key: cord-0307127-eyxrd8zq authors: Niazi, Sadegh; Philp, Lisa K.; Spann, Kirsten; Johnson, Graham R. title: Characterization of the utility of three nebulizers in investigating infectivity of airborne viruses date: 2021-03-12 journal: bioRxiv DOI: 10.1101/2021.03.11.435057 sha: 94a33194873f09e957d34055bcb0666b0e597cbb doc_id: 307127 cord_uid: eyxrd8zq Laboratory-generated bioaerosols are widely used in aerobiology studies of viruses, however few comparisons of alternative nebulizers exist. We compared aerosol production and virus survival for a Collison nebulizer, vibrating mesh nebulizer (VMN), and hydraulic spray atomizer (HAS). We also measured the dry size distribution of the aerosols produced, calculated the droplet sizes before evaporation and the dry size distribution from normal saline solution. Dry count median diameters of 0.25, 0.63 and 0.76 µm were found for normal saline from the Collison nebulizer, VMN and HSA, respectively. The volume median diameters were 2.91, 3.2 and 2.43 µm, respectively. The effect of nebulization on the viability of two influenza A viruses (IAVs) (H1N1, H3N2) and human rhinovirus (HRV)-16, was assessed by direct nebulization into an SKC Biosampler. The HSA had least impact on surviving fractions (SFs) of H1N1 and H3N2 (89±5%, 94±3%), followed by the Collison nebulizer (82±2%, 82±3%). The VMN yielded SFs of 78±2% and 76±2%, respectively. Conversely, for HRV-16, the VMN produced higher SFs (86±15%). Our findings indicate that although the VMN had the greatest impact on IAV survival, it produced higher aerosol concentrations within the airborne-size range making it more suitable where high aerosol mass production is required. Importance Viral respiratory tract infections cause millions of lost days of work and physician visits globally, accounting for significant morbidity and mortality. Respiratory droplet and droplet nuclei from infected hosts are the substantial potential carriers of such viruses within indoor environments. Laboratory-generated bioaerosols are applied in understanding the transmission and infection of viruses, simulating the physiological aspects of bioaerosol generation in a controlled environment. However, little comparative characterization exists for nebulizers used in infectious disease aerobiology, including Collison nebulizer, Vibrating mesh nebulizer, and hydraulic spray atomizer. This study characterized the physical features of aerosols generated by laboratory nebulizers, and their performance in producing aerosols at a size relevant to airborne transmission used in infectious disease aerobiology. We also determined the impact of nebulization mechanisms of these nebulizers on the viability of human respiratory viruses, including IAV H1N1, IAV H3N2 and HRV-16. Respiratory viruses are responsible for significant morbidity and mortality as well as 49 millions of lost days of work and physician visits globally (1, 2). Evidence suggests that the 50 airborne mode of transmission plays a significant role in the spread of respiratory viruses (3, 4) . 51 Respiratory droplets and droplet nuclei generated from the human host respiratory system are 52 potential carriers of pathogens within indoor environments (5, 6). However, there is no consistent 53 explanation that fully addresses the ability of these viruses to survive in an airborne state (7). It 54 has been reported that a combination of environmental and biological factors can affect the 55 efficiency of airborne transmission of viruses in indoor environments (8). In modelling airborne 56 transmission, laboratory-generated pathogen-laden aerosols are broadly used to investigate the 57 transmission, infection and toxicology of respiratory viruses (8) (9) (10) . Techniques used to generate 58 virus-laden aerosols in the laboratory enable greater control over aerosol characteristics, 59 including their droplet concentration, size and efficacy for carrying viruses (11). Animal models 60 have also been used to investigate the infection course and pathogenesis of inhaled 61 microorganisms (12). Here, the route of exposure, aerosol size, and infectious dose can influence 62 the infection development and pathogenic outcomes. In these models, the method of 63 aerosolization is critical, as reduced virus viability due to mechanical preparation would 64 influence study outcomes. Considering this critical factor, the field lacks a comprehensive study 65 that fully characterizes the physical and biological properties of carrier aerosols generated by 66 laboratory-use nebulizers, particularly for the simulation of respiratory virus-laden aerosols in a 67 clinical environment. The Collison nebulizer, as the gold standard, has dominated bioaerosol generation research 69 since its invention in 1932 (13) (14) (15) . It applies Bernoulli's theory, impaction of a liquid 70 suspension against the interior of a glass to generate small size aerosols (16) HSA concentrates the suspension into a stream by forcing it through a very small hole. There is a 91 one-way valve in the nozzle that maintains air from flowing back into the pump and allows for 92 suction within the pump so that liquid can be pulled up the tube. However, these latter two types 93 of nebulizer VMN and HSA have rarely been applied