Ozone inactivation of airborne influenza and lack of resistance of respiratory syncytial virus to aerosolization and sampling processes

Marie-Eve Dubuis; Étienne Racine; Jonathan M Vyskocil; Nathalie Turgeon; Christophe Tremblay; Espérance Mukawera; Guy Boivin; Nathalie Grandvaux; Caroline Duchaine

doi:10.1371/journal.pone.0253022

. 2021 Jul 12;16(7):e0253022. doi: 10.1371/journal.pone.0253022

Ozone inactivation of airborne influenza and lack of resistance of respiratory syncytial virus to aerosolization and sampling processes

Marie-Eve Dubuis ^1,², Étienne Racine ³, Jonathan M Vyskocil ^1,², Nathalie Turgeon ¹, Christophe Tremblay ², Espérance Mukawera ^4,⁵, Guy Boivin ^6,⁷, Nathalie Grandvaux ^4,⁵, Caroline Duchaine ^1,^2,^*

Editor: Sander Herfst⁸

PMCID: PMC8274922 PMID: 34252093

Abstract

Influenza and RSV are human viruses responsible for outbreaks in hospitals, long-term care facilities and nursing homes. The present study assessed an air treatment using ozone at two relative humidity conditions (RHs) in order to reduce the infectivity of airborne influenza. Bovine pulmonary surfactant (BPS) and synthetic tracheal mucus (STM) were used as aerosols protectants to better reflect the human aerosol composition. Residual ozone concentration inside the aerosol chamber was also measured. RSV’s sensitivity resulted in testing its resistance to aerosolization and sampling processes instead of ozone exposure. The results showed that without supplement and with STM, a reduction in influenza A infectivity of four orders of magnitude was obtained with an exposure to 1.70 ± 0.19 ppm of ozone at 76% RH for 80 min. Consequently, ozone could be considered as a virucidal disinfectant for airborne influenza A. RSV did not withstand the aerosolization and sampling processes required for the use of the experimental setup. Therefore, ozone exposure could not be performed for this virus. Nonetheless, this study provides great insight for the efficacy of ozone as an air treatment for the control of nosocomial influenza A outbreaks.

Introduction

Viral outbreaks, including those caused by SARS-CoV-2, influenza and respiratory syncytial virus (RSV), are a major concern for hospitals, long-term care facilities and nursing homes [1–3]. Seasonal outbreaks of influenza occur annually and the threat of a pandemic is always present [4, 5]. The risk of developing complications from an influenza infection is greater for young children, pregnant women, adults that are 65 years and older, and people suffering from chronic diseases or immunosuppression [6]. Moreover, RSV is of great concern because almost every child is infected with this virus by the time they are 2 years old [7]. Adults can also be infected by RSV [8], particularly elderly individuals and those with underlying health problems, such as immunosuppression and cardiopulmonary diseases [9–12]. Complications from RSV such as bronchiolitis and pneumonia are common [7, 13] and outbreaks can occur in hospital settings, pediatric care units and neonatal intensive care units [14–17].

Previous studies have shown that airborne influenza genomes are present in hospital settings [18, 19], sometimes at concentrations of up to 10⁵ copies/m³ (unpublished data from authors). RSV genomes have been detected in the air of an emergency clinic [20], in hospital rooms with RSV-infected patients [21] and in a pediatric acute care ward [22]. Only a small number of positive samples (2.3%) were collected in the air of the pediatric acute care ward, which suggests that airborne RSV transmission is not likely [22]. On the other hand, another study detected airborne infectious RSV in the air around infants that were hospitalized with bronchiolitis in a pediatric ward and an intensive care. Mean concentrations were 3.71 X 10⁵ PFU/m³ in the pediatric ward and 4.09 X 10⁵ PFU/m³ in the intensive care unit [23].

Because of the significant risks associated with influenza and RSV, a means to control the potential aerosol transmission of these diseases would be beneficial for health-care facilities. For this purpose, we have tested an air disinfection protocol using ozone. The experimental setup has previously been used for phages and murine norovirus [24]. The results that were obtained were promising, even when using lower concentrations of ozone than those used by Tseng and Li (2006) for the inactivation of airborne phages [25].

According to Vejerano and Marr (2018), a single human-produced evaporating respiratory droplet of 60 μm (roughly 0.1 nl) is estimated to compose 1 ng of salt, 1 ng of protein and 0.06 ng of surfactant. They have also suggested that the surfactant of the droplet may protect the lipid membrane of viruses [26]. Additionally, evidence suggests that RSV is protected when sucrose is added to the media [27–30].

The aim of this study was to assess the virulence of two enveloped viruses, influenza A and RSV, after being aerosolized in the laboratory and exposed to an air disinfection protocol using ozone at two relative humidity conditions (RHs) in a rotating aerosol chamber. Bovine pulmonary surfactant (BPS) and synthetic tracheal mucus (STM) were added to influenza A aerosols in order to test any protective effects they may have on the virus. Due to the sensitivity of RSV to various testing conditions, only the preliminary experiments were conducted for this virus. These experiments tested the ability of RSV to withstand the aerosolization and sampling processes.

Materials and methods

Influenza A virus and host cells

Influenza A/Michigan/45/2015 (clinical strain A/Quebec/22578/2016 provided by Guy Boivin) was propagated in MDCK (CCL-34, ATCC) and ST6GalI-MDCK cells. Host cells were cultivated in Minimum Essential Medium (MEM; Gibco, Grand Island, USA) with 10% Fetal Bovine Serum (FBS; Wisent, Saint-Jean-Baptiste, CANADA). Two viral lysate stocks were prepared: the first contained 9.22 X 10⁷ TCID₅₀/ml and the second contained 1.68 X 10⁷ TCID₅₀/ml. Viral lysate subsamples were stored at -80°C in volumes of 30 ml or 27 ml.

Respiratory syncytial virus and host cells

The RSV-A2 strain (10-247-000; Advanced Biotechnologies Inc.) was amplified in HEp-2 cells (CCL-23; ATCC), purified on a 30% sucrose cushion [31, 32], and resuspended in 50mM Hepes and 5% sucrose in Opti-MEM (Gibco Grand Island, USA). Aliquots of 150 μl were stored at -80°C until use. The stock was titrated in 6-well plates at a concentration of 2.52 X 10⁸ PFU/ml. For all other experiments, the titration was performed using the TCID₅₀ technique, as described by Sun and López [33].

Influenza A aerosolization, exposure to ozone and sampling

One viral lysate subsample was thawed prior to each nebulization. Ten microliters of Antifoam A concentrate (Sigma-Aldrich, St-Louis, USA) was added to the nebulizing liquid to prevent foaming. Based on the estimated composition of a human droplet, we selected two substances to supplement the nebulizing liquid: BPS (lipid-based) and STM (protein-based). In order to compare the effectiveness of each supplement at protecting the airborne virus, they were each tested separately and added to the nebulizing solution at a final concentration of 10%. This method was used by Kormuth et al. (2018) with extracellular material produced by human bronchial epithelial cells (HBE ECM) [34]. However, we could not use pooled washes of HBE ECM because a volume of 60 ml of the supplement was needed for our experiments. Depending on the test conditions, the nebulizing liquid was either left as is (no treatment; NT) or supplemented with 10% of BPS (BPS treatment; BPST) (provided by BLES Biochemicals Inc.) or 10% of STM (STM treatment; STMT). The STM was composed of 0.3 g of albumin (05470-1G; Sigma, St-Louis, USA) and 1.2 g of pig gastric mucin-type III (M1778-10G, Sigma) in 28.2 ml of buffer solution (154 mM NaCl, 3 mM CaCl₂, 15 mM NaH₂PO₄/Na₂HPO₄; pH 7.4) [35].

Aerosolization was performed as described in [24]. Briefly, the virus stock was nebulized at 20 psi with a 6-jet Collison nebulizer (BGI, Waltham, USA). The Collision nebulizer was connected to compressed medical-grade air for 10 minutes inside a 55-L rotative environmental aerosol chamber [36], which was placed in a biosafety level (BSL) II cabinet. Diffusion dryer tubes of different lengths were used to adjust the RH inside the chamber. RH values were monitored with a probe (model STH-ID 300, KIMO Instruments, Montpon, FRANCE). Resulting RH values were 31.6 ± 3.9% and 80.6 ± 1.9% for the NT and BPST experiments. For the STMT experiments, RH values of 34.3 ± 3.6% and 75.8 ± 2.2% were obtained. The nebulizing step was followed by a 10-minute waiting time to ensure an even mix of aerosols in the chamber. Aerosol distribution size and number were assessed using an Aerodynamic Particle Sizer (APS) (model 3321; TSI Inc., Shoreview, USA). Particle dilution was necessary for accurate reads, and therefore a 1/100 dilutor with a 1/20 capillary (model 3302A, TSI Inc.) was used with the APS. The mass median aerodynamic diameters (MMAD) for the NT experiments were 1.22 ± 0.03 μm at 32% RH and 1.26 ± 0.03 μm at 81% RH. For the BPST, the MMADs were 1.18 ± 0.03 μm at 32% RH and 1.35 ± 0.02 μm at 81% RH, and the MMADs for the STMT were 1.27 ± 0.02 μm at 34% RH and 1.51 ± 0.07 μm at 76% RH.

