Application of a Photocatalyst as an Inactivator of Bovine Coronavirus

Bovine coronavirus (BCoV), a major causative pathogen of bovine enteric and respiratory diseases and a zoonotic pathogen transmissible between animals and humans, has led to severe economic losses in numerous countries. BCoV belongs to the genus Betacoronavirus, which is a model of a pathogen that is threatening human health and includes severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus. This study aimed to determine whether photocatalytic material effectively reduces CoVs in the environment. Using the film adhesion method of photocatalytic materials, we assessed its antiviral activity and the effect of visible light irradiation according to methods defined by the International Organization for Standardization. Consequently, photocatalytic material was found to have antiviral activity, reducing the viral loads by 2.7 log TCID50 (tissue culture infective dose 50)/0.1 mL (500 lux), 2.8 log TCID50/0.1 mL (1000 lux), and 2.4 log TCID50/0.1 mL (3000 lux). Hence, this photocatalytic material might be applicable not only to reducing CoVs in the cattle breeding environment but also perhaps in other indoor spaces, such as offices and hospital rooms. To our knowledge, this study is the first to evaluate the antiviral activity of a photocatalytic material against CoV.


Virus and Cell Culture
The Hokkaido/9/03 strain, originally isolated from nasal swab samples of cattle with pneumonia at a farm in Hokkaido prefecture, Japan, was used in this study [36].
The Hokkaido/9/03 strain was inoculated in 75 cm 2 flasks (10 6 cells) of HRT-18G cells (ATCC: CRL-11663) and incubated at 37 • C for 1 h in a humidified 5% CO 2 incubator. Thereafter, cells were washed twice with 20 mL of sterilized phosphate buffered saline (PBS), added to 20 mL of Dulbecco's modified Eagle's medium (DMEM) (Nissui, Tokyo, Japan) without fetal bovine serum (FBS), and incubated at 37 • C for 3 days. Thereafter, cells were harvested and centrifuged at 1200× g for 5 min to eliminate debris. The virus titer of the supernatant (tissue culture infective dose 50 (TCID 50 ) / 0.1 mL) was determined using the method of Reed and Muench [37] and stored at −80 • C until use. Although the Viruses 2020, 12, 1372 3 of 10 virus titer of the stored Hokkaido/9/03 strain approached 10 7.0 TCID 50 /0.1 mL, the stored viruses were diluted in half with PBS to prepare a virus suspension to be used in subsequent experiments.

Film Adhesion of Photocatalytic Material and Test Equipment
Photocatalytic material was manufactured by Wako Filter Technology Co., Ltd. (Ibaraki, Japan). The photocatalytic material was composed of a peroxotitanium acid solution (70%) and a peroxo-modified anatase solution (30%), which is responsive to visible light.
The antiviral activity of this photocatalytic material was assessed using the film adhesion method. A transparency film (overhead projector film VF-1410N, KOKUYO Co., Ltd., Osaka, Japan) was cut into 50 × 50 mm 2 squares and sprayed with the photocatalytic material with a spray gun (LPH-50-062G, ANEST IWATA Corporation, Yokohama, Japan). Thereafter, the film surface was dried with hot air at 60-70 • C. Spraying and drying were repeated alternately to adjust to a 0.2 mg/m 2 coating weight. A transparency film uncoated with photocatalytic material was used as a negative control.
The testing equipment was set up in accordance with ISO 18071, a guideline of the International Organization for Standardization [38], facilitating the assessment of the antiviral activity of photocatalytic material under an indoor lighting environment. The equipment comprised a light source and a moisture chamber with a test film ( Figure 1).  [37] and stored at −80 °C until use. Although the virus titer of the stored Hokkaido/9/03 strain approached 10 7.0 TCID50/0.1 mL, the stored viruses were diluted in half with PBS to prepare a virus suspension to be used in subsequent experiments.

