2. Materials and Methods

2.1. SARS-CoV-2 Stock
The SARS-CoV-2 isolate, England 02/2020 (EPI_ISL_407073) passage 3 (P3) used in this study was propagated by the High Containment Microbiology Department at UKHSA (formerly PHE), Porton Down, Salisbury, UK. The virus was isolated from a clinical sample taken during acute phase illness, using Vero E6 cells (ECACC, 85020206). A P2 master bank was produced using Vero E6 cells (BEI Resources, NR-596) and a P3 working bank produced in Vero/hSLAM cells (ECACC 04091501). Cell lines were infected at 95% confluence with an of MOI 0.0005–0.01 and maintained in 1× Minimal Essential Medium GlutaMax (Gibco, Grand Island, New York, USA), 4% heat-treated foetal bovine serum(Sigma, St Louis, MO, USA), 1× non-essential amino acids (Gibco, Paisley, Scotland, UK), 25 mM HEPES buffer (Gibco, Paisley, Scotland, UK); additionally, Vero/hSLAM cells were also maintained in the presence of 0.4 mg/mL geneticin (Gibco, Grand Island, New York, USA ). Virus was harvested 3 to 6 days post-infection and supernatant clarified by centrifugation (3000 rpm, 10 min). Virus was aliquoted and stored at −80 °C. The titre of the P3 virus stock was determined to be 2.0 × 107 PFU (plaque forming units)/mL by plaque assay. All work handling SARS-CoV-2 was performed within a containment level 3 laboratory.

2.2. Nebulisers
Six different nebulisers, with four types of nebulising action, were selected to aerosolise SARS-CoV-2 under different relative humidities (RH). The nebulisers are listed in Table 1. in an order relevant to the degree of physical impact of the aerosolization process on the pathogen (shear forces generated from pressure, from highest to lowest):
SLAG operation: To avoid damaging the perforated disc elements, the pressure was gradually increased by increasing the air flow applied in increments for each run. Similarly, the volume of virus inoculum applied during operation was altered to produce visible, vigorous bursting bubbles without excessive foaming, by increasing and decreasing the speed of the peristaltic pump used to aliquot the liquid. It was found, especially for the 90 mm SLAG, that pre-wetting the perforated 90 mm disc moments before applying compressed air to the system, achieved maximal levels of bubble bursting (sparging). Due to limitations on time, the air pressure and inoculum volume were not fully optimised for the 1 inch SLAG, however the ranges applied to both disc sizes appear in Table 1. All other nebulisers were used according to the manufacturer’s instructions.

2.3. Henderson Apparatus
A Henderson apparatus allows closed-circuit containment and direction of microbial aerosols generated from a nebuliser at a controlled relative humidity (Figure 1). The Henderson apparatus was used to deliver aerosols of SARS-CoV-2 at 40 L/min (balancing the total flow between the nebuliser and the Biaera unit) and the aerosols were collected at the end of the apparatus spray tube using biological samplers. The apparatus was contained within a flexible film isolator (FFI) within Containment Level 3 facilities. The challenge system is controlled by an AeroMP control unit (Biaera, Hagerstown, MD, USA). The AeroMP is a platform system designed to manage the aerosol generation, characterisation and sampling processes via a dashboard software laptop system. The aerosol management platform controls, monitors, and records all relevant parameters during an aerosol procedure including air flow rates, generated pressures, temperature and relative humidity. The software automatically monitors the conditions in the apparatus and balances the system airflows. The mean temperature during all experiments was 23.4 °C, range 21 °C to 25 °C.

2.4. Aerodynamic Particle Sizing Spectrophotometer
An APS device (model 3321, TSI Inc., Shoreview, MN, USA) was used to determine the size distributions of particles in aerosolised viruses, nebulised, and routed through the Henderson Apparatus. The APS device was set to sample 1:100 dilution, for two 30 s periods at 30 s and 4 min post-initiation of nebulisation. The sample was taken at the end of the spray tube and in an equivalent location to where animals would theoretically be exposed to the aerosol. See Figure 1.

