key: cord-1017257-l018fd28
authors: Rathnasinghe, Raveen; Jangra, Sonia; Miorin, Lisa; Schotsasert, Michael; Yahnke, Clifford; Garcίa-Sastre, Adolfo
title: The virucidal effects of 405 nm visible light on SARS-CoV-2 and influenza A virus
date: 2021-09-01
journal: bioRxiv
DOI: 10.1101/2021.03.14.435337
sha: 7aacdc00d2e5c7bd98f24bd753930333aa485b98
doc_id: 1017257
cord_uid: l018fd28

Germicidal potential of specific wavelengths within the electromagnetic spectrum is an area of growing interest. While ultra-violet (UV) based technologies have shown satisfactory virucidal potential, the photo-toxicity in humans coupled with UV associated polymer degradation limit its use in occupied spaces. Alternatively, longer wavelengths with less irradiation energy such as visible light (405 nm) have largely been explored in the context of bactericidal and fungicidal applications. Such studies indicated that 405 nm mediated inactivation is caused by the absorbance of porphyrins within the organism creating reactive oxygen species which result in free radical damage to its DNA and disruption of cellular functions. The virucidal potential of visible-light based technologies has been largely unexplored and speculated to be ineffective given the lack of porphyrins in viruses. The current study demonstrated increased susceptibility of lipid-enveloped respiratory pathogens of importance such as SARS-CoV-2 (causative agent of COVID-19) as well as the influenza A virus to 405nm, visible light in the absence of exogenous photosensitizers indicating a potential porphyrin-independent alternative mechanism of visible light mediated viral inactivation. These results were obtained using less than expected irradiance levels which are generally safe for humans and commercially achievable. Our results support further exploration of the use of visible light technology for the application of continuous decontamination in occupied areas within hospitals and/or infectious disease laboratories, specifically for the inactivation of respiratory pathogens such as SARS-CoV-2 and Influenza A.

nm mediated inactivation is caused by the absorbance of porphyrins within the 23 organism creating reactive oxygen species which result in free radical damage to its 24 DNA and disruption of cellular functions. The virucidal potential of visible-light based 25 technologies has been largely unexplored and speculated to be ineffective given the 26 lack of porphyrins in viruses. The current study demonstrated increased susceptibility of 27 lipid-enveloped respiratory pathogens of importance such as SARS-CoV-2 (causative 28 agent of COVID-19) as well as the influenza A virus to 405nm, visible light in the 29 absence of exogenous photosensitizers indicating a potential porphyrin-independent 30 alternative mechanism of visible light mediated viral inactivation. These results were 31 obtained using less than expected irradiance levels which are generally safe for humans 32 and commercially achievable. Our results support further exploration of the use of 33 visible light technology for the application of continuous decontamination in occupied 34 areas within hospitals and/or infectious disease laboratories, specifically for the 35 inactivation of respiratory pathogens such as SARS-CoV-2 and Influenza A. For the range of output used in this study, multiple discrete levels were created using 153 pulse width modulation within the LED driver itself. These levels were made to be 154 individually selectable using a simple knob on the attached control module. 155

As expected, the amount of visible light within the 400nm-420nm bandwidth, measured 156 in mWcm -2 , is a measurement of the "dose" delivered to the target organism and is used 157 to quantify this relationship similar to that used in UV disinfection applications. 158

To fully examine this effect, a range of irradiance values were used representing actual 159 product deployment conditions in occupied rooms. The lowest value (0.035 mWcm -2 ) 160 represents a single-mode, lower wattage used in general lighting applications while the 161 highest value (0.6 mWcm -2 ) represents a dual-mode, higher wattage used in critical care 162 applications such as an operating room. 163

