Vero cells (ATCC, CCL-81) and Vero-E6 cells (ATCC, CRL-1586) were maintained in DMEM medium supplemented with 10% FCS and 1% penicillin–streptomycin at 37 °C, under an atmosphere containing 5% CO2. A clinical isolate of SARS-CoV-2 was isolated from a nasopharyngeal swab collected from a patient suffering from COVID-19 at the Tours University Hospital. The virus was amplified using Vero cells expressing Transmembrane Protease Serine 2 (TMPRSS2) to prepare a viral stock in DMEM medium supplemented with 2% FCS and 1% penicillin–streptomycin. This supplemented DMEM medium was defined as infection medium. Viral titer was expressed in 50% tissue culture infective dose (TCID50). This measurement reflects the dilution at which 50% of wells show cytopathic effect. This was quantified by endpoint dilution, using the Spearman and Kärber formula [9, 10], previously used for similar applications [8]. The limit of detection was 4 TCID50/mL. Results below 4 TCID50/mL were considered as equivalent to 1 TCID50/mL. Concentration of the viral stock used in this study was 3 × 105 TCID50/mL (5.5-log10 TCID50/mL). Absorbance of this viral stock in DMEM was 2.39 cm−1 and 2.53 cm−1 at 254 and 265 nm, respectively (Denovix DS11 spectrophotometer). Viral stock was stored at – 80 °C.

The first UV-C lamp was the Puritec HNS-L 2G11 UV-C germicidal mercury lamp (OSRAM, Rosny-sous-bois, France), emitting at 254 nm with a nominal wattage of 18 W. Lamp dimensions were 31.5 cm × 4 cm. The second UV-C lamp was the KL265-50 V-SM-WD UV-C LED (Klaran, Green Island, USA), emitting between 260 and 270 nm with a peak at 265 nm and consuming 70 mW of power. LED dimensions were 3.5 × 3.5 mm. Irradiations were performed at 3, 5 or 10 cm with the lamp positioned directly above the irradiated well for durations of 5, 15, 30 or 60 s. UV-C irradiances received by the sample were measured with an optometer X1-5 and a detector UV-3726-5 (Gigahertz-Optik, GmbH). This device was calibrated at 254 nm and a spectral mismatch correction was calculated for measurements at 265 nm. UV-C irradiances (254 nm) received by the sample after irradiation with the mercury lamp at 3, 5 and 10 cm were 3.10 mW/cm2, 3.25 mW/cm2 and 1.43 mW/cm2, respectively. UV-C irradiances (265 nm) received by the sample after irradiation with the LED at 3, 5 and 10 cm were 1.49 mW/cm2, 1.47 mW/cm2 and 0.64 mW/cm2, respectively. UV-C doses (mJ/ cm2) for each duration (seconds) were calculated as follows: \(UV-C dose =UV-C irradiance \times duration of exposure\)

The SARS-CoV-2 inoculum (200 µL at 3 × 105 TCID50 inoculum/ml) was deposited as a drop at a single spot using a pipettor on relevant surfaces in 12-well plates. Plastic (bottom of the well), steel (1 cm2 steel ring), tissue (1 cm2 punch of cotton cloth), paper (1 cm2 punch of 80 g/m2 white paper), and cardboard (1 cm2 punch of solid unbleached board) were used in this experiment.

A five minutes contact time between the inoculum and all tested surfaces was applied. It allowed complete absorption on absorbent surfaces. Viruses were then directly exposed to UV-C irradiation at 3, 5 or 10 cm (on top, without cover) for 5, 15, 30 or 60 s. A control condition without UV-C exposure was considered for each material in duplicate for each experiment. The exposed inoculum (200 µL) was directly collected for non-absorbent surfaces or eluted for absorbent surfaces by 5 min soaking in 200 µL of infection medium (described above). It was then deposited on Vero-E6 cells to amplify remaining infectious viruses. These cells had previously been plated at 3 × 105 cells / well in 12-well plates, 24 h before the experiment. After 1 h of infection, the viral suspension was removed, the cells were washed with 500 µL of PBS and 1 ml of infection medium was added to each well. Twenty-four hours later, the supernatant was collected and the viral titer was determined by endpoint dilution and calculation of the TCID50 (TCID50 UV-C). This amplification step was necessary to ensure optimal sensitivity of the SARS-CoV-2 concentration measurement. A standard curve was performed with tenfold serial dilutions of the stock concentrations titrated before and after amplification in a single experiment to confirm that linearity was maintained after amplification. SARS-CoV-2 concentration after mock-irradiation and amplification (TCID50 control) was used as a control to take into account the amplification step and the intrinsic properties of each surface (absorption and inactivation). SARS-CoV-2 inactivation (%) was calculated as follows: Inactivation (%) = (\(1-\frac_UV-C}_ control}\))*100. SARS-CoV-2 inactivation experiments were performed in duplicate in three independent experiments to obtain six individual values used to calculate the mean and standard deviation. SARS-CoV-2 inactivation efficiencies were compared between different UV-C sources, different distance and different durations of irradiation, using the Mann–Whitney (two groups) or the two-way ANOVA tests (more than two groups) with the GraphPad 9 software.