key: cord-0937995-fg8dun52
authors: De Santis, Riccardo; Luca, Vincenzo; Näslund, Jonas; Ehmann, Rosina K.; De Angelis, Marta; Lundmark, Eva; Nencioni, Lucia; Faggioni, Giovanni; Fillo, Silvia; Amatore, Donatella; Regalbuto, Elisa; Molinari, Filippo; Petralito, Giancarlo; Wölfel, Roman; Stefanelli, Paola; Rezza, Gianni; Palamara, Anna Teresa; Antwerpen, Markus; Forsman, Mats; Lista, Florigio
title: Rapid inactivation of SARS-CoV-2 with LED irradiation of visible spectrum wavelengths
date: 2021-10-28
journal: J Photochem Photobiol
DOI: 10.1016/j.jpap.2021.100082
sha: bc4073965ed8172d546d645dbe342cd52d8ca98b
doc_id: 937995
cord_uid: fg8dun52

Difficulty in controlling SARS-CoV-2 transmission made the ability to inactivate viruses in aerosols and fomites to be an important and attractive risk reduction measure. Evidence that light frequencies have the ability to inhibit microorganisms has already been reported by many studies which, however, focused on ultraviolet (UV) wavelengths, which are known to induce potential injury in humans. In the present study, the effect on suspensions of SARS-CoV-2 of a Light Emitting Diode (LED) device capable of radiating frequencies in the non-hazardous visible light spectrum (VIS) was investigated. In order to evaluate the efficiency of viral inactivation, plaque assay and western blot of viral proteins were performed. The observed results showed a significant reduction in infectious particles that had been exposed to the LED irradiation of visible light. Furthermore, the analysis of the intracellular expression of viral proteins confirmed the inactivating effect of this irradiation technology. This in vitro study revealed for the first time the inactivation of SAR-CoV-2 through LED irradiation with multiple wavelengths of the visible spectrum. However additional and more in-depth studies can aim to demonstrate the data obtained during these experiments in different matrices, in mutable environmental conditions and on other respiratory viruses such as the influenza virus, the type of LED technology can decisively contribute on reducing virus transmission through the continuous sanitation of common environments without risks for humans and animals.

Difficulty in controlling SARS-CoV-2 transmission made the ability to inactivate viruses in aerosols and 23

fomites to be an important and attractive risk reduction measure. Evidence that light frequencies have 24 the ability to inhibit microorganisms has already been reported by many studies which, however, 25 focused on ultraviolet (UV) wavelengths, which are known to induce potential injury in humans. In the 26 present study, the effect on suspensions of SARS-CoV-2 of a Light Emitting Diode (LED) device capable 27 of radiating frequencies in the non-hazardous visible light spectrum (VIS) was investigated. In order to 28 evaluate the efficiency of viral inactivation, plaque assay and western blot of viral proteins were 29

performed. The observed results showed a significant reduction in infectious particles that had been 30 exposed to the LED irradiation of visible light. Furthermore, the analysis of the intracellular expression 31 of viral proteins confirmed the inactivating effect of this irradiation technology. This in vitro study 32 revealed for the first time the inactivation of SAR-CoV-2 through LED irradiation with multiple 33 wavelengths of the visible spectrum. The experiments were performed using a LED strip supplied by Nextsense Srl and powered with 117

Biovitae ® technology ( Figure 1A ). The LED device under investigation tested uses a special 118 combination of frequencies covering the visible spectrum, with energy humps at 400-420nm, 430-119 460nm, 500-780nm, and a main peak at 413 nm ± 5 nm Figure 1B) . In order to prevent any sample 120 heating during exposure, the lamp is set up with a heat sink for thermal management. The plaque assay (Figure 2 ) demonstrated that LED irradiation treatment with given wavelengths of 159 the visible light spectrum (LED light) was able to significantly reduce the amount of infectious particle 160 in standardized virus suspensions. Different concentrations of SARS-CoV-2 were irradiated at a 161 distance of 25 cm over the course of 60 minutes with different subsampling time points (Figure 3 ). 162