The device was placed in a rig to ensure a consistent distance (10") between the fixture 164 and the samples. The output of the fixture in the test rig was measured using a Stellar-165 RAD Radiometer from StellarNet configured to make wavelength and irradiance 166 measurements from 350nm-1100nm with < 1nm spectral bandwidth using a NIST 167 traceable calibration. To ensure that the regular white light portion of the illumination 168 (which is non-disinfecting) was not measured, the measurement was electronically 169 limited to a 1nm bandwidth over the 400nm-420nm range. The normalized spectral 170 profile is shown in Fig. 3 below. The absolute value of the measurement was 171 determined using a NIST traceable calibration as previously described. Samples were irradiated with the range of wavelengths depicted in Figure 3 . This was 177 deliberately done for two reasons: 1) prior work had shown that visible light disinfection 178 was primarily active at 405nm 33 and 2) to emphasize the applied science associated 179 with actual clinical use where a virus in the environment could be exposed to both 180 405nm and regular white light in an occupied room. 181

To isolate the contribution of 405nm light, the control samples were placed outside the 182 field of irradiation created by the disinfection product but within the biosafety hood. 183

Accordingly, these samples were exposed to the overhead lights within the room which 184 contained virtually no 405nm light (< 0.001 mWcm -2 ). 185

In any assessment of viral inactivation, thermal denaturing of the organism is a concern. 186 Older lighting technologies such as incandescent sources heat a resistive element and 187

were widely used in a variety of applications. This creates heat at both the source and 188 to objects within its field. Fortunately, the disinfecting light (sample) and the overhead 189 lights in the room (control) did not use this technology and therefore contain no infrared 190 emissions (> 800 nm) a commonly known benefit associated with LED lighting. As a 191 confirmation, the temperature beneath the disinfecting light was measured using a are commonly used in the overhead lights within buildings, the lights in the BSL-3 199 laboratory were traditional fluorescent. As shown in Figure 2 , the controls were 200 exposed only to traditional fluorescent lighting with a negligible amount of disinfecting 201 light (< 0.001 mWcm -2 ) between 400nm and 420nm. Due to the inherent differences 202 between fluorescent and LED lighting, the standard LED spectra has a small, but 203 measurable amount of disinfecting light (0.006 mWcm-2) between 400nm and 420nm. 204

As will be later shown, this amount of light can have a measurable disinfecting effect. 205 penicillin-streptomycin at 37°C with 5% CO2 for 72 hours. Influenza A virus used here 224 was generated using plasmid based reverse genetics system as previously described 34 . 225

The backbone used in the study was A/Puerto Rico/8/34/Mount Sinai(H1N1) under the 226 GenBank accession number AF389122. IAV-PR8 virus was grown and titrated in MDCK 227 as previously described 34 . As a non-enveloped virus, the cell culture adapted murine 228

Vero-CCL81 cells with DMEM and 2% HI-FBS and penicillin-streptomycin at 37°C with 230 5% CO2 for 48 hours 35 . 231

The SARS-CoV-2 virus was exclusively handled at the Icahn school of Medicine BSL-3 233 and studies involving IAV and EMCV were handled in BSL-2 conditions. Indicated PFU 234 amounts were mixed with sterile 1X PBS and were irradiated in 96 well format cell 235 culture plates in triplicates. In these studies, A starting dose of 5x10 5 PFU for SARS-236

CoV-2 and starting doses of 1x10 5 PFU for IAV and EMCV were used. The final 237 volumes for inactivation were 250 μl per replicate. The untreated samples were 238 prepared the same way and were left inside the biosafety cabinet isolated from the 239 inactivation device at room temperature. The plates were sealed with qPCR plate 240 transparent seal and an approximate 10% reduction of the intensity was observed due 241 to the sealing film. The distance from the lamp and the samples was measured to be 242 10". All samples were extracted at indicated times and were frozen at -80°C and were 243 thawed together for titration via plaque assays. 244

For SARS-CoV-2 studies, confluent monolayers of Vero-E6 cells in 12-well plate format 246 were infected (with an inoculum volume 150μl) with 10-fold serially diluted samples in 247 

The stability of IAV virus at room temperature for a period of 8 hours was found to be 311 the negligible in untreated IAV spiked PBS samples ( Figure 6A ). 312

