Evaluation of Environmental Stability and Disinfectant Effectiveness for Human Coronavirus OC43 on Human Skin Surface

ABSTRACT The environmental stability of human coronavirus OC43 (HCoV-OC43) on the surface of human skin and the effectiveness of disinfectant against HCoV-OC43, which are important to prevent contact transmission, have not been clarified in previous studies. Using previously generated models, we evaluated HCoV-OC43 stability and disinfection effectiveness. Then we compared the results with those for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The median survival time of HCoV-OC43 on the surface of human skin was 24.6 h (95% confidence interval, 19.7 to 29.6 h), which was higher than that of SARS-CoV-2 (10.8 h). Although the in vitro disinfectant effectiveness evaluation showed that HCoV-OC43 has a higher ethanol resistance than SARS-CoV-2, HCoV-OC43 on the skin surface was completely inactivated by a minimum of 50% ethanol within 5 s (the log reduction values were >4.0). Moreover, 1.0% chlorhexidine gluconate and 0.2% benzalkonium chloride showed relatively high disinfectant effectiveness, and the log reduction values when these disinfectants were applied for 15 s were >3.0. HCoV-OC43 is highly stable on the skin surface, which may increase the risk of contact transmission. Although HCoV-OC43 has relatively high ethanol resistance, appropriate hand hygiene practices with current alcohol-based disinfectants sufficiently reduce the risk of contact transmission. IMPORTANCE This study revealed the environmental stability of HCoV-OC43 and disinfectant effectiveness against HCoV-OC43, which had not been demonstrated in previous studies. HCoV-OC43 is highly stable on the surface of human skin, with a survival time of approximately 25 h. High stability of HCoV-OC43 may increase the risk of contact transmission. Furthermore, the in vitro disinfectant effectiveness evaluation showed that HCoV-OC43, which is classified as an envelope virus, has a relatively high ethanol resistance. This finding suggests that disinfectant effectiveness may vary greatly depending on the virus and that each virus targeted for infection control should be evaluated individually. HCoV-OC43 on the skin surface was rapidly inactivated by 50% ethanol, which suggests that appropriate hand hygiene practices with current alcohol-based disinfectants can sufficiently reduce the risk of HCoV-OC43 contact transmission.

IMPORTANCE This study revealed the environmental stability of HCoV-OC43 and disinfectant effectiveness against HCoV-OC43, which had not been demonstrated in previous studies. HCoV-OC43 is highly stable on the surface of human skin, with a survival time of approximately 25 h. High stability of HCoV-OC43 may increase the risk of contact transmission. Furthermore, the in vitro disinfectant effectiveness evaluation showed that HCoV-OC43, which is classified as an envelope virus, has a relatively high ethanol resistance. This finding suggests that disinfectant effectiveness may vary greatly depending on the virus and that each virus targeted for infection control should be evaluated individually. HCoV-OC43 on the skin surface was rapidly inactivated by 50% ethanol, which suggests that appropriate hand hygiene practices with current alcohol-based disinfectants can sufficiently reduce the risk of HCoV-OC43 contact transmission.
Contact and droplet transmission are the primary transmission pathways for HCoV-OC43 (13,14), and hand hygiene is important for prevention. The viral stability and disinfectant effectiveness against HCoV have been reported in several studies on HCoV-229E (15)(16)(17)(18). Since the conventional method for titrating HCoV-OC43 (using the indirect immunoperoxidase assay [19][20][21][22][23][24]) was cumbersome, the stability of HCoV-OC43 and the efficacy of disinfectants against HCoV-OC43 have scarcely been evaluated (18,25). Additionally, the stability on human skin and the disinfectant effectiveness against all HCoVs have not been evaluated, although this information is very important for disease prevention. Ethanol (EA), isopropanol (IPA), chlorhexidine gluconate (CHG), benzalkonium chloride (BAC), and povidone-iodine (PVP-I) are commonly used on human skin for hand hygiene, and these were evaluated in this study.
For this study, we established a model to evaluate viral stability and disinfection effectiveness against viruses on human skin obtained from forensic autopsy specimens in our previous studies (26,27). Additionally, we developed a simple and accurate 50% tissue culture infectious dose (TCID 50 ) assay by cytopathic effect (CPE) observation with HCoV-OC43 infecting VeroE6 cells expressing transmembrane protease serine 2 (TMPRSS2) (28). These models were used to evaluate the stability of HCoV-OC43 and disinfection effectiveness of various disinfectants against HCoV-OC43 on human skin.
Moreover, the ethanol concentration that was required to achieve a logarithmic decrease of 3.5 in virus titer concentration in a 15-s disinfection reaction (defined as required EA concentration) was calculated. The required EA concentrations for HCoV-OC43 and SARS-CoV-2 were 52.39% (95% CI, 48.38 to 57.47) and 27.94% (95% CI, 27.45 to 28.44), respectively. These values showed that HCoV-OC43 had significantly higher EA resistance than SARS-CoV-2 ( Fig. 2).

DISCUSSION
The stability of HCoV and the disinfectant effectiveness against HCoV on the surface of human skin, which are important data for the prevention of contact transmission, had not been previously investigated and were unknown. This study revealed the stability and disinfectant effectiveness using our previously established evaluation model (26)(27)(28).
Our environmental stability assessment showed that the survival time of HCoV-OC43 on the surface of human skin was approximately 25 h, which was more than 2fold longer than that of SARS-CoV-2. Although SARS-CoV-2 reportedly demonstrates higher environmental stability than influenza virus (26), our study suggests that HCoV-OC43 demonstrates even higher environmental stability. Therefore, HCoV-OC43 may have a higher contact transmission risk than SARS-CoV-2. The mechanism by which differences in environmental stability occur has not been clarified at this stage. However, a previous study on SARS-CoV-2 reported different environmental stability in each variant (29), and the environmental stability might differ depending on the spike protein. Therefore, the difference in environmental stability between SARS-CoV-2 and HCoV-OC43 might also be due to the difference in spike proteins. However, further research is needed to test this hypothesis.
Previous studies have demonstrated the efficacy of alcohol-based disinfectants against SARS-CoV-2 (30)(31)(32). In addition, our previous study showed that influenza virus and SARS-CoV-2 were completely inactivated in vitro with 40% EA for 5 s (27). However, the in vitro disinfectant effectiveness evaluation showed that HCoV-OC43 was not inactivated after reaction with 40% EA for 60 s and that the required EA concentration for HCoV-OC43 disinfection was significantly higher than that for SARS-CoV-2. Thus, HCoV-OC43 was shown to have higher EA resistance than SARS-CoV-2. Ethanol is known to be highly effective against enveloped viruses, as opposed to nonenveloped viruses (33). Nevertheless, HCoV-OC43 showed a relatively high EA resistance among the enveloped viruses compared to influenza virus and SARS-CoV-2. These findings suggest that the disinfectant effectiveness against each virus targeted for infection control should be evaluated and confirmed individually.
However, unlike the results of the in vitro evaluation, HCoV-OC43 on the skin surface was rapidly inactivated with 50% EA. Rubbing 52% (wt/wt) or more ethanol onto the hands for at least 15 s is a hand hygiene practice recommended by the World Health Organization (33). Therefore, the current EA-based disinfectants for hand hygiene can adequately inactivate HCoV-OC43 on the surface of human skin. HCoV-OC43 was also rapidly inactivated by 70% IPA and 10% PVP-I. While 50% or more EA-based disinfectants are effective are suitable for hand hygiene targeted against HCoV-OC43, our results suggest that 70% IPA-based disinfectants and 10% PVP-I-based disinfectants are suitable alternatives. Additionally, high concentrations of CHG and BAC, such as 1.0% CHG and 0.2% BAC, showed a relatively high disinfectant effectiveness at reaction times of 15 s or longer but were slightly less effective than 50% or more EA. Therefore, the high concentrations of CHG and BAC-based disinfectants may be used as alternatives to alcohol-based disinfectants. This study had some limitations that should be mentioned. First, HCoV-OC43 was diluted with phosphate-buffered saline (PBS) for evaluation. Changing the solvent used for diluting the virus may affect the results of the evaluation. Second, since the HCoV-OC43 strain used in this study may have adapted during passaging in cell lines or in mice at the ATCC, the environmental stability of this strain may differ from that of primary isolates.
In conclusion, this study elucidated the stability of HCoV-OC43 on the surface of human skin and examined the disinfectant effectiveness of multiple agents against HCoV-OC43. The high stability of HCoV-OC43 on the surface of human skin may increase the risk of contact transmission. Furthermore, although HCoV-OC43 has relatively high ethanol resistance, appropriate hand hygiene practices can sufficiently reduce the contact transmission risk.

MATERIALS AND METHODS
Viruses and cells. VeroE6 cells expressing TMPRSS2 (VeroE6/TMPRSS2 cells; number JCRB1819) were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS) and G418 (Nacalai Tesque, Kyoto, Japan) (34,35). HCoV-OC43 (ATCC VR-1558) was purchased from the American Type Culture Collection (Manassas, VA, USA). HCoV-OC43 was cultured in Vero E6/TMPRSS2 cells and stored as a working stock at 280°C. HCoV-OC43 was concentrated and purified as follows. VeroE6/TMPRSS2 cells cultured in DMEM supplemented with 10% FBS and G418 were inoculated with HCoV-OC43 and incubated at 33°C for 7 days, and the culture medium was harvested. The harvested culture medium was centrifuged at 4°C and 3,000 rpm for 5 min to remove cell debris. After centrifugation, the supernatants were centrifuged again at 1,500 rpm for 20 min at 4°C and sterilized by passage through a 0.45-mm filter. Thereafter, virions in the supernatant were sedimented through a 20% (wt/wt) sucrose cushion in PBS with ultracentrifugation at 28,000 rpm for 2.5 h at 4°C in a Beckman SW28 rotor (27,36).
The titer of infectious HCoV-OC43 virions was measured by the TCID 50 assay using Vero E6/TMPRSS2 cells as reported in our previous studies (28,37). Specifically, VeroE6/TMPRSS2 cells cultured in 96-well plates were infected with HCoV-OC43, and CPE was observed using an inverted light microscope (Olympus IX71; Olympus, Tokyo, Japan) 4 to 5 days postinfection.
Collection of human skin specimens and preparation of the evaluation model for disinfectant effectiveness and viral stability. Human skin was collected from forensic autopsy specimens obtained from the Department of Forensic Medicine, Kyoto Prefectural University of Medicine, within 24 h after patient death. Abdominal skin autopsy specimens from subjects aged 20 to 70 years were excised into rectangles greater than 5 by 10 cm 2 , which were used for subsequent evaluation (38). Additionally, the disinfectant effectiveness against viruses and viral stability on human skin were evaluated using a previously established human skin model (26,27). Specifically, the subcutaneous tissue from human skin was rapidly removed, and the treated skin (mainly epidermal and dermal layers) was rinsed with PBS and placed onto a culture insert with an 8.0-mm-pore-size membrane (Corning, Corning, NY, USA). The culture inserts were placed into 12-well plates containing 1.0 mL of DMEM in each well.
Evaluation of HCoV-OC43 stability on the surface of human skin. The survival time of HCoV-OC43 on the skin was evaluated using our previously reported protocol (26). The same evaluation was performed on SARS-CoV-2 as a comparison. Specifically, 2 mL of HCoV-OC43 or SARS-CoV-2 (2.0 Â 10 5 TCID 50 s in 2 mL of PBS) was applied to skin samples and incubated at 25°C under a relative humidity of 45 to 55% for 0 to 24 h. After incubation, residual viruses on the skin were recovered with 1 mL of DMEM and then titrated. The survival time was defined as the time until the virus on the skin was not detected. The titer detection limit of each virus was 3.2 Â 10 0 TCID 50 s.

Disinfectant Effectiveness against HCoV-OC43
Microbiology Spectrum The in vitro evaluation protocol was based on a previous study (27). Briefly, HCoV-OC43 was mixed with PBS to adjust the viral titer to 5.0 Â 10 7 TCID 50 s/mL before exposure to each disinfectant. Next, 36 mL of each disinfectant was added to 4 mL of the virus mixture (2.0 Â 10 5 TCID 50 s in 4 mL of the virus mixture) and incubated at 25°C for 5, 15, and 60 s. After incubation, 360 mL of soybean-casein digest broth with lecithin and polysorbate 80 (SCLDP; Nihon Pharmaceutical, Tokyo, Japan) and 400 mg of nonpolar polystyrene adsorbent (Bio-Beads SM-2 resin; Bio-Rad, Hercules, CA, USA) were added to each tube to neutralize the disinfectant and then mixed at 25°C for 30 min. Finally, 1,600 mL of DMEM was added and the remaining viral titers in final solutions were measured. During the evaluation of PVP-I, 0.5% sodium thiosulfate (Nacalai Tesque) was used instead of SCLDP. The titer detection limit of HCoV-OC43 was 6.3 Â 10 0 TCID 50 s. For evaluation of disinfectant effectiveness, the log reduction was calculated using the viral titer when PBS was added as a control, instead of each disinfectant. Three independent experiments were conducted, and the results were expressed as means 6 standard errors.