in the infectious disease aerobiology 94 research field. In this study, we hypothesized that a VMN or an HSA is superior to the 95 commonly used Collison nebulizer, as they result in less mechanical stress on respiratory viruses 96 and therefore enable a higher viable dose to be delivered for experimental purposes. This study was therefore designed to determine the influence of nebulization methods of 98 the 1-jet Collison nebulizer, VMN and HSA on the viability of human respiratory viruses 99 Influenza A virus (IAV) H1N1, IAV H3N2 and Human rhinovirus-16 (HRV-16). We also 100 characterized the size and weight of aerosols produced by these three nebulizers. were calculated for a solution composed of 9 g L -1 NaCl (dashed curves). Table 1 summarizes 110 the detailed properties of the aerosols produced. The Collison generated smaller aerosols 111 compared to the other two nebulizers, with count median diameters (CMD) of 0.045 µm and 0.25 112 µm for 0.05 g L -1 and 9 g L -1 NaCl solutions, respectively. However, the corresponding volume 113 median diameters (VMD) of these two NaCl solutions were 0.50 µm and 2.91 µm. VMN 114 generated larger aerosols than the Collison with a CMD of 0.11 µm and 0.63 µm for 0.05 g L -1 115 and 9 g L -1 NaCl solutions, and VMD of 0.56 µm and 3.2 µm, respectively. HSA generated the 116 largest aerosols with CMD of 0.13 µm and 0.76 µm, although the VMD were 0.43 µm and 2.43 117 µm, for 0.05 g L -1 and 9 g L -1 NaCl solutions, respectively. The geometric standard deviations 118 (GSDs) of the aerosols produced by the Collison, VMN and the HSA were 2.47, 2.09 and 1.86, 119 respectively, indicating that HSA generated a more uniform aerosol size compared to the other 120 two nebulizers. differences in SFs of the tested viruses between three mentioned nebulizers. 131 We also tested the shear and impact forces delivered by the high-velocity air streams of the 132 1-jet Collison nebulizer on the viability of viruses suspended during 30 min run times ( Figure 133 3). There was a gradual decrease in SFs for all three viruses over 30 min. SFs of IAVs H1N1 and 134 H3N2 declined from 0.82±0.02 at 5 min to 0.67±0.03 at 30 min, and 0.82±0.03 at 5 min to 135 0.68±0.02 at 30 min, respectively, these differences between 5 and 30 min operating times were 136 statistically significant (P=0.003 and P=0.012, respectively), suggesting that the Collison may 137 not be an appropriate option for studies that require longer nebulizer operating time or high 138 concentration of viable virus in aerosols. However, the SF of HRV-16 decreased less, from 139 0.83±0.08 at 5 min to 0.73±0.05 at 30 min, which was not significantly different, suggesting that 140 HRV is less susceptibility to mechanical stress compared to IAVs. (23) and IAVs (24) are 30 and 80-120 nm , respectively, therefore, any aerosols smaller than this 161 would not be able to carry viral particles, although could carry non-infectious viral fragments. Previous studies reported that a Collison nebulizer produces high concentrations of aerosols 163 which were monodisperse with a MMD between 1-2 µm (25) . Based on the particles size 164 distributions generated from a Collison nebulizer, May et al. stated that only 1 percent of the 165 mass of producing aerosols is larger than 5 µm (26, 27) . A study conducted by Ibrahim et al. 166 reported that the CMD of the aerosols produced by the Collison nebulizer is between 33 and 38 167 nm, which is not large enough to carry an Influenza virus particle (> 80 nm) (28). However, fluid 168 physiochemical properties such as viscosity, surface tension (29) derived from a solution of 9 g L -1 NaCl, which is larger than most viruses, suggesting that VMN 173 is suited to investigations of airborne virus-laden aerosols in terms of aerosol's physical size. HSA atomized aerosols were larger than those generated by the two other nebulizers, but were 175 still within the airborne range size, with a CMD of 0.76 µm for 9 g L -1 NaCl solution. However, HSA nebulization produced a lower concentration of aerosols in the same running time, followed 177 by 1-jet Collison nebulizer. This property could disadvantage studies that require a higher Table 2 . 201 This study was conducted to investigate the characteristics of aerosols generated by strains; however, the gravitational loss of aerosols was higher due to the generation of large size 210 droplets. Our results indicated that VMN is the best nebulizer for infectious disease aerobiology 211 research due to its production of aerosols in a usable size range (200nm-5000nm), higher aerosol 212 mass injection rate (2 g min -1 ) which allows short duration injection and good aging time 213 resolution. The VMN does not inject air, therefore it is suitable for studies that require 214 modulation of RH and temperature control within enclosed chambers. HSA were measured using a scanning mobility particle sizer (SMPS 3034, TSI Inc., Shoreview, 228 MN, USA). A dilute sample solution (0.05 g L -1 ) was used for measurements to ensure that the 229 dry aerosols were within the size range of the SMPS which is between 9 to 1000 nm, depending 230 on operating conditions. We then calculated the initial droplet sizes. 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