In order to better understand the effect of the lipid-envelope in viral inactivation by 405 315 nm irradiation, we used a non-lipid enveloped RNA virus derived from the 316 Picornaviridae family. EMCV virus was irradiated at a high dose of 0.6 mWcm -2 similar 317 to SARS-CoV-2 and IAV. 318 

In this case however, a total reduction of 0.0969 (approximately 2 times) in comparison 327

to the untreated after 8 hours of irradiation was observed (Fig 7A and 7B) The ongoing SARS-CoV-2 pandemic has affected the day-to-day functions in the entire 342 world, raising concerns not only with regards to therapeutics but also in the context of 343 virus survivorship and decontamination 36 . Taking into consideration the rapid spread of 344 SARS-CoV-2 from person to person by droplets, aerosols, and fomites, whole-room 345 disinfection systems can be viewed as a supplement to best practices for interrupting 346 transmission of the virus. 347

Given the ongoing COVID-19 pandemic, we wanted to explore the impact of 405 nm 348 enriched visible light technology on inactivation of respiratory pathogens such as SARS-349

Without the use of exogenous photosensitizers, we were able to show that irradiation 351 with low intensity (0.035 mWcm -2 ) visible light yielded a reduction of log10 0.3288 352 inactivation after four hours (0.5 Jcm -2 ) and a total log10 1.0325 inactivation of SARS-353

CoV-2 after 24 hours (3.02 Jcm -2 ). A slightly higher dose (0.076 mWcm -2 ) resulted in 354 log10 1.5393 reduction after 24 hours (6.56 Jcm -2 ) while an irradiation dose of 0.150 355 mWcm -2 showed a reduction log10 2.0056 after 24 hours (12.96 Jcm -2 ) of irradiation. 356

Finally, increasing the dose to 0.6 mWcm -2 yielded log10 2.3010 reduction only after 357 eight hours (12.96 Jcm -2 ), indicating a both time and dose dependent inactivation of 358 infectious viruses. 359

The irradiations using standard LED lighting raise some interesting questions for further 360 discussion. With nearly 6x the amount of disinfecting light as compared to traditional 361 fluorescent lighting but 1/6 th the lowest amount of 405nm used in the study; it is 362 conceivable that a reduction could be observed. The magnitude of this effect at less than 24h was larger than expected based on other irradiations performed in this study. conditions. While both viruses have lipid envelopes, there is clearly a difference here 384 that will require further study. One possible explanation is the difference in the virion 385 size creating a physically smaller cross-section for absorption. (IAV ~120 nm and SARS-CoV-2 ~200 nm) 37, 38 . Nevertheless, both viruses were largely inactivated after Philanthropy Project (research grant 2020-215611 (5384)) and anonymous donors to 501 AG-S, and by a research contract from Kenall Manufacturing to the AG-S lab. 502

The García-Sastre Laboratory has received research support from Pfizer, Senhwa 504

Biosciences, 7Hills Pharma, Avimex, Blade Therapeutics, Dynavax, ImmunityBio, 505

Nanocomposix and Kenall Manufacturing. Adolfo García-Sastre has consulting 506 agreements for the following companies involving cash and/or stock: Vivaldi 507

Biosciences, Pagoda, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Accurius, Pfizer and 508

Esperovax. RR, CY and AGS have filed for a provisional patent based upon these 509 results. 510