Graphs A and B show the activity of LED light on viral concentrations of 8x10 1 pfu/ml and 8x10 2 163 pfu/ml, respectively. In both cases, it was observed that viral inactivation reaches more than 2-log 164 reduction (99%) after 60 minutes of exposure. Graph C shows that even at higher concentration 165 (7x10 3 PFU/ml and 3x10 4 PFU/ml) the LED light is still able to inactivate more than 99% of the viral 166 particles. A viral inactivation of approximately 96% was also achieved using a dose of 1.7x10 5 PFU/ml. 167 168 169

The inactivating properties of LED light was also confirmed by analysis of the expression of the viral 171

proteins. Vero E6 cells were infected with SARS-CoV-2 (hCoV-19/Italy/CDG1/2020/EPI_ISL_412973), 172 controls (6x10 3 PFU) that had been both exposed and unexposed to the LED light and harvested at 8 Sars-Cov-2 disease represents a global threat for both public health and economic outlook worldwide. 183

Inhalation of aerosolized droplets from infectious people is considered to be the main way of virus 184

transmission, but despite social distancing measures, virus circulation is still sustained. Thus, indirect 185 infection through contact with contaminated surfaces seems to contribute to the route of transmission. 186

Recent studies performed under controlled experimental conditions, have shown the stability of Sars-187

CoV-2 for days on high-touch surfaces [21] , with short persistence on surfaces with low porosity as 188 compared to other highly porous surfaces [22] . In this context, the search for new methods aimed to 189 inactivate virus in the environment, could represent a good weapon to counteract Sars-Cov-2 190 diffusion. 191

The viral inactivation by UV irradiation based on lamps emitting UV radiation C (UVC) around 254 nm 192

is one of the most adopted methods [23], although is known to cause harmful effects on humans with 193 short-and long-term effects [9; 15] and therefore it should be not used in inhabited environments. 194 study through irradiation with visible spectrum wavelengths has to be elucidated to clarify the 219 underlying virucidal mechanism(s) of visible light. It could be hypothesized that reactive species 220 generated by LED irradiation may alter the viral membrane or S protein stability, thus affecting the 221 virus ability to entry host cells. However, it is also known that cell culture medium itself can influence 222 ROS levels: the common medium component riboflavin could lead/enhance photoinactivation through 223 generation of ROS as its absorbance spectrum also includes peaks in the LED white light spectrum [27; 224 28]. Therefore, for evaluating the effects of the culture medium on both light absorbance and SARS-225

CoV-2 infectivity, and exclude a possible role of riboflavin, an experiment was performed by exposing 226

to LED light the virus diluted in PBS 1x. The results showed the same extent of inactivation (data not 227 shown). 228

However, further studies are needed to clarify the virucidal underlying mechanism of visible light. 229

The use of lamps emitting in the UVC spectrum provides an efficient treatment of air, liquids and 230 surfaces as demonstrated by studies showing complete viral inactivation in a time ranging from few 231 seconds to 15 minutes depending on experimental conditions. But the limit of UVC disinfection 232 systems is represented by its use only in unoccupied spaces. Therefore, it is noteworthy that these 233 white light emitting devices can be used for continuous decontamination and viral reduction in 234 virtually all human environments without the dangerous side effects and protective measures 235 associated with the use of UV light frequencies [29] . Further additional and more in-depth studies can 236 aim to demonstrate the data obtained during these experiments in different matrices, in mutable 237 environmental conditions, and on other respiratory viruses such as the influenza virus. 238

This type of LED technology could decisively contribute to reducing virus transmission through the 239 continuous sanitation of common environments without risks for humans and animals; and 240

confirming the antiviral effects of such LED technology in common indoor environments will be an 241 essential step to predict its potential impact on virus transmission via fomites. 

Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach

260 2. WHO Coronavirus Disease (COVID-19) Dashboard

Aerosol and surface stability of SARS-CoV-2 as 263 compared with SARS-CoV-1

World Health Organization 265 declares global emergency: A review of the 2019 novel coronavirus (COVID-19)

Aerosol and surface distribution of severe acute respiratory syndrome 269 coronavirus 2 in hospital wards

Disinfection by ultraviolet irradiation

Ultraviolet irradiation doses for coronavirus inactivation -review and 274 analysis of coronavirus photoinactivation studies

Safety of upper-room ultraviolet germicidal air disinfection for room occupants: results from the Tuberculosis 278

Control Measures for SARS-CoV-2: A Review on Light-Based Inactivation of Single-280

Stranded RNA Viruses. Pathogens

Susceptibility of SARS-CoV-2 to UV irradiation

Fast inactivation 285 of SARS-CoV-2 by UV-C and ozone exposure on different materials

Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays

Irradiation 292 by ultraviolet light-emitting diodes inactivates influenza a viruses by inhibiting replication and transcription of viral 293 RNA in host cells

Simulated 296 Sunlight Rapidly Inactivates SARS-CoV-2 on Surfaces

Accidental exposure to UV radiation 298 produced by germicidal lamp: case report and risk assessment

All you need is light: 300 antimicrobial photoinactivation as an evolving and emerging discovery strategy against infectious disease

In vitro photodynamic antimicrobial activity of methylene blue and endoscopic white light 303 against Helicobacter pylori 26695

Inactivation of Bacterial Pathogens following Exposure to Light 305 from a 405-Nanometer Light-Emitting Diode Array

Pulsed blue light inactivates two strains of human coronavirus

Influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by 311 NOX4-derived ROS

Effectiveness of 222-nm ultraviolet light 313 on disinfecting SARS-CoV-2 surface contamination

Stability of SARS-CoV-2 and other coronaviruses in the environment and on 316 common touch surfaces and the influence of climatic conditions: A review

Direct and indirect effects of UV radiation on DNA and its components

The role of oxygen in the visible-light 321 inactivation of Staphylococcus aureus

High-intensity narrow-spectrum light inactivation 323 and wavelength sensitivity of Staphylococcus aureus

Inactivation of viruses by chemically and photochemically 325 generated singlet molecular oxygen

The influence of riboflavin photochemistry on 327 plasma coagulation factors

Light-dependent generation of reactive oxygen species in cell culture media

Safety Evaluation of a 405-nm LED Device for Direct Antimicrobial 331 Treatment of the Murine Brain

The strip used for the experiments, consisting of 13 Biovitae ® LEDs emitting wavelengths in the 400-420nm range and 335 delivering three different peaks within, and 37 conventional Osram Oslon (R) Square GW CSSRM2.EM-MFN2-XXX5-1 white light LEDs

LEDs are powered at a constant current of 500mA, guaranteed by a TCI MP 80/500 SLIM power supply. B. Relative radiometric spectral 337 distribution of the Biovitae ® strip

Inhibition of SARS-Cov2 (hCoV-19/Italy/CDG1/2020/EPI_ISL_412973) by LED-irradiation with given wavelengths of the visible 339 light spectrum (LED light). a-c I, Positive control. Plaque formation in Vero E6 cells. (a) 10 5 PFU/ml; (b) 10 4 PFU/ml

Virus irradiated with LED light for 60 minutes and inoculated in Vero E6 cells. (d) 2x 10 2 PFU/ml

Viral inhibition of SARS-CoV-2 after 15, 30, 45 and 60 minutes of LED light exposition at 25 cm distance. Panel A: viral 343 concentration 8x10 1 PFU/ml (∆)

Panel B: viral concentration 8x10 2 PFU/ml (□)

Panel C: viral concentrations 7x10 3 PFU/ml (○)

Experiments were conducted by three different laboratories indicated as follows: Scientific Department

Data points represent the average of duplicate samples from at least two single experiments

Western blot analysis of SARS-CoV-2 proteins (spike, S and nucleocapsid, N) in Vero E6 cells infected with viral stock exposed 348 (I+LED) or not (I) to LED light. The expression of both proteins was analyzed at 8 hours or 24 hours p.i

Blot is representative of three experiments performed. CTR, Uninfected cells

I, positive control; I+LED, virus-irradiated infected cells