Next, aerosols were exposed to air (designated as the reference conditions) or a mixed gas of approximately 21% of ozone in air for 0, 30 or 60 minutes at low or high RHs. Ozone was produced by a generator (model 201705004A210Y, EMO3) and injected into the chamber at a flowrate of 0.4 L/min for 30 seconds. Ozone injection continued until a concentration of 0.23 ppm ± 0.03 ppm was obtained for the NT and BPST experiments. The injection continued for 50 seconds for the STMT experiments, and a concentration of 1.70 ± 0.19 ppm was reached.

Samples were collected using a SKC BioSampler (SKC Inc., Eighty Four, USA) filled with 20 ml of MEM and connected to an SKC vacuum pump (model 228 ± 9605; SKC Inc.) for 20 minutes at a flowrate of 12.5 L/min. As mentioned in Dubuis et al. (2020), due to the 20-minute sampling step, a 10-minute exposure period was added to the tested exposure times of 0, 30 and 60 minutes. Consequently, the exposure times for the NT and BPST experiments were 10, 40 and 70 minutes.

For the STMT experiments, a new ozone probe (PortaSens III; Analytical Technology Inc., USA) coupled with two sensors (500–2000 ppb; 00–1163 and 1–5 ppm: 00–1008) was used. The sensors could be quickly switched depending on the ozone concentration during the experiments. The sensitivity of the sensors is considered to be 1% of their ranges, which are 20 ppb (500–2000 ppb) and 50 ppb (1–5 ppm), with a resolution of 1 ppb and 0.01 ppm, respectively. Because the probe was able to directly measure the ozone concentration inside the chamber, readings were performed every 10 minutes, which allowed us to monitor the residual ozone concentration over time. After the nebulizing step, ozone was injected into the chamber for 50 seconds. Following this step, even distribution of aerosols was obtained within a 10-minute waiting period. The ozone concentration was then measured, followed by an APS reading. Because of the 10-minute mixing of aerosols prior to the ozone and APS readings, a 10-minute period needed to be added to the exposure times, as well as a 10-minute period from the sampling step. Total exposure times for the STMT experiments were 20, 50 and 80 minutes.

As explained in previously published work, control samples were drawn from the nebulizing stock to monitor the viral concentrations throughout the experiments [24]. The effect of ozone in the SKC BioSampler was also assessed and considered negligible [24].

RSV nebulizing and sampling assays

Virus resistance in air sampler

Solutions of DMEM + 5% sucrose and DMEM + 20% sucrose were prepared and stored at 4°C until use. Final sucrose concentrations of 5% and 20% were obtained by following the methods from a previous report by Grosz et al. [30]. A conical tube was filled with 50 ml of the 5% sucrose solution and spiked with three aliquots of the frozen RSV stock. The solution was vortexed and divided between two SKC BioSamplers (20 ml each). An aliquot of the spiked virus solution (initial concentration) was stored on ice. The SKC BioSamplers were placed inside a BSL II cabinet and were not connected to an aerosol chamber. The BioSamplers sampled clean air from the BSL II cabinet for a period of 20 minutes. Aliquots for both SKC BioSamplers were extracted (final concentration) and kept on ice. TCID₅₀ was performed immediately for each aliquot. The same procedure was repeated with the spiked 20% sucrose solution.

Virus resistance in two nebulizers

An assay was conducted to determine whether the nebulizing process alone could inactivate RSV. The 6-jet Collison nebulizer and a gentler device, the Aeroneb Lab nebulizer (Aerogen Inc., Galway, Ireland) were used.

The 6-jet Collison nebulizer was filled with 20 ml of DMEM mixed with one aliquot of the frozen viral stock. An aliquot that represented the initial concentration inside the nebulizer was extracted and stored on ice. The 6-jet Collison nebulizer was connected to the same experimental setup as the one for influenza A aerosolization described above, and run for 10 minutes. A second aliquot was extracted and kept on ice, this one containing the final nebulizer concentration. TCID₅₀ was performed on these two aliquots. The 6-jet Collison experiment was performed in sextuplicate.

For the Aeroneb nebulizer, two different solutions were prepared: one with 5% sucrose and the other with 20%. The 5% sucrose solution was spiked with the frozen RSV stock (three aliquots of frozen RSV in 50 ml of solution) and 7 ml of this mixture was used to fill the nebulizer. An aliquot was immediately drawn and kept on ice (initial concentration). The Aeroneb was connected to a desiccant tube with a collection cup, which was then connected to the aerosol chamber setup. The collection cup was disinfected with 70% ethanol and placed in an ice bath. Air dilution was performed at a rate of 2 L/min to push the produced aerosols into the chamber and the nebulizer was run until empty (18 minutes). An aliquot from the collection cup (final concentration) was kept on ice and immediately titrated using the TCID₅₀ method. This experiment was performed once, after which the 20% sucrose solution assay was performed using the same method.

RNA extraction, RT-qPCR and influenza A titration

Three aliquots of each sample were kept at -80°C: one for culture, one for RT-qPCR and one as a backup. Two aliquots from the nebulizing liquid were also frozen. To better conserve the virus, each aliquot was supplemented with bovine serum albumin factor V (BSA; Gibco, Canada) following the proportions described by Turgeon et al. (2019).

The QIAamp Viral RNA Kit (QIAGEN, Hilden, GERMANY) was used for RNA extraction without the RNA carrier. Elution was performed in two 40-μl volumes of TE buffer (80 μl total). One-step Real-Time PCR (RT-qPCR) was conducted with the Bio-Rad CFX384 thermocycler (Bio-Rad Laboratories, Mississauga, CANADA) for 5 μl of the extracted RNA. For the controls, 1/10 000 dilutions were required to fit the standard curve. The following conditions were used: 50°C for 10 min, 94°C for 3 min, 40 cycles at 94°C for 15 s and 60°C for 1 min. Each plate contained a standard curve of a 10-fold dilution series of influenza A plasmid preparation, as well as no template controls (NTC). Specific influenza A primers and a probe were used [37].

For infectious virus titration, TCID₅₀ was performed using MDCK or ST6GalI-MDCK cells. Plates were incubated at 37°C with 5% CO₂ for 72h.

Calculations and statistical analysis

Calculations were performed as described in Dubuis et al. (2020) [24]. Briefly, infectious ratios (IRs) for the reference conditions (air) and the ozone conditions were obtained by dividing mean culture counts (PFU/ml) with mean qPCR results. The normalized infectious ratios (NIRs) were then obtained by normalizing IRs with the 6-jet Collison nebulizer’s initial viral concentrations. Relative infectious ratios (RIRs) were calculated using the ozone NIRs and the corresponding median air NIRs. Consequently, the aerosolization, humidity and aerosol-aging effects are not included in the RIR values.

Data were analyzed using a three-way ANOVA. The three fixed effects were treatments, temperature and humidity with three, three and two levels, respectively. Statistical models included two-by-two interaction terms. Univariate normality was verified by applying the Shapiro-Wilk tests on the error distribution from the statistical models. The Brown-Forsythe variation of Levene’s test statistic was used to verify equal variances. A logarithm transformation was used for all statistical analyses, because normality and variance assumptions were not fulfilled. In order to perform comparisons between RH and exposure times, low RH values (32% and 34%) and high RH values (76% and 81%) were considered to be identical. Exposure times were combined into three groups: short (10 min and 20 min), medium (40 min and 50 min) and long (70 min and 80 min). These groups were then compared with each other. For these statistical analyses, results with P-values < 0.05 were considered significant. Analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC).

Ozone doses were estimated by calculating the area under concentration-vs-time curves, for RH values of 34% and 76%. Concentration-vs-time curves were fitted to observed data by regression analysis for exposure times between 10 min and 70 min (see S1 File for details). Regression analyses were performed in RStudio (version 1.2.5033). The areas under the curves were calculated numerically with a trapezoid scheme coded in C++ and validated against analytical computations. For exposure times between 0 and 10 min, extrapolation of the fitted concentration-vs-time curves was deemed too unreliable for dose calculations. Accumulated dose between 0 and 10 min was instead estimated by assuming that ozone concentration was constant over that time interval and equal to the fitted concentration value at 10 min. Consequently, calculated ozone doses represent floor values. Confidence intervals were not reported for dose calculations because the uncertainty associated to the constant concentration assumption was much larger than the width of the confidence bands around the regressions curves.

Results

Ozone concentrations and doses

The ozone probe was used to monitor the concentration of ozone inside the aerosol chamber throughout the exposure periods. The initial readings were performed 10 min after ozone injection and subsequent readings were conducted at 10-min intervals. Therefore, the ozone concentrations measured with the PortaSens III probe do not include the additional 10-min sampling period. Although ozone concentrations between the two RHs were at times highly varied, the same quantity of ozone was injected for both RHs. The injected ozone concentration for all experiments was 1.70 ± 0.19 ppm, which was determined based on the ozone measurements at 34% RH after the 10-min mixing period. As shown in Fig 1, the ozone concentration decreased gradually over time at 34% RH. At 76% RH, the initial concentration was much lower, at 0.44 ± 0.12 ppm, and it decreased to close to zero after 30 min.

