key: cord-302160-4yfvspaq
authors: Ruetalo, Natalia; Businger, Ramona; Schindler, Michael
title: Rapid and efficient inactivation of surface dried SARS-CoV-2 by UV-C irradiation
date: 2020-10-07
journal: bioRxiv
DOI: 10.1101/2020.09.22.308098
sha: 
doc_id: 302160
cord_uid: 4yfvspaq

The SARS-CoV-2 pandemic urges for cheap, reliable and rapid technologies for disinfection and decontamination. We here evaluated the efficiency of UV-C irradiation to inactivate surface dried SARS-CoV-2. Drying for two hours did not have a major impact on the infectivity of SARS-CoV-2, indicating that exhaled virus in droplets or aerosols stays infectious on surfaces at least for a certain amount of time. Strikingly, short exposure of high titer surface dried virus (3*10^6 IU/ml) with UV-C light (16 mJ/cm2) resulted in a total reduction of SARS-CoV-2 infectivity. Together, our results demonstrate that SARS-CoV-2 is rapidly inactivated by relatively low doses of UV-C irradiation. Hence, UV-C treatment is an effective non-chemical possibility to decontaminate surfaces from high-titer infectious SARS-CoV-2.

Introduction 57 SARS-CoV-2 has spread globally and there is an urgent need for rapid, highly efficient, 58 environmentally friendly, and non-chemical disinfection procedures. Application of UV-C light 59 is an established technology for decontamination of surfaces and aerosols (1-3). This 60 procedure has proven effective to inactivate SARS-CoV-1 (4-6), several other enveloped and 61 non-enveloped viruses as well as bacteria (7). Recently, it has also been shown that SARS-62

CoV-2 is sensitive to inactivation by UV-C irradiation (8-10). However, doses and exposure 63 times necessary for total inactivation of SARS-CoV-2 were in a range precluding efficient 64 application of UV-based methods to be employed for large-scale decontamination of surfaces 65 and aerosols (10). We hence conducted a "real-life" application approach simulating the 66 inactivation of dried surface residing infectious SARS-CoV-2 by a mobile handheld UV-C 67 emitting device and an UV-C box designed to decontaminate medium-size objects. Our data 68

shows that surface dried SARS-CoV-2 retains infectivity for at least two hours. Short 69 exposure of high-titer surface dried SARS-CoV-2 to UV-C light lead to a total reduction of 70 UV-C light inactivation treatment. 35 uL of virus stock, corresponding to ~4*10 6 infectious 88 units (IU) of icSARS-CoV-2-mNG were spotted (in triplicates) in 6-well plates and dried for 89 two hours at RT. 6-well plates spotted with dried virus were treated with UV-C-light using the 90 Soluva® pro UV Disinfection Chamber (Heraeus) for 60 seconds or the Soluva® pro UV 91 Disinfection Handheld (Heraeus) for 2 seconds in a fix regime at 5 and 20 cm plate distance. 92

In addition, a moving regime using a slow (3.75 cm/s) and fast (12 cm/s) speed at 20 cm 93 distance was tested. As control, 6-well plates were spotted with the virus and dried, but not 94 UV-treated. After UV-treatment, the spotted virus was reconstituted using 1 mL of infection 95 media (culture media with 5% FCS). As control, 35 uL of the original virus stock were diluted 96 to 1 ml with infection media and used as virus stock infection control. We set up an experimental approach to evaluate the effect of UV-C treatment on the stability 114 of SARS-CoV-2. Simulating the situation that exhaled droplets or aerosols from infected 115 individuals contaminate surfaces, we produced a high-titer SARS-CoV-2 infectious stock and 116 dried 35µL of this stock corresponding to ~4*10^6 IU/ml in each well of a 6-well plate. The 117 plates were then either non-treated or exposed to five UV-C regimens (Fig. 1a) . These 118 include inactivation for 60 s in a box designed to disinfect medium-size objects, 2 s exposure 119 at 5 cm or 20 cm distance with a handheld UV-C disinfection device and finally an approach 120 simulating decontamination of surfaces via the handheld UV-C device. For this, we performed 121 slow and fast-moving at a distance of ~20 cm, with "slow" corresponding to a speed of ~3.75 122 cm/s (supplemental movie 1) and "fast" at ~12 cm/s (supplemental movie 2). UV-C irradiance 123 (254 nm) in the box with an exposure time of 60 seconds corresponds to an irradiation dose 124 of 800 mJ/cm²; for the handheld (HH) at 5 cm the UV-C dose at two second irradiation time is 125 80 mJ/cm² and at 20 cm is 16 mJ/cm². From the speed of the "slow" and "fast" moving 126 regimens we calculate a UV-C dose of 2.13 mJ/cm² (slow) and 0.66 mJ/cm² (fast), assuming 127 a focused intensity beam. However, taking into consideration the UV-C light distribution 128 underneath the handheld device the integrated UV-C dose accumulates to 20 mJ/cm² for the 129 fast regimen. 130 Subsequently, dried virus was reconstituted with 1 mL infection media and used to inoculate 131 naïve Caco-2 cells at serial dilutions to calculate viral titers. Taking advantage of an infectious 132 SARS-CoV-2 strain expressing the chromophore mNeonGreen (11), we quantified infected 133 (mNG+) and total (Hoechst+) cells by single-cell counting with an imaging multiplate reader. 134

Of note, even short UV-C treatment of the dried virus in the context of the moving "fast" 135 regimen completely inactivated SARS-CoV-2, as no infected cells were detected based on 136 fluorescence protein expression (Fig. 1b) . Titration of two-fold series dilutions of the UV-137 treated and non-treated control samples, as well as the freshly thawed strain as reference, 138 revealed that (i) drying for two hours does not have a major impact on the infectivity of SARS-139

CoV-2 and (ii) all five UV-C treatment regimens effectively inactivate SARS-CoV-2 (Fig. 1c) . 140 6 Calculation of viral titers based on the titration of the reconstituted virus stocks revealed a 141 loss of titer due to drying from ~4*10^6 to ~3*10^6 IU/ml and effective 6-log titer reduction of 142 SARS-CoV-2 by all employed UV-C treatment regimens (Fig. 1d) . Altogether, our data 143 demonstrate that UV-C regimens that expose high-titer SARS-CoV-2 to doses down to 16 144 mJ/cm² are sufficient to achieve complete inactivation of the virus. 145 146 Discussion 147

Disinfection of surfaces and aerosols by UV-C irradiation is an established, safe and non-148 chemical procedure used for the environmental control of pathogens (1-3, 12) . UV-C 149 treatment has proven effective against several viruses including SARS-CoV-1 (4-6) and other 150 coronaviruses i.e. Canine coronaviruses (13). Hence, as recently demonstrated by others (8-151 10) and now confirmed by our study it was expected that SARS-CoV-2 is permissive for 152 inactivation by UV-C treatment. One critical question is the suitability of this technology in a 153 "real-life" setting in which the exposure time of surfaces or aerosols should be kept as short 154 as possible to allow for a realistic application, for example in rooms that need to be used 155 frequently as operating rooms or lecture halls. Furthermore, in such a setting, we assume that 156 the virus is exhaled from an infected person by droplets and aerosols, dries on surfaces and 157 hence represents a threat to non-infected individuals. We simulated such a situation and first 158 evaluated if surface dried SARS-CoV-2 is infectious. Drying for two hours, in agreement with 159 previous work (14), did not result in a significant reduction of viral infectivity indicating smear-160 infections could indeed play a role in the transmission of SARS-CoV-2 (Fig. 1) . On the other 161 hand, our virus-preparations are dried in cell culture pH-buffered medium containing FCS, 162 which might stabilize viral particles. Hence, even though this is not the scope of the current 163 study, it will be interesting to evaluate if longer drying or virus-preparations in PBS affect the 164 environmental stability of SARS-CoV-2. Irrespective of the latter, UV-C-exposure of dried 165 high-titer SARS-CoV-2 preparations containing ~3*10^6 IU/ml resulted in a complete 166 reduction of viral infectivity (Fig. 1) . In this context, it is noteworthy that we achieved a 6-log 167 virus-titer reduction in a setting simulating surface disinfection with a moving handheld device. 168

Effect of ultraviolet germicidal irradiation on viral aerosols

Role of ultraviolet (UV) disinfection in infection control and 208 environmental cleaning

Role of Ultraviolet Disinfection in 210 the Prevention of Surgical Site Infections

Stability of SARS 212 coronavirus in human specimens and environment and its sensitivity to heating and UV 213 irradiation

Large-scale preparation of UV-inactivated SARS coronavirus 215 virions for vaccine antigen

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UV Dose)

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Rapid inactivation of 223 SARS-CoV-2 with deep-UV LED irradiation

225 Effectiveness of 222-nm ultraviolet light on disinfecting SARS-CoV-2 surface contamination

228 Susceptibility of SARS-CoV-2 to UV irradiation

An 230 Infectious cDNA Clone of SARS-CoV-2

No touch' technologies for environmental 232 decontamination: focus on ultraviolet devices and hydrogen peroxide systems

Williamson 237 BN, et al. Aerosol and Surface Stability of SARS-CoV-2 as

With the "fast"-moving protocol (see supplemental video 1) we were exposing surfaces at a 169 distance of 20 cm with a speed of 12.5 cm/s resulting in an calculated integrated UV-C dose 170 of 20 mJ/cm² at 254 nm. This is substantially less than the previously reported 1048 mJ/cm² 171 necessary to achieve a 6-log reduction in virus titers when exposing aqueous SARS-CoV-2 to 172 UV-C (10). In another study, using a 222 nm UV-LED source, 3 mJ/cm² lead to a 2.51-log 173 (99.7 %) reduction of infectious SARS-CoV-2 when irradiating for 30 s, however inactivation 174 did not be increase with extended irradiation regimens up to 300 s (9). In addition, 20 s deep-175 ultraviolet treatment at 280 nm corresponding to a dose of 75 mJ/cm² reduced SARS-CoV-2 176 titer up to 3-logs (8). Comparing these values to other pathogens, SARS-CoV-2 seems 177 particularly sensitive towards UV-C light. To achieve a 3-log titer reduction, 75-130 mJ/cm² 178 are necessary for adenovirus, 11-28 mJ/cm² for poliovirus, and bacteria as for instance 179Bacillus subtilis require 18-61 mJ/cm² (7). This is in-line with susceptibility of SARS-CoV 180 towards UV-C in aerosols at 2.6 mJ/cm², whereas adenovirus or MS2-bacteriophages were 181 resistant to such a treatment (1). 182Altogether, we establish the effectiveness of UV-C treatment against SARS-CoV-2 in a 183 setting designed to simulate realistic conditions of decontamination. The easy, rapid, 184 chemical-free, and high efficacy of UV-C treatment to inactivate SARS-CoV-2 demonstrates 185 the applicability of this technology in a broad range of possible settings.