Inactivation of SARS-CoV-2 and influenza A virus by spraying hypochlorous acid solution and hydrogen peroxide solution in the form of Dry Fog

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is transmitted by droplet and 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 spraying aerosolized hypochlorous acid solution and hydrogen peroxide solution in the form of Dry Fog (fog that does not wet objects even if touched). SARS-CoV-2 and influenza virus were dried on plastic plates and placed into a test chamber for inactivation by the Dry Fog spraying of disinfectants. The results obtained showed that Dry Fog spraying inactivated SARS-CoV-2 and influenza A virus in time- and exposed disinfectant amount-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 spray solutions 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 spraying.


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Coronavirus disease 2019  continues to spread worldwide, with 41 more than 266 million individuals being infected and more than 5.26 million dying to  [4,5], and its stability was shown to be higher than those of 54 SARS-CoV and influenza A virus [6,7]. Therefore, the disinfection of environmental 55 surfaces is indispensable as an infection control measure. However, it is not realistic to 56 frequently and manually disinfect the surfaces of large spaces, such as a train station or 5 57 airport, because of the manpower and time required. Although the spraying of a 58 disinfectant is an alternative spatial disinfection method, it is not recommended by WHO 59 due to its effects on the human body [8]. Furthermore, the Center for Disease Control and 60 Prevention of the United States of America (USA) does not recommend the spraying of 61 disinfectants in hospital rooms because due to the lack of scientific verification on 62 effective, reliable, and safe spatial spraying methods for disinfectants, it is not regarded 63 as an adequate method for decontaminating air and surfaces [9]. 64 Therefore, the present study examined the effectiveness of inactivating SARS-65 CoV-2 virus adhering to plastic microplates by spraying disinfectant in the form of Dry 66 Fog, which is defined as an aerosol with a Sauter mean droplet diameter ≤10 μm and 67 maximum droplet diameter ≤50 μm. Its virucidal effects on influenza A virus, which is a 68 common envelope virus transmitted through droplets and contact transmission worldwide, 69 were also investigated. In consideration of the effects of residues after spraying on the 70 human body, we tested hypochlorous acid solution and hydrogen peroxide solution, 71 which leave almost no residue on environmental surfaces after spraying. respectively, for viral titration, and median tissue culture infectious doses (TCID 50 ) were 113 measured following the fixation of cells with 5% formaldehyde in phosphate-buffered 114 saline (PBS) and staining with 0.5% crystal violet in 20% EtOH. TCID 50 values below 115 the detection limit (3.16 × 10 2 TCID 50 /ml or 2.5 log10 TCID 50 /ml) were assigned to half 116 of the detection limit, equivalent to 1.58 × 10 2 TCID 50 /ml or 2.2 log10 TCID 50 /ml, 117 because substituting the value to half of the detection limit was previously shown to be 118 less biased than substitution to zero or the detection limit [12]. SARS-CoV-2 (1.2 × 10 5 119 TCID 50 in 5 µl) and influenza A virus (2.8 × 10 6 TCID 50 in 5 µl) were added to 96-well 120 flat-bottomed microplates (Corning Japan, Shizuoka, Japan), air-dried for 10-15 minutes 121 using a small electric fan, and subjected to inactivation experiments. In addition, for some 122 experiments, 5 µl of artificial saliva (Saliveht aerosol; Teijin Pharma, Tokyo, Japan) or 123 PBS was mixed with 5 µl of viral supernatants prior to air-drying samples.  A virus (Fig. 3D). The Dry Fog spraying of distilled water did not reduce the viral 210 infectivity of SARS-CoV-2 or influenza A virus regardless of the elapsed time (Fig. 3).

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Relationship between spray concentrations and exposed disinfectant amounts 213 To calculate the exposed disinfectant amount in viral samples in the wells of 96-well 214 microplates, we measured their dissolved concentrations in 20 ml of distilled water in 90-215 mm Petri dishes during virus inactivation experiments. We then estimated the exposed 216 disinfectant amount in the wells (diameter of 6.5 mm) of a 96-well microplate using an 217 area conversion. Regarding the Dry Fog spraying of hypochlorous acid solution (125,250,218 and 8,700 ppm) and hydrogen peroxide solution (1,410, 2,820, 5,640, 11,280, and 56,400 14 219 ppm), the total spraying time versus the exposed disinfectant amount as well as the 220 concentration of sprayed disinfectant versus the exposed disinfectant amount per unit   differed, the infectivities of SARS-CoV-2 and influenza A virus were both reduced to 276 below the detection limit over time with the spraying of disinfectants (Fig. 3). Previous 277 studies reported the high stability of SARS-CoV-2 on environmental surfaces [4][5][6][7].   The results of the Dry Fog spraying of hypochlorous acid solution and hydrogen 301 peroxide solution using the test chamber confirmed that the exposed disinfectant amount 302 of FAC and hydrogen peroxide per unit spray time increased according to the spray 303 amount and sprayed disinfectant concentration (Fig. 4). Furthermore, SARS-CoV-2 and 304 influenza A virus were inactivated in a manner that was dependent on the exposed 305 amounts of disinfectants (Fig. 5). The present results were obtained from a spraying it is inadequate to simply calculate the amount of disinfectant needed from the volume of 312 the space to be sprayed. It may be necessary to spray more or higher concentrations of a 313 disinfectant in actual spaces. Since the above factors of spraying in actual spaces reflects 314 the exposed disinfectant amount, it is important to measure the exposed disinfectant 315 amount at the site of use in order to confirm its effectiveness.

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There are a number of limitations that need to be addressed. Only the SARS-

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CoV-2 Wuhan strain and influenza A virus H1N1 strain were tested in the present study.

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SARS-CoV-2 variants of concern, including the delta variant, are continually emerging.  calculating the TCID 50 value, as described in the Materials and methods. Horizontal 489 dotted lines in the graphs show the detection limit of the viral titer (3.16 × 10 2 TCID 50 /ml 490 or 2.5 log 10 TCID 50 /ml). A viral titer below the detection limit was plotted as half of the 491 detection limit (1.58 × 10 2 TCID 50 /ml or 2.2 log 10 TCID 50 /ml). The significance of 492 differences was analyzed using a two-way ANOVA. P < 0.05 was considered to be 493 significant. *P < 0.0001 was estimated for comparisons between DW and HAS 8,700  titer was then measured by calculating the TCID 50 value, as described in the Materials 517 and methods. In addition, the exposed disinfectant amounts of free available chlorine 518 (FAC) (A and B) and hydrogen peroxide (H 2 O 2 ) (C and D) in the wells of a 96-well 519 microplate were calculated. A scatter plot was created from each dataset of the TCID 50 520 value and exposed disinfectant amount for the combination of the virus and disinfectant.

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R squared (R 2 ) values, equations, and p values were estimated using a simple linear 522 regression analysis and reported on the graphs. Horizontal dotted lines in the graphs show 523 the detection limit of the viral titer (3.16 × 10 2 TCID 50 /ml or 2.5 log 10 TCID 50 /ml). A viral 32 524 titer below the detection limit was plotted as half of the detection limit (1.58 × 10 2 525 TCID 50 /ml or 2.2 log 10 TCID 50 /ml).  The viral titer was measured by calculating the TCID 50 value, as described in the 533 Materials and methods. Horizontal dotted lines in the graphs show the detection limit of 534 the viral titer (3.16 × 10 2 TCID 50 /ml or 2.5 log10 TCID 50 /ml). The significance of 535 differences between artificial saliva-and PBS-containing samples was analyzed using an 536 unpaired t-test. No significant differences were observed between the samples (P.>0.05). 537 n.s. not significant. Each data point represents the average and standard deviation 538 obtained from at least two independent experiments.