UV-C irradiation is highly effective in inactivating SARS-CoV-2 replication

The potential virucidal effects of UV-C irradiation on SARS-CoV-2 were experimentally evaluated for different illumination doses and virus concentrations (1000, 5, 0.05 MOI). At a virus density comparable to that observed in SARS-CoV-2 infection, an UV-C dose of just 3.7 mJ/cm2 was sufficient to achieve a more than 3-log inactivation without any sign of viral replication. Moreover, a complete inactivation at all viral concentrations was observed with 16.9 mJ/cm2. These results could explain the epidemiological trends of COVID-19 and are important for the development of novel sterilizing methods to contain SARS-CoV-2 infection.


Scientific Reports
| (2021) 11:6260 | https://doi.org/10.1038/s41598-021-85425-w www.nature.com/scientificreports/ hospital rooms), the second one corresponds to the average concentration found in the sputum of COVID-19 infected patients, and the third one is a very large concentration, corresponding to that observed in terminally diseased COVID-19 patients 29 . After UV-C exposure, viral replication was assessed by culture-polymerase chain reaction (C-RT-PCR) targeting two regions (N1 and N2) of the SARS-CoV-2 nucleocapsid gene, as well as by analyzing SARS-CoV-2-induced cytopathic effect. Analyses were performed in the culture supernatant of infected cells at three different time points (24,48 and 72 h for SARS-CoV-2 at MOI 1000 and 5; 24, 48 h and 6 days for SARS-CoV-2 at MOI 0.05), as well as on cell lysates at the end of cellular culture (72 h: MOI 1000 and 5; 6 days: MOI 0.05). This approach allows to follow the kinetic of viral growth and to verify whether the used dose is sufficient to completely inactivate the virus over time. This is useful from a practical point of view, when UV-C devices are used to disinfect surfaces and the environment. The effect of the UV-C exposure on SARS-CoV-2 replication was extremely evident and independent from the MOI employed; dose-response and time-dependent curves were observed. Figures 1, 2 and 3 report for different MOI the number of SARS-CoV-2 copies for the three concentrations as a function of the UV-C dose and time, quantified on a standard curve from a plasmid control. The corresponding normalised curves of the virus copies are reported in the same figures.
Viral replication was not observed at the lowest viral concentration (0.05 MOI) in either untreated or in UV-C-irradiated samples in the initial 48 h (Fig. 1). However, 6 days after infection, viral replication was distinctly evident in the UV-C unexposed condition, but was completely absent following UV-C irradiation even at 3.7 mJ/ cm 2 both in cell culture supernatants (Fig. 1A,B) and in cell lysate (Fig. 1C). A two-way ANOVA analysing the effect of UV-C dose and time of incubation failed to identify a significant effect of the UV exposure on viral replication. This is due to the fact that at very low MOI relevant increases in N1 and N2 copy numbers were detectable only in a single condition-at six days in the absence of UV-C exposure-thus hampering the statistical power of the analysis.
At the intermediate viral concentration (5 MOI), a significant reduction of copy number starting from the 3.7 mJ/cm 2 dose with a decrease of a factor of 2000 (> 3-log decrease) after 24 h was observed (Fig. 2D). A two-way ANOVA confirmed that this UV-C dose significantly dampened viral replication (p = 0.000796, and p = 0.000713 for N1 and N2 copies respectively). Even more important, the copy number did not increase over time, suggesting an effective inactivation of the virus, which was further confirmed by cytopathic effect assessment ( Fig. 3A-C).
Using a high viral input (MOI = 1000), the two-way ANOVA confirmed that all the tree UV doses analysed resulted in a significant suppression of viral replication for both N1 (3.7 mJ/cm 2 : p = 0.008455; 16.9 mJ/ cm 2 : p = 0.004216; and 84.4 mJ/cm 2 : p = 0.000202) and N2 copies (3.7 mJ/cm 2 : p = 6.43E−05; 16.9 mJ/cm 2 : p = 1.68E−05; and 84.4 mJ/cm 2 : p = 1.68E−05) (Fig. 4). Notably, a different course of infection was observed, in which the inhibitory effect was not accompanied by viral suppression for the UV-C dose of 3.7 mJ/cm 2 www.nature.com/scientificreports/ ( Fig. 4A,B,D). Indeed, a relevant reduction in N1 e N2 copy numbers was observed in a UV-C dose-dependent manner as early as 24 h (by a factor of 10 3 at 3.7 mJ/cm 2 and 10 4 at 16.9 mJ/cm 2 , Fig. 4A,B,D), but longer culture times resulted in an increase in N1 and N2 copy numbers for the UV-C dose of 3.7 mJ/cm 2 . This indicates that the residual viral input left by the 3.7 mJ/cm 2 was able to replicate and sufficient to generate an effective infection. This is not the case in cultures exposed to higher UV-C doses, as no replication could be detected in these conditions. All the results were further confirmed by 2-ANOVA statistical analyses performed on viral replication at intracellular level ( We compared our results with data available in the literature and observed that our inactivating dose is much smaller than that reported in Heilingloh et al. 26 (1000 mJ/cm 2 for the complete inactivation). This discrepancy is likely to be the consequence of the UV-C absorption by the medium used in Heilingloh et al., which has a fourfold higher thickness compared to the one used in our experiments. This possibility is supported by the observation that 200 mJ/cm 2 of UV-A, which is not absorbed by the medium, was sufficient to reduce viral replication of 1-log. As UV-A light is significantly less efficient (order of magnitudes) than UV-C, the reported UV-C inactivating dose (100 mJ/cm 2 ) seems to be questionable.
Two other papers measured the effect of UV-C light on SARS-CoV-2. In Ruetalo et al. 25 , the illumination of 254 nm light was employed on a dried sample of SARS-CoV-2. Complete inactivation was obtained with 20 mJ/ cm 2 , a value greater than ours, but in the same range. It has to be underlined that in the dried film a shielding   28 , who demonstrated that the dose required to obtain a similar degree of viral inactivation was twice in dried samples from gMEM compared to the ones resuspended in simulated saliva. Notably, the two mediums differ for their composition, mainly in terms of protein and solid percentage, with higher values for the gMEM. Inagaki et al. used a 285 nm UV LED and showed that a dose of about 38 mJ/cm 2 was sufficient to completely inactivate SARS-CoV-2. This dose is greater compared to the one we established; this discrepancy can be explained by the observation that the 285 nm is less efficient than the 254 nm wavelength 24 . Finally, in an elegant study Storm et al. 27 compared the virucidal effect of UV-C in wet and dry systems. Results were based on the use of a very small volume of viral stock in DMEM (5 μl) and showed that a dose of 3.4 mJ/cm 2 inactivated wet samples, whereas a dose that twice as high was needed in dried samples. These results are comparable to the ones herein, and the shielding effect in dried samples is almost evident. Such comparisons show how the experimental conditions adopted significantly impact on the definition of the dose of UV-C resulting in virus inactivation. It is therefore crucial to accurately describe all the details of the experiments to perform a reliable comparison.
In conclusion, we report the results of a highly controlled experimental model that allowed us to identify the UV-C radiation dose sufficient to inactivate SARS-CoV-2. The response depends on both the UV-C dose and the virus concentration. Indeed, for virus concentrations typical of low-level contaminated closed environment and sputum of COVID-19 infected patients, a very small dose of less than 4 mJ/cm 2 was enough to achieve full inactivation of the virus. Even at the highest viral input concentration (1000 MOI), viral replication was totally inactivated with a dose ≥ 16.9 mJ/cm 2. These results show how the SARS-CoV-2 is extremely sensitive to UV-C light and they are important to allow the proper design and development of efficient UV based disinfection methods to contain SARS-CoV-2 infection.