Film Adhesion of Photocatalytic Material and Test Equipment
Photocatalytic material was manufactured by Wako Filter Technology Co., Ltd. (Ibaraki, Japan). The photocatalytic material was composed of a peroxotitanium acid solution (70%) and a peroxomodified anatase solution (30%), which is responsive to visible light.
The antiviral activity of this photocatalytic material was assessed using the film adhesion method. A transparency film (overhead projector film VF-1410N, KOKUYO Co., Ltd., Osaka, Japan) was cut into 50 × 50 mm 2 squares and sprayed with the photocatalytic material with a spray gun (LPH-50-062G, ANEST IWATA Corporation, Yokohama, Japan). Thereafter, the film surface was dried with hot air at 60-70 °C. Spraying and drying were repeated alternately to adjust to a 0.2 mg/m 2 coating weight. A transparency film uncoated with photocatalytic material was used as a negative control.
The testing equipment was set up in accordance with ISO 18071, a guideline of the International Organization for Standardization [38], facilitating the assessment of the antiviral activity of photocatalytic material under an indoor lighting environment. The equipment comprised a light source and a moisture chamber with a test film ( Figure 1).

Assessment of Antiviral Activity of Photocatalytic Material with Visible Light Irradiation
A sterilized moist paper filter was placed at the bottom of a sterilized Petri dish containing 2 mL of sterilized water. Two glass rods were intercalated to avoid contact between the test film and the paper filter. The test film, which was UV irradiated with an irradiance of 1 mW/cm 2 (380 nm) for 24 h with a black light (06947604, NEC Corporation, Tokyo, Japan) to inactivate organic matter before the test, was placed on them with the photocatalytic-coated surface. In total, 150 µL of the virus suspension (10 6.7 TCID 50 /0.1 mL) was harvested with a sterilized pipette tip and dropped onto the test film. A 40 × 40 mm 2 square of sterilized film was placed on top of the dripped suspension and lightly pushed into the suspension to spread it throughout the entire film surface. Thereafter, the lid of the Petri dish was put in place to conserve moisture. The Petri dishes containing the six test films (three photocatalytic-coated films and three photocatalytically uncoated films) with the virus suspension were irradiated with 500, 1000, and 3000 lux visible light with a white fluorescent lamp (600113, Mitsubishi Electric Lighting Corporation, Kanagawa, Japan) for 4 h. The Petri dishes containing the six test films (three photocatalytic-coated films and three photocatalytically uncoated films) with the virus suspension were maintained in the dark for the same duration at 25 ± 3 • C.
After 4 h with or without visible light irradiation, both test films and cover films were washed with 5 mL of DMEM without FBS in a Stomacher bag, and these washout solutions were used to determine the virus titers.

Determination of TCID 50 of Virus Suspensions on Test Films after 4 h with or without Visible Light Irradiation
Virus titers in suspensions on test films after 4 h with or without visible light irradiation were determined using the TCID 50 assay on confluent monolayers of HRT-18G cells in 96-well plates (Techno Plastic Products AG, Trasadingen, Switzerland). Cell monolayers were prepared by adding 100 µL of 10 4.2 cells/mL in DMEM with 5% FBS to each well, and then plates were incubated at 37 • C for 24 h in a humidified 5% CO 2 incubator. Each collected solution was serially 10-fold diluted in DMEM without FBS. After 100 µL of each dilution was inoculated with confluent cells in 5 wells, the mixture was incubated at 37 • C for 7 days. On day 7 postinoculation, the TCID 50 /0.1 mL of each solution was determined using the aforementioned method [37].

Criteria for Assessment of Antiviral Activity of Photocatalyst and Effect of Visible Light Irradiation
The values of antiviral activity and the effects of visible light irradiation were determined in accordance with ISO 18071 [38] as follows: Value of antiviral activity of photocatalyst with visible light irradiation: Value of antiviral activity of photocatalyst without visible light irradiation: Effect of visible light irradiation: where BL-means of the virus titers of three photocatalytically uncoated films after visible light irradiation with a constant illuminance; SL-means of the virus titers of three photocatalytic-coated films after visible light irradiation with a constant illuminance; BD-means of the virus titers of three photocatalytically uncoated films after being kept in the dark for the same duration as visible light irradiation; SD-means of the virus titers of three photocatalytic-coated films after being kept in the dark for the same duration as visible light irradiation.

of 10
The performance of the photocatalytic material was determined to be effective when the antiviral activity of the photocatalyst with visible light irradiation (V L ) was ≥2.0 and the effect of visible light irradiation ( ∆ V) was ≥0.3, based on the criteria of the Photocatalysis Industry Association of Japan (PIAJ) [39]. V L of 2.0 and ∆ V of 0.3 correspond with a mean reduction of 1/100 and 1/2 of viruses with visible light irradiation, respectively.