2.5. Biological Samplers
To assess viability of nebulised viruses, all-glass impingers (AGI-30) containing 10 mls of cMEM (complete Minimal Essential Medium [12]) and operating at 12 L/min were connected to the Henderson apparatus down-stream of the spray tube (see Figure 1) for the duration of 5-min sprays [34].
A 6-stage Andersen sampler was used to fractionate the aerosols generated by the 6-jet Collison nebuliser into different sizes during sampling [35]. Relative humidity was maintained within a range of 45 to 60%. The Andersen was operated for 5 min at 28.3 L/min and was placed at the same point as the AGI-30 in the previous experiments, see Figure 1. Three different collection methods were employed in the Andersen’s stages (differing from the normal solid agar media used) to ensure the viral particles could be captured and enumerated, using glass petri dishes to reduce the effects of static charge: (a) 27 mL of cMEM (stages 1–5 only); (b) 20 mL 2% agarose plus 7 mL cMEM (6 stages); or (c) Gelatine Membranes (6 stages), 0.2 µm pore size (Sartorius, Goettingen, Germany). Four sterilised glass microscope slides were stacked under each gelatine membrane, to raise them to the correct height for size-based impaction to occur. Post-exposure, gelatine membranes were dissolved in 10 mL warmed cMEM for 1 min with agitation, before collecting for assay.
All biological sampling liquids described above were transferred to a cryotube for storage at −80 °C before analysis. The process of storing samples at −80 °C then thawing before processing was not found to significantly affect viability of the virus [12].

2.6. Plaque Assays
Plaque assays were completed according to protocols described previously [12]. Briefly, the thawed collection fluid was serially diluted 1 in 10 to an appropriate dilution, then plaque assayed on Vero-E6 cells, before staining and enumeration of plaques. The number of plaques in each well was determined and expressed as PFU.

2.7. Data Analysis
Virus titre in samples collected from nebulisers, AGI and Andersen samplers was calculated by taking averages of technical replicates (plaque assay performed in duplicate), to give PFU/mL; this was then multiplied by the total liquid volume of the sample to give total PFU.
Viable fraction (VF) values were calculated for each nebuliser assessed. The VF is the relationship of viable virus particles arising from the total number of particles nebulised. It is found from the total PFU collected in the AGI, divided by the total number of particles counted by the APS during the 5-min spray. The units are PFU/particle.
Spray Factors (SF) were calculated for each nebuliser assessed. The SF is a unitless ratio that defines a relationship between the viability of a challenge suspension in the nebulizer (Cneb, PFU/L) and the concentration of viable virus in the circulating aerosol (Caero, PFU/L) and is commonly used in animal aerosol challenge models [23,29,36]. The value obtained is system dependent, but it allows comparisons of stability between different agents used:SF=CaeroCneb
The concentration of virus in the circulating aerosol (Caero) is calculated by the formula:Caero=Cimp× VimpQimp× texp where Cimp is the concentration of virus (PFU/L), Vimp is the volume of collection fluid in the AGI (mL), Qimp is the sample flow rate of the AGI (L/min), and texp is the exposure time (min). Virus concentrations in Caero are converted to PFU/L to allow calculation against Cneb in PFU per litre of air sampled.
Calculation of Mass Median Aerodynamic Diameter (MMAD) size from Andersen data: Total PFU was used to calculate (in Microsoft Excel) cumulative percentage collected at each stage 1 to 6 as:Cumulative % of stage (n)=Σ[total pfu of stages (n→6)]Σ[total pfu stages (1→6)]×100
These percentages were plotted against the size cut-off for each stage, here the cut-offs for stages 1 to 6 are 7 µm, 4.7 µm, 3.3 µm, 2.1 µm, 1.1 µm, and 0.65 µm, respectively; and log10 transformed on both axes. A linear regression line was added and the particle size at 50% is then taken as the MMAD for that run. This took place in GraphPad Prism v9.