Virucidal efficacy of hypochlorous acid water for aqueous phase and atomization against SARS-CoV-2

ACC

available chlorine concentration

COVID-19

coronavirus disease 2019

HOCl

hypochlorous acid water

MPO

myeloperoxidase

ROS

reactive oxygen species

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

TCID₅₀

50% tissue culture infectious dose

The new pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative virus of coronavirus disease 2019 (COVID-19), which emerged at the end of 2019 and has since spread rapidly worldwide (Zhu et al. 2020). The clinical symptoms of COVID-19 vary widely from asymptomatic to severe, and it can be fatal (Chen et al. 2020; Guan et al. 2020). As of 28 August 2023, the World Health Organization (WHO) confirmed 770 million cases and >6.9 million deaths (WHO 2023).

SARS-CoV-2 is an enveloped positive-sense single-stranded RNA virus, which can spread through asymptomatic and symptomatic individuals through ejected respiratory droplets and aerosols when breathing, coughing, sneezing, and speaking under insufficient ventilation (Hu et al. 2021; Leung 2021). The virus can survive from a few hours to several days on various material surfaces, such as glass, plastic, natural rubber, and metal (Riddell et al. 2020; van Doremalen et al. 2020; Hirose et al. 2021; Wiktorczyk-Kapischke et al. 2021). Therefore, the droplet infection and fomite transmission are feasible routes of infection. Consequently, to prevent human-to-human viral transmission, it is important to inactivate SARS-CoV-2 in the form of released droplets or on the surface of fomites. In addition, handwashing and hand disinfection are also important to prevent SARS-CoV-2 infection.

For this purpose, the use of chemical disinfectants and sanitizers can reduce the high risk of pathogen transmission. We have explored or developed various disinfectants and sanitizers to prevent infectious diseases. The main components used in disinfectants and sanitizers contain alcohol, quaternary ammonium salts, and amphoteric surfactants (McDonnell & Russell 1999; Tischer et al. 2012; Gerba 2015; Tsujimura et al. 2015; Falk 2019; Xiao et al. 2022). At the beginning of the COVID-19 pandemic, the demand for alcohol disinfectants increased. Therefore, alternative disinfectants were evaluated in Japan. Ionless hypochlorous acid water (HOCl) is one of the several reactive oxygen species produced by phagocytes, such as macrophages and neutrophils, playing a key role in disinfection of bacteria, fungi, protozoa, and viruses (Aratani 2018). HOCl is a strong oxidizing agent with antipathogen efficacy. HOCl has rapid and broad-spectrum antimicrobial efficacy against clinically relevant microorganisms in vitro and in vivo (Wang et al. 2007). HOCl was reportedly superior to hypochlorite or hydrogen peroxide in terms of therapeutic index, which is an indicator of the safety margin of a disinfectant (Wang et al. 2007). HOCl can inactivate Gram-negative and positive bacteria, fungi, and viruses (WHO 2021). HOCl exhibits high virucidal efficacy by interacting with viral structural proteins (capsid or surface compound), lipid envelope, and DNA/RNA (Block & Rowan 2020; Hawkins & Davies 2021; Qiao et al. 2022). HOCl can react better with thiol-containing compounds, such as cysteine, methionine, and glutathione, than other cellular components and therefore induces protein unfolding and aggregation (Gray et al. 2013; da Cruz Nizer et al. 2020). Hawkins & Davies (2002) reported that HOCl-induced DNA damage. HOCl reacts with unsaturated fatty acids and cholesterol and induces a change in membrane properties (Spickett et al. 2000; da Cruz Nizer et al. 2020). Previous studies have reported the viricidal efficacy of HOCl solution against influenza virus, herpes simplex virus, human immunodeficiency virus 1, and coronavirus in the aqueous phase (Tachikawa et al. 1999; Miyaoka et al. 2021). According to Hatanaka et al. (2022) and Takeda et al. (2021), HOCl solution containing 28 and 51–56 ppm of chlorine concentration is efficient against SARS-CoV-2. A study reported that >35 ppm of HOCl is efficient against SARS-CoV-2 infection (NITE: National Institute of Technology and Evaluation 2022). In the study, each experimental condition by five facilities was different, for example, the producing method of HOCl solution (electrolyzed or not electrolyzed), the concentration of fetal bovine serum (FBS; 1–5%) in the virus solution, and reaction rates (1:9 or 1:19) with HOCl and virus solution.

