncertain and the corresponding curves are therefore shown as faded. S-curves and ISORROPIA calculations are only performed once the concentrations in the particle and gas phases have stabilized.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9020228/v1/9d33ae7736f41a9e3e2b3757.png"},{"id":104779925,"identity":"95d5a224-6ffb-4417-8db7-8bbba2ebc6a9","added_by":"auto","created_at":"2026-03-17 07:48:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1434283,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9020228/v1/75b0c61d-9d36-4451-9596-9ddab94bd49e.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Sensitivity of aerosol-borne Influenza A Virus to gaseous nitric acid and carbon dioxide","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInfluenza A virus (IAV) poses a social and environmental burden, affecting humans, birds, and mammals through seasonal epidemics and pandemics. The World Health Organization (WHO) recognizes IAV as a major public health challenge and is actively preparing for a future influenza pandemic\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It is widely accepted that IAV transmission also occurs via the air\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Tools to prevent or mitigate the spread of IAV include vaccination and antiviral treatments, the use of personal protective equipment, ventilation, ultraviolet irradiation, air filtration, and isolation of infected patients. However, these measures are either insufficiently accepted or difficult to implement at the level of the general population. To achieve a better understanding of how influenza spreads, further work is needed across several research areas, such as epidemiology, immunology, building ventilation, transmission modelling, but also studies on the role of environmental factors regarding the ability of infectious respiratory particles to maintain their infectivity and ultimately transmit the disease.\u003c/p\u003e \u003cp\u003eThe physicochemical evolution of exhaled infectious respiratory particles is an important aspect for understanding the transmission of infectious viruses, as conditions of salinity, acidity, and other factors change greatly after exhalation and influence the inactivation of viruses. Yang and Marr\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e were the first to hypothesize that concentration changes in H\u003csup\u003e+\u003c/sup\u003e ions during particle evaporation after exhalation could lead to a reduction in pH. Conversely, experiments by Oswin et al.\u003csup\u003e6,7\u003c/sup\u003e using virus transport media concluded that exhaled particles would become alkaline due to CO\u003csub\u003e2\u003c/sub\u003e outgassing from the bicarbonate buffer. Again, conversely, Klein et al.\u003csup\u003e8\u003c/sup\u003e and Luo et al.\u003csup\u003e9\u003c/sup\u003e argued, based on laboratory experiments and biophysical modeling, that exhaled submicron aerosol particles can quickly become acidic due to the condensation of trace acids from the ambient air. Irreversible structural changes in viral surface proteins have recently been shown to be the acid-induced inactivation mechanism for IAV and SARS-CoV-2\u003csup\u003e10,11\u003c/sup\u003e, but it remains unclear which aerosol particle sizes are responsible for transmission and how fast they take up acids from the ambient air. Particles indoors may have lower acidity levels than outdoors, as trace acids and bases concentrations are different\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eControlled exposure of virus-laden aerosol particles to different levels of gas phase compounds allows to assess the sensitivity of viruses to pH variations, compared to the effects of other environmental factors, and study potential interactions and synergies between factors. To date, this has been done in two studies, both of which for SARS-CoV-2 in large (50 \u0026micro;m initial diameter) particles\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The results indicated increased aerostability with increased CO\u003csub\u003e2\u003c/sub\u003e concentration, which the authors explained by the reduction in CO\u003csub\u003e2\u003c/sub\u003e outgassing required for the particles to reach equilibrium with the gas phase, thus minimizing pH variations. Further, they found that the addition of nitric acid vapours slightly enhanced virus stability.\u003c/p\u003e \u003cp\u003eHere, we investigate the effect of gas phase composition on IAV in submicrometer particles (dry size) whose composition mimics particles exhaled during breathing. Using a medium that mimics lung fluid, we first show evidence of particle acidification due to the action of gaseous nitric acid down to concentrations of a few ppb. By injecting controlled amounts of nitric acid in the aerosol chamber of the LAPI BREATH facility (Laboratory of Atmospheric Processes and their Impacts - Bioaerosol Research \u0026amp; Environmental Airborne Transmission Hub\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e), we then observe how gas phase concentrations correlate with IAV aerostability. We also carry out experiments with variable levels of carbon dioxide, a gas that accumulates inside occupied buildings and is widely used as an indicator of pathogen transmission risk.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cp\u003e\u003cb\u003e2.1 Experimental setup and procedure.\u003c/b\u003e We utilize the LAPI BREATH facility (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), described in detail by Motos et al.\u003csup\u003e15\u003c/sup\u003e. Briefly, the facility consists of a 1.6 m\u003csup\u003e3\u003c/sup\u003e polytetrafluoroethylene (PTFE) chamber, in which we inject IAV-containing particles using a nebulizer based on bubble-bursting (the sparging liquid aerosol generator, SLAG) which are sampled over the course of an experiment with a condensational growth instrument, the BioSpot-VIVAS, and subsequently analyzed via virus titration and genomic copies quantification. Chamber temperature, relative humidity (RH) and gas phase composition are measured and controlled. The study of Motos et al.\u003csup\u003e15\u003c/sup\u003e provides the aerosol particle size distribution generated by the SLAG, the effect of aerosolization on IAV inactivation, and the rate of particle and virus losses to the chamber walls. PBS was chosen as the aerosol medium for its minimal complexity, allowing isolation of the effect of particle salinity, modulated by RH, on IAV inactivation. The effect of adding organic compounds to the medium was also studied. Building upon these findings, we increase the level of complexity to better mimic human exhalation by moving to a physiologically more relevant medium, synthetic lung fluid (SLF; see Supplementary Methods S1). The H1N1 IAV strains used in this study are A/Puerto Rico/8/34 (PR8) and A/Netherlands/602/2009 (Neth/09).\u003c/p\u003e \u003cp\u003eThe aerosolization and sampling procedures followed for the aerosol exposure experiments in the present study are detailed in Motos et al.\u003csup\u003e15\u003c/sup\u003e. Briefly, SLF powder is resuspended in the appropriate volume of autoclaved Milli-Q water and kept at 4\u0026deg;C for a maximum of 14 days. SLF solution is diluted 50 times in autoclaved Milli-Q water to generate the medium; 22 ml of which is spiked with IAV to a targeted titer of ~\u0026thinsp;10\u003csup\u003e8\u003c/sup\u003e-10\u003csup\u003e9\u003c/sup\u003e PFU/ml for PR8 and ~\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e-10\u003csup\u003e8\u003c/sup\u003e PFU/ml for Neth/09 and placed in a 100-ml screw-capped container. To verify the concentration of the viral inoculum, 25 \u0026micro;l are sampled into 2.5 ml PBSi (PBS for infection; see Supplementary Methods S1) and kept in the fridge during the experiment. We aerosolize IAV for 1 minute with the SLAG at a flow rate of 30 liters per minute. Samples are collected in a 35 x 10 mm Petri dish filled with 2.5 ml PBSi every 5 minutes (4 minutes for sampling and 1 minute for changing the Petri dish in the BioSpot-VIVAS) for the first 35 minutes of the experiment. Three additional samples are collected between 40\u0026ndash;45 min, 50\u0026ndash;55 min and 60\u0026ndash;65 min. Once collected, samples are transferred into 50-ml centrifuge tubes and stored on ice until the end of the experiment. Finally, the samples are aliquoted in 96-well plates and 1.5 ml tubes and stored at -20\u0026deg; C. Samples are enumerated for infectious titer and genome copy concentration within 14 days. Virus and SLF preparation as well as titration of infectious viruses and quantification of viral genomes follow the procedures from Schaub et al.\u003csup\u003e16\u003c/sup\u003e and Luo et al.\u003csup\u003e9\u003c/sup\u003e and are detailed in Supplementary Methods S1.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2 Metric for displaying data.\u003c/b\u003e Following Motos et al.\u003csup\u003e15\u003c/sup\u003e, we express our infectivity results in terms of 99% IAV inactivation time (\u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e), i.e., the time required for a 2-log inactivation, assuming first-order inactivation kinetics. Briefly, we fit infectivity data assuming first-order kinetics, using the midpoint of sampling intervals as time values. The fits are then corrected for physical losses on the chamber walls measured as the decrease in the number of viral genome copies over time by digital polymerase chain reaction (dPCR). The \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e metric provides infectivity results independent of the time of exposure and related to other timescales of importance for transmission risk, such as particle settling time or the room ventilation/filtration timescale.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3 Particle acidification procedure.\u003c/b\u003e To investigate the impact of aerosol particle acidification on the conservation of IAV infectivity, we precondition the chamber air by adding nitric acid prior to injection of infectious respiratory particles. Condensation of acid vapors to the particle phase reduces the pH of the particles, which in turn affects IAV infectivity. The LAPI BREATH is equipped with an acid injection system, based on the evaporation of an acid solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Once the chamber air has been conditioned to the desired RH, the airflow is directed to an Erlenmeyer flask containing 250 mL of 3% nitric acid through a tight-fitting cap, with a flow rate of 20 liters per minute; the outlet flow is directed to the chamber via the injection port.\u003c/p\u003e \u003cp\u003eThe choice of nitric acid for the aerosol particle acidification experiments is made after a rigorous selection of the ideal compound, based on 4 criteria: solubility, volatility, acid strength and occupational exposure limit value. High solubility at 20\u003cem\u003e\u0026deg;\u003c/em\u003eC is crucial because the injection system we use is based on evaporation. The volatility of the compound, i.e., its propensity to remain in the gas phase or to condense to the particle phase, must be neither too low, in order to avoid adsorption on the PTFE lines and walls of the chamber before condensing on the aerosol particles, nor too high, as this would cause the acid to remain in the gas phase without affecting the particles. We estimated the volatility of acids based on their Henry\u0026rsquo;s law coefficient, following the approach of Liu et al.\u003csup\u003e17\u003c/sup\u003e. This study showed a relationship between this characteristic of volatile organic compounds and small polar compounds and their instrument response time, or in other words, the delay times in measurements caused by the partitioning of these compounds between the gas phase and the surface of the inlet tubing used for measurement. The third criterion, acid strength, indicates how effectively the condensed phase of the acid can reduce particle pH. Finally, the occupational exposure limit value, provided by the Health Institute for Occupational Safety and Health (NIOSH), provides information on the risk of exposure to the compound for human health. Based on this selection, nitric acid, a strong water miscible acid (pKa = -1.38) with intermediate volatility (Henry\u0026rsquo;s law constant of 89,166 M/atm\u003csup\u003e18\u003c/sup\u003e, corresponding to a response time of approximately 335 s detected by a mass spectrometer) and a safety limit of 2 ppm averaged over a 10-hour workshift\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, appears to be the most suitable acid for our study. The fact that nitric acid is ubiquitous in the atmosphere, and a widely used, well characterized acid in atmospheric studies is a further argument in favor of its use.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.4 Gas phase composition measurements.\u003c/b\u003e Gas phase nitric acid is detected by a VOCUS chemical ionization mass spectrometer with iodide-adduct ions (hereafter VOCUS, Tofwerk AG, Thun, BE, Switzerland). It is equipped with AIM (adduct ionization mechanism) ion-molecule reactor (IMR), allowing real-time monitoring of volatile compounds in the ppt range\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e with little fragmentation and memory effect. We connect the instrument to the chamber directly through a sealed opening in one of the chamber walls, with PTFE tubing as short as possible to minimize nitric acid losses. To avoid sampling infectious viruses by the VOCUS, we measure the nitric acid concentration before and after each experiment and interpolate the data to estimate the concentration during the experiments. The decay rate of nitric acid concentration with time during an experiment is characterized based on a test experiment with virus-free SLF. Details concerning the calibration of the Vocus can be found in Supplementary Fig. S1 and Methods S2.\u003c/p\u003e \u003cp\u003eCarbon dioxide is injected directly into the aerosol chamber from the laboratory gas network supplied via PTFE tubing connected to the chamber injection port. CO\u003csub\u003e2\u003c/sub\u003e concentration is measured using an Aranet4 household sensor (Aranet, Riga, Latvia) attached to the stainless-steel tubes connected to the chamber sampling port.\u003c/p\u003e \u003cp\u003eIn experiments carried out without injection of nitric acid or carbon dioxide, the gas phase only includes what is hereafter referred to as \u0026ldquo;filtered air\u0026rdquo;, namely compressed outside air captured on the building roof, filtered for particles by a high-efficiency particle air (HEPA) filter and for organic substances by an activated carbon filter.\u003c/p\u003e \u003cp\u003eIn experiments conducted without the addition of nitric acid or carbon dioxide, the chamber is filled with compressed outdoor air collected on the roof of the building, previously HEPA-filtered for particles and activated carbon-filtered for organic substances. The concentration values measured in this filtered air are between 390 and 450 ppm CO\u003csub\u003e2\u003c/sub\u003e and mass spectrometric measurements of the HNO\u003csub\u003e3\u003c/sub\u003e background concentration in the chamber show values between 0.03 and 0.05 ppb. This background is irrelevant for our experiments with \u0026ge;\u0026thinsp;5 ppb HNO\u003csub\u003e3\u003c/sub\u003e, but is important for our experiments without HNO\u003csub\u003e3\u003c/sub\u003e addition, which we will revisit in the discussion.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.5 Particle-phase composition measurements.\u003c/b\u003e High-resolution time-of-flight aerosol mass spectrometer (HTOF-AMS, thereafter \u0026ldquo;AMS\u0026rdquo;, Aerodyne Research, Inc.) with a mass resolution of \u0026sim;\u0026thinsp;2000 over the range of \u003cem\u003em/z\u003c/em\u003e 100 to \u003cem\u003em/z\u003c/em\u003e 400 is deployed for online, non-refractory aerosol particle characterization. Aerosols are continuously sampled through a PM\u003csub\u003e2.5\u003c/sub\u003e aerodynamic lens. The ionization efficiency of nitrate is calibrated with 300 nm ammonium nitrate particles against simultaneous scanning mode particle sizer (SMPS, DMA model TSI long and CPC model 3772 TSI Inc., Shoreview, MN, USA) measurements to convert ion counts to particle mass concentration. The relative ionization efficiency of ammonium, sulfate, and sodium is calibrated in the same approach. We connect a HEPA filter to the inlet of the AMS and sampled air for 15 minutes to determine the following limits of detection (in \u0026micro;g/m\u003csup\u003e3\u003c/sup\u003e): 0.013 for SO\u003csub\u003e4\u003c/sub\u003e, 0.0034 for NO\u003csub\u003e3\u003c/sub\u003e, 0.00096 for NH\u003csub\u003e4\u003c/sub\u003e and 0.35 for NaCl\u003csup\u003e+\u003c/sup\u003e. A detailed description of the AMS can be found in DeCarlo et al.\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.6 pH retrievals.\u003c/b\u003e To observe changes in pH caused by aerosol particle exposure to nitric acid, we perform two chamber experiments, one with and one without gaseous nitric acid. Virus-free SLF is aerosolized in the chamber, while nitric acid in the gas phase and organic and inorganic compounds in the particle phase are continuously measured (together with chamber temperature and RH, respectively 24.5\u0026deg;C and 55% RH with negligible variations). We retrieve the pH value of particles based on the thermodynamic equilibrium of gas- and particle-phase compounds measured simultaneously by VOCUS and AMS. While we used a biophysical model (Respiratory Aerosol Model, ResAM) in our previous work\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, the partitioning of HNO\u003csub\u003e3\u003c/sub\u003e between the gas and particle phases due to the small size of the aerosol particles quickly leads to thermodynamic equilibrium, which is also confirmed by VOCUS and AMS measurements. This greatly simplifies pH determination. First, we utilize the ISORROPIA-Lite thermodynamic model, which calculates the composition and phase state of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e-NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e-Na\u003csup\u003e+\u003c/sup\u003e-organics-water solutions, allowing for metastable aerosol states and assuming gas-particle equilibrium. The pH is then determined from pH\u0026thinsp;=\u0026thinsp;log\u003csub\u003e10\u003c/sub\u003e(\u003cem\u003ea\u003c/em\u003e\u003csub\u003eH+\u003c/sub\u003e) and \u003cem\u003ea\u003c/em\u003e\u003csub\u003eH+\u003c/sub\u003e = \u003cem\u003eH\u0026middot;p\u003c/em\u003e\u003csub\u003eHNO3\u003c/sub\u003e/\u003cem\u003ea\u003c/em\u003e\u003csub\u003eNO3\u0026minus;\u003c/sub\u003e, where \u003cem\u003ea\u003c/em\u003e\u003csub\u003eH+\u003c/sub\u003e is the H\u003csup\u003e+\u003c/sup\u003e activity, \u003cem\u003eH\u003c/em\u003e is the Henry\u0026rsquo;s Law constant for HNO\u003csub\u003e3\u003c/sub\u003e(g) \u0026harr; H\u003csup\u003e+\u003c/sup\u003e(l) + NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e(l) and \u003cem\u003ea\u003c/em\u003e\u003csub\u003eNO3\u0026minus;\u003c/sub\u003e = \u003cem\u003ec\u003c/em\u003e\u003csub\u003eNO3\u0026minus;\u003c/sub\u003e \u0026sdot; γ\u003csub\u003eNO3\u0026minus;\u003c/sub\u003e is the nitrate activity. Detailed descriptions of this model can be found in Nenes et al.\u003csup\u003e22\u003c/sup\u003e and Fountoukis and Nenes\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Second, we apply the S curves method\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, which relies on a theoretical relationship between nitrate partitioning, water content and pH and can be visualized using sigmoid-shaped curves calculated from solubility and dissociation of nitrate in water, including activity coefficients.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003eWe first describe the dependence of IAV aerostability on air composition and then present evidence of aerosol particle acidification by detailing pH measurements of particles nebulized in the chamber in the presence or absence of nitric acid.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.1 Infectivity experiments in filtered, nitric acid-enriched and carbon dioxide-enriched air.\u003c/b\u003e The inactivation of IAV in aerosolized SLF medium is measured over a RH range from 10% to 95% RH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and compared with previous data obtained using the same experimental setup with PBS and sucrose-supplemented PBS as media (Supplementary Fig. S2). In general, the infectivity decay measured in these three media are very similar (Supplementary Fig. S2) and much less variable than many dependencies reported in the literature\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Infectivity decreases monotonically with increasing RH in SLF medium, with a decline of more than one log level in \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e between 10% and 95% RH. This behavior is probably due to the decreasing viscosity of SLF with increasing humidity. While we found a slight minimum in \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e as a function of RH in PBS as a medium (\u0026ldquo;U-shape\u0026rdquo;), this was hardly present in PBS+sucrose, which we attributed to the protective effect of the organics\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. With SLF, this minimum disappears completely. In the following, we use SLF as the medium most similar to body fluids to investigate the influence of air composition on IAV infectivity.\u003c/p\u003e \u003cp\u003eStarting with filtered air (0.03\u0026ndash;0.05 ppb HNO3 and 420\u0026ndash;440 ppm CO\u003csub\u003e2\u003c/sub\u003e), we investigate the influence of increasing the concentration of each compound on the aerostability of an IAV strain PR8 (H1N1), which was also used in Motos et al.\u003csup\u003e15\u003c/sup\u003e. Nitric acid inactivates IAV very effectively, as shown by the cascading anticorrelation between \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e and HNO\u003csub\u003e3\u003c/sub\u003e concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At 55% RH, the sensitivity of IAV to gaseous HNO\u003csub\u003e3\u003c/sub\u003e is high: 5 ppb reduces \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e by almost a factor of 2, and 30 ppb by almost an order of magnitude. It should be noted that this latter concentration remains 67 times lower than the 8-hour time-weighted average threshold limit value (TWA TLV) for HNO\u003csub\u003e3\u003c/sub\u003e, 2 ppm, as shown by the green vertical bar in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. However, this sensitivity shows a clear dependence on RH. Under dry conditions (25% RH), 30 ppb HNO\u003csub\u003e3\u003c/sub\u003e only reduces \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e by 25%, while 80% RH leads to results below the limit of quantification (LOQ) in the PFU titer. We compare the sensitivity of IAV PR8 to acidic conditions with that of the more clinically relevant Neth/09 strain and find that HNO\u003csub\u003e3\u003c/sub\u003e concentrations of 30 ppb at 25% RH and 9 ppb at 55% RH lead to infectivity below the LOQ, although reference measurements (without acid injection) show similar \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e values as for the PR8 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eInjecting large concentrations of CO\u003csub\u003e2\u003c/sub\u003e into the chamber (4000 and 6500 ppm, values attainable in poorly ventilated indoor environments but which surround the 8-hour TWA TLV of 5000 ppm; see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) bears no impact on IAV inactivation at low and high RH, but provides some limited protection at 55% RH, with a near doubling of \u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e99\u003c/em\u003e\u003c/sub\u003e values compared to the reference case. We also performed an experiment with an extremely high CO\u003csub\u003e2\u003c/sub\u003e level (about 250000 ppm, i.e., 25% v/v CO\u003csub\u003e2\u003c/sub\u003e in air), resulting in inactivation kinetics comparable to the 4500 and 6500 ppm cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Overall, our results show that IAV infectivity is very sensitive to the concentration of nitric acid present in the air, moderated by relative humidity, but largely insensitive to carbon dioxide concentration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Evidence of particle acidification due to exposure to gaseous nitric acid.\u003c/b\u003e To confirm that the inactivation of IAV described in Section 3.1 is indeed caused by a low pH of the aerosol particles resulting from the uptake of nitric acid, we performed two experiments, which differ solely by the injection of nitric acid as chamber preconditioning before the aerosolization of virus-free SLF. The nitric acid concentration (~\u0026thinsp;30 ppb) and the RH (55%) are chosen to reproduce the conditions under which IAV inactivation occurs rapidly (\u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;4 min; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e indicates that the presence of nitric acid vapour results in very low levels of nitrate in the particles (the particle-phase NO\u003csub\u003e3\u003c/sub\u003e mass fraction drops from ~\u0026thinsp;0.6 to ~\u0026thinsp;0.002 based on gas- and particle-phase nitrate measurements; see panels C and G). Since the particles emitted by the SLAG are in the submicron range after drying\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, they quickly reach equilibrium with the gaseous nitric acid, leading to a rapid reduction in particle pH from approximately 5 to 2. Note that direct pH measurements of our diluted SLF solution using a pH-meter provided a value of approximately 6.4, slightly higher than the particle pH retrieved in the absence of nitric acid (after aerosolization in the chamber; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cb\u003e4.1 IAV inactivation in SLF particles.\u003c/b\u003e In addition to a bubble-bursting nebulizer (SLAG) simulating aerosol generation in the alveolar region, a chamber reproducing an indoor environment with sufficient dimensions to account for aerosol deposition, and a virus sampler reproducing the liquid-to-liquid deposition of aerosol particles in the respiratory tract of a host, our experimental setup now also includes a medium reproducing the essential properties of the respiratory tract fluid. The main limitations to a perfect imitation of the natural situation concern the absence of very large particles produced by the upper respiratory tract (larynx, tongue and lips) that the SLAG cannot generate, and the relatively small amount of bicarbonates present in our SLF compared to body fluids (which helps avoiding carbonate precipitation, but in small particles equilibrium is attained readily irrespective of the initial carbonate concentration by fast outgassing of CO\u003csub\u003e2\u003c/sub\u003e). The results obtained with LAPI BREATH using SLF as medium indicate that the aerostability of IAV decreases with increasing RH. This clearly contradicts the WHO rating that high humidity prolongs airborne virus infectivity, without however formulating guidelines on RH management due to the lack of relevant data\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. While RH levels above ~\u0026thinsp;80% should be avoided indoors, as they promote the growth of mold, the risk of IAV transmission is maximized in dry environments. These results confirm previous findings\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e but disagree with recent studies that found IAV infectivity to be independent of RH\u003csup\u003e31,32\u003c/sup\u003e. The different media, air compositions, and virus strains used in these studies, which may contribute to different experimental outcomes, could explain this discrepancy.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.2 Effect of air composition on IAV infectivity.\u003c/b\u003e While the dependence of virus aerostability on RH and medium composition has been studied for decades, often with contradicting results\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, its dependence on gas phase compounds has only recently attracted scientific interest\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In particular, acids and bases are known to affect indoor air quality\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. They are often semi-volatile, partition to aerosol particles exhaled by people, and affect the pH and potentially the infectivity of viruses contained in these particles. Luo et al.\u003csup\u003e9\u003c/sup\u003e calculated the evolution of particle pH and virus inactivation after exhalation using ResAM, which they constrained by the thermodynamic and diffusion kinetic properties of SLF determined from levitated SLF droplets and by pH-dependent IAV inactivation rate from plaque measurements. They showed that the condensation of trace acids typically present in indoor air is sufficient to lower the pH of fine aerosol particles to pH 4 within minutes, leading to rapid inactivation of IAV but not SARS-CoV-2. They found that enriching the air with non-hazardous levels of HNO\u003csub\u003e3\u003c/sub\u003e could effectively reduce the aerostability of both viruses. When 50 ppb of HNO\u003csub\u003e3\u003c/sub\u003e was added to the air, they calculated that \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e fell to a few seconds.\u003c/p\u003e\u003cp\u003eOur results here provide experimental confirmation of the general trend of these model results for IAV inactivation and indicate that IAV infectivity is indeed anticorrelated to the HNO\u003csub\u003e3\u003c/sub\u003e concentration and that this anticorrelation is modulated by RH. At 55% RH, our pH retrieval (pH\u0026thinsp;=\u0026thinsp;log\u003csub\u003e10\u003c/sub\u003e(\u003cem\u003eH\u0026middot;p\u003c/em\u003e\u003csub\u003eHNO3\u003c/sub\u003e/(\u003cem\u003ec\u003c/em\u003e\u003csub\u003eNO3\u0026minus;\u003c/sub\u003e\u0026sdot; γ\u003csub\u003eNO3\u0026minus;\u003c/sub\u003e)), see Section 2.6) from the measured \u003cem\u003ep\u003c/em\u003e\u003csub\u003eHNO3\u003c/sub\u003e and \u003cem\u003ec\u003c/em\u003e\u003csub\u003eNO3\u0026minus;\u003c/sub\u003e shows that SLF particles become slightly acidic even when no nitric acid is injected into the chamber (~\u0026thinsp;pH 5, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), which is due to the background level of HNO\u003csub\u003e3\u003c/sub\u003e of ~\u0026thinsp;0.03 ppb (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). When HNO\u003csub\u003e3\u003c/sub\u003e is increased to 30 ppb (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), \u003cem\u003ec\u003c/em\u003e\u003csub\u003eNO3\u0026minus;\u003c/sub\u003e increases by about a factor of 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,E), pH decreases to about 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), and the corresponding inactivation time \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e decreases from 50 to about 6 minutes (brown and green circles in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At low RH of 25, the SLF becomes much more viscous than at higher RH and the HNO\u003csub\u003e3\u003c/sub\u003e uptake is limited by liquid phase diffusion (see Fig. S9D of Luo et al.\u003csup\u003e9\u003c/sup\u003e). This may explain why \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e at 25% RH is by 1\u0026ndash;2 orders of magnitude longer than at higher RH.\u003c/p\u003e \u003cp\u003eThe intermediate levels with 5 ppb and 17 ppb HNO\u003csub\u003e3\u003c/sub\u003e and the concomitant monotonous reduction in \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e at 55% RH indicate the increasing impact of acidity on IAV. In contrast to Haddrell et al.\u003csup\u003e13\u003c/sup\u003e, we find no evidence of alkaline states to play a role. They found an enhancement of aerostability of SARS-CoV-2 after the addition of nitric acid to their moisture injection system, which they explained as a transition from alkaline to neutral pH. However, they used aerosol particles with much larger size (~\u0026thinsp;14-micron equilibrated diameter compared to 250 nm diameter in the peak of the equilibrated volume distribution in the present study; see Motos et al.\u003csup\u003e15\u003c/sup\u003e). The characteristic time for uptake of ambient trace gases by freshly emitted or exhaled particles scales roughly with the square of the diameter. Therefore, for our small particles the HNO\u003csub\u003e3\u003c/sub\u003e uptake and acidification take only seconds and alkalinity due to the loss of bicarbonate from the particles cannot prevail\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinally, we note that the agreement of our experiments with Luo et al.\u003csup\u003e9\u003c/sup\u003e is only qualitative. They reported \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;3 s for SLF droplets smaller than 0.5 \u0026micro;m at 50% RH and 50 ppb HNO\u003csub\u003e3\u003c/sub\u003e. In the present experiments, the inactivation time is much longer, namely \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;360 s for 55% RH and 35 ppb HNO\u003csub\u003e3\u003c/sub\u003e. This can be due to a small fraction of large particles (radius\u0026thinsp;\u0026gt;\u0026thinsp;2 \u0026micro;m), for which the uptake of HNO\u003csub\u003e3\u003c/sub\u003e takes more time than for smaller particles, also due to slow liquid phase diffusion of nitrate ions in SLF\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Precise measurements of the entire size distribution is a topic of further investigation.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.3 Perspectives and final remarks.\u003c/b\u003e Dry environments concentrate the risk of indoor IAV transmission by increasing the stability of the virus in airborne particles. Assuming that the LAPI BREATH is able to reproduce the conditions that lead to the transmission of IAV, mainly in terms of aerosol particle size and composition, this translates in our measurements to \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e values that are a factor 3 to 5 higher at 25% RH compared to 55%. Furthermore, \u003cem\u003et\u003c/em\u003e\u003csub\u003e99\u003c/sub\u003e shows a systematic dependence on the HNO\u003csub\u003e3\u003c/sub\u003e concentration in indoor air.