Immunofluorescence Assay
To visualize the antiviral activity of the photocatalytic material irradiated with 1000 lux visible light for 4 h, an immunofluorescence assay (IFA) in addition to the TCID 50 assay was performed using virus suspensions harvested every 1 h (from 0 to 4 h) after visible light irradiation. Cell monolayers were prepared by adding 300 µL of 10 4.1 cells/mL in DMEM with 5% FBS per well in an 8-well chamber slide (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37 • C for 24 h in a humidified 5% CO 2 incubator. The harvested solutions were inoculated into confluent cells and then maintained at 37 • C for 1 h. Thereafter, the solutions were replaced with DMEM without FBS. Approximately 22 h later, cells were washed with PBS, fixed with acetone, and incubated with anti-BCoV rabbit serum (originally generated in our laboratory) for 1 h. Subsequently, cells were washed thrice with PBS and incubated with goat FITC (fluorescein isothiocyanate) conjugate anti-rabbit IgA, IgG, and IgM (Cappel Laboratories, Cochranville, PA, USA) for 1 h. Thereafter, cells were washed thrice with PBS and incubated with DAPI (4,6-diamidino-2-phenylindole) solution (Dojindo Laboratories, Kumamoto, Japan) for 10 min. After three washes with PBS, the coverslips were sealed with Mountant Perma Fluor (Thermo Fisher Scientific, Waltham, MA, USA) and observed using a fluorescence microscope (Axiovert200/40 LD-1; Carl Zeiss AG, Oberkochen, Germany).

Immunofluorescence Assay
The virus titers of virus suspensions from photocatalytic uncoated and coated films harvested every 1 h with or without 1000 lux visible light irradiation for 4 h are summarized in Table 2  IFA images using virus suspensions from photocatalytically uncoated films revealed no differences in the levels of green fluorescence, regardless of visible light irradiation and the duration of irradiation ( Figure 2). The IFA image using virus suspensions from photocatalytic-coated films after 4 h in the dark further decreased the levels of green fluorescence compared with those of the photocatalytically uncoated film after 4 h in the dark. Furthermore, the levels of green fluorescence markedly decreased in IFA images using virus suspensions from photocatalytic-coated films, consistent with the effects of Viruses 2020, 12, 1372 7 of 10 visible light irradiation, compared with those from photocatalytic-coated films in the dark. These results are almost identical to those of the TCID 50 assay.
Viruses 2020, 12, x FOR PEER REVIEW 7 of 11 after 4 h in the dark further decreased the levels of green fluorescence compared with those of the photocatalytically uncoated film after 4 h in the dark. Furthermore, the levels of green fluorescence markedly decreased in IFA images using virus suspensions from photocatalytic-coated films, consistent with the effects of visible light irradiation, compared with those from photocatalyticcoated films in the dark. These results are almost identical to those of the TCID50 assay.

Discussion
The values of antiviral activity of photocatalysts with visible light irradiation (VL) using virus suspensions on test films after visible light irradiation for 4 h at 500, 1000, and 3000 lux yielded 2.4-2.8 log TCID50/0.1 mL. Furthermore, the effects of visible light irradiation (ΔV) at the three values of illumination yielded 0.5-0.8 log TCID50/0.1 mL. Both values of antiviral activity of photocatalysts with visible light irradiation (VL) and the effects of visible light irradiation (ΔV) at all three illuminations exceeded the criteria defined by the PIAJ [39]. Hence, the photocatalytic material used in this study appears to have high antiviral activity against BCoV.
Habibi-Yangjeh et al. reported three mechanisms of viral inactivation by a photocatalyst consisting of a peroxotitanium complex as follows: physical damage of viruses, metal ion toxicity obtained from metal-including photocatalysts, and chemical oxidation by reactive oxygen species generated over the photocatalysts [35]. Moreover, a previous study demonstrated that the adenovirus was destroyed by a photocatalyst under transmission electron microscopy observation [40]. Therefore, the reduction of BCoV loads presented herein might have been caused by these three mechanisms because the photocatalyst used in this study was made from a peroxotitanium complex.
Furthermore, this study shows that the photocatalytic material used here was completely effective under visible light irradiation of 500 lux. The illuminance of solar light is usually >90,000 lux during the daytime [41]; hence, this photocatalytic material can be fully useful outdoors. On the other

Discussion
The values of antiviral activity of photocatalysts with visible light irradiation (V L ) using virus suspensions on test films after visible light irradiation for 4 h at 500, 1000, and 3000 lux yielded 2.4-2.8 log TCID 50 /0.1 mL. Furthermore, the effects of visible light irradiation ( ∆ V) at the three values of illumination yielded 0.5-0.8 log TCID 50 /0.1 mL. Both values of antiviral activity of photocatalysts with visible light irradiation (V L ) and the effects of visible light irradiation ( ∆ V) at all three illuminations exceeded the criteria defined by the PIAJ [39]. Hence, the photocatalytic material used in this study appears to have high antiviral activity against BCoV.
Habibi-Yangjeh et al. reported three mechanisms of viral inactivation by a photocatalyst consisting of a peroxotitanium complex as follows: physical damage of viruses, metal ion toxicity obtained from metal-including photocatalysts, and chemical oxidation by reactive oxygen species generated over the photocatalysts [35]. Moreover, a previous study demonstrated that the adenovirus was destroyed by a photocatalyst under transmission electron microscopy observation [40]. Therefore, the reduction of BCoV loads presented herein might have been caused by these three mechanisms because the photocatalyst used in this study was made from a peroxotitanium complex.
Furthermore, this study shows that the photocatalytic material used here was completely effective under visible light irradiation of 500 lux. The illuminance of solar light is usually >90,000 lux during the daytime [41]; hence, this photocatalytic material can be fully useful outdoors. On the other hand, the illuminance in indoor environments, including offices, hospital rooms, laboratories, and libraries, is generally recommended to be 500 lux in accordance with ISO 8995 [42]; hence, this photocatalyst Viruses 2020, 12, 1372 8 of 10 is potentially useful also under indoor lighting environments. Therefore, this material is potentially applicable as a viral inactivator to reduce not only viral pathogens in environments where cattle are maintained but also the risk of CoV contact infections by spraying various surfaces including walls, floors, knobs, chairs, and handrails inside human living and community spaces.
We observed a reduction in the virus titer on photocatalytic-coated films after 3 h with 1000 lux visible light irradiation based on the criteria defined by the PIAJ [39]. Furthermore, IFA revealed a decrease in the levels of green fluorescence with a reduction in the virus titer. These results indicate that the photocatalytic material used in this study displayed antiviral activity for at least 3 h with visible light irradiation. In a previous study, the load of avian influenza virus (H1N1) on glass coated with platinum-loaded tungsten oxide (Pt-WO 3 ) as a photocatalytic material was clearly decreased with 1000 lux visible light irradiation for 2 h [33]. These facts might support photocatalytic materials as a useful tool for viral inactivation to prevent the transmission of enveloped RNA viruses associated with respiratory disorders.
In conclusion, this study shows that the photocatalytic material used here exhibits high performance with visible light irradiation of different illuminances for 4 h. Especially, it is valuable that this material has effective antiviral activity against BCoV with visible light irradiation of 500 lux, an illuminance recommended in indoor environments. These results suggest the possibility of this photocatalyst having antiviral activity against SARS-CoV-2 because BCoV belongs to the same genus as that of SARS-CoV-2, which has propagated and transmitted worldwide. Although further studies are required to analyze the antiviral activity of this material against SARS-CoV-2, this material might be of interest, when properly used, to prevent and reduce CoV infection indoors; however, a possible increase in the concentration of some volatile organic compounds due to photocatalysis may be a concern, as reported in [43].