Far-UVC light efficiently and safely inactivates airborne human coronaviruses CURRENT STATUS: UNDER REVIEW

A direct approach to limit airborne transmission of pathogens is to inactivate them within a short time of their production. Germicidal ultraviolet light (UV), typically at 254 nm, is effective in this context, but it is a health hazard to the skin and eyes. By contrast, far-UVC light (207-222 nm) efficiently kills pathogens without harm to exposed human cells or tissues. We previously demonstrated that 222-nm UV light efficiently kills airborne influenza virus (H1N1); here we extend the far-UVC studies to explore efficacy against human coronaviruses from subgroups alpha (HCoV-229E) and beta (HCoV-OC43). We found that low doses of, respectively 1.7 and 1.2 mJ/cm 2 inactivated 99.9% of aerosolized alpha coronavirus 229E and beta coronavirus OC43. Based on these results for the beta HCoV-OC43 coronavirus, continuous far-UVC exposure in public locations at the currently recommended exposure limit (3 mJ/cm 2 /hour) would result in 99.9% viral inactivation in ~ 25 minutes. Increasing the far- UVC intensity by, say, a factor of 2 would halve these disinfection times, while still maintaining safety. As all human coronaviruses have similar genomic size, a key determinant of radiation sensitivity, it is realistic to expect that far-UVC light will show comparable inactivation efficiency against other human coronaviruses, including SARS-CoV-2. The , infection incubated for three or four days at 34ºC. Cytopathic effects (CPE) were scored at a bright field microscope (10x) as vacuolization of cytoplasm, cell rounding and sloughing. The TCID 50 was calculated with the Reed and Muench method 28,35 . To confirm the CPE scores, the samples were fixed in 100% methanol for five minutes and stained with 0.1% crystal violet. The results are reported as the estimate of plaque forming units (PFU)/ml using the conversion PFU/ml = 0.7 TCID 50 by applying the Poisson distribution 29


Introduction
Coronavirus disease 2019 (COVID- 19) was first reported in December 2019 and then characterized as a pandemic by the World Health Organization on March 11, 2020. Despite extensive efforts to contain the spread of the disease, it has spread worldwide with over 1.9 million confirmed cases and over 130,000 confirmed deaths as of April 16, 2020 1 . Transmission of SARS-CoV-2, the beta coronavirus causing COVID-19, is both through direct contact and airborne routes, and studies of SARS-CoV-2 stability have shown viability in aerosols for at least 3 hours 2 . Given the rapid spread of the disease, including through asymptomatic carriers 3 , it is of clear importance to explore practical mitigation technologies that can inactivate the airborne virus in public locations and thus limit airborne transmission.
Ultraviolet (UV) light exposure is a direct antimicrobial approach 4 and its effectiveness against different strains of airborne viruses has long been established 5 . The most commonly employed type of UV light for germicidal applications is a low pressure mercury-vapor arc lamp, emitting around 254 nm; more recently xenon lamp technology has been used, which emits broad UV spectrum 6 .
However, while these lamps can be used to disinfect unoccupied spaces, operation of these conventional germicidal UV lamps in occupied public spaces -critical to prevent person-to-person transmission -is not possible since exposure to these wavelengths is a health hazard, causing both skin cancer and eye diseases [7][8][9][10] .
By contrast far-UVC light (207 to 222 nm) has been shown to be as efficient as conventional germicidal UV light in killing microorganisms 11 , but it does not have the human health issues associated with conventional germicidal UV light. In short (see below) the reason is that far-UVC has a range in biological materials of only a few micrometers, and thus it cannot reach living human cells in the skin or eyes. But because viruses (and bacteria) are extremely small, far-UVC light can still penetrate and kill them. Thus far-UVC light has about the same highly effective germicidal properties of UV light, but without the associated human health risks [12][13][14][15] . Several groups have thus proposed that far-UVC light (207 or 222 nm), which can be generated using inexpensive excimer lamps, is a potential safe and efficient anti-microbial technology [12][13][14][15][16][17][18] which can be deployed in occupied public locations.
The physics-based mechanistic basis to this far-UVC approach 12 is that light in this wavelength range has a very limited penetration depth. Specifically, far-UVC light (207-222 nm) is very strongly absorbed by proteins through the peptide bond, and other biomolecules 19,20 , so its ability to penetrate biological materials is very limited compared with, for example, 254 nm (or higher) conventional germicidal UV light 21,22 . This limited penetration is still much larger than the size of viruses and bacteria, so far-UVC light is as efficient in killing these pathogens as conventional germicidal UV light 12-14. However, unlike germicidal UV light, far-UVC light cannot penetrate either the human stratum corneum (the outer dead-cell skin layer), nor the ocular tear layer, nor even the cytoplasm of individual human cells. Thus, far-UVC light cannot reach or damage living cells in the human skin or the human eye, in contrast to the conventional germicidal UV light which can reach these sensitive cells [7][8][9][10] .
In summary far-UVC light is anticipated to have about the same anti-microbial properties as conventional germicidal UV light, but without producing the corresponding health effects. Should this be the case, far-UVC light has the potential to be used in occupied public settings to prevent the airborne person-to-person transmission of pathogens such as coronaviruses.
We have previously shown that a very small dose (2 mJ/cm 2 ) of far-UVC light at 222 nm was highly efficient in inactivating aerosolized H1N1 influenza virus 23 . In this work we explore the efficacy of 222 nm light against two airborne human coronaviruses: alpha HCoV-229E and beta HCoV-OC43.
Both were isolated over 50 years ago and are endemic to the human population, causing 15-30% of respiratory tract infections each year 24 . Like SARS-CoV-2, the HCoV-OC43 virus is from the beta subgroup 25 .
Here we measured the efficiency with which far-UVC light inactivates these two human coronaviruses when exposed in aerosol droplets of sizes similar to those generated during sneezing and coughing 26 . As all coronaviruses have comparable physical and genomic size, a critical determinant of radiation response 27 , we hypothesized that both viruses would respond similarly to far-UVC light, and indeed that all coronaviruses will respond similarly.

