Inactivation of SARS-CoV-2 and influenza A virus by dry fogging hypochlorous acid solution and hydrogen peroxide solution.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is transmitted mainly by droplet or aerosol infection; however, it may also be transmitted by contact infection. SARS-CoV-2 that adheres to environmental surfaces remains infectious for several days. We herein attempted to inactivate SARS-CoV-2 and influenza A virus adhering to an environmental surface by dry fogging hypochlorous acid solution and hydrogen peroxide solution. SARS-CoV-2 and influenza virus were air-dried on plastic plates and placed into a test chamber for inactivation by the dry fogging of these disinfectants. The results obtained showed that the dry fogging of hypochlorous acid solution and hydrogen peroxide solution inactivated SARS-CoV-2 and influenza A virus in CT value (the product of the disinfectant concentration and contact time)-dependent manners. SARS-CoV-2 was more resistant to the virucidal effects of aerosolized hypochlorous acid solution and hydrogen peroxide solution than influenza A virus; therefore, higher concentrations of disinfectants or longer contact times were required to inactivate SARS-CoV-2 than influenza A virus. The present results provide important information for the development of a strategy that inactivates SARS-CoV-2 and influenza A virus on environmental surfaces by spatial fogging.

Introduction

Coronavirus disease 2019 (COVID-19) continues to spread worldwide, with more than 424 million individuals being infected and more than 5.88 million dying to date [1]. The World Health Organization (WHO) has recommended a number of countermeasures to the public, including getting vaccinated, avoiding 3Cs (spaces that are closed, crowded, or involve close contact), wearing a properly fitting mask when physical distancing is not possible and in poorly ventilated settings, and frequently cleaning hands with an alcohol-based hand rub or soap and water [2]; however, the pandemic continues.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Viral transmission is established by inhaling droplets or aerosols containing the virus that are excreted from infected individuals in a 3Cs setting or by touching the eyes, nose, or mouth with hands contaminated with the virus adhering to environmental surfaces [3]. Previous studies reported the high stability of SARS-CoV-2 adhering to environmental surfaces [4, 5], and its stability was shown to be higher than those of SARS-CoV and influenza A virus [6, 7]. Therefore, the disinfection of environmental surfaces is indispensable as an infection control measure. However, it is not realistic to frequently and manually disinfect the surfaces of large spaces, such as a train station or airport, because of the manpower and time required. Although the fogging of a disinfectant is an alternative spatial disinfection method, it is not recommended by the WHO due to its effects on the human body [8]. In addition, the Center for Disease Control and Prevention of the United States of America (USA) does not recommend the fogging of disinfectants in hospital rooms in the 2003 and 2008 guidelines [9]. Newer technologies for performing disinfectant fogging were assessed in the 2011 guidelines for the prevention and control of norovirus gastroenteritis outbreaks in healthcare settings; nevertheless, further research is required to clarify the effectiveness and reliability of disinfectant fogging [9].

Therefore, the present study examined the effectiveness of inactivating SARS-CoV-2 virus adhering to plastic microplates by dry fogging disinfectants. Dry fog is defined as an aerosol with a Sauter mean droplet diameter ≤10 μm and maximum droplet diameter ≤50 μm [10], and it does not wet objects even if touched. Its virucidal effects on influenza A virus, which is a common envelope virus transmitted through droplets and contact transmission worldwide, were also investigated. In consideration of the effects of residues after dry fogging on the human body, we tested hypochlorous acid solution and hydrogen peroxide solution, which leave almost no residue on environmental surfaces after dry fogging. Hypochlorous acid solution is an aqueous solution that contains hypochlorous acid (HOCl) as the main component. Hypochlorous acid solution may be prepared by dissolving sodium hypochlorite in water with adjustments to a weak acidic pH. The main component of the weakly acidic solution is HOCl, while that of the alkaline solution is hypochlorite ions (OCl-). HOCl exerts stronger bactericidal effects than OCl- [11]. Therefore, a weakly acidic (pH 6.5) hypochlorous acid solution was dry fogged in the present study.

Materials and methods

Preparation of disinfectants

Hypochlorous acid solution and hydrogen peroxide solution were prepared as disinfectants to be dry fogged for the inactivation of viruses. Commercially available, weakly acidic (pH 6.5) hypochlorous acid solution with a free available chlorine (FAC) concentration (the sum of HOCl and OCl- concentrations) [11] of 250 ppm (Super Jiasui; HSP Corporation, Okayama, Japan) and a solution diluted by distilled water with a FAC concentration of 125 ppm were used. To prepare a solution with a FAC concentration of 8,700 ppm, sodium hypochlorite (Hayashi Pure Chemical Ind., Ltd., Osaka, Japan) was dissolved in distilled water, followed by an adjustment of the pH of the solution to 6.5 with HCl (Hayashi Pure Chemical Ind., Ltd.) using the pH meter SK-620PHⅡ (Sato Keiryoki Mfg. Co., Ltd, Tokyo, Japan). Commercially available hydrogen peroxide solution (56,400 ppm; Part 1, Decon7; Decon7 Systems LLC., Texas, USA) and solutions diluted by distilled water with hydrogen peroxide concentrations of 11,280, 5,640, 2,820, and 1,410 ppm were prepared. Regarding the negative control, distilled water was dry fogged.

Cells and viruses

VeroE6/TMPRESS2 cells (JCRB1819) [12] were maintained in Dulbecco’s modified Eagle medium (DMEM) (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Merck, Kenilworth, New Jersey, USA) and 1 mg/ml of G418 (Sigma-Aldrich) (complete DMEM), while MDCK cells were maintained in Eagle’s minimum essential medium (MEM) (MEM1, Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% FBS in a CO2 incubator. The SARS-CoV-2 Wuhan strain, SARS-CoV-2/Hu/DP/Kng/19-020 was propagated by infecting VeroE6/TMPRESS2 cells and cultured in FBS-free DMEM supplemented with 1 mg/ml of G418 for 24 hours. The influenza A H1N1 strain, A/Puerto Rico/8/1934 was propagated by infecting MDCK cells and cultured in FBS-free MEM supplemented with 2 μg/ml of acetylated trypsin (Sigma-Aldrich) for 3 days. Viral supernatants were clarified by centrifugation, aliquoted, and stored at -85°C. Ten-fold serially diluted viral supernatants were incubated with VeroE6/TMPRESS2 or MDCK cells for 3 or 4 days, respectively, for viral titration, and median tissue culture infectious doses (TCID50) were measured using the Spearman-Kaber method following the fixation of cells with 5% formaldehyde in phosphate-buffered saline (PBS) and staining with 0.5% crystal violet in 20% EtOH. TCID50 values below the detection limit (3.16 × 102 TCID50/ml or 2.5 log10 TCID50/ml) were assigned to half of the detection limit, equivalent to 1.58 × 102 TCID50/ml or 2.2 log10 TCID50/ml, because substituting the value to half of the detection limit was previously shown to be less biased than substitution to zero or the detection limit [13].

Preparation of air-dried virus samples

Viral solutions (5 μl) containing SARS-CoV-2 (1.2 × 105 TCID50) or influenza A virus (2.8 × 106 TCID50) were applied using a micropipette to the bottom of 5 wells (per virus inactivation experiment) on a 96-well flat-bottomed microplate (Corning Japan, Shizuoka, Japan), air-dried for 10–15 minutes using a small electric fan, and subjected to a virus inactivation experiment. As a negative control for the experiment, an air-dried viral sample in a well was re-suspended with 200 μl of DMEM containing a neutralizer of the disinfectant prior to dry fogging, as described below. In addition, in some experiments, 5 μl of artificial saliva (Saliveht aerosol; Teijin Pharma, Tokyo, Japan) or PBS was mixed with 5 μl of viral supernatants prior to air-drying samples.

Preparation of the test chamber for dry fogging

A closed test chamber was prepared to fill the space with dry fog. The detailed settings of the chamber are shown in Fig 1. The chamber size was 500 × 700 × 300 mm (height × width × depth), made of acrylic, and set in a biosafety cabinet of class II type A/B3 or class II type A1. A sliding door was set on the front of the chamber for the handling of samples inside the chamber. A fogger equipped with an impinging-jet atomizing nozzle [14] [AE-1 (03C), AKIMist®”E”; H. Ikeuchi & Co., Ltd., Osaka, Japan] was used to fog disinfectants into the space. To generate aerosolized disinfectants in the form of dry fog, 0.3 MPa compressed air was supplied to the fogger from a compressor (0.2LE-8SB0; Hitachi Industrial Equipment Systems Co., Ltd., Tokyo, Japan). The fogging capacity was 2.3 liters per hour and the Sauter mean droplet diameter was 7.5 μm. In the virus inactivation experiment using dry fogging, four 90-mm Petri dishes containing 20 ml of distilled water, a temperature and humidity sensor (Model RHT-3 Temperature and Humidity Sensor; Sensatec Co., Ltd., Kyoto, Japan), and a 96-well microplate containing air-dried viral samples in 5 wells were placed in the chamber (Fig 1). Four 90-mm Petri dishes containing distilled water were placed to measure the concentrations of disinfectants trapped in water during the virus inactivation experiment and also to calculate the product of the concentrations of disinfectants and contact times (CT value).

Fig 1

Schematic illustration of the test chamber.

The composition of the chamber is described in detail in the Materials and methods.

Virus inactivation experiment by dry fogging and measurements of concentrations of disinfectants trapped in distilled water

The times at which spatial fogging, the termination of the virus inactivation operation, and measurements of the concentrations of disinfectants trapped in 20 ml of distilled water were performed as indicated in Fig 2. Spatial fogging was conducted as follows. A disinfectant was dry fogged for 5 seconds at the start of the experiment and left to stand for 4 minutes. Dry fogging was then repeated 3 more times for 2.5 seconds each and left to stand for 4 minutes after each fogging. Dry fogging was performed 4 times, namely, 0, 4, 8, and 12 minutes after the initiation of the experiment, and the total experimental period was 16 minutes. This dry fogging operation allowed for the continuous generation of dry fog without unnecessarily moistening the environmental surface in the chamber, and dry fog did not fade. The virus inactivation reaction by dry fogging disinfectants was terminated by resuspending air-dried viral samples in 200 μl of DMEM containing a neutralizer of the disinfectant being tested. DMEM containing 0.1 M sodium thiosulfate (Hayashi Pure Chemical Ind., Ltd.) was used as the neutralizer for hypochlorous acid solution, while DMEM containing 0.1 mg/ml of catalase (Nacalai Tesque, Inc.) was used as that for hydrogen peroxide solution. These neutralizers did not affect cell growth under experimental conditions (data not shown). The residual infectious titer of viral samples after the inactivation experiment was evaluated by measuring the TCID50 value, as described above. The concentration of the dry fogged disinfectant was assessed by collecting the droplet-shaped disinfectant that had fallen into 20 ml of distilled water in the 90-mm Petri dish placed in the chamber, and measuring the concentration of the disinfectant dissolved in distilled water. The FAC concentration when hypochlorous acid solution was dry fogged was measured by DPD (N,N-dimethyl-p-phenylenediamine) absorptiometry using a residual chlorine meter (HI96771; Hanna Instruments, Chiba, Japan). The hydrogen peroxide concentration when hydrogen peroxide solution was dry fogged was measured by 4-aminoantipyrine absorptiometry using an enzyme with a hydrogen peroxide concentration meter (DPM2-H2O2; Kyoritsu Chemical-Check Lab., Corp., Kanagawa, Japan). Concentrations in appropriately diluted samples with distilled water were within the detection ranges of the measuring instruments and reagents. In addition, air temperature and humidity in the chamber were monitored during experiments.

Fig 2

Flow diagram of the virus inactivation experiment.

A detailed procedure is described in the Materials and methods.

A set of viral inactivation experiments was performed as follows. Four 90-mm Petri dishes containing distilled water, a temperature and humidity sensor, and a 96-well microplate containing air-dried viral samples in 5 wells were placed in the test chamber (Fig 1), as described above. Prior to the dry fogging of disinfectants, a dried viral sample in the first well was resuspended with DMEM (200 μl) containing a neutralizer by pipetting. The sliding door was then closed, and the first dry fogging of the disinfectant was performed for 5 seconds. After 4 minutes, the sliding door of the chamber was opened, the dried viral sample in the second well was resuspended with DMEM (200 μl) containing the neutralizer, and the first Petri dish containing distilled water was removed from the chamber. The sliding door was closed, and the second dry fogging of the disinfectant was performed for 2.5 seconds. After 4 minutes, the sliding door was opened, the dried viral sample in the third well was resuspended with DMEM (200 μl) containing the neutralizer, and the second Petri dish containing distilled water was removed from the chamber. The sliding door was again closed, and the third dry fogging of the disinfectant was performed for 2.5 seconds. After 4 minutes, the sliding door was opened, the dried viral sample in the fourth well was resuspended with DMEM (200 μl) containing the neutralizer, and the third Petri dish containing distilled water was removed from the chamber. The sliding door was closed, and the fourth dry fogging of the disinfectant was performed for 2.5 seconds. After 4 minutes, the sliding door was opened, the dried viral sample in the fifth well was resuspended with DMEM (200 μl) containing the neutralizer, and the fourth Petri dish containing distilled water was removed from the chamber. The concentrations of the droplet-shaped disinfectant that fell into distilled water in the Petri dishes were measured between dry fogging. Therefore, the concentrations of the disinfectant trapped in distilled water were measured 4 times in each set of experiments, i.e., at 4 minutes (total of 5 seconds of dry fogging), 8 minutes (total of 7.5 seconds of dry fogging), 12 minutes (total of 10 seconds of dry fogging), and 16 minutes (total of 12.5 seconds of dry fogging). Virus inactivation experiments under the same conditions were repeated more than three times, except for those to evaluate artificial saliva, which were repeated twice.

Statistical analysis

Statistical analyses were performed using the standard function of GraphPad Prism 8 software (GraphPad Software, San Diego, California) with a 2-way ANOVA, unpaired t-test, non-linear regression (curve fit) analysis, or simple linear regression analysis.

Results

The dry fogging of hypochlorous acid solution and hydrogen peroxide solution inactivated SARS-CoV-2 and influenza A virus over time

We examined changes in the viral infectious titer upon dry fogging with various concentrations of hypochlorous acid solution (FAC concentrations of 8,700, 250, and 125 ppm), hydrogen peroxide solution (56,400, 11,280, 5640, 2820, and 1410 ppm of hydrogen peroxide), or distilled water. Dry fogging experiments were initially conducted using a commercially available hypochlorous acid solution (250 ppm; Super Jiasui; HSP); however, SARS-CoV-2 was not inactivated (Fig 3A). A previous study reported that when viral culture fluid was mixed with 35 ppm hypochlorous acid solution, SARS-CoV-2 was effectively inactivated [15]; therefore, we calculated the FAC concentration needed to inactivate an air-dried virus that settled in the wells (0.32 cm2) of 96-well microplates. The result obtained revealed that 8,700 ppm hypochlorous acid solution in the form of dry fog was required to inactivate SARS-CoV-2 under our experimental conditions. Fig 3 shows the time course of changes in virus infectious titers under the experimental conditions employed in the present study. The viral titer of SARS-CoV-2 was not reduced by 250 ppm hypochlorous acid solution (Fig 3A), whereas that of influenza A virus was effectively decreased over time (Fig 3B). Furthermore, 8,700 ppm hypochlorous acid solution effectively reduced the infectious titer of SARS-CoV-2 (Fig 3A). Moreover, 56,400 ppm hydrogen peroxide solution reduced the infectious titer of SARS-CoV-2 (Fig 3C), while 11,280 ppm hydrogen peroxide solution decreased the viral titer of influenza A virus (Fig 3D). It is important to note that since dry fogging was performed 4 times at 4-minute intervals in the virus inactivation experiment, the concentration of the disinfectant increased over time. Nevertheless, viruses were inactivated over time under the experimental conditions used. In contrast, the dry fogging of distilled water did not reduce the viral infectivity of SARS-CoV-2 or influenza A virus regardless of the elapsed time (Fig 3).

Fig 3

Inactivation of viruses by the dry fogging of disinfectants.

Changes in the viral infectious titers (TCID50 values) of SARS-CoV-2 (A and C) and influenza A virus (B and D) upon dry fogging with hypochlorous acid solution (HAS) (A and B), hydrogen peroxide solution (H2O2) (C and D), or distilled water (DW) (A, B, C, and D) were evaluated, as described in the Materials and methods. Horizontal dotted lines in the graphs show the detection limit of the viral titer. *P < 0.0001 compared with DW using a two-way ANOVA. n.s., not significant. Each data point represents the average and standard deviation obtained from more than three repeated experiments.

Relationship between dry fogged disinfectant concentrations and concentrations of disinfectants trapped in distilled water

To calculate the CT value of a disinfectant, we measured its dissolved concentration in 20 ml of distilled water in 90-mm Petri dishes during virus inactivation experiments. Raw data on the concentrations of disinfectants that dissolved in distilled water are shown in S1 Table. Regarding the dry fogging of hypochlorous acid solution (125, 250, and 8,700 ppm) and hydrogen peroxide solution (1,410, 2,820, 5,640, 11,280, and 56,400 ppm), the total fogging time versus the concentrations of disinfectants trapped in distilled water as well as the concentrations of dry fogged disinfectants versus the concentrations of disinfectants trapped in distilled water per unit fogging time are plotted in Fig 4.

Fig 4

Relationship between dry fogged disinfectant concentrations and concentrations of disinfectants trapped in 20 ml of distilled water in Petri dishes.

The dry fogging of hypochlorous acid solution (A) or hydrogen peroxide solution (B) was performed as described in the Materials and methods. The concentrations of free available chlorine FAC (C) and hydrogen peroxide (H2O2) per unit fogging time (D) were calculated and plotted. R squared (R2) values were estimated using a non-linear regression (curve fit) analysis and reported on the graphs (A and B). In addition, R2 values, equations, and p values were estimated using a simple linear regression analysis and reported on the graphs (C and D).

The results obtained confirmed that the dry fogging of various concentrations of hypochlorous acid solution and hydrogen peroxide solution increased their concentrations in distilled water in proportion to the total fogging time (Fig 4A and 4B). In addition, the concentrations of disinfectants in distilled water per unit fogging time increased in proportion to the dry fogged disinfectant concentration of each disinfectant (Fig 4C and 4D). On one hand, these were expected results because the amount of the droplets of disinfectants from the fogger was proportional to the total fogging time, and the concentrations of disinfectants contained in the droplets was proportional to disinfectant concentrations. On the other hand, the slope of hypochlorous acid solution was approximately 0.59-fold smaller than that of hydrogen peroxide solution (Fig 4C and 4D). Since the fogging time was the same for hypochlorous acid solution and hydrogen peroxide solution, the number of droplets that fell and dissolved into distilled water in Petri dishes was equivalent. Therefore, the data obtained indicated that the concentration of FAC in droplets decreased more than that of hydrogen peroxide when they travelled in the form of a dry fog. The temperature inside the chamber was maintained at approximately 19–26°C, while humidity was maintained at 59–99% during dry fogging experiments.

Relationship between virucidal effects of dry fogged disinfectants and the CT value

The model of Eq (1) calculated from Chick Watson’s law is often used to show the bactericidal effects of a disinfectant on microorganisms [11].

In the equation, N0 is the initial bacterial count, N is the viable bacterial count at the contact time (T) of bacteria to the disinfectant, C is the disinfectant concentration, and k is the inactivation rate constant of the bacteria. In the present study, we evaluated the virucidal effects of dry fogged disinfectants by CT values, the product of disinfectant concentrations and contact times, similar to the bacteria inactivation experiment. The concentration of the disinfectant trapped in 20 ml of distilled water in Petri dishes was used as the concentration (C). Fig 5 shows the logarithmic values of viral infectious titers at various CT values. The viral titers of SARS-CoV-2 and influenza A virus linearly decreased with increases in CT values in the dry fogging of hypochlorite acid solution. With the dry fogging of hydrogen peroxide solution, viral titers also linearly decreased. It is important to note that since the logarithmic values of viral infectious titers at various CT values were plotted and fit by a simple linear regression analysis, the solid lines in Fig 5 are shown by Eq (2), which is a modification of Eq (1).

Fig 5

The relationship between virucidal effects of a disinfectant and the CT value.

Changes in the viral infectious titers (TCID50 values) of SARS-CoV-2 (A and C) and influenza A virus (B and D) upon dry fogging with hypochlorous acid solution (A and B) or hydrogen peroxide solution (C and D) were evaluated, as described in the Materials and methods. The CT value of a dry fogged disinfectant was calculated, and a scatter plot was created from each dataset of the viral infectious titer and CT value for the combination of the virus and disinfectant. R squared (R2) values, equations, and p values were estimated using a simple linear regression analysis and reported on the graphs. Horizontal dotted lines in the graphs show the detection limit of the viral titer.

In the equation, I0 is the viral titer before dry fogging, I is the viral titer at the contact time (T) of the virus to a dry fogged disinfectant, and C is the concentration of a disinfectant trapped in 20 ml of distilled water.

In formula (2), k is the inactivation rate constant of the virus. With the dry fogging of hypochlorous acid solution, the inactivation constant of SARS-CoV-2 was approximately 46-fold smaller than that of influenza A virus. In addition, with the dry fogging of hydrogen peroxide solution, the inactivation constant of SARS-CoV-2 was approximately 8.8-fold smaller than that of influenza A virus. Therefore, SARS-CoV-2 was more resistant to hypochlorous acid solution and hydrogen peroxide solution than influenza A virus.

Effects of saliva on the inactivation of SARS-CoV-2 and influenza A virus by the dry fogging of hypochlorous acid solution and hydrogen peroxide solution

As shown in Figs 3 and 5, the dry fogging of hypochlorous acid solution and hydrogen peroxide solution inactivated SARS-CoV-2 and influenza A virus. We prepared air-dried viral samples using viral supernatants for these experiments, and viral solutions differed from body fluids. Therefore, to assess the effects of saliva components on the virucidal effects of dry fogged disinfectants, air-dried viral samples were prepared by mixing viral supernatants and artificial saliva solution or PBS as the negative control. Hypochlorous acid solution and hydrogen peroxide solution were then dry fogged for viral samples. The results obtained revealed no significant differences in the levels of virus inactivation upon the dry fogging of disinfectants between air-dried viral samples prepared with artificial saliva and PBS (Fig 6).

Fig 6

Effects of saliva on the inactivation of SARS-CoV-2 and influenza A virus by the dry fogging of disinfectants.

SARS-CoV-2 (A and C) or influenza A virus (B and D) was mixed with artificial saliva or PBS, and air-dried. Changes in the viral infectious titers (TCID50 values) upon dry fogging with hypochlorous acid solution (HAS) (A and B) or hydrogen peroxide solution were then evaluated, as described in the Materials and methods. Horizontal dotted lines in the graphs show the detection limit of the viral titer. n.s., not significant between artificial saliva- and PBS-containing samples using an unpaired t-test. Each data point represents the average and standard deviation obtained from two repeated experiments.

Discussion

The inactivation of pathogenic viruses, including SARS-CoV-2, influenza A virus, norovirus, and adenovirus, by the dry fogging of a mixture of peracetic acid and hydrogen peroxide was previously demonstrated [16-21]. Therefore, the dry fogging of disinfectants is considered to effectively inactivate pathogenic viruses on environmental surfaces in laboratories, safety cabinets, and health care facilities. In this report, we investigated whether SARS-CoV-2 and influenza A virus that had been air-dried and adhered to an environmental surface were inactivated by the dry fogging of hypochlorous acid solution or hydrogen peroxide. Hypochlorous acid is decomposed into hydrochloric acid and oxygen, while hydrogen peroxide is decomposed into water and oxygen with time. Since decomposition products other than water are gases at a normal temperature and pressure, they do not remain on environmental surfaces after dry fogging with appropriate ventilation. To the best of our knowledge, the inactivation of SARS-CoV-2 by the dry fogging of hypochlorous acid solution or hydrogen peroxide has not yet been reported. The present results revealed that even though the concentration of the disinfectant required for virus inactivation by dry fogging differed, the infectivities of SARS-CoV-2 and influenza A virus were both reduced to below the detection limit over time with the dry fogging of disinfectants (Fig 3). A previous study reported the higher stability of SARS-CoV-2 than influenza A virus on environmental surfaces [6]. We speculated that the higher stability of SARS-CoV-2 than influenza A virus may be related to our results showing that with dry fogging for the same duration, higher concentrations of hypochlorous acid solution and hydrogen peroxide solution were required to inactivate SARS-CoV-2 than influenza A virus. SARS-CoV-2 and influenza A viruses are both enveloped RNA viruses; however, experiments to elucidate differences in their resistance to the dry fogging of these disinfectants were not conducted in the present study.

The merits of dry fogging are 1) it does not wet the environmental surface and 2) it is diffusible and has a small droplet size; therefore, an object will not be excessively wet unless fogging is performed in the same place for a long time. With dry fogging, it is not necessary to wipe off the disinfectant after fogging, and it is also possible to use it in an environment that cannot become wet. Regarding diffusivity, since the droplet size is small, droplets are assumed to fall at a low speed and float in the air for a long time. By utilizing this property of dry fog, it is also possible to diffuse droplets using a fan. Furthermore, droplets reach the backs of objects, such as desks and chairs, as well as gaps that cannot be accessed; therefore, dry fogging is considered to be suitable for spatial fogging. On the other hand, one of the disadvantages of dry fogging is the risk of the inhalation of droplets. Therefore, several factors need to be considered, such as the concentration of droplets in space, the staying time, the amount of air being inhaled by the number of breaths, the concentration of the solution in droplets, and the toxicity of the chemical to the human body. In the present study, we assumed an unmanned space and, thus, did not consider risks to the human body; however, when fogging a disinfectant in the form of dry fog in a manned environment, it is extremely important to consider risks to the human body. Therefore, if someone has to be present, they need to be wearing appropriate personal protective equipment and respiratory protection.

The results of the dry fogging of hypochlorous acid solution and hydrogen peroxide solution using the test chamber confirmed that the concentrations of FAC and hydrogen peroxide trapped in distilled water increased according to the total fogging time and fogged disinfectant concentration (Fig 4). Furthermore, SARS-CoV-2 and influenza A virus were inactivated in a manner that was dependent on CT values (Fig 5). However, the present results were obtained from a fogging experiment conducted in a small space (approximately 0.11 m3), and, thus, various factors need to be considered when dry fogging in actual spaces, such as the vaporization of droplets and reductions in disinfectant concentrations in droplets due to the longer distance travelled by droplets [22, 23]. In the present study, a maximum of approximately 8 ml of disinfectant was dry fogged in a space of approximately 0.11 m3, and it is inadequate to simply calculate the amount of disinfectant needed for the volume of the space to be fogged. It may be necessary to fog for a longer duration or with higher concentrations of a disinfectant in actual spaces. Since the above factors of fogging in actual spaces reflects the concentration of a dry fogged disinfectant, it is important to measure it at the site of use in order to confirm its effectiveness.

Three important factors for the occurrence of infectious diseases are infection sources, transmission routes, and susceptible hosts; therefore, countermeasures need to be taken against these factors. Among them, as a countermeasure against infection sources, patient isolation and quarantine are currently being performed for COVID-19. Regarding more active countermeasures against infection sources, the inactivation of SARS-CoV-2 by UV or ozone irradiation has been examined at basic research levels as part of the attempt to inactivate the virus adhering to environmental surfaces [24-26]. The inactivation of influenza A virus adhering to environmental surfaces using an ultrasonic atomizer of hypochlorous acid solution has been reported [27]. In the present study, we revealed that the dry fogging of hypochlorous acid solution and hydrogen peroxide solution effectively inactivated SARS-CoV-2 and influenza A virus that had been adhered to plastic microplates, suggesting that it is an active countermeasure against infection sources on environmental surfaces.

There are a number of limitations that need to be addressed. Only the SARS-CoV-2 Wuhan strain and influenza A virus H1N1 strain were tested in the present study. SARS-CoV-2 variants of concern, including the delta variant, are continually emerging. Furthermore, there is a wide variety of influenza A viral strains. Inactivation experiments were not performed on these viral strains in the present study. However, a previous study suggested that hypochlorous acid attacks multiple components of microorganisms, including the plasma membrane and nucleic acids, as its germicidal effect [11]. Although the structures of viral proteins due to genetic mutations may differ between the Wuhan strain and other SARS-CoV-2 variants, there may be a commonality in the basic structures and components of virions, such as the lipid bilayer (envelope) and structural and non-structural proteins. A lipid bilayer is the least resistant component of enveloped viruses to disinfection [9]; therefore, dry fogging is expected to act effectively not only against the Wuhan strain and H1N1 strain tested in the present study, but also against other virus strains. As another limitation, we only examined the effects of dry fogging on viruses dried on the surfaces of plastic microplates. Further studies are warranted to investigate its effects on viruses adhering to the surfaces of other materials. Furthermore, it is important to examine whether dry fogging of disinfectants inactivates viruses in a space. Nevertheless, we consider spatial fogging to be an effective method for inactivating SARS-CoV-2 and influenza A virus on environmental surfaces. The accumulation of more information in the future and the development of methods that inactivate pathogens, such as viruses, in the environment for practical use are desired.

FAC and H2O2 concentrations in 20 ml of distilled water in Petri dishes.

(XLSX)

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12 Jan 2022

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Reviewer #1: In title and throughout paper, the term “dry fog” does not need to be capitalized, since it is not a proper noun. this may be an issue with English

Line 21: you should clarify that the current thinking is that the predominant mechanism for transmission is via air (small particles, up to droplets), but that contact with contaminated surfaces may be a possibility.

57: confusing “spraying” for “fogging” they are not the same.

59-63: the sentence is a bit long, and somewhat inaccurate. Check the reference again, but after looking at it myself, it seems the CDC is ok with spraying, and using some types of foggers. Maybe the authors are confusing fogging with spraying, due to English being their second language.

