Understanding the factors that influence the airborne survival of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in aerosols is important for identifying routes of transmission and the value of various mitigation strategies for preventing transmission. We present measurements of the stability of SARS-CoV-2 in aerosol droplets (∼5 to 10 µm equilibrated radius) over timescales spanning 5 s to 20 min using an instrument to probe survival in a small population of droplets (typically 5 to 10) containing ∼1 virus/droplet. Measurements of airborne infectivity change are coupled with a detailed physicochemical analysis of the airborne droplets containing the virus. A decrease in infectivity to ∼10% of the starting value was observable for SARS-CoV-2 over 20 min, with a large proportion of the loss occurring within the first 5 min after aerosolization. The initial rate of infectivity loss was found to correlate with physical transformation of the equilibrating droplet; salts within the droplets crystallize at relative humidities (RHs) below 50%, leading to a near-instant loss of infectivity in 50 to 60% of the virus. However, at 90% RH, the droplet remains homogenous and aqueous, and the viral stability is sustained for the first 2 min, beyond which it decays to only 10% remaining infectious after 10 min. The loss of infectivity at high RH is consistent with an elevation in the pH of the droplets, caused by volatilization of CO 2 from bicarbonate buffer within the droplet. Four different variants of SARS-CoV-2 were compared and found to have a similar degree of airborne stability at both high and low RH.

The airborne transmission of infection relies on the ability of pathogens to survive aerosol transport as they transit between hosts. Understanding the parameters that determine the survival of airborne microorganisms is critical to mitigating the impact of disease outbreaks. Conventional techniques for investigating bioaerosol longevity in vitro have systemic limitations that prevent the accurate representation of conditions that these particles would experience in the natural environment. Here, we report a new approach that enables the robust study of bioaerosol survival as a function of relevant environmental conditions. The methodology uses droplet-on-demand technology for the generation of bioaerosol droplets (1 to greater than 100 per trial) with tailored chemical and biological composition. These arrays of droplets are captured in an electrodynamic trap and levitated within a controlled environmental chamber. Droplets are then deposited on a substrate after a desired levitation period (less than 5 s to greater than 24 h). The response of bacteria to aerosolization can subsequently be determined by counting colony forming units, 24 h after deposition. In a first study, droplets formed from a suspension of Escherichia coli MRE162 cells (10 8 ml 21 ) with initial radii of 27.8 + 0.08 mm were created and levitated for extended periods of time at 30% relative humidity. The time-dependence of the survival rate was measured over a time period extending to 1 h. We demonstrate that this approach can enable direct studies at the interface between aerobiology, atmospheric chemistry and aerosol physics to identify the factors that may affect the survival of airborne pathogens with the aim of developing infection control strategies for public health and biodefence applications.royalsocietypublishing.org/journal/rsif J. R. Soc. Interface 16: 20180779

Understanding the factors that influence the airborne survival of viruses such as SARSCoV2 in aerosols is important for identifying routes of transmission and the value of various mitigation strategies for preventing transmission. We present measurements of the stability of SARSCoV2 in aerosol droplets (5 to 10 micrometres equilibrated radius) over timescales spanning from 5 seconds to 20 minutes using a novel instrument to probe survival in a small population of droplets (typically 5-10) containing ~1 virus/droplet. Measurements of airborne infectivity change are coupled with a detailed physicochemical analysis of the airborne droplets containing the virus. A decrease in infectivity to 10 % of the starting value was observable for SARS-CoV-2 over 20 minutes, with a large proportion of the loss occurring within the first 5 minutes after aerosolisation. The initial rate of infectivity loss was found to correlate with physical transformation of the equilibrating droplet; salts within the droplets crystallise at RHs below 50% leading to a near instant loss of infectivity in 50 to 60% of the virus. However, at 90% RH the droplet remains homogenous and aqueous, and the viral stability is sustained for the first 2 minutes, beyond which it decays to only 10% remaining infectious after 10 minutes. The loss of infectivity at high RH is consistent with an elevation in the pH of the droplets, caused by volatilisation of CO2 from bicarbonate buffer within the droplet. Three different variants of SARS-CoV-2 were compared and found to have a similar degree of airborne stability at both high and low RH.