Fig 1 — The boxes at 10 minutes of exposure represent a total of 18 readings for both RHs. Boxes at 20, 30 and 40 min represent twelve readings each and boxes at 50, 60 and 70 min represent six readings each.

The calculated ozone doses floor values for both RH levels at 10-minute intervals are presented in Table 1. Even if the ozone injection was identical, after an exposure time of 70 min, the ozone dose floor value at 34% was much higher, at 58.67 ppm ∙ min, than the dose at 76%, at 7.41 ppm ∙ min. Furthermore, the values increase by a factor of two between 10 and 70 minutes at 76%, while they increase by a factor of three at 34% RH.

Table 1. Ozone doses floor value (ppm ∙ min) for each exposure time at 34% and 76% RH.

Exposure Time (min)	Ozone dose (ppm ∙ min)
Exposure Time (min)	34% RH	76% RH
10	16.90	3.69
20	31.11	5.72
30	41.05	6.49
40	48.01	6.89
50	52.88	7.13
60	56.29	7.29
70	58.67	7.41

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Reference conditions for influenza A

Fig 2 shows the NIRs for influenza A for the NT (Fig 2A), BPST (Fig 2B), and STMT (Fig 2C) experiments. As explained in Dubuis et al. (2020), normalized infectious ratio (NIR) values close to one mean that the reference conditions (exposure to air) do not cause a decrease in infectivity compared to the nebulizer content. NIR values below one indicate a reduction in infectivity for aerosols exposed to the reference conditions [24]. It is worth mentioning that the nebulized concentrations of infectious viruses for the NT, BPST and STMT experiments were 6.96 X 10⁷ ± 9.40 X 10⁷ PFU/ml, 1.58 X 10⁷ ± 5.35 X 10⁶ PFU/ml and 1.58 X 10⁷ ± 4.54 X 10⁶ PFU/ml, respectively. The concentrations of sampled viruses were lower, at 1.58 X 10⁵ ± 1.92 X 10⁵ PFU/ml for the NT, 2.20 X 10⁴ ± 3.49 X 10⁴ for the BPST and 1.58 X 10⁴ ± 2.26 X 10⁴ for the STMT.

Interactions with exposure times did not affect the NIR (p = 0.93) for both supplement and non-supplement conditions, meaning that the NIR values remained stable throughout the experiments. Additionally, the interactions between RH and both the NT (p = 0.74) and the STMT (p = 0.41) did not affect the NIRs. Indeed, the infectivity loss was equal to or less than one order of magnitude for both RHs. However, the interaction between BPST and RH (p < 0.01) was significant. When BPS was added to the nebulizer solution, there was an immediate decrease in infectivity of one to two orders of magnitude when RH was at 32%, and a decrease of three to four orders of magnitude when RH was at 81%. These NIR values reveal differences between the BPST and the NT (p < 0.01) and the BPST and the STMT (p < 0.01). The BPST significantly reduced the infectivity of Influenza A compared to the NT or compared to the STMT. Therefore, the NT and STMT conditions are more appropriate than the BPST for the conservation of influenza’s infectivity throughout the reference condition.

Relative infectious ratios

The relative infectious ratios (RIRs) are presented in Fig 3, which represents the only effects that were observed as a result of ozone exposure. There was no significant difference (p = 0.31) between the NT (Fig 3A) and the BPST (Fig 3B). This means that the BPS did not protect the remaining infectious airborne influenza A from an exposure to 0.23 ppm ± 0.03 ppm of ozone. Significant differences were observed between interactions at 81% RH (p = 0.02) and at exposure times of 40 min (p < 0.01) and 70 min (p < 0.01), during which RIRs decreased by one to two orders of magnitude.

RIR results for the NT samples (Fig 3A and 3C) exhibited strong differences between those with ozone exposures of 0.23 ppm ± 0.03 ppm and those with exposures of 1.70 ± 0.19 ppm (p < 0.01). The maximum reduction in infectivity was four orders of magnitude, which was obtained after 80 min at 76% RH for an ozone exposure of 1.70 ± 0.19 ppm (ozone dose floor value of 7.41 ppm ∙ min). The efficacy of this treatment was higher at 76% RH compared to 34% RH, with notable differences after 50 min (p < 0.01) and 80 min (p < 0.01) of exposure time.

For an ozone exposure to 1.70 ± 0.19 ppm at 34% RH, the STMT (Fig 3D) did not increase airborne influenza A infectivity after 20 min (p = 0.14) and 50 min (p = 0.29) of exposure time. However, at 80 min of exposure (p < 0.01) (Fig 3D), the STMT helped protect the viral aerosols from the large (two orders of magnitude) decrease in infectivity that was observed for the NT (Fig 3C). At 76% RH, the viral aerosols exposed to the STMT exhibited a loss in infectivity of one order of magnitude after 20 min (p = 0.01) and 50 min (p < 0.01), meaning that the STMT did not protect aerosols from the ozone exposure of 1.70 ± 0.19 ppm. After 80 min, no difference in infectivity was observed between the NT and STMT aerosols (p = 0.95).

When combining the maximum infectivity loss observed from the reference condition (Fig 2; four orders of magnitude) and the ozone exposure (Fig 3; two orders of magnitude), the BPST resulted in the highest decrease of infectivity. Because the majority of the loss happened when influenza aerosols were exposed to the reference condition, the exposure to ozone becomes less powerful. Therefore, the combination of BSPT and ozone cannot be chosen as the more effective air treatment. The opposite was observed for the NT and STMT, with a maximum infectivity loss of one or four orders of magnitude when exposed to the reference condition and the ozone treatment, respectively. The high RH (76%) and longer exposure time (80 min) also exhibited the highest reduction of infectious influenza. Overall, an exposure of 1.70 ± 0.19 ppm of ozone at 76% RH for 80 min resulted in the highest infectivity loss for the NT and STMT.

RSV infectivity assay

Virus resistance in air sampler

The spiked RSV solution with 5% sucrose contained a concentration of 3.98 X 10⁵ TCID₅₀/ml. The spiked RSV solution with 20% sucrose had a concentration of 1.26 X 10⁶ TCID₅₀/ml.

After 20 minutes of sampling clean air with the SKC BioSampler, a loss of infectivity of one and two orders of magnitude was observed in the 5% and 20% sucrose solutions, respectively. The viral titers left in the two SKC BioSamplers with 5% sucrose were 3.98 X 10³ TCID₅₀/ml and 2.24 X 10⁴ TCID₅₀/ml, for a mean concentration of 1.32 X 10⁴ TCID₅₀/ml. The viral titers that remained after sampling with a 20% sucrose solution were 1.26 X 10⁴ TCID₅₀/ml and 7.08 X 10³ TCID₅₀/ml, resulting in a mean concentration of 9.83 X 10³ TCID₅₀/ml.

Virus resistance in two nebulizers

The results from the infectivity assays performed with both the 6-jet Collison and the Aeroneb nebulizers are shown in Fig 4. The detection limit for the titration method was calculated with one positive well and corresponds to a concentration of 2.24 X 10¹ TCID₅₀/ml. The initial infectious concentration in the 6-jet Collison nebulizer was 5.87 X 10⁵ ± 3.95 X 10⁵ TCID₅₀/ml. After nebulization, the viral titer dropped considerably. Of the six replicates, two had concentrations of 3.98 X 10¹ TCID₅₀/ml, two others were at the detection limit of 2.24 X 10¹ TCID₅₀/ml, and the remaining two were below the detection limit. This suggests that when the RSV passes through the 6-jet Collison nebulizer, it seems to almost completely inactivate the virus.

When RSV infectivity was examined using the Aeroneb, the initial concentrations were 3.98 X 10⁵ TCID₅₀/ml and 1.26 X 10⁶ TCID₅₀/ml for the 5% and 20% sucrose solutions, respectively. After nebulization, 1.26 X 10² TCID₅₀/ml remained infectious in the 5% sucrose solution, while the concentration was below the detection limit in the 20% sucrose solution. Therefore, despite the Aeroneb using a gentler nebulizing method, the RSV did not retain infectivity even with the sucrose supplement.

After assessing the results for RSV resistance to air sampling and nebulizing, it is clear that this virus does not maintain enough infectivity for reliable experimentation in the rotating aerosol chamber. For each condition, the combined infectivity loss from sampling and nebulizing was at least four orders of magnitude. Furthermore, aerosol loss is expected in the experimental setup, which can occur in the diffusion dryer tubes, at the junction of tubing and barb fittings and while sampling with the SKC BioSampler. From previous non-published experiments performed in our lab, it has been noted that there can be an aerosol loss of two to three orders of magnitude for every aerosol experiment. Consequently, the overall infectivity and aerosol loss would be around six orders of magnitude, which is too large to conduct experiments on the exposure to air and ozone for RSV, as was done for Influenza A.

