Cell and virus preparation
Vero (ATCC CCL-81) and Vero E6 (ATCC CRL-1586) cells were cultured and maintained in EMEM containing 10% heat-inactivated FBS (Biosera, Nuaille, France), 2 mM L-glutamine, and antibiotics (REF 10378-016, Thermo Fisher Scientific Inc., MA, USA). SARS-CoV-2 isolate (SARS-CoV-2/Hu/DP/Kng/19-020, Genbank: LC528232) was obtained from Kanagawa Prefectural Institute of Public Health, Chigasaki, Kanagawa, Japan. The virus was inoculated into Vero cells (80-90% confluency) in EMEM containing 2% FBS, 2 mM L-glutamine, and antibiotics. On day 2 post-infection, the culture supernatant was harvested and filtered through a 0.45 μm Minisart Syringe Filter (Sartorius Stedim Biotech GmbH, Goettingen, Germany) for virus stocks. Aliquots were stored at -80°C until use.
Vero E6 cells (2.0×105 cells) were seeded on a 12-well plate and cultured overnight. Cells were infected with 10-fold serial dilutions of a virus stock in EMEM containing 2% FBS and were incubated at 37°C for 1 hour. Inocula were replaced with 1 mL of a 0.5% suspension of methyl cellulose #4000 (REF 11675-82, Nacalai Tesque Inc., Kyoto, Japan) in EMEM containing 1% FBS, 2 mM L-glutamine, and antibiotics. Cells were incubated at 37°C for 3 days, fixed, and stained with 10% formaldehyde (REF 068-03841, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 0.5% crystal violet (REF 031-04852, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The titer of the SARS-CoV-2 stocks was 1.3-2.6×105 PFU/mL.
Just before DUV-LED irradiation, the virus stocks were appropriately diluted by EMEM containing 2% FBS (1.0×105 PFU/mL), and then by PBS containing 2% FBS or EMEM containing 2% FBS to generate virus inoculum (1.0×104 PFU/mL). For each irradiation experiment, 100 µL of virus inoculum was placed in a defined well (E4) of a 96-well plate, and the plate was exposed to the designated irradiation wavelength and time. After irradiation, each virus inoculum was subjected to the plaque assay.
Absorbance spectra of samples in a quartz cell with 10-mm path length were measured using a UV-visible spectrometer (UV-1280, Shimadzu, Kyoto, Japan). Since the absorbance of EMEM containing 10% FBS and PBS containing 10% FBS were beyond the upper detection limit of the spectrometer, these media were diluted with distilled water, and the corresponding absorbance spectra were corrected according to the dilution ratios.
DUV-LED irradiation apparatus
A home-built DUV-LED irradiation apparatus was used in this study. The DUV-LED irradiation apparatus consisted of a DUV-LED unit (CCS Inc., Kyoto, Japan), a virus medium chamber (REF3595, Corning Inc., NY, USA), and chamber alignment jigs made of aluminum alloy bars. The DUV-LED unit and a chamber alignment jig were fixed on an acrylic plate. The other chamber alignment jig was freely movable. The virus medium chamber was placed just under the DUV-LED unit and aligned by the fixed and freely movable jigs.
Irradiation power density measurement
The irradiation power density of DUV-LED at each wavelength was obtained by measuring the transmitted light through a well of a virus medium chamber. We perforated a hole (the same diameter, 6.4 mm, with the bottom of the well) at the defined well (E4). The total power of the transmitted DUV-LED light was monitored by a power meter (detection size of 9.5 mm in diameter; S120VC, Thorlabs Inc., NJ, USA) that was set just under the hole. The irradiation power density was calculated by the total power of the transmitted light divided by the area of the well bottom.
Because SARS-CoV-2 infectivity appeared to be exponentially reduced in inverse correlation with the total dose of DUV-LED energy, the dose-response curve can be fitted with the following equation:
N = Ninit exp(-αItotal), (1)
where N, Ninit, and Itotal represent the number of plaques, the initial number of plaques, and the total dose of the DUV-LED energy, respectively. We defined the inactivation coefficient as α in Eq. 1.
Electron spin resonance (ESR) spectroscopy
Radical species in the culture media generated by DUV irradiation were measured by ESR spectroscopy with the spin-trapping method [38,39], and 5,5-dimethyl-1-pyrrorine-N-oxide (DMPO, Labotec Co., Ltd., Tokyo, Japan) was used as the spin trapping reagent. For ESR spectroscopy, 95 µL of EMEM containing 2% FBS, diluted with PBS containing 2% FBS by 10-fold, was mixed with 5 µL of 100 mM DMPO. The mixed solution was put in a chamber of a 96-well plate and was irradiated by the DUV-LED irradiation apparatus developed in this study. The irradiation power densities and the exposure time of the DUV-LEDs were set at 0.94 mW/cm2 and 160 s, respectively, for all the wavelengths of 265, 280, and 300 nm; and thus, the total dose of DUV-LED energy was 150 mJ/cm2 at all wavelengths. After the DUV-LED irradiation, the mixed solution was immediately transferred to three sections of glass capillaries (10 µL, Drummond Co., Broomall, PA, USA) and set into the ESR spectrometer (EMXPlus, Bruker Corp., MA, USA) with an X-band cavity (ER 4103TM, Bruker Corp., MA, USA). Hyperfine coupling constants were obtained using the computer program Winsim (version 0.96; NIEHS, NIH, Research Triangle Park, NC, USA, https://www.niehs.nih.gov) . The following ESR parameters were used: microwave frequency of 9.8497 GHz, microwave power of 10 mW, sweep width of 5 mT around the center magnetic field of 351 mT, modulation frequency of 100 kHz, modulation amplitude of 0.2 mT, time constant of 164 ms, conversion time of 230 ms, and total scan time of 240 s.