Atomization with HOCl reduces bacteria on surfaces in vitro and in the hospital room (Hakim et al. 2016; Miyazaki et al. 2022). The virucidal efficacy of HOCl atomization was reported against norovirus, avian influenza virus, and SARS-CoV-2 on surfaces (Park et al. 2007; Hakim et al. 2015; Urushidani et al. 2022). Details of the appropriate use of HOCl against SARS-CoV-2 in the aqueous phase and under atomization are unclear.

In the present study, we investigated whether HOCl treatment would influence the infectivity of SARS-CoV-2 to susceptible cells in the presence of the different concentrations of organic compounds and variants. We also explored whether HOCl atomization would provide effective disinfection to prevent SARS-CoV-2 infection.

VeroE6 cells expressing the transmembrane serine protease TMPRSS2 (VeroE6/TMPRSS2: JCRB1819) were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition (JCRB Cell Bank, Osaka, Japan). VeroE6/TMPRSS2 cells were cultured in Dulbecco's modified Eagle's medium (D-MEM) GlutaMAX (Thermo Fisher Scientific, Tokyo, Japan) containing 5% heat-inactivated FBS (Vitromex, Vilshofen, Germany), 100 units/mL penicillin, and 100 mg/mL streptomycin (Nacalai Tesque, Inc., Kyoto, Japan), and 1 mg/mL G-418 solution (Roche Diagnostics GmbH, Mannheim, Germany) under biosafety level 2.

The Pango lineage A virus strain that was prevalent in the early stages of the COVID-19 pandemic (JPN/TY/WK-521) was provided by the National Institute of Infectious Diseases. To compare more pathogenic and transmissible variants of concern with Pango lineage A virus, the Pango lineage B.1.617.2 virus strain (KUH355) was isolated clinically and provided by the Kitasato University Hospital. Both viruses were used to infect VeroE6/TMPRSS2 cells for virus expansion, and virus-infected cells were cultured in D-MEM containing 2% FBS. After 48 h of incubation, each virus-infected cell culture supernatant was collected and centrifuged at 3,000 rpm for 10 min at room temperature (23 °C) to eliminate cells and debris. Culture supernatants were divided and stocked at −80 °C until use. All viruses were treated in the biological safety cabinet class II type A2 (LAL-1500XPA2 + : Oriental Giken Inc., Tokyo, Japan) of biosafety level 3 (BSL3).

HOCl was provided by NIPRO Corporation (Osaka, Japan). HOCl was produced by the electrolysis of the saturated sodium chloride (NaCl) solution. The products were purified by reverse osmosis filtration to remove ions, such as Na⁺. The conductivity and pH of 500 ppm HOCl used in this study were 280.1 mS/cm and 6.24, respectively.

The average value of TCID₅₀ was used as the viral infectivity. All experiments were performed in the biological safety cabinet of BSL3.

The test chamber used for the spray tests was placed into the biological safety cabinet in BSL3. KUH355 (1.5 × 10⁷ pfu/mL) virus solutions diluted 10- and 100-fold with double distilled water (ddw) were placed in plastic plates and placed on a wet towel in the test chamber (Figure 3). 30–500 ppm and 10–300 ppm of HOCl or distilled water were sprayed for 5 h using two ultrasonic atomizers. Each of the three plastic plates was treated with HOCl or distilled water at the same time. Plastic plates were collected at 1, 3, and 5 h, and 0.95 mL of recovery liquid (D-MEM) was added to the plastic plates, and the virus was suspended. Ten-fold serial dilutions of the virus solutions were prepared using D-MEM. Each diluted mixture was added into VeroE6/TMPRSS2 cells. After 3 days of incubation, the culture supernatants were eliminated. Then, VeroE6/TMPRSS2 cells were fixed with cold methanol and stained with 0.5% of methylene blue solution for the calculation of TCID₅₀ by the Behrens–Karber method. All experiments were performed in the biological safety cabinet of BSL3.

Statistical analyses were performed with GraphPad Prism 8.2 (GraphPad Software, La Jolla, CA). Statistical significance was analyzed using one-way analysis of variance (ANOVA) with the Bonferroni post hoc test. Differences were considered statistically significant at P < 0.05.

All infectious experiments were reviewed and approved by the Biosafety Committee of Kitasato University (approval numbers: A06-116 and 199) and the Kitasato University Medical Ethics Organization (approval number: COVID-19-CT001).

This study was performed to determine whether HOCl, produced by electrolysis of saturated chloride solution and filtrated through a reverse osmosis membrane to remove ions, had antiseptic efficacy against SARS-CoV-2 infectivity in the presence of organic compounds and under conditions of atomization. In the suspension test, we demonstrated a reduction of SARS-CoV-2 infectivity by contact with the HOCl solution for 20 s and 5 min. We found that the presence of organic compounds affected SARS-CoV-2 infectivity depending on the level of the ACC in the HOCl solution. We also observed that HOCl atomization significantly reduced SARS-CoV-2 infectivity in the spray test. The presence of organic compounds, including FBS, greatly affected the virucidal efficacy against SARS-CoV-2 in the suspension test and atomization test.

HOCl, a reactive chlorine species similar to ClO⁻ and Cl₂, is a high-potency oxidizing agent (Deborde & von Gunten 2008). In the innate immune system, HOCl is generated from hydrogen peroxide and chloride ions by myeloperoxidase (MPO) in phagocytes (Pattison et al. 2012; Winterbourn & Kettle 2013). Then, neutrophils and monocytes utilize HOCl to eliminate invading microorganisms (Gray et al. 2013; Da Cruz Nizer et al. 2020). MPO-knockout mice physiologically demonstrate increased susceptibility to infection with Candida albicans and Klebsiella pneumoniae in comparison with wild-type mice (Hirche et al. 2005; Homme et al. 2013). The HOCl solution shows powerful antibacterial efficacy against Gram-positive bacteria (Bacillus spp., Clostridium spp., Staphylococcus spp., Streptococcus spp.) and Gram-negative bacteria (Bordetella spp., Campylobacter spp., Vibrio spp., Pseudomonas spp.); Actinomycetaceae (Corynebacterium spp.); and Enterobacteriaceae (Escherichia spp., Shigella spp., Yersinia spp.), as well as virucidal efficacy against DNA (Adenoviridae, Herpesviridae) and RNA (Orthomyxoviridae, Coronaviridae) viruses (Tachikawa et al. 1999; Tagawa et al. 2000; Huang et al. 2008; Taharaguchi et al. 2014; Tamaki et al. 2014; Takeda et al. 2020; Miyaoka et al. 2021).

Previous studies using the suspension test have reported that treatment with HOCl solution can inactivate SARS-CoV-2 in cultured cells, such as VeroE6/TMPRSS2 cells. Hatanaka et al. (2022) have reported that 28.1 ppm HOCl solution achieved a 4-log reduction of SARS-CoV-2 infectivity within 10 s. In addition, 29.7 and 59.4 ppm HOCl solution achieved a 3.7–3.9- and 5-log reduction of SARS-CoV-2 infectivity within 10 s to 3 min in the presence of 0.1% FBS. Takeda et al. (2021) have reported that a residual chlorine concentration of 51–56 ppm in HOCl solution achieved >4-log reduction of SARS-CoV-2 infectivity within 20 s in the presence of 0.05% FBS. NITE summarized the results of the virucidal efficacy against SARS-CoV-2 at five facilities. They showed that a 19–200 ppm HOCl solution achieved a 3- and 4-log reduction of SARS-CoV-2 infectivity within 20 s to 5 min in the presence of 0.05–0.25% FBS. These studies have reported that viral solutions were prepared in the viral growth medium containing 1–5% FBS. The reaction rates of the virus solution and HOCl were 1:9 or 1:19. Then, they evaluated virucidal efficacy at a final concentration of 0.05–0.25% FBS. Moreover, previous studies have reported that the amount of protein in saliva, which is the cause of the droplet infection, is usually 100 times lower than that in plasma. Saliva contains 2% of proteins and 98% of water (Nunes et al. 2011; Kang & Kho 2018). Therefore, we chose the concentration of 0.2% and 0.02% FBS. In the present study, we found that 30 ppm HOCl solution achieved >5-log reduction of JPN/TY/WK-521 strain infectivity within 20 s in the presence of 0.2% FBS. In contrast, we demonstrated that 30 ppm HOCl solution achieved a 1.1-log reduction in KUH355 strain infectivity within 20 s in the presence of 0.2% FBS. This difference in the virucidal efficacy might affect the Delta variant, which is more transmissible than the original variant (Mistry et al. 2022). We also indicated that 10 ppm HOCl solution achieved sufficient reduction (>5 log) of JPN/TY/WK-521 and KUH355 virus strains infectivity within 20 s in the presence of 0.02% FBS. Thus, 30 ppm HOCl solution for 20 s may be sufficient to prevent the infection of the original SARS-CoV-2 strain. In contrast, the more transmissible mutant SARS-CoV-2 strain may require 50 ppm HOCl solution for > 20 s.

To reduce the risk of airborne transmission of pathogens, the bactericidal and virucidal efficacies of HOCl solution have been previously evaluated using atomization. Hakim et al. (2016) have reported that 50 and 100 ppm HOCl solution achieved an approximately 4-log reduction of Escherichia coli and Salmonella infantis in dry conditions within 5 min of atomization. Benedusi et al. (2022) have also reported that 300 ppm HOCl solution showed 69–99.9% reduction of E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa with 10 min of atomization, although bactericidal activities were affected by the differences in material surfaces, such as semi-porous, flat, and porous. They also showed that 300 ppm HOCl solution achieved >95% reduction of human coronavirus 229E and adenovirus with 5–10 min of atomization. Urushidani et al. (2022) intermittently sprayed 250 or 8,700 ppm HOCl solutions for 2.5–5 s (each 4 min) in the test chamber and reported that 250 ppm HOCl solution did not neutralize air-dried SARS-CoV-2 infectivity after 16 min HOCl exposure. However, 8,700 ppm HOCl solution reduced air-dried SARS-CoV-2 infectivity to decrease the limit of detection within 12 min for 16 min HOCl exposure. In the present study, we found that atomized HOCl at 300 and 500 ppm for 3 and 5 h achieved a 1.4–2.3 and 2.3–5.7 log reduction of KUH355 virus solution infectivity in the presence of 0.2% FBS. We also observed that 30 ppm HOCl atomization for 3 and 5 h achieved 2.8–4.1 log reduction of KUH355 viral solution infectivity in the presence of 0.02% FBS. Moreover, SARS-CoV-2 infectivity in the presence of 0.02% FBS was reduced by >4-log by 300 ppm of HOCl atomization for 1, 3, and 5 h. Our results suggest that the HOCl solution may have effective virucidal efficacy against SARS-CoV-2, although a longer atomization time compared with a previous report may be needed. Our data also showed that atmospheric ACC is constantly maintained at <0.40 ppm in 200-L test chamber after atomization with 500 ppm HOCl. Our test chamber was almost twofold in volume as that reported in another study, in which the chlorine concentration was approximately 10 ppm with 8,700 ppm atomization. Therefore, differences in chlorine concentration in the experimental chamber may have influenced the virucidal efficacies against SARS-CoV-2 we observed in this study.

The WHO does not recommend the disinfection of COVID-19 by fogging environmental surfaces with disinfectant, as it may increase the risk of adverse effects to the eyes, respiratory tract, and skin. Because HOCl reportedly did not cause these adverse effects, the adverse effects of HOCl are probably related to the chlorine concentration and pH of the exposed HOCl. In addition, no adverse events were observed by drinking 20–60 ppm HOCl water in animal studies, washing wounds with 200 ppm HOCl solution, and mouth washing with 80 and 200 ppm HOCl solution in clinical studies (Morita et al. 2011; Kubota et al. 2015; Block & Rowan 2020; Alzahrani et al. 2022; Sevinç Güll et al. 2022). Although the effect of the high purity of HOCl on adverse events is unclear, HOCl should be atomized under controlled conditions at the appropriate concentration. Our results, which prove the relationship between the virucidal efficacy and the ACC of HOCl, suggest that HOCl atomization for disinfection may be useful for cleaning unmanned hospital rooms, schools, daycare centers, and various facilities at night.

We have clarified that both aqueous and atomized HOCl solutions are effective in neutralizing SARS-CoV-2 infectivity. The virucidal efficacy of HOCl was not influenced by the difference in SARS-CoV-2 variants (Pango lineage A and B virus strains). In addition, we showed the virucidal efficacy of HOCl atomization against SARS-CoV-2. HOCl atomization may reduce the burden of cleaning and disinfection work in various facilities such as hospital rooms, daycare centers, and schools.

The authors thank laboratory staffs of Kitasato University and NIPRO Corporation for technical and experimental supports.

This study was supported by the COVID-19 Kitasato Project Study and Research on Emerging and Re-emerging Infectious Diseases and Immunization on Health, Labour and Welfare Policy Research Grants (grant number 21HA2017). This study was funded by a research grant from NIPRO Corporation.

Author M.K. received research support from NIPRO Corporation. All authors except M.K. have no financial interests.

M.K., R.E., H.F., N.S., and N.K. performed the study concept and design. M.K. and R.E. performed writing the original draft. M.K., R.E., S.M., H.F., N.S., N.K., H.K., and H.H. performed the development of methodology and writing, review, and revision of the manuscript. M.K., R.E., H.F., and N.S. provided acquisition, analysis and interpretation of data, and statistical analysis. H.F., N.S., and N.K. provided reagents, materials, test chamber, and instrumentations. N.K., H.K., and H.H. supervised and provided the financial support for this project. All authors have read and approved the final manuscript.

All relevant data are included in the paper or its Supplementary Information.

M.K. received research support from NIPRO Corporation. All authors except M.K. have no financial interests.