Moreover, the effect of EA on HCoV-OC43 was evaluated in detail. The same evaluation was performed on SARS-CoV-2 as a comparison. To evaluate EA effectiveness, we used 80, 60, 55, 50, 45,40,35,30,25, and 20% (wt/wt) EA. Briefly, HCoV-OC43 and SARS-CoV-2 were mixed with PBS to adjust the viral titer to 5.0 Â 10 7 TCID 50 s/mL before exposure to each disinfectant. Next, 36 mL of each disinfectant was added to 4 mL of the virus mixture (2.0 Â 10 5 TCID 50 s in 4 mL of virus mixture) and incubated at 25°C for 15 s. After incubation, 360 mL of SCLDP and 400 mg of nonpolar polystyrene adsorbent were added to each tube to neutralize the disinfectant, and then the sample was mixed at 25°C for 30 min. Finally, 1,600 mL of DMEM was added, and the remaining viral titers in final solutions were measured. The titer detection limits of HCoV-OC43 and SARS-CoV-2 were 6.3 Â 10 0 TCID 50 s.
Evaluation of disinfectant effectiveness against HCoV-OC43 on human skin. The same disinfectants for the in vitro assay were evaluated on the surface of human skin. The evaluation protocol was based on a previous study (27). Before exposure to each disinfectant, HCoV-OC43 was mixed with PBS to adjust the virus titer to 1.0 Â 10 8 TCID 50 s/mL. The viral mixture (2.0 Â 10 5 TCID 50 s in 2 mL of virus mixture) was applied to the epidermis of the skin, and the samples were incubated for 10 min at 25°C under a relative humidity of 45 to 55% to dry the viral mixture on the skin completely. Next, 40 mL of each disinfectant was applied to the skin, incubated for 5, 15, and 60 s, and then air dried for 2 to 3 min. After drying, the remaining viruses on the skin were recovered using 500 mL of neutralizing medium (SCLDP or 0.5% sodium thiosulfate), and the neutralizing medium and 400 mg of nonpolar polystyrene adsorbent were then mixed at 25°C for 30 min. Finally, 1,500 mL of DMEM was added and then titrated. The titer detection limit of HCoV-OC43 was 6.3 Â 10 0 TCID 50 s. For evaluation of disinfectant effectiveness, the log reduction was calculated using the viral titer when PBS was added as a control, instead of each disinfectant. Three independent experiments were conducted, and the results were expressed as means 6 standard errors.
Ethical considerations. The protocol for this study was approved by the Ethics Committee of Kyoto Prefectural University of Medicine (ERB-C-1593). Written informed consent was obtained from the bereaved families of the study subjects.
Statistical analysis. Data were analyzed using GraphPad Prism 7 software (GraphPad, La Jolla, CA, USA). For the evaluation of EA effectiveness, EA concentration was defined as the explanatory variable (x axis) and the log viral titer as the explained variable (y axis). A nonlinear regression analysis was performed using a four-parameter logistic model (45). The EA concentration required to exceed a log reduction of 3.5 in a 15-s reaction with a disinfectant was defined as the x value when the y value of the regression curve was 1.3. For the evaluation of the survival time and half-life, the elapsed time was defined as the explanatory variable (x axis) and the log viral titer of HCoV-OC43 and SARS-CoV-2 as the explained variables (y axis). A least-squares linear regression analysis was performed using the logarithmic link function for each virus to create a regression curve. The survival time of each virus was defined as the x value when the y value of the regression curve was 0.5. The half-life was calculated from the slope of each regression curve when the residual viral titers on the skin were 2.0 logTCID 50 s and 3.0 logTCID 50 s.
Data availability. All data included in this study are available from the corresponding author on request.