Emerging outbreaks of airborne pathogenic infections worldwide, such as the current SARS-CoV-2 pandemic, have raised the need to understand parameters affecting the airborne survival of microbes in order to develop measures for effective infection control. We report a novel experimental strategy, TAMBAS (Tandem Approach for Microphysical and Biological Assessment of Airborne Microorganisms Survival), to explore the synergistic interactions between the physicochemical and biological processes that impact airborne microbe survival in aerosol droplets. This innovative approach provides a unique and detailed understanding of the processes taking place from aerosol droplet generation through to equilibration and viability decay in the local environment, elucidating decay mechanisms not previously described. The impact of evaporation kinetics, solute hygroscopicity and concentration, particle morphology and equilibrium particle size on airborne survival are reported, using Escherichia coli (MRE162) as a benchmark system. For this system, we report that the particle crystallisation does not directly impact microbe longevity, bacteria act as crystallization nuclei during droplet drying and equilibration, and the kinetics of size and compositional change appear to have a larger effect on microbe longevity than equilibrium solute concentration. IMPORTANCE A transformative approach to identify the physicochemical processes that impact the biological decay rates of bacteria in aerosol droplets is described. It is shown that the evaporation process and changes in the phase and morphology of the aerosol particle during evaporation impact microorganism viability. The equilibrium droplet size was found to affect airborne bacterial viability. Furthermore, the presence of Escherichia coli (MRE162) in a droplet does not affect aerosol growth/evaporation, but influences the dynamic behaviour of the aerosol through processing the culture media prior to aerosolization affecting the hygroscopicity of the culture medium; this highlights the importance of the inorganic and organic chemical composition within the aerosolised droplets that impact hygroscopicity. Bacteria act also as a crystallisation nucleus. The novel approach and data has implications for increased mechanistic understanding of aerosol survival and infectivity in bioaerosol studies spanning medical, veterinary, farming, and agricultural fields, including the role of micro-organisms in atmospheric processing and cloud formation.

Methods Virus Strains and Culture MethodsSeventeen clone one (17Cl-1) mouse fibroblast cells (Sturman and Takemoto 1972) were cultured at 37 o C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM, high glucose; Sigma, UK) supplemented with 10% foetal bovine serum (FBS, Sigma), 100 units/ml penicillin (Gibco, UK), 100 µg/ml streptomycin (Gibco, UK), non-essential amino acids (NEAA, Gibco), and L-glutamine (Gibco, UK). To generate MHV-A59 (Sawicki et al. 2005) viral stocks, T75 tissue culture flasks (Corning) seeded with 17CL-1 cells, were infected at a multiplicity of infection (MOI) of 0.01, and then incubated for 48 hours at 37 o C and 5% CO2. The virally infected cells were then subjected to a freeze-thaw cycle by placing the T75 flask into a -80 o C freezer overnight and then the culture thawed and centrifuged at 1200g before being aliquoted and stored at -80 o C. Airborne Longevity MeasurementsThe environmental conditions are set by adjusting the Peltier voltage and polarity to set the temperature and the ratio of dry to wet air to set the humidity. MHV suspension is drawn into a 1 ml syringe which is then attached to the instrument and used to feed the virus solution to the droplet dispenser via a remotely operated motor. The dispenser is remotely operated by computer and used to generate a population of droplets of the desired size within the electric field. Both the initial and final frequency of the electric field are set by computer as well as the rate at which the field frequency changes, to allow for the droplet to be kept within the trap as it equilibrates to the conditions. Once the desired time is reached, an isolation plate is retracted causing the electric field to be set to zero; then, the droplets are pulled down into a plate containing 5-10 ml of DMEM so that the remaining virus can be quantified. For each measurement, two levitations are carried out. First a short

Respiratory pathogens can be spread though the transmission of aerosolised expiratory secretions in the form of droplets or particulates. Understanding the fundamental aerosol parameters that govern how such pathogens survive whilst airborne is essential to understanding and developing methods of restricting their dissemination. Pathogen viability measurements made using Controlled Electrodynamic Levitation and Extraction of Bioaerosol onto Substrate (CELEBS) in tandem with a comparative kinetics electrodynamic balance (CKEDB) measurements allow for a direct comparison between viral viability and evaporation kinetics of the aerosol with a time resolution of seconds. Here, we report the airborne survival of mouse hepatitis virus (MHV) and determine a comparable loss of infectivity in the aerosol phase to our previous observations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the addition of clinically relevant concentrations of mucin to the bioaerosol, there is a transient mitigation of the loss of viral infectivity at 40% RH. Increased concentrations of mucin promoted heterogenous phase change during aerosol evaporation, characterised as the formation of inclusions within the host droplet. This research demonstrates the role of mucus in the aerosol phase and its influence on short-term airborne viral stability.

The mechanistic factors hypothesized to be key drivers for the loss of infectivity of viruses in the aerosol phase often remain speculative. Using a next-generation bioaerosol technology, we report measurements of the aero-stability of several SARS-CoV-2 variants of concern in aerosol droplets of well-defined size and composition at high (90%) and low (40%) relative humidity (RH) upwards of 40 min. When compared with the ancestral virus, the infectivity of the Delta variant displayed different decay profiles. At low RH, a loss of viral infectivity of approximately 55% was observed over the initial 5 s for both variants. Regardless of RH and variant, greater than 95% of the viral infectivity was lost after 40 min of being aerosolized. Aero-stability of the variants correlate with their sensitivities to alkaline pH. Removal of all acidic vapours dramatically increased the rate of infectivity decay, with 90% loss after 2 min, while the addition of nitric acid vapour improved aero-stability. Similar aero-stability in droplets of artificial saliva and growth medium was observed. A model to predict loss of viral infectivity is proposed: at high RH, the high pH of exhaled aerosol drives viral infectivity loss; at low RH, high salt content limits the loss of viral infectivity.

The airborne transport of bacteria has a wide range of impacts, from disease transmission to cloud formation. By understanding the factors that influence the airborne stability of bacteria, we can better understand these processes.