Discussion

The ozone probe used in this experiment allowed for the continued measurement of ozone concentrations inside the aerosol chamber at two RHs over a 70 min period (Fig 1) and the calculations of the ozone dose floor values. These additional informations obtained from the ozone probe are interesting since it was previously suggested that ozone interacts with water to form free radicals [38] and that high levels of humidity in the air could increase the production of free radicals and lead to virus inactivation [39, 40]. As seen in Fig 1 and Table 1, there is a notable difference in the initial ozone concentration measurements and dose floor values between the two tested RHs. Because all setup parameters were identical apart from the RH inside the chamber, one may conclude that the differences were caused by RH alone. The majority of the difference in ozone concentration between 34% and 76% also occurred during the first 10 min, which could indicate that the reaction with humidity is quite fast. Furthermore, the ozone concentration was still detectable at low RHs after 70 min, indicating that ozone was present during the entire exposure period. On the other hand, even if the ozone concentration was close to zero after 40 min at high RHs and that the ozone dose floor value only increased from 6.89 ppm ∙ min (40 min) to 7.41 ppm ∙ min (70 min), the RIR values (Fig 3) show that the inactivation of airborne influenza was superior after longer exposure times. Additionally, for all tested conditions, the effect of ozone treatment is greater at high RHs than at low RHs, indicating that the free radicals that are produced have higher virucidal properties than ozone alone. This is consistent with previous studies [25, 40, 41]. Moreover, the addition of STM seems to have a protective effect at 34% RH after 80 min, while a disrupting effect is noted at 76% RH after 50 min. Although free radicals are produced when ozone interacts with water and organic compounds, this outcome could be the result of a synergy between high RH and the presence of proteins. Additional testing could be useful to verify if a synergistic inactivation effect is observed while using different supplements at high or low RH.

The NIRs that resulted from the NT and the STMT are comparable with those from Kormuth et al. (2018), in which HBE ECM was added. This confirmed that airborne influenza A maintains infectivity at low and high RHs for one hour [34]. However, our findings show that the BPST did not provide protection for influenza A aerosols. Indeed, it was responsible for NIR decreases of one to two (32% RH) and three to four (81% RH) orders of magnitude for the reference conditions (Fig 2B). One hypothesis for these results is that the surfactant interacts with the lipid membrane of the virions, which causes structural damage. This differs from the study by Vejerano and Marr (2018) study, which suggested that it could act as a protective viral coating [26]. When STM was added (Fig 2C), the infectivity loss was negligible when influenza A aerosols were exposed to the reference conditions. Influenza A therefore seems to be a resistant virus when airborne.

BPST experiments where influenza A aerosols were exposed to 0.23 ppm of ozone were carried out prior to the STMT experiments. NIR results indicated that the BPST led to more inactivated airborne influenza A compared to the NT. These observations led to the selection of the other supplement, STM, and exposure to a higher ozone concentration was not tested with the BPST.

We hypothesized that adding a supplement could cause a change in particle size, thus affecting the response of influenza A when aerosolized and altering its resistance to air or ozone. Particle MMADs were at 1.22 ± 0.03 μm at 32% RH and 1.26 ± 0.03 μm at 81% RH, nearly the same diameters as those observed when there was no supplement. The MMADs were also similar at low RHs for the BPST or STMT, at 1.18 ± 0.03 μm and 1.27 ± 0.02 μm, respectively. At high RHs, larger MMADs were observed for the BPST (1.35 ± 0.02 μm) and the STMT (1.51 ± 0.07 μm). The larger particle sizes do not explain the differences in infectivity that were observed as a result of exposure to the reference conditions (Fig 2), nor the differences observed due to ozone exposure (Fig 3).

RSV infectivity was assessed during two preliminary tests: the 6-jet Collison test and the infectivity assay. The information obtained from these tests was crucial because the experimental set up is not leak-proof and a loss of two to three orders of magnitude can be expected. Therefore, a minimum concentration of 10⁵–10⁶ TCID₅₀/ml must be preserved in the nebulizing liquid in order to perform the exposure experiments for air and ozone. As shown in Fig 4, almost all RSV infectivity was lost during the nebulization step using the 6-jet Collison nebulizer. This nebulizer has been able to successfully aerosolize multiple viruses, including four phages [42], the murine norovirus (MNV-1) [24] and influenza A, suggesting that RSV may be more fragile than these other viruses. Since resistance behaviors from multiple aerosolized viruses are somewhat unpredictable [24, 43], the different behaviors of influenza A and RSV were not surprising. An infectivity assay verified whether the loss of infectivity was caused by the Aeroneb while nebulizing or the BioSampler while sampling. Despite the addition of sucrose to the RSV suspension, infectivity was lost through both devices. The number of infectious viruses that remained after being nebulized in the Aeroneb (1.26 X 10² or < 2.24 X 10¹ TCID₅₀/ml) was not sufficient to perform the subsequent exposure experiments. The SKC BioSampler also resulted in a loss of infectivity of one to two orders of magnitude. Previous work from Grosz et al. (2014) showed that RSV maintains its infectivity when aerosolized with the PARI Sprint^TM nebulizer [30]. This gentler device uses a compressor to produce aerosols instead of the vibrating membrane used in the Aeroneb, which could explain the higher infectious titers obtained in their study. In conclusion, RSV seems to be very sensitive to the nebulization processes of the 6-jet Collison and the Aeroneb nebulizers, as well as the sampling process associated with the SKC BioSampler.

Our experimental setup has been designed to allow us to work with level II viruses. Every part has been carefully chosen for that purpose, but compromises had to be made on some aspects. The aerosol chamber is constructed in aluminum to minimize particle loss due to static and it rotates to reduce particle deposition. It is also airtight and therefore maintains the level of relative humidity. Aerosol concentrations and MMAD over 18h have been studied in a previous publication [36]. The ozone is injected directly inside the chamber. The valves are shut after the injection to prevent the ozone from escaping the chamber. The ozone concentrations are then measured with a probe connected directly to the chamber. Consequently, the ozone concentration measures in the chamber are accurate. However, the aerosol path between the nebulizer and the chamber could lead to significant particle loss. As a safety measure, the material used to make the diffusion dryer tubes had to be shatterproof. It also had to be transparent to monitor the color of the silica beads. Therefore these tubes were made with polycarbonate. This plastic is known to have static properties, which could cause aerosol loss. These tubes are also not completely airtight because they protect the other parts of the setup by acting as breaking points in case of an accidental pressure increase. For the setup to fit inside the BSL II cabinet, the aerosol path includes 180° elbows in a few spots. The impaction of aerosols could occur in these elbows. The aerosol tubing used to connect the nebulizer, the diffusion dryers and the aerosol chamber is made of static dissipative silicon coated with carbon. However, since it the aerosol path (nebulizer–up to five diffusion dryers—chamber), particles could be lost by diffusion. As for the SKC BioSampler, this device preserves the virus infectivity and has a great collection efficiency of 1 μm particles but the re-aerosolization phenomenon has previously been documented [44]. While all these features could cause aerosol loss, we only evaluate the portion reaching the chamber by normalizing the infectious ratios. The drawbacks of this loss is that we are unable to test viruses that are sensitive or that do not produce high viral titers, as was the case for RSV.

The bacteriophage Phi6 has been used in multiple studies as a surrogate for influenza due to its similar RNA genome and envelope [24, 42, 45]. Phi6 was therefore a good candidate to be used as a model virus for RSV. The results obtained in our study for influenza A and RSV resistance to aerosolization and ozone exposure can be compared to those of Phi6. Dubuis et al. (2020) have shown that Phi6 loses its infectivity at low RH levels and is inactivated at high RHs when exposed to 1.13 ± 0.26 ppm of ozone [24]. In our study, even using 1.70 ± 0.19 ppm of ozone instead of 1.13 ± 0.26 ppm did not result in the inactivation of influenza A at low RHs. However, ozone was able to reduce its infectivity by four orders of magnitude at high RHs. Influenza A is significantly more resistant than Phi6 when aerosolized. Compared to RSV, which could not sustain nebulization and air sampling in our experiments, Phi6 is more robust when aerosolized. Therefore, the use of Phi6 as a surrogate for either airborne influenza A and RSV is not supported by our findings.

According to a guideline for virucidal chemical disinfectants published by the German Association for the Control of Virus Diseases and the Robert Koch Institute, a virucidal disinfectant must reduce viral infectivity by at least four orders of magnitude [46]. In this study, the effect of ozone as a disinfectant is presented using RIR values. For all the tested conditions, the required infectivity reduction was achieved after exposure to 1.70 ± 0.19 ppm of ozone at 76% RH for 80 min (Fig 3C and 3D) which also corresponds to an ozone dose floor value of 7.41 ppm ∙ min. The STMT did not protect airborne influenza A from the virucidal activity of ozone (Fig 3D), especially when compared to the NT (Fig 3C).