65: change “spraying” to “dispensing”… this comment applies throughout the paper.

67: add reference for your dry fog definition.

85: indicate how pH was measured

118: suggest starting a new paragraph here in discussing inoculation of the viruses into the well plates. Also need to add some more details about this. For example, need to address how were the viruses added, how many well plates were inoculated with virus each experiment, how the positive controls were handled, etc.

130: so a sprayer was used and not a fogger? It’s confusing.

136: explain what the 4 petri plates of water were used for

162: what is DPD? Confirm that the chlorine meter measures free available chlorine and not just other species of chlorine

169: indicate what was used to measure temp and relative humidity

170-180: the paragraph about how the experiments were conducted is not very clear.

171: need to clarify how many viral samples were resuspended with the DMEM/neutralizer. I’m assuming maybe ¼ of samples were treated this way… and then another ¼ after each 4 minute dwell time? Please fix this.

174: are the concentrations of the disinfectants from the petri dishes reported anywhere, and how do they compare to what was spread?

189-210: I’m not sure it is scientifically correct to report the data as a function of time, since what you’re really doing is increasing dose or mass over time. If you had fogged the chamber one time, then taken samples after that at discrete time points, then it would be ok to report inactivation as a function of time. But you have the two variables (time and mass of disinfectant dispensed or dose) intertwined.

212: regarding this subsection, that you would get increased mass of disinfectant over cumulative time seems a bit obvious, so authors need to clarify what is new about this.

241: you should indicate here what the inactivation rates are, instead of just saying they are derived from the slopes.

253: clarify what you mean by viral solutions differed by physiological conditions

258: as mentioned before, be careful about using the word kinetics (implying rate over time), since it’s also about inactivation as a function of mass applied or dose.

268: PAA is always in equilibrium with hydrogen peroxide.

1st paragraph in Discussion: The authors need to clarify what was new/significant about their research. If no one has looked at using fogging of hydrogen peroxide or hypochlorous acid to inactivate SARS CoV 2 or influenza, then state this. If there have been studies looking at that, then you need to reference those and further differentiate your study from them.

269: if dry fogging is considered to be effective based on previous research, then explain why the current study was conducted. This sentence needs some work.

272: rather than say they won’t remain in the environment, you should discuss their reaction byproducts and how long those will remain in the environment. HP decays to water and O2, but what about HOCl?

272: again, to reiterate throughout the article, you need to differentiate between spraying and fogging. They are not the same.

277-278: do those studies compare influenza and SARS CoV-2 stability? If not, then referencing them doesn’t really help here with respect to saying they’re consistent or not with your study.

279: you need to clarify that to effectively inactivate both the flu and SARS virus using a similar contact time, you needed to use a higher concentration of disinfectant for the flu virus. You might have been able to inactivate the flu virus effectively with a lower concentration of disinfectant, but it may have required a longer contact time.

285: indicate whether you saw excessive condensation on the chamber walls when you were doing your experiments. Did opening the chamber door every 4 minutes help to remove condensation?

290: it depends on the drop size if they behave like air. They need to be really small to behave like air

294-299: you may want to clarify that fogging of disinfectant chemicals should be done with no people present in the space. If they have to be present, they should be wearing appropriate PPE and respiratory protection.

300-303: again, this is quite obvious.

316-318: this sentence makes no sense. Please rewrite.

316-328: this paragraph doesn’t really have any relevance to the study. Delete or fix.

343: I think the lipid envelope is one of the more critical physiological factors involved in viral inactivation; you may want to emphasize that more in your discussion. Lipid enveloped viruses are the least resistant microbes to disinfection: https://www.cdc.gov/infectioncontrol/guidelines/disinfection/tables/figure1.html

Figure 4: need to indicate the units for Y axis dose for A and B. Also, in Figure 4C and 4D, the X-axis says concentration, but your units say micrograms. Concentration is usually mass/volume. So need to clarify these.

497: at least 3 independent experiments? Or were they replicates? The number of replicates and/or experiments should be explained in the Materials and Methods section

General comment about figure legends: They all need to be more concise; they are too long and wordy. For example, no need to refer to and or repeat info already discussed in M and M section. If info about how the data or chart was developed or m

Reviewer #2: excellent work, well designed and thought through; see below

Line 61: correct the sentence

Line 112: specify what method used to calculate TCID50 (Reed-Muench or Spearman-Kaber)

Line 128: specify what type of BSC (Class II type A2, Class II type B2, Class III, etc.)

Show data demonstrating the effectiveness and/or interference of the neutralizers used to neutralize HAS and H2O2 on viral growth and/or tissue culture

Any thoughts on why HAS is more resistant to SARS-CoV-2 (in comparison to Influenza A) while less so against H2O2

Line 295: did you mean, “the amount of air being inhaled by the number of breaths” instead of the amount of suction…”

Line 296: consider writing “and the toxicity of the chemical to the human body”

Line 311: did you mean “the amount of disinfectant needed for the volume…”

Line 496: “n.s. not significant” are you planning to add this in the actual figure? If not, no need for this sentence here

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Reviewer #2: No

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21 Feb 2022

Thank you for the opportunity to revise our manuscript, PONE-D-21-38754 entitled “Inactivation of SARS-CoV-2 and influenza A virus by spraying hypochlorous acid solution and hydrogen peroxide solution in the form of dry fog” by Urushidani, M. et al. We highly appreciate the detailed review and constructive suggestions. We consider the quality of the manuscript to have been markedly improved by the suggested edits. Changes made in the revised manuscript are marked using track changes.

We herein present the Reviewers’ comments followed by our point-by-point responses, including their locations (line numbers) in the revised manuscript.

Reviewer #1:

In title and throughout paper, the term “dry fog” does not need to be capitalized, since it is not a proper noun. this may be an issue with English

Thank you for your comment. We corrected this term throughout the manuscript.

Line 21: you should clarify that the current thinking is that the predominant mechanism for transmission is via air (small particles, up to droplets), but that contact with contaminated surfaces may be a possibility.

According to the Reviewer’s suggestion, we revised the sentence on lines 20-22, as follows. “Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is transmitted mainly by droplet or aerosol infection; however, it is also transmitted by contact infection.”

57: confusing “spraying” for “fogging” they are not the same.

Thank you for your insight. We replaced “spraying” with “fogging” or “dry fogging” throughout the text.

59-63: the sentence is a bit long, and somewhat inaccurate. Check the reference again, but after looking at it myself, it seems the CDC is ok with spraying, and using some types of foggers. Maybe the authors are confusing fogging with spraying, due to English being their second language.

Thank you for your insight. We checked the reference again, and revised the sentences on lines 61-67, as follows. “In addition, the Center for Disease Control and Prevention of the United States of America (USA) does not recommend the fogging of disinfectants in hospital rooms in the 2003 and 2008 guidelines [9]. Newer technologies for performing disinfectant fogging were assessed in the 2011 guidelines for the prevention and control of norovirus gastroenteritis outbreaks in healthcare settings; nevertheless, further research is required to clarify the effectiveness and reliability of disinfectant fogging [9].”

65: change “spraying” to “dispensing”… this comment applies throughout the paper.

Thank you for your suggestion. We changed “spraying” to “fogging” or “dry fogging” throughout the text.

67: add reference for your dry fog definition.

Thank you for your comment. We added reference #10 for the dry fog definition.

85: indicate how pH was measured

We measured the pH of the solution using the pH meter SK-620PHⅡ (Sato Keiryoki mfg. Co., Ltd, Tokyo, Japan). We added this information to the Materials and methods section on lines 95-96, as follows. “using the pH meter SK-620PHⅡ (Sato Keiryoki Mfg. Co., Ltd, Tokyo, Japan).”

118: suggest starting a new paragraph here in discussing inoculation of the viruses into the well plates. Also need to add some more details about this. For example, need to address how were the viruses added, how many well plates were inoculated with virus each experiment, how the positive controls were handled, etc.

Thank you for your advice. According to the Reviewer’s suggestion, we made a new paragraph “Preparation of air-dried virus samples” and added more detailed experimental methods to lines 125-132, as follows.

“Preparation of air-dried virus samples

Viral solutions (5 µl) containing SARS-CoV-2 (1.2 × 105 TCID50) or influenza A virus (2.8 × 106 TCID50) were applied using a micropipette to the bottom of 5 wells (per virus inactivation experiment) on a 96-well flat-bottomed microplate (Corning Japan, Shizuoka, Japan), air-dried for 10-15 minutes using a small electric fan, and subjected to a virus inactivation experiment. As a negative control for the experiment, an air-dried viral sample in a well was re-suspended with 200 µl of DMEM containing a neutralizer of the disinfectant prior to dry fogging, as described below.”

We also added more information on the handling of air-dried virus samples in virus inactivation experiments to a later paragraph (lines 146-150), as follows. “In the virus inactivation experiment using dry fogging, four 90-mm Petri dishes containing 20 ml of distilled water, a temperature and humidity sensor (Model RHT-3 Temperature and Humidity Sensor; Sensatec Co., Ltd., Kyoto, Japan), and a 96-well microplate containing air-dried viral samples in 5 wells were placed in the chamber (Fig. 1).”

130: so a sprayer was used and not a fogger? It’s confusing.

We replaced “sprayer” with “fogger” on line 141.

136: explain what the 4 petri plates of water were used for

We added an explanation on the purpose of the 4 petri dishes on lines 150-153, as follows. “Four 90-mm Petri dishes containing distilled water were placed to measure the concentrations of disinfectants trapped in water during the virus inactivation experiment and also to calculate the product of the concentrations of disinfectants and contact times (CT value).”

162: what is DPD? Confirm that the chlorine meter measures free available chlorine and not just other species of chlorine

We added an explanation for the abbreviation DPD to line 178, as follows. “DPD (N,N-dimethyl-p-phenylenediamine)” We measured free available chlorine (FAD) according to the procedure mentioned in the CDC website , https://www.cdc.gov/safewater/chlorine-residual-testing.html/. We did not measure other species of chlorine in the present study.

169: indicate what was used to measure temp and relative humidity

We added information on the equipment used to measure temperature and humidity to lines 148-149, where the procedure was described for first time in the manuscript, as follows. “a temperature and humidity sensor (Model RHT-3 Temperature and Humidity Sensor; Sensatec Co., Ltd., Kyoto, Japan)”

170-180: the paragraph about how the experiments were conducted is not very clear.

171: need to clarify how many viral samples were resuspended with the DMEM/neutralizer. I’m assuming maybe ¼ of samples were treated this way… and then another ¼ after each 4 minute dwell time? Please fix this.

Thank you for your comments. We agree with the Reviewer that the experimental procedure was not clearly described. In addition, we found some errors in the description. Therefore, we completely rewrote the experimental procedure on lines 187-213, as follows. “A set of viral inactivation experiments was performed as follows. Four 90-mm Petri dishes containing distilled water, a temperature and humidity sensor, and a 96-well microplate containing air-dried viral samples in 5 wells were placed in the test chamber (Fig. 1), as described above. Prior to the dry fogging of disinfectants, a dried viral sample in the first well was resuspended with DMEM (200 µl) containing a neutralizer by pipetting. The sliding door was then closed, and the first dry fogging of the disinfectant was performed for 5 seconds. After 4 minutes, the sliding door of the chamber was opened, the dried viral sample in the second well was resuspended with DMEM (200 µl) containing the neutralizer, and the first Petri dish containing distilled water was removed from the chamber. The sliding door was closed, and the second dry fogging of the disinfectant was performed for 2.5 seconds. After 4 minutes, the sliding door was opened, the dried viral sample in the third well was resuspended with DMEM (200 µl) containing the neutralizer, and the second Petri dish containing distilled water was removed from the chamber. The sliding door was again closed, and the third dry fogging of the disinfectant was performed for 2.5 seconds. After 4 minutes, the sliding door was opened, the dried viral sample in the fourth well was resuspended with DMEM (200 µl) containing the neutralizer, and the third Petri dish containing distilled water was removed from the chamber. The sliding door was closed, and the fourth dry fogging of the disinfectant was performed for 2.5 seconds. After 4 minutes, the sliding door was opened, the dried viral sample in the fifth well was resuspended with DMEM (200 µl) containing the neutralizer, and the fourth Petri dish containing distilled water was removed from the chamber. The concentrations of the droplet-shaped disinfectant that fell into distilled water in the Petri dishes were measured between dry fogging. Therefore, the concentrations of the disinfectant trapped in distilled water were measured 4 times in each set of experiments, i.e., at 4 minutes (total of 5 seconds of dry fogging), 8 minutes (total of 7.5 seconds of dry fogging), 12 minutes (total of 10 seconds of dry fogging), and 16 minutes (total of 12.5 seconds of dry fogging).”

174: are the concentrations of the disinfectants from the petri dishes reported anywhere, and how do they compare to what was spread?

Thank you for the comment. We added a supplementary table (S1 Table) showing raw data on disinfectant concentrations dissolved in 20 ml of distilled water in Petri dishes, and described it in the Results section on lines 253-255, as follows. “Raw data on the concentrations of disinfectants that dissolved in distilled water are shown in S1 Table.” We hope that our response to this comment is adequate.

189-210: I’m not sure it is scientifically correct to report the data as a function of time, since what you’re really doing is increasing dose or mass over time. If you had fogged the chamber one time, then taken samples after that at discrete time points, then it would be ok to report inactivation as a function of time. But you have the two variables (time and mass of disinfectant dispensed or dose) intertwined.

Thank you for your insight. We agree with the Reviewer that it is not adequate to interpret the data as “the viruses were inactivated by dry fogging of disinfectant in a time-dependent manner”. Therefore we removed the word “time-“ from lines 29 and 553 and replaced the words “in a time-dependent manner” with “over time” on lines 224 and 239. It is clear that the inactivation of viruses was observed under the experimental conditions used; therefore, we consider this modification to improve the accuracy of the interpretation of data obtained. In addition, we added sentences to explain how we conducted the experiments to lines 243-246, as follows. “It is important to note that since dry fogging was performed 4 times at 4-minute intervals in the virus inactivation experiment, the concentration of the disinfectant increased over time. Nevertheless, viruses were inactivated over time under the experimental conditions used.”

212: regarding this subsection, that you would get increased mass of disinfectant over cumulative time seems a bit obvious, so authors need to clarify what is new about this.

We agree with the Reviewer that it is obvious that the amount of exposure increased as the fogging time became longer. We described the results as “expected results” on lines 265-268. Our new result is that when we compared the dry fogged and collected (in distilled water in Petri dishes) concentrations of disinfectants, the concentration of hypochlorous acid in droplets appeared to be lower than that of hydrogen peroxide during travelling in the form of dry fog. Therefore, we added sentences to explain this in more detail to lines 270-274, as follows. “Since the fogging time was the same for hypochlorous acid solution and hydrogen peroxide solution, the number of droplets that fell and dissolved into distilled water in Petri dishes was equivalent. Therefore, the data obtained indicated that the concentration of FAC in droplets decreased more than that of hydrogen peroxide when they travelled in the form of a dry fog.”

241: you should indicate here what the inactivation rates are, instead of just saying they are derived from the slopes.

Thank you for your insight. We reconsidered how to evaluate the virucidal effects of disinfectants, and revised the sentences in the Results section on lines 278-305, as follows.

“Relationship between virucidal effects of dry fogged disinfectants and the CT value

The model of Eq (1) calculated from Chick Watson’s law is often used to show the bactericidal effects of a disinfectant on microorganisms [11].

log (N / N0) = -kCT (1)

In the equation, N0 is the initial bacterial count, N is the viable bacterial count at the contact time (T) of bacteria to the disinfectant, C is the disinfectant concentration, and k is the inactivation rate constant of the bacteria. In the present study, we evaluated the virucidal effects of dry fogged disinfectants by CT values, the product of disinfectant concentrations and contact times, similar to the bacteria inactivation experiment. The concentration of the disinfectant trapped in 20 ml of distilled water in Petri dishes was used as the concentration (C). Figure 5 shows the logarithmic values of viral infectious titers at various CT values. The viral titers of SARS-CoV-2 and influenza A virus linearly decreased with increases in CT values in the dry fogging of hypochlorite acid solution. With the dry fogging of hydrogen peroxide solution, viral titers also linearly decreased. It is important to note that since the logarithmic values of viral infectious titers at various CT values were plotted and fit by a simple linear regression analysis, the solid lines in Figure 5 are shown by Eq (2), which is a modification of Eq (1).

In the equation, I0 is the viral titer before dry fogging, I is the viral titer at the contact time (T) of the virus to a dry fogged disinfectant, and C is the concentration of a disinfectant trapped in 20 ml of distilled water.

In formula (2), k is the inactivation rate constant of the virus. With the dry fogging of hypochlorous acid solution, the inactivation constant of SARS-CoV-2 was approximately 46-fold smaller than that of influenza A virus. In addition, with the dry fogging of hydrogen peroxide solution, the inactivation constant of SARS-CoV-2 was approximately 8.8-fold smaller than that of influenza A virus. Therefore, SARS-CoV-2 was more resistant to hypochlorous acid solution and hydrogen peroxide solution than influenza A virus.”

We decided to evaluate the viricidal effects of dry fogged disinfectants by comparing viral infectious titers at various CT values calculated by the concentrations of disinfectants trapped in 20 ml of distilled water in Petri dishes and contact times. Accordingly, we replaced the term “exposed disinfectant amount” with “concentration of a disinfectant” or an equivalent throughout the text. In addition, we revised Figures 4 and 5 as well as the figure legends. We hope the Reviewer agrees with the revised method for evaluating the virucidal effects of dry fogged disinfectants.

253: clarify what you mean by viral solutions differed by physiological conditions

We modified the sentence on lines 310-312, as follows. “We prepared air-dried viral samples using viral supernatants for these experiments, and viral solutions differed from body fluids.”

258: as mentioned before, be careful about using the word kinetics (implying rate over time), since it’s also about inactivation as a function of mass applied or dose.

Thank you for your comments. We modified the sentence on lines 316-318, as follows. “The results obtained revealed no significant differences in the levels of virus inactivation upon the dry fogging of disinfectants between air-dried viral samples prepared with artificial saliva and PBS (Fig. 6).”

268: PAA is always in equilibrium with hydrogen peroxide.

Thank you for your insight. We corrected the sentence on lines 326-327, as follows. “the dry fogging of a mixture of peracetic acid and hydrogen peroxide was previously demonstrated [16-21].”

1st paragraph in Discussion: The authors need to clarify what was new/significant about their research. If no one has looked at using fogging of hydrogen peroxide or hypochlorous acid to inactivate SARS CoV 2 or influenza, then state this. If there have been studies looking at that, then you need to reference those and further differentiate your study from them.

269: if dry fogging is considered to be effective based on previous research, then explain why the current study was conducted. This sentence needs some work.

Thank you for your comment. We consider this to be the first study to demonstrate the inactivation of SARS-CoV-2 by the dry fogging of hypochlorous acid solution or hydrogen peroxide alone. Therefore, we added this information to lines 334-336, as follows. “To the best of our knowledge, the inactivation of SARS-CoV-2 by the dry fogging of hypochlorous acid solution or hydrogen peroxide has not yet been reported.”

272: rather than say they won’t remain in the environment, you should discuss their reaction byproducts and how long those will remain in the environment. HP decays to water and O2, but what about HOCl?

Thank you for your insight. We modified the sentences on lines 329-334, as follows. “Hypochlorous acid solution and hydrogen peroxide solution were employed as disinfectants to be dry fogged in the present study. Hypochlorous acid is decomposed into hydrochloric acid and oxygen, while hydrogen peroxide is decomposed into water and oxygen with time. Since decomposition products other than water are gases at a normal temperature and pressure, they do not remain on environmental surfaces after dry fogging with appropriate ventilation.”

272: again, to reiterate throughout the article, you need to differentiate between spraying and fogging. They are not the same.

Thank you for the comment. We replaced “spraying” with “fogging” or “dry fogging” throughout the text.

277-278: do those studies compare influenza and SARS CoV-2 stability? If not, then referencing them doesn’t really help here with respect to saying they’re consistent or not with your study.

279: you need to clarify that to effectively inactivate both the flu and SARS virus using a similar contact time, you needed to use a higher concentration of disinfectant for the flu virus. You might have been able to inactivate the flu virus effectively with a lower concentration of disinfectant, but it may have required a longer contact time.

Thank you for your insight. We agree that the studies cited in references 4, 5 and 7 did not compare the stabilities of influenza A virus and SARS-CoV-2. In addition, although the study cited in reference 6 compared their stabilities on human skin as well as on environmental surfaces, their findings and the present results were not similar. Furthermore, our description of fogging was insufficient. Therefore, we modified the sentences on lines 340-344, as follows. “A previous study reported the higher stability of SARS-CoV-2 than influenza A virus on environmental surfaces [6]. We speculated that the higher stability of SARS-CoV-2 than influenza A virus may be related to our results showing that with dry fogging for the same duration, higher concentrations of hypochlorous acid solution and hydrogen peroxide solution were required to inactivate SARS-CoV-2 than influenza A virus.”

285: indicate whether you saw excessive condensation on the chamber walls when you were doing your experiments. Did opening the chamber door every 4 minutes help to remove condensation?

Thank you for your comment. We did not see excessive condensation on the chamber walls under the experimental conditions used. However, the floor of the chamber was a little wet after the experiments. We attributed this to the falling of dry fog to the floor. We do not know the extent to which opening of the chamber door affected condensation.

290: it depends on the drop size if they behave like air. They need to be really small to behave like air

Thank you for your insight. We remove the words “behave like air” and modified the sentence on lines 355-356, as follows. “Furthermore, droplets reach the backs of objects, such as desks and chairs, as well as gaps that cannot be accessed;”

294-299: you may want to clarify that fogging of disinfectant chemicals should be done with no people present in the space. If they have to be present, they should be wearing appropriate PPE and respiratory protection.

Thank you for your insight. We added the sentence on lines 364-365, as follows. “Therefore, if someone has to be present, they need to be wearing appropriate personal protective equipment and respiratory protection.”

300-303: again, this is quite obvious.

Thank you for the comment. We wish to keep sentences because we would like to show the context to the general readers.

316-318: this sentence makes no sense. Please rewrite.

Thank you for the comment. We rewrote the sentences on lines 382-384, as follows. “Three important factors for the occurrence of infectious diseases are infection sources, transmission routes, and susceptible hosts; therefore, countermeasures need to be taken against these factors.”

316-328: this paragraph doesn’t really have any relevance to the study. Delete or fix.

Thank you for your insight. Although the Reviewer considers the paragraph to be unnecessary, we would like to explain 3 important factors involved in the occurrence of infectious diseases to the general readers. In addition, we would like to mention that the inactivation of pathogens on environmental surfaces is a countermeasure against an infection source. Therefore, please let us keep this paragraph. We hope that the Reviewer agrees with our opinion.

343: I think the lipid envelope is one of the more critical physiological factors involved in viral inactivation; you may want to emphasize that more in your discussion. Lipid enveloped viruses are the least resistant microbes to disinfection: https://www.cdc.gov/infectioncontrol/guidelines/disinfection/tables/figure1.html

Thank you for your insight. We agree with you, and added a sentence to lines 410-411 in order to emphasize it, as follows. “A lipid bilayer is the least resistant component of enveloped viruses to disinfection [9];”

Figure 4: need to indicate the units for Y axis dose for A and B. Also, in Figure 4C and 4D, the X-axis says concentration, but your units say micrograms. Concentration is usually mass/volume. So need to clarify these.

Thank you for pointing out the lack of an explanation in Fig. 4. We revised the Figure accordingly.

497: at least 3 independent experiments? Or were they replicates? The number of replicates and/or experiments should be explained in the Materials and Methods section

Thank you for the suggestion. We added an explanation to the Materials and methods section on lines 213-215, as follows. “Virus inactivation experiments under the same conditions were repeated more than three times, except for those to evaluate artificial saliva, which were repeated twice.” Since most of the viral inactivation experiments conducted under the same conditions were performed more than three times, please let us keep the explanation in the Figure legends.

General comment about figure legends: They all need to be more concise; they are too long and wordy. For example, no need to refer to and or repeat info already discussed in M and M section. If info about how the data or chart was developed or m

Thank you for your comment. We shortened the figure legends. We hope the general readers understand the context of the figures easily without repeatedly reading the main text. We hope the Reviewer agrees with our opinion.

Reviewer #2:

excellent work, well designed and thought through; see below

Thank you for your comments.

Line 61: correct the sentence

We rewrote the sentences on lines 61-67 as to be more accurate, as follows. “In addition, the Center for Disease Control and Prevention of the United States of America (USA) does not recommend the fogging of disinfectants in hospital rooms in the 2003 and 2008 guidelines [9]. Newer technologies for performing disinfectant fogging were assessed in the 2011 guidelines for the prevention and control of norovirus gastroenteritis outbreaks in healthcare settings; nevertheless, further research is required to clarify the effectiveness and reliability of disinfectant fogging [9].”

Line 112: specify what method used to calculate TCID50 (Reed-Muench or Spearman-Kaber)

Thank you for pointing out the lack of an explanation. We used the Spearman-Kaber method. The sentence on lines 116-119 was revised, as follows. “median tissue culture infectious doses (TCID50) were measured using the Spearman-Kaber method following the fixation of cells with 5% formaldehyde in phosphate-buffered saline (PBS) and staining with 0.5% crystal violet in 20% EtOH.”

Line 128: specify what type of BSC (Class II type A2, Class II type B2, Class III, etc.)

Thank you for your comment. We used a biosafety cabinet class II type A/B3 in the P3 room, while a biosafety cabinet class II type A1 in the P2 room. Therefore, the sentence on lines 139-140 was modified, as follows. “set in a biosafety cabinet of class II type A/B3 or class II type A1.”

Show data demonstrating the effectiveness and/or interference of the neutralizers used to neutralize HAS and H2O2 on viral growth and/or tissue culture

Thank you for your comment. These neutralizers did not affect cell growth, at least under our experimental conditions; therefore, we added the sentence “These neutralizers did not affect cell growth under experimental conditions (data not shown).” to lines 171-172. The effects of the neutralizers on viral growth were not examined; however, since we included the reagents in all virus inactivation experiments, including a negative control one, we consider the effects on viral growth to not have affected the experimental outcomes. We hope the Reviewer agrees with our opinion.

Any thoughts on why HAS is more resistant to SARS-CoV-2 (in comparison to Influenza A) while less so against H2O2

Thank you for your comment. We consider the different stabilities of those viruses to reflect their susceptibilities to the disinfectants examined, as mentioned in the Discussion section, lines 340-344, as follows. “A previous study reported the higher stability of SARS-CoV-2 than influenza A virus on environmental surfaces [6]. We speculated that the higher stability of SARS-CoV-2 than influenza A virus may be related to our results showing that with dry fogging for the same duration, higher concentrations of hypochlorous acid solution and hydrogen peroxide solution were required to inactivate SARS-CoV-2 than influenza A virus.” However, we were unable to elucidate this in more detail in the present study.

Line 295: did you mean, “the amount of air being inhaled by the number of breaths” instead of the amount of suction…”

Line 296: consider writing “and the toxicity of the chemical to the human body”

Thank you for your comments. According to your suggestion, we revised the sentences on lines 358-361, as follows. “Therefore, several factors need to be considered, such as the concentration of droplets in space, the staying time, the amount of air being inhaled by the number of breaths, the concentration of the solution in droplets, and the toxicity of the chemical to the human body.”

Line 311: did you mean “the amount of disinfectant needed for the volume…”

Thank you for pointing out the unclear description. We revised the sentence on lines 376-377, as follows. “it is inadequate to simply calculate the amount of disinfectant needed for the volume of the space to be fogged.”

Line 496: “n.s. not significant” are you planning to add this in the actual figure? If not, no need for this sentence here

Thank you for your comment. Since Fig. 3A contains “not significant” results, we would like to keep it.

Submitted filename: PONE-D-21-38754_Response to reviewers.docx

Click here for additional data file.

9 Mar 2022

22 Mar 2022

Thank you for the opportunity to revise our manuscript, PONE-D-21-38754R1 entitled “Inactivation of SARS-CoV-2 and influenza A virus by dry fogging hypochlorous acid solution and hydrogen peroxide solution” by Urushidani, M. et al. We appreciate the detailed review and suggestions made by the reviewer #1. We herein present the Reviewers’ comments, followed by our point-by-point responses. Changes made in the revised manuscript are marked using track changes.

Reviewer #1: I think you still need to make the figure captions more concise. A lot of the verbiage in the captions I believe is already described in the materials/methods section, or described in the Results section.

(Response) Thank you for your suggestion. We shortened the figure legends.

I think the Discussion section could be made more concise as well, especially the last two paragraphs. this seems like a repeat of the Introduction.

(Response) Thank you for your suggestion. We removed some sentences that overlapped with the Introduction section, and made the Discussion section more concise. Please let us remain some sentences “overlapped” with the Results section, because we would like to emphasize the results. We hope the reviewer agrees with our opinion.

In the abstract and elsewhere, where you say that it is also transmitted by contact infection, i think maybe you should say it may be transmitted by surface contact. Use the word "may".

(Response) Thank you for the suggestion. We added “may” in the corresponding sentence in the Abstract section.

Submitted filename: PONE-D-21-38754R1_Response to reviewers.docx

Click here for additional data file.

24 Mar 2022

Inactivation of SARS-CoV-2 and influenza A virus by dry fogging hypochlorous acid solution and hydrogen peroxide solution

PONE-D-21-38754R2

Dear Dr. Kameoka,

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.

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Etsuro Ito

Academic Editor

PLOS ONE

29 Mar 2022

PONE-D-21-38754R2

Inactivation of SARS-CoV-2 and influenza A virus by dry fogging hypochlorous acid solution and hydrogen peroxide solution

Dear Dr. Kameoka:

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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on behalf of

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