Methods
In vitro SARS-CoV-2 infection assay. 3   UV illumination test. The illumination of the virus solution was conducted using a low-pressure mercury lamp mounted in a custom designed holder, which consist in a box with a circular aperture 50 mm in diameter placed at approximately 220 mm from the source. The aperture works as a spatial filter to make the illumination of the area behind more uniform. A mechanical shutter is also present to start the illumination process. The plate is placed 30 mm below the circular aperture and a single dwell (34.7 mm in diameter), centered in respect to the 50 mm aperture, has been irradiated from the top. The dwell was filled with 0.976 ml of the virus suspended in Dulbecco's Modified Eagle's Medium (DMEM) in order to have a 1 mm thick liquid layer. After the irradiation, the sample was treated as described in the previous section.
The intensity of the lamp and its spectral properties have been measured using an Ocean Optics HR2000 + spectrometer (Ocean Optics Inc., Dunedin, USA). The HR2000 + spectrometer was calibrated against a reference deuterium-halogen source (Ocean Optics Inc. Winter Park, Winter Park, Florida) and in compliance with National Institute of Standards and Technology (NIST) practices recommended in NIST Handbook 150-2E, Technical guide for Optical Radiation Measurements. The last calibration was performed in March 2019. The detector of our spectrometer is a high-sensitivity 2048-element Charge-Coupled Device (CCD) array from Sony. The spectral range is 200-1100 nm with a 25 μm wide entrance slit and an optical resolution of 1.4 nm (FWHM). The cosine-corrected irradiance probe, model CC-3-UV-T, is attached to the tip of a 1 m long optical fibre and couples to the spectrometer. The intensity of the lamp has been measured by positioning the spectrometer in five positions: in the center and at the ends of a 20 mm cross arm after a warming up time of 30 s. The spectra in the five positions are reported in Fig. 5A together with a scheme of the dwell and illuminated area.
As expected, the emission is dominated by the UV-C line (Fig. 5A) and its intensity was uniform in the area with an average value of 1.082 mW/cm 2 . The stability of the lamp was evaluated in ± 11E−3 mW/cm 2 during a 130 s measurement. According to this value, three exposure times were set: 5, 23 and 114 s (with an accuracy of 0.2 s), which correspond to following doses: 5.4, 25.0, 123.4 mJ/cm 2 . This is the nominal UV doses provided to the dwell, but we were interested in the effective doses (D e ) reaching the virus. It was necessary to calculate the effective irradiance (I e ). This step was performed considering both the reflection losses at the air/water interface (R w ) and the Transmittance (T s ) of the DMEM solution at 254 nm (from the spectrum in Fig. 5B, considering the cuvette losses, T s = 0 0.70). It is important to notice that the spectrum was measured in a quartz cuvette (1 mm thick) by means of a Jasco V770 spectrophotometer and this thickness was the same of the solution in the dwell during the UV irradiation step.
The reflection loss was computed as follow: where n w = 1.375 is the refractive index of water at 254 nm. Then, I e was calculated: (1) R w = (n w − 1) 2 (n w + 1) 2 , (2) I e = I n (1 − R w ) × T s .