To the best of our knowledge, this study is the first to test an air disinfection protocol that uses ozone to inactivate airborne influenza A. The STMT has also provided great insight into its protective role for airborne influenza A. This supplement is only effective against ozone exposure at low RHs, and does not prevent the inactivation of aerosolized influenza A at high RHs.

The influenza virus is a persistent burden on hospitals, and air treatment could be an effective way to reduce its nosocomial transmission. The results from this study can be used as a basis for air decontamination in different settings. Since the studied ozone concentrations are above the threshold limit value–ceiling of 0.1 ppm [47], the decontamination protocol should be performed in leak-proof rooms in the absence of occupants for at least 80 min. This issue could be problematic for some settings that cannot evacuate the infected rooms for the proper amount of time required for air disinfection. Another element to consider is the reaction of ozone with some materials including rubber and its derivatives [48] which can cause the degradation of some mattresses [49]. Disinfection of recycled air inside the heating, ventilation and air-conditioning (HVAC) plenum could be an alternative to limit the material degradation and allow the occupants to stay in the rooms. One downside of this approach is the contact time, which cannot be performed over an 80-min period. The calculated ozone dose floor value required to achieve a reduction of four orders of magnitude was 7.41 ppm∙min and was obtained at 76% RH. Since the real ozone dose to which viruses were exposed might be significantly higher than the calculated ozone dose floor value, the exposure in the HVAC plenum should aim for an ozone dose of at least 7.41 ppm∙min at an RH close to 76%. Depending on the contact time in the HVAC plenum, ozone generation could be modulated to achieve this minimal dose. The other limitation of a disinfection in the HVAC plenum is that the furniture and areas hard to reach inside the infected rooms would not be decontaminated. Considering these elements, each setting should assess which option is the most appropriate to meet its needs before implementing an air disinfection protocol using ozone.

The emergence of the SARS-CoV-2 pandemic has shed light on the transmission of viruses through air. Evidence suggests that aerosols in hospital rooms can contain SARS-CoV-2 viruses [50, 51], making the implementation of ozone air treatments a practical way to reduce viral transmission and to ensure a safe environment for patients. Although ozone disinfection has not been tested directly on SARS-CoV-2 aerosols, evidence from the inactivation of other airborne viruses (four phages, murine norovirus and influenza) suggest that ozone could be a useful disinfectant for different types of viruses. As further evidence, a recent study has suggested that ozone could be a potential disinfectant for SARS-CoV-2 by targeting its spike protein and envelope lipids [52].

Conclusion

In conclusion, the efficacy of an air treatment protocol using ozone to target airborne influenza A was evaluated. RSV did not survive the aerosolization and sampling processes in our laboratory setup, and therefore ozone exposure experiments could not be conducted using this virus. In order to better reflect aerosols produced by infected people, which can contain surfactants and proteins, the nebulizing liquid was supplemented with BPS and STM for experimentation. These various conditions provided clues to understanding the protective roles of BPS and STM for influenza A when aerosolized and exposed to ozone. The air treatment that resulted in a reduction in infectivity of four orders of magnitude was the exposure to 1.70 ± 0.19 ppm of ozone at 76% RH for 80 min, and was effective for both the STMT and the NT, indicating that ozone could be qualified as a virucidal disinfectant.

This study provides robust results regarding the efficacy of air treatment, which could be useful for the control of nosocomial transmission of influenza A. Further tests should be conducted in order to determine how air disinfection could be implemented in hospital settings and other environments that could benefit from this technology.

Supporting information

S1 File. Regression analyses.

(DOCX)

Click here for additional data file.^{(2MB, docx)}

Acknowledgments

The authors would like to thank Liva Checkmahomed and Marie-Eve Hamelin for technical support as well as Serge Simard for statistical analysis and Amanda Toperoff and Michi Waygood for English revision.

Data Availability

All relevant data are within the paper.

Funding Statement

CD: Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (RGPIN-2020-04284). https://www.nserc-crsng.gc.ca CD and NG: Quebec Respiratory Health Network team grant. https://rsr-qc.ca MED and CT: Natural Sciences and Engineering Research Council of Canada MED: Institut de Recherche Robert-Sauvé en Santé et en Sécurité du Travail. irsst.qc.ca The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Ozone inactivation of airborne influenza and lack of resistance of respiratory syncytial virus to aerosolization and sampling processes

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2. Please provide the source of the Influenza A/Michigan/45/2015 sample you used in this study.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors of this manuscript elaborated on previous work that described the inactivation of aerosolized phages and murine norovirus by ozone (Dubuis et al., 2020). Here the authors used the same approach to study the inactivation of an influenza- and respiratory syncytial virus. This study provides new insights in the aerosol stability of influenza viruses in relation with relative humidity and ozone concentration, which are placed in perspective with other studies. However, some points are unclear to the reviewer and need to be clarified prior to publication.

1. What is the composition of the non-treated nebulizing liquid (is it a buffer or a medium?) and do the authors expect hardly any inactivation after incubation in air for 60 min under these conditions?

2. The nebulizing liquid is supplemented with either BPS or STM, of which the authors stated that this was as previously done by Kormuth et al., 2018 with extra cellular material from human bronchial epithelial cells (HBE ECM) (Line 108). However although 10% supplementation is used in both studies, this does not necessarily result in comparable conditions in terms of e.g. protein/lipid content. STM for example contain albumin and pig gastric mucin, which are absent in the HBE ECM. Can the authors elaborate more on the rationale behind the supplementation of the nebulizing liquid?

3. The inactivation of the viruses are all presented as normalized infectious ratio’s or relative infectious ratio’s, which is understandable as two variables are tested: treatment of the nebulizing liquid and the effect of ozone on virus inactivation under each condition. The downside of this approach is however that the reader does not have any idea on the number of virus particles that were nebulized and were collected in each experiment and whether these numbers were comparable between the different experiments. The reviewer would recommend to show this data at least for the reference conditions for more clarity.

4. Line 279: ‘addition of BPS did not offer additional protection of airborne influenza A …”. This sentence is quite odd in the reviewers opinion as the authors previously showed that BPS affects virus infectivity with 2-3 logs (Fig. 2B) upon aging in air in absence of ozone.

5. Can the authors explain why the addition of STM is protective after 80 min expose at an RH of 34%, but resulted more inactivation at a RH of 75% after 50 minute exposure?

Textual comments

Line 72: Maybe specify the droplet size/volume for more clarity

Line 285-286, please mention which ozone dose for clarity.

Line 289: Maybe it is good to make immediately clear that this observation is obtained at 1.7 ppm ozone. Now it is stated at the end.

Line 406-407. Can the authors provide references for this claim?

Reviewer #2: The Dubuis et al. manuscript describes efforts to evaluate the virucidal efficacy of ozone against Influenza A and Respiratory Syncytial Virus (RSV) containing aerosols. This addresses the important topic of methods to reduce nosocomial transmission of respiratory viruses. The authors utilize a test system encompassing a 6-jet collison nebulizer introducing virus containing aerosols into a rotating drum aerosol chamber with periodic sampling with an SKC BioSampler. The authors show that 1.7+/-0.19ppm ozone at 76% relative humidity (RH) for 80 minutes resulted in a four orders of magnitude loss of infectious Influenza A. They conclude that this treatment could be used as a virucidal disinfectant for the control of nosocomial transmission of Influenza A. However, additional efforts to ensure that the loss in infectivity of Influenza A is due to ozone inactivation and not through losses to the system are required. Also, the addition of RSV aerosolization and sampling efforts adds confusion to the manuscript and disrupts its flow.

Major Issues:

(1) The authors were able to achieve sufficient detection of infectious Influenza A using the collison nebulizer and SKC sampler system to evaluate ozone treatment as an aerosol disinfection method. However, the authors were unable to achieve such success in detecting sufficient amounts of infectious RSV to perform similar evaluations. While the authors made progress on evaluating the stability of RSV during aerosolization and sampling, much work needs to be done in further characterizing aerosolization and sampling methods for publication. The addition of these efforts adds unnecessary confusion for the readers and detracts from the Influenza A findings.

(2) In the discussion, the authors state that “the experimental set up is not leak-proof and a loss of two to three orders of magnitude can be expected.” The authors should consider additional methods to reduce and/or control this loss for several reasons. The loss in recovery over time should be reported directly, at least as a representative plot, to show readers that a significant amount of virus is lost over time due to physical loss to the chamber wall and leaks, and the loss of infectivity of Influenza A in aerosols (which has been reported in Schuit et al. JID 2020). Adding this data would also help in detailing the range in concentration of infectious virus that is being used to calculate the NIR and RIR ratios. The leakiness of the system is also likely contributing to the significant decrease in ozone levels over time in the chamber. While it is likely unavoidable in a hospital setting, ensuring the correct concentration of ozone in a closed, experimental setup is crucial before attempting to implement this type of strategy in a real-world scenario. While the authors take efforts to normalize these losses to the experimental setup through PCR and concentration within the nebulizer at the start of the experiments, additional efforts must be made to ensure that the loss in infectivity is due to ozone.

Minor Issues:

(1) The concentration of ozone at 76% RH is noted to approach zero after 30 minutes, however, the experiments with the greatest efficiency in inactivation are carried out for 80 minutes. Also, the mean RIR for NT +1.70+/-0.19ppm at 76% RH for 80 minutes is much lower than that of 50 minutes, even though the ozone concentrations at these time points is negligible, if present at all. Additional discussion, if meaningful, should be included to rationalize the impact of ozone at these longer time-points when the concentration are so low, especially in light of point 2 above.

Reviewer #3: Comments/Critiques

- Is there a reason that ozone concentration and ozone exposure time are not combined into a single "dose" parameter as is used in Chick-Watson-type considerations of disinfection? This is the approach used by Tseng and Li, which is cited in the manuscript. Such a combined parameter seems particularly important in this study since the ozone concentrations were not constant and decreased over time. Therefore trials listed in the manuscript, for example, as representing aerosol exposure to 1.7 ppm of ozone are actually exposures starting at 1.7 ppm and gradually and continuously decreasing over 70 min to less than 0.3 ppm. It is the dose - the integral under this concentration-time curve - that should be reported and should be used to infer the resulting effects.

- Line 350: In discussing chamber testing, is it an accurate description to say "the chamber leaks"? Is there evidence of this that can be referenced? Is the chamber leaking or are the aerosols simply being lost to the chamber interior surfaces? The assertion of a leak in a process intended to conserve aerosol mass is troubling but can be easily assessed using a tracer gas. Such assessment is recommended if the authors maintain the view that chamber leakage is a systematic bias of the system.

- Although the study cites the opportunity to introduce air treatments for preventing the spread of airborne infectious viral diseases as its motivation, the manuscript does not suggest what form such treatments might take. The ozone concentrations examined in the study are higher than generally allowed for human occupied spaces, and the exposure times examined in the study are generally longer than would be required to evacuate a(n unoccupied) room of contaminated air using the ventilation system (hospital rooms operate at ventilation rates comparable to several complete changes of room air per hour). If the motivation is air treatment, then the manuscript should provide some discussion of how this might be implemented in a way that is at least comparable to these incumbent approaches. Beyond this minimum, there are material degradation concerns that can limit ozone use that may not be most suitable for this manuscript, but are of concern and warrant some mention.

Minor Critiques/Typographic and Grammatical Errors:

- Line 59: The previous sentence that focuses on influenza makes clear that the authors' inference about airborne transmission stems from data resulting from measurement of airborne samples. However, for RSV (starting at Line 59), none of the various sample locations described refer to airborne samples; therefore, in the next sentence ("Only a small number....") it is not clear whether the authors are arguing that airborne transmission is unlikely due to the scarcity of *airborne* samples or whether airborne transmission is unlikely due to the scarcity of *any* samples. Please clarify.

- Line 63: Manuscript would be improved if additional context were given to the characterization of "high" used to describe genome copies per cubic meter of air. Clearly, this depends on distance from the source, air changes per hour imposed by ventilation in the room, etc.

- Line 71: Similar critique as above: the characterization of "low concentrations of ozone" would be greatly improved if additional context were provided. There are multiple contexts to consider. There is the context of prior studies of ozonation for sterilization as well as the limits on ozone concentrations in air for protection of human health. The latter is much lower and therefore likely would undercut any unqualified assertion that the ozone concentrations of the present studies are "low".

- Line 120: The sentence beginning "Diffusion dryer tubes...." is missing a verb.

- Line 133: Dilute (i.e., ppm-level) concentrations of ozone in air are inaccurately described as "ozone" (solely).

- Line 145: Why was a new ozone monitor introduced?

- Line 148: The second phrase in this sentence lacks a verb ("....which 20 ppb and 50 ppb, with .....")

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2021 Jul 12;16(7):e0253022. doi: 10.1371/journal.pone.0253022.r002

Author response to Decision Letter 0

21 May 2021

Dear Editor and Reviewers

We are very thankful for the thorough revision of our manuscript and fruitful comments provided. Here is a point-by-point response to each reviewers’ comments and details about the changes made. We hope this revised version meets your expectations.

________________________________________________________________________

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

RESPONSE: We have updated the style requirements according to the templates. We also have renamed the figure files.

2. Please provide the source of the Influenza A/Michigan/45/2015 sample you used in this study.

RESPONSE: We have added the reference “Influenza A/Michigan/45/2015 (clinical strain A/Quebec/22578/2016 provided by Guy Boivin) was propagated in MDCK (CCL-34, ATCC) and ST6GalI-MDCK cells.”

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

RESPONSE: The nebulizing liquid is a viral lysate from the cell culture. We have added this precision in the sections “influenza A virus and host cells” and “Influenza A aerosolization, exposure to ozone and sampling”.

RESPONSE: Since the aerosols produced by the human respiratory tract contain other elements such as protein, lipids and salts, we wanted to represent this content when aerosolizing viruses. We initially thought that the droplet’s contents concentrated into the droplet nuclei could provide a protection from ozone. Therefore, aerosolizing viruses from lysate would not be as representative as real aerosols produced by infected humans. We agree with your comment that the STM main ingredients are not the same as the HBE ECM composition. However, we needed almost 60 ml of this supplement to conduct our experiments, which is a volume way above what we could produce by pooling 150 µl washes of HBE ECM as performed by Kormuth et al. (2018). Therefore, we had to find a solution that could be made in higher volumes. The STM created by Hamed and Fiegel (2014) has chemical and physical properties similar to native mucus and we could prepare 60 ml for our experiments. We added a sentence in the “Influenza A aerosolization, exposure to ozone and sampling” section: “However, we could not use pooled washes of HBE ECM because a volume of 60 ml of the supplement was needed for our experiments.”

RESPONSE: Thank you for your comment. We added this specification in the reference conditions section. “It is worth mentioning that the nebulized concentrations of infectious viruses for the NT, BPST and STMT experiments were 6.96 X 107 ± 9.40 X 107 PFU/ml, 1.58 X 107 ± 5.35 X 106 PFU/ml and 1.58 X 107 ± 4.54 X 106 PFU/ml, respectively. The concentrations of sampled viruses were lower, at 1.58 X 105 ± 1.92 X 105 PFU/ml for the NT, 2.20 X 104 ± 3.49 X 104 for the BPST and 1.58 X 104 ± 2.26 X 104 for the STMT.”

RESPONSE: We understand your concern. Here is the new sentence: “This means that the BPS did not protect the remaining infectious airborne influenza A from an exposure to 0.23 ppm ± 0.03 ppm of ozone.”

5. Can the authors explain why the addition of STM is protective after 80 min expose at an RH of 34%, but resulted more inactivation at a RH of 75% after 50 minute exposure?

RESPONSE: We agree that this observation deserves an explanation. We have added these sentences in the discussion at the end of the first paragraph: “Moreover, the addition of STM seems to have a protective effect at 34% RH after 80 min, while a disrupting effect is noted at 75% RH after 50 min. Although free radicals are produced when ozone interacts with water and organic compounds, this outcome could be the result of a synergy between high RH and the presence of proteins. Additional testing could be useful to verify if a synergistic inactivation effect is observed while using different supplements at high or low RH.”

Textual comments

6. Line 72: Maybe specify the droplet size/volume for more clarity

RESPONSE: We have added these elements to the article. “According to Vejerano and Marr (2018), a single human-produced evaporating respiratory droplet of 60 µm (roughly 0.1 nl) is estimated to compose 1 ng of salt, 1 ng of protein and 0.06 ng of surfactant.”

7. Line 285-286, please mention which ozone dose for clarity.

RESPONSE: We have added this information. “The maximum reduction in infectivity was four orders of magnitude, which was obtained after 80 min at 76% RH for an ozone exposure of 1.70 ± 0.19 ppm.”

8. Line 289: Maybe it is good to make immediately clear that this observation is obtained at 1.7 ppm ozone. Now it is stated at the end.

RESPONSE: We have added the concentration at the beginning of the paragraph. “For an ozone exposure to 1.70 ± 0.19 ppm at 34% RH, the STMT (Fig 3D) did not increase airborne influenza A infectivity after 20 min (p = 0.14) and 50 min (p = 0.29) of exposure time.”

9. Line 406-407. Can the authors provide references for this claim?

RESPONSE: We have modified this sentence and included references for the new claim: “Since resistance behaviors from multiple aerosolized viruses are somewhat unpredictable [24, 43], the different behaviors of influenza A and RSV were not surprising.”

Major Issues:

1. The authors were able to achieve sufficient detection of infectious Influenza A using the collison nebulizer and SKC sampler system to evaluate ozone treatment as an aerosol disinfection method. However, the authors were unable to achieve such success in detecting sufficient amounts of infectious RSV to perform similar evaluations. While the authors made progress on evaluating the stability of RSV during aerosolization and sampling, much work needs to be done in further characterizing aerosolization and sampling methods for publication. The addition of these efforts adds unnecessary confusion for the readers and detracts from the Influenza A findings.

RESPONSE: Thank you for your insight. We understand that the results obtained for RSV are not as satisfying as those with Influenza. However, we strongly feel that it is important to present negative or inconclusive results since they have scientific value. They also provide a comparison between influenza and RSV. This kind of comparison is not described in the literature and this information could be really useful for other teams who would like to study airborne RSV.

2. In the discussion, the authors state that “the experimental set up is not leak-proof and a loss of two to three orders of magnitude can be expected.” The authors should consider additional methods to reduce and/or control this loss for several reasons. The loss in recovery over time should be reported directly, at least as a representative plot, to show readers that a significant amount of virus is lost over time due to physical loss to the chamber wall and leaks, and the loss of infectivity of Influenza A in aerosols (which has been reported in Schuit et al. JID 2020). Adding this data would also help in detailing the range in concentration of infectious virus that is being used to calculate the NIR and RIR ratios. The leakiness of the system is also likely contributing to the significant decrease in ozone levels over time in the chamber. While it is likely unavoidable in a hospital setting, ensuring the correct concentration of ozone in a closed, experimental setup is crucial before attempting to implement this type of strategy in a real-world scenario. While the authors take efforts to normalize these losses to the experimental setup through PCR and concentration within the nebulizer at the start of the experiments, additional efforts must be made to ensure that the loss in infectivity is due to ozone.

RESPONSE: Since another reviewer also made a comment regarding the leakages, we have decided addressed both requests at the same time. We have modified the paragraph describing the issue of aerosol losses in the experimental set up (results section) but we have not given too much details there. We have added a paragraph in the discussion explaining the pros and cons of our experimental set up so that the reader can understand why some materials were chosen and why we cannot do some experiences using sensitive viruses. We have also addressed the issue of the ozone concentration.

“Furthermore, aerosol loss is expected in the experimental setup, which can occur in the diffusion dryer tubes, at the junction of tubing and barb fittings and while sampling with the SKC BioSampler. From previous non-published experiments performed in our lab, it has been noted that there can be an aerosol loss of two to three orders of magnitude for every aerosol experiment.”

“Our experimental setup has been designed to allow us to work with level II viruses. Every part has been carefully chosen for that purpose, but compromises had to be made on some aspects. The aerosol chamber is constructed in aluminum to minimize particle loss due to static and it rotates to reduce particle deposition. It is also airtight and therefore maintains the level of relative humidity. Aerosol concentrations and MMAD over 18h have been studied in a previous publication [36]. The ozone is injected directly inside the chamber. The valves are shut after the injection to prevent the ozone from escaping the chamber. The ozone concentrations are then measured with a probe connected directly to the chamber. Consequently, the ozone concentration measures in the chamber are accurate. However, the aerosol path between the nebulizer and the chamber could lead to significant particle loss. As a safety measure, the material used to make the diffusion dryer tubes had to be shatterproof. It also had to be transparent to monitor the color of the silica beads. Therefore these tubes were made with polycarbonate. This plastic is known to have static properties, which could cause aerosol loss. These tubes are also not completely airtight because they protect the other parts of the setup by acting as breaking points in case of an accidental pressure increase. For the setup to fit inside the BSL II cabinet, the aerosol path includes 180° elbows in a few spots. The impaction of aerosols could occur in these elbows. The aerosol tubing used to connect the nebulizer, the diffusion dryers and the aerosol chamber is made of static dissipative silicon coated with carbon. However, since it the aerosol path (nebulizer – up to five diffusion dryers - chamber), particles could be lost by diffusion. As for the SKC BioSampler, this device preserves the virus infectivity and has a great collection efficiency of 1 µm particles but the re-aerosolization phenomenon has previously been documented [43]. While all these features could cause aerosol loss, we only evaluate the portion reaching the chamber by normalizing the infectious ratios. The drawbacks of this loss is that we are unable to test viruses that are sensitive or that do not produce high viral titers, as was the case for RSV.”

Minor Issues:

3. The concentration of ozone at 76% RH is noted to approach zero after 30 minutes, however, the experiments with the greatest efficiency in inactivation are carried out for 80 minutes. Also, the mean RIR for NT +1.70+/-0.19ppm at 76% RH for 80 minutes is much lower than that of 50 minutes, even though the ozone concentrations at these time points is negligible, if present at all. Additional discussion, if meaningful, should be included to rationalize the impact of ozone at these longer time-points when the concentration are so low, especially in light of point 2 above.

RESPONSE: We have modified one sentence in the discussion to point out this outcome. “On the other hand, even if the ozone concentration was close to zero after 40 min at high RHs, the RIR values (Fig 3) show that the inactivation of airborne influenza was superior after longer exposure times. Additionally, for all tested conditions, the effect of ozone treatment is greater at high RHs than at low RHs, indicating that the free radicals that are produced have higher virucidal properties than ozone alone.”

Reviewer #3: Comments/Critiques

1. Is there a reason that ozone concentration and ozone exposure time are not combined into a single "dose" parameter as is used in Chick-Watson-type considerations of disinfection? This is the approach used by Tseng and Li, which is cited in the manuscript. Such a combined parameter seems particularly important in this study since the ozone concentrations were not constant and decreased over time. Therefore trials listed in the manuscript, for example, as representing aerosol exposure to 1.7 ppm of ozone are actually exposures starting at 1.7 ppm and gradually and continuously decreasing over 70 min to less than 0.3 ppm. It is the dose - the integral under this concentration-time curve - that should be reported and should be used to infer the resulting effects.

RESPONSE: Thank you for pointing this out. We decided to use concentration values instead of single doses for field application purposes. The initial (and highest) concentration of 1.70 ppm is important for healthcare settings because of the impacts of ozone exposure on human health. Furthermore, ozone exposure for humans is always reported in concentration and not with a dose.

As you suggested, we have calculated the ozone doses using the integral under the concentration-time curve. These new results are below the concentration results (Fig 1) in the “Ozone concentrations and doses” section. We present the doses in a table (Table 1) with the appropriate explanations. Because these are new results, we have added a paragraph in the “Calculations and statistical analysis” section and have also added sentences in the discussion. We have uploaded a supporting information file with the regression analyses information. Here are the main additions to the manuscript:

A. Calculations and statistical analysis: Ozone doses were estimated by calculating the area under concentration-vs-time curves, for RH values of 34% and 76%. Concentration-vs-time curves were fitted to observed data by regression analysis for exposure times between 10 min and 70 min (see supporting information for details). Regression analyses were performed in RStudio (version 1.2.5033). The areas under the curves were calculated numerically with a trapezoid scheme coded in C++ and validated against analytical computations. For exposure times between 0 and 10 min, extrapolation of the fitted concentration-vs-time curves was deemed too unreliable for dose calculations. Accumulated dose between 0 and 10 min was instead estimated by assuming that ozone concentration was constant over that time interval and equal to the fitted concentration value at 10 min. Consequently, calculated ozone doses represent floor values. Confidence intervals were not reported for dose calculations because the uncertainty associated to the constant concentration assumption was much larger than the width of the confidence bands around the regressions curves.

B. Ozone concentrations and doses: The calculated ozone doses floor values for both RH levels at 10-minute intervals are presented in Table 1. Even if the ozone injection was identical, after an exposure time of 70 min, the ozone dose floor value at 34% was much higher, at 58.67 ppm ∙ min, than the dose at 76%, at 7.41 ppm ∙ min. Furthermore, the values increase by a factor of two between 10 and 70 minutes at 76%, while they increase by a factor of three at 34% RH.

Table 1. Ozone doses floor value (ppm ∙ min) for each exposure time at 34% and 76% RH.

Exposure Time (min) Ozone dose (ppm ∙ min)

34% RH 76% RH

10 16.90 3.69

20 31.11 5.72

30 41.05 6.49

40 48.01 6.89

50 52.88 7.13

60 56.29 7.29

70 58.67 7.41

C. Discussion: The calculated ozone dose floor value required to achieve a reduction of four orders of magnitude was 7.41 ppm∙min and was obtained at 76% RH. Since the real ozone dose to which viruses were exposed might be significantly higher than the calculated ozone dose floor value, the exposure in the HVAC plenum should aim for an ozone dose of at least 7.41 ppm∙min at an RH close to 76%. Depending on the contact time in the HVAC plenum, ozone generation could be modulated to achieve this minimal dose.

2. Line 350: In discussing chamber testing, is it an accurate description to say "the chamber leaks"? Is there evidence of this that can be referenced? Is the chamber leaking or are the aerosols simply being lost to the chamber interior surfaces? The assertion of a leak in a process intended to conserve aerosol mass is troubling but can be easily assessed using a tracer gas. Such assessment is recommended if the authors maintain the view that chamber leakage is a systematic bias of the system.

RESPONSE: We agree with your comment that there are differences between leaks and loss. Another reviewer made a comment regarding the leaks, therefore we have addressed both requests at the same time. We have modified the paragraph describing the issue of aerosol losses in the experimental set up (results section) but we have not given too much details there. We have added a paragraph in the discussion explaining the pros and cons of our experimental set up so that the reader can understand why some materials were chosen and why we cannot do some experiences using sensitive viruses. We have also addressed the issue of the ozone concentration.

3. Although the study cites the opportunity to introduce air treatments for preventing the spread of airborne infectious viral diseases as its motivation, the manuscript does not suggest what form such treatments might take. The ozone concentrations examined in the study are higher than generally allowed for human occupied spaces, and the exposure times examined in the study are generally longer than would be required to evacuate a(n unoccupied) room of contaminated air using the ventilation system (hospital rooms operate at ventilation rates comparable to several complete changes of room air per hour). If the motivation is air treatment, then the manuscript should provide some discussion of how this might be implemented in a way that is at least comparable to these incumbent approaches. Beyond this minimum, there are material degradation concerns that can limit ozone use that may not be most suitable for this manuscript, but are of concern and warrant some mention.

RESPONSE: Thank you for your comment. We have added a paragraph in the discussion debating the kind of air treatment protocols that could be implemented (pros and cons). We have also added the calculated ozone dose floor value that would be required to achieve a reduction of four orders of magnitude. Here is the new paragraph: “The Influenza virus is a persistent burden on hospitals, and air treatment could be an effective way to reduce its nosocomial transmission. The results from this study can be used as a basis for air decontamination in different settings. Since the studied ozone concentrations are above the threshold limit value – ceiling of 0.1 ppm [47], the decontamination protocol should be performed in leak-proof rooms in the absence of occupants for at least 80 min. This issue could be problematic for some settings that cannot evacuate the infected rooms for the proper amount of time required for air disinfection. Another element to consider is the reaction of ozone with some materials including rubber and its derivatives [48] which can cause the degradation of some mattresses [49]. Disinfection of recycled air inside the heating, ventilation and air-conditioning (HVAC) plenum could be an alternative to limit the material degradation and allow the occupants to stay in the rooms. One downside of this approach is the contact time, which cannot be performed over an 80-min period. The calculated ozone dose floor value required to achieve a reduction of four orders of magnitude was 7.41 ppm∙min and was obtained at 76% RH. Since the real ozone dose to which viruses were exposed might be significantly higher than the calculated ozone dose floor value, the exposure in the HVAC plenum should aim for an ozone dose of at least 7.41 ppm∙min at an RH close to 76%. Depending on the contact time in the HVAC plenum, ozone generation could be modulated to achieve this minimal dose. The other limitation of a disinfection in the HVAC plenum is that the furniture and areas hard to reach inside the infected rooms would not be decontaminated. Considering these elements, each setting should assess which option is the most appropriate to meet its needs before implementing an air disinfection protocol using ozone.”

Minor Critiques/Typographic and Grammatical Errors:

4. Line 59: The previous sentence that focuses on influenza makes clear that the authors' inference about airborne transmission stems from data resulting from measurement of airborne samples. However, for RSV (starting at Line 59), none of the various sample locations described refer to airborne samples; therefore, in the next sentence ("Only a small number....") it is not clear whether the authors are arguing that airborne transmission is unlikely due to the scarcity of *airborne* samples or whether airborne transmission is unlikely due to the scarcity of *any* samples. Please clarify.

RESPONSE: Thank you for your insight, we agree that it was confusing. We have modified the sentences to make it clear that we are talking about air samples. Here is the new version: “RSV genomes have been detected in the air of an emergency clinic (20), in hospital rooms with RSV-infected patients (21) and in a pediatric acute care ward (22). Only a small number of positive samples (2.3%) were collected in the air of the pediatric acute care ward, which suggests that airborne RSV transmission is not likely (22).”

5. Line 63: Manuscript would be improved if additional context were given to the characterization of "high" used to describe genome copies per cubic meter of air. Clearly, this depends on distance from the source, air changes per hour imposed by ventilation in the room, etc.

RESPONSE: Since “high” is somewhat arbitrary we modified the two sentences to remove this claim. Here is the new version: “On the other hand, another study detected airborne infectious RSV in the air around infants that were hospitalized with bronchiolitis in a pediatric ward and an intensive care. Mean concentrations were 3.71 X 105 PFU/m3 in the pediatric ward and 4.09 X 105 PFU/m3 in the intensive care unit [23].

6. Line 71: Similar critique as above: the characterization of "low concentrations of ozone" would be greatly improved if additional context were provided. There are multiple contexts to consider. There is the context of prior studies of ozonation for sterilization as well as the limits on ozone concentrations in air for protection of human health. The latter is much lower and therefore likely would undercut any unqualified assertion that the ozone concentrations of the present studies are "low".

RESPONSE: We agree that we only refer to the concentrations used by other studies and not human health. The only study we found that inactivated airborne phages was by Tseng and Li, which we have cited in the modified sentence: “The results that were obtained were promising, even when using lower concentrations of ozone than those used by Tseng and Li (2006) for the inactivation of airborne phages (25).

7. Line 120: The sentence beginning "Diffusion dryer tubes...." is missing a verb.

RESPONSE: Thank you for pointing this out. We have added a verb. “Diffusion dryer tubes of different lengths were used to adjust the RH inside the chamber.”

8. Line 133: Dilute (i.e., ppm-level) concentrations of ozone in air are inaccurately described as "ozone" (solely).

RESPONSE: Thank you for your comment. You are right, the ozone generator converts the oxygen present in the air (around 21%) in ozone. Therefore, the “ozone” is not pure ozone, but a mixed gas of approximately 21% of ozone in air. This has been corrected in the text: “Next, aerosols were exposed to air (designated as the reference conditions) or a mixed gas of approximately 21% of ozone in air for 0, 30 or 60 minutes at low or high RHs.”

9. Line 145: Why was a new ozone monitor introduced?

RESPONSE: The ozone monitor that we previously used could not be connected with fittings and tubing to the aerosol chamber. Therefore, the ozone concentration had to be assessed with another aerosol chamber that had a window, which was necessary for the ozone probe readings. The acquisition of a new ozone probe allowed us to measure the ozone concentrations directly inside the rotative drum because it could be connected with fittings and tubing.

10. Line 148: The second phrase in this sentence lacks a verb ("....which 20 ppb and 50 ppb, with .....")

RESPONSE: Here is the sentence with the verb “The sensitivity of the sensors is considered to be 1% of their ranges, which are 20 ppb (500-2000 ppb) and 50 ppb (1-5 ppm), with a resolution of 1 ppb and 0.01 ppm, respectively.”

Attachment

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PLoS One. doi: 10.1371/journal.pone.0253022.r003

Decision Letter 1

Sander Herfst

27 May 2021

Ozone inactivation of airborne influenza and lack of resistance of respiratory syncytial virus to aerosolization and sampling processes

PONE-D-21-02103R1

Dear Dr. Duchaine,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Additional Editor Comments:

Please explain why a co-author was added to the manuscript (what was their contribution) and what the reason is that a funding agency was removed from the acknowledgements section? Especially the latter is rather unusual.

PLoS One. doi: 10.1371/journal.pone.0253022.r004

Acceptance letter

Sander Herfst

2 Jul 2021

PONE-D-21-02103R1

Ozone inactivation of airborne influenza and lack of resistance of respiratory syncytial virus to aerosolization and sampling processes

Dear Dr. Duchaine:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Kind regards,

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on behalf of

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Data Availability Statement

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PERMALINK

Ozone inactivation of airborne influenza and lack of resistance of respiratory syncytial virus to aerosolization and sampling processes

Marie-Eve Dubuis

Étienne Racine

Jonathan M Vyskocil

Nathalie Turgeon

Christophe Tremblay

Espérance Mukawera

Guy Boivin

Nathalie Grandvaux

Caroline Duchaine

Roles

Abstract

Introduction

Materials and methods

Influenza A virus and host cells

Respiratory syncytial virus and host cells

Influenza A aerosolization, exposure to ozone and sampling

RSV nebulizing and sampling assays

Virus resistance in air sampler

Virus resistance in two nebulizers

RNA extraction, RT-qPCR and influenza A titration

Calculations and statistical analysis

Results

Ozone concentrations and doses

Fig 1. Ozone concentrations measured at 34% and 76% RH at 10-min intervals over a maximum exposure time of 70 min.

Table 1. Ozone doses floor value (ppm ∙ min) for each exposure time at 34% and 76% RH.

Reference conditions for influenza A

Fig 2. Normalized Infectious Ratios (NIRs) for influenza A at three exposure times and two Relative Humidity (RH) levels.

Relative infectious ratios

Fig 3. The effect of ozone on influenza A infectivity at two Relative Humidity (RH) levels and three exposure times.

RSV infectivity assay

Virus resistance in air sampler

Virus resistance in two nebulizers

Fig 4. Initial and final infectious RSV concentrations in the 6-jet Collison and the Aeroneb nebulizers.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Sander Herfst

Roles

Author response to Decision Letter 0

Decision Letter 1

Sander Herfst

Roles

Acceptance letter

Sander Herfst

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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