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387 eight hours achieving more 1.5 log10 reduction. Intriguingly, it was observed that both 388 RNA viruses were able to remain stable at room temperature for at least 24 hours, 389indicating minimal decay which is consistent with previous studies 36, 39 . We next 390 irradiated a non-enveloped RNA virus, EMCV. Previous results for visible light against 391 non-enveloped viruses demonstrated the need for external photosensitizers such as 392 artificial saliva, blood, feces, etc 30, 36 . Without a porphyrin containing medium, we 393 expected little to no inactivation when this virus was irradiated with visible light. For 394 these measurements, we used the highest available irradiance of 0.6 mWcm -2 . As 395 anticipated, we observed only a log10 0.0969 reduction after eight hours, however, this 396 appears to be with the statistical precision of the measurement based on the results 397 obtained from shorter irradiations (1, 2, and 4 hours). For comparison, a study involving 398 the M13-bacteriophage virus (a non-enveloped virus) showed a 3-Log reduction using 399 an irradiance of 50mWcm -2 (almost 100 times greater than the highest irradiance used 400 in this study) for 10 hours at 425 nm further supporting the idea that non-enveloped 401 viruses may require higher doses of visible light 40 . 402Our study was conducted using a neutral liquid media composed of PBS without any 403 photosensitizers and we were able to show that visible light can indeed inactivate lipid-404 enveloped viruses, differing from the theory that states that photosensitizers are a 405 requirement for inactivation. While these results provide insight into the basic science 406 involved, they were performed within the context of the applied science needed to show 407 the potential impact of this technology upon the current COVID-19 pandemic. By using 408 safe, commercially practical irradiance levels, our results are more directly translatable 409 to occupied rooms in the clinical environment. impossible. It is interesting to note that they did observe viral inactivation in their 425 controls from wavelengths less than 420nm confirming the qualitative findings of our 426 study without confirming the specific use of 405nm. This suggests that the viral 427 inactivation is a likely a broad response (> 20nm) with relative contributions unique to 428 the chemistry of each organism. They also considered much longer exposures (~7 429 days) and much higher illuminance (> 200 lux) than that used in our study although this 430 is again difficult to compare given the lack of radiometric quantification of their light 431 source. It is important to note that the control samples used in our study were exposed 432 to the same overhead (non-405nm) lights as the irradiated samples and our results are 433 the observed difference between the two demonstrating the contribution from 405nm 434 over and above that potentially from 420-430nm. Future experiments can further 435 quantify the potential effect. 436The other theory, potential UV-A irradiation, was historically applied to lamp-based 437 sources with broad spectral (> 100nm) outputs. Again, the use of LED technology 438 addresses this question as the peak irradiance at 390nm of the device used in this 439 study was < 1% of its peak irradiance at 405nm without the need for any additional 440 filtration. Future experiments can further quantify the potential effect. 441Another consideration to be addressed is thermal heating of the virus by the LED 442 source. Tsen and Achilefu used a pulsed laser method at 425nm 43 with ~100 mWcm -2 443 average power density for < 2h while simultaneously measuring the sample temperature 444 with a thermocouple. They detected less than a 2°C demonstrating minimal temperature 445 impact even under a power density nearly 9 orders of magnitude larger than that used 446 in this study. This was confirmed by our thermocouple measurements as stated earlier. 10s, nearly 1000 times that used in this study 44 . While the total energy delivered was 449 more comparable to that used in our study, they did not make explicit temperature 450 measurements, their analysis ruled out any potential thermal effects. This question could serve as the basis for a range of future studies. 456The results obtained suggest that the performance of visible light against SARS-CoV-2 457 is similar to other organisms commonly found in the environment such as S. aureus. 458Previous studies have shown that the visible light irradiance levels used in this study 459 (0.035 mWcm -2 to 0.6 mWcm -2 ) reduce bacteria levels in occupied rooms and improve 460 outcomes for surgical procedures. It is therefore reasonable to conclude that visible light 461 might be an effective disinfectant against SARS-CoV-2. More importantly, this 462 disinfection can operate continuously as it is safe for humans based upon the exposure 463 guidelines in IEC 62471 45 . This means that once it has been in use for a period of time, 464 the environment will be cleaner and safer the next time it is occupied by humans. 465One limitation of this study is that the inactivation assays were performed in static liquid 

We thank Kenall Manufacturing for supplying the M4DLIC6 fixture and test rig used in 495 this study. We thank Randy Albrecht for support with the BSL3 facility and procedures 496 at the ISMMS, and Richard Cadagan for technical assistance. This research was partly 497 funded by CRIP (Center for Research for Influenza Pathogenesis), a NIAID supported 498Center of Excellence for Influenza Research and Surveillance (CEIRS, contract # 499 HHSN272201400008C); by the generous support of the JPB Foundation, the Open