\u003c/p\u003e \u003cp\u003eCould this contribute to future epidemic risk prevention? A variety of pharmaceutical and non-pharmaceutical risk reduction strategies have been investigated\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Ventilation with outdoor air appears to be the simplest and most effective of all measures, but its application becomes problematic at low outdoor temperatures. Next, air filtration and ultraviolet disinfection (especially far-UVC) have been shown effective, but they respectively influence aerosol pH\u003csup\u003e9\u003c/sup\u003e and increase ozone and ultra-fine particle levels\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, so that advantages and disadvantages must also be evaluated\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Finally, there have been recent advances in research into the inactivation of IAV through exposure to gaseous species via the oxidative pathway, e.g., through the use of solution sprays containing hypochlorous acid\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the same way that chlorine, a pollutant that is toxic to human health at high aqueous concentrations, is added to drinking water to combat infectious viruses and bacteria, one could consider disinfecting the air with nitric acid, under the strict supervision of experts, during acute transmission risk events such as transport and large indoor gatherings. Although we do not currently recommend injecting nitric acid into buildings, we advocate research into air acidification for disinfection in order to combat airborne pathogens by means of very low acid concentrations considered safe for human health. To this end, studies such as the one presented here should be extended to other virus strains and other environmental conditions, with particular attention paid to the cross-sensitivity of various factors influencing the stability of IAV, as shown here for the interaction between RH and acidification. A combination of laboratory experiments and process-oriented modeling can show whether airborne acids could contribute to future epidemic prevention strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor contribution\u003c/h2\u003e \u003cp\u003eGM wrote the original draft. GM, CT, JZ, KV, FC and AB performed formal analysis. GM, CT, BL, IG, MP, AB, UK, SS, TP, TK and AN discussed the methodology. SS, TP, TK and AN conceptualized the study and acquired funding.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe acknowledge the SNSF through the support of the IVEA (CRSII5_189939) and AirTRAC (IC00I0-228106) projects, and the support by an SNSF R\u0026rsquo;Equip grant for the purchase of the AMS and VOCUS. We thank Prof. Spyros Pandis for his support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization (2022) Pandemic influenza preparedness framework for the sharing of influenza viruses and access to vaccines and other benefits, 2nd ed. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/publications/i/item/9789240024854\u003c/span\u003e\u003cspan address=\"https://www.who.int/publications/i/item/9789240024854\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTellier R (2009) Aerosol transmission of influenza A virus: a review of new studies. J Royal Soc Interface 6:S783\u0026ndash;S790\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao S et al (2018) Probable transmission routes of the influenza virus in a nosocomial outbreak. 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Food Environ Virol 18:4\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9020228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9020228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTransmission via aerosols is an important route of transmission of influenza A virus (IAV) in humans, whose control remains a global challenge. After exhalation, aerosol particles undergo rapid physicochemical changes that lead to substantial aerosol pH variations, which can impair the infectivity of IAV. Here, we investigate the aerostability of IAV, a key step of the transmission chain, using an aerosol chamber that allows assessing the influence of air composition on aerosol pH. At 55% relative humidity, we observe high sensitivity of IAV to nitric acid concentrations as low as 5 ppb, i.e., 400 times lower than the permissible exposure limit, with the time required to achieve a 99% reduction in active viruses being halved. In contrast, even an increase in carbon dioxide concentration to 250,000 ppm has a negligible effect on IAV. Our results underscore the crucial role of trace acids in transmitting aerosolized respiratory viruses, including strong synergies with air humidification.\u003c/p\u003e","manuscriptTitle":"Sensitivity of aerosol-borne Influenza A Virus to gaseous nitric acid and carbon dioxide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-10 06:27:54","doi":"10.21203/rs.3.rs-9020228/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"10f3b1d6-8c9a-4776-8033-f06f5d531f43","owner":[],"postedDate":"March 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63916060,"name":"Biological sciences/Microbiology/Environmental microbiology/Air microbiology"},{"id":63916061,"name":"Health sciences/Diseases/Infectious diseases/Influenza virus"},{"id":63916062,"name":"Biological sciences/Biological techniques/Mass spectrometry"}],"tags":[],"updatedAt":"2026-03-11T09:20:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-10 06:27:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9020228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9020228","identity":"rs-9020228","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}