Results
Inactivation of human coronaviruses after exposure to 222 nm light in aerosols Infectivity Assay. We used a standard approach to measure viral inactivation, assaying coronavirus infectivity in human host cells (normal lung cells), in this case after exposure in aerosols to different doses of far-UVC light. We quantified virus infectivity with the 50% tissue culture infectious dose TCID 50 assay 28 , and estimated the corresponding plaque forming units (PFU)/ml using the conversion PFU/ml = 0.7 TCID 50 29 . Fig. 1 shows the fractional survival of aerosolized coronaviruses HCoV-229E and HCoV-OC43 expressed as PFU UV /PFU controls as a function of the incident 222-nm dose. Robust linear regression (  2) and HCoV-OC43 (Fig. 3), which had been exposed in aerosolized form to different far-UVC doses. The viral solution was collected from the BioSampler after running through the aerosol chamber while being exposed to 0, 0.5, 1 or 2 mJ/cm 2 of 222-nm light. Cells were incubated with the exposed virus for one hour, the medium was replaced with fresh infection medium, and immunofluorescence was performed 24 hours later. We assessed the human cell lines for expression of the viral spike glycoprotein, whose functional subunit S2 is highly conserved among coronaviruses 31,32 . In Figs

Discussion
The severity of the 2020 COVID-19 pandemic warrants the rapid development and deployment of effective countermeasures to reduce person-to-person transmission. We have developed a promising approach using single-wavelength far-UVC light at 222 nm generated by filtered excimer lamps, which inactivate viruses and bacteria, without inducing biological damage in exposed human cells and tissue 11-16. The approach is based on the biophysically-based principle that far-UVC light, because of its very limited penetration in biological materials, can traverse and kill viruses and bacteria which are typically micrometer dimensions or smaller, but it cannot penetrate even the outer dead-cell layers of human skin, nor the outer tear layer on the surface of the human eye 12 .
In this work we have used an aerosol irradiation chamber to test the efficacy of 222-nm far-UVC light to inactivate two aerosolized human coronaviruses, beta HCoV-OC43 and alpha HCoV-229E. As shown in Fig values we have obtained for coronaviruses is shown in Table 2, together with our earlier results for aerosolized H1N1 influenza virus 23 .
The results suggest that both of the studied coronavirus strains have similar high sensitivity to far-UVC inactivation. Robust linear regression produced overlapping 95% confidence intervals for the inactivation rate constant, k, of 2.5 to 4.8 cm 2 /mJ and 3.8 to 7.1 cm 2 /mJ respectively for the 229E and OC43 strains. As all human coronaviruses have similar genomic sizes which is a primary determinant of UV sensitivity 27 , it is reasonable to expect that far-UVC light will show similar inactivation efficiency against all human coronaviruses, including SARS-CoV-2. The data obtained here are consistent with this hypothesis.
It is useful to compare the performance of far-UVC light with conventional germicidal (peak 254 nm) UVC exposure. We are aware of only one such study 33 , which used an aerosolized murine beta coronavirus. The study reported a D 88 of 0.599 mJ/cm 2 , which others 4 have used to estimate the D 90 for the virus with 254 nm light as 0.6 mJ/cm 2 . This value is similar to those estimated in the current work (see Table 2), suggesting similar inactivation efficiency of 222 nm far-UVC and conventional germicidal 254 nm UVC for aerosolized coronavirus, and providing further support for the suggestion that all coronaviruses have similar sensitivities to UV light.
The sensitivity of the coronaviruses to far-UVC light, together with extensive safety data even at much higher far-UVC exposures [12][13][14][15][16][17][18] , suggests that it will be feasible and safe to have the lamps providing continuous far-UVC exposure in public places to significantly reduce the probability of  Benchtop Aerosol Irradiation Chamber. A one-pass, dynamic aerosol/virus irradiation chamber was used to generate, expose, and collect aerosol samples as previously described 23 . Viral aerosols were generated by adding a virus solution in a high-output extended aerosol respiratory therapy nebulizer (Westmed, Tucson, AZ) and operating using an air pump with an input flow rate of 11 L/min. Virus flowed into the chamber and was mixed with dry and humidified air to maintain humidity between approximately 50-70%. The relative humidity, temperature, and aerosol particle size distribution were monitored throughout operation. Aerosol was exposed to far-UVC light and finally collected using a Biosampler (SKC Inc., Eighty Four, PA).
The far-UVC lamp was positioned approximately 22 cm away from the UV exposure chamber and directed at the 26 cm × 25.6 cm × 254 µm UV-transmitting plastic window (Topas 8007x10, Topas Advanced Polymers, Florence, KY). Consistent with our previous experiments using this chamber 23 , the flow rate through the system was 12.5 L/min. The volume of the UV exposure region was 4.2 L so each aerosol was exposed for approximately 20 seconds as it traversed the window. The entire irradiation chamber was contained in a biosafety level 2 cabinet and all air inputs and outputs were equipped with HEPA filters (GE Healthcare Bio-Sciences, Pittsburgh, PA) to prevent unwanted contamination from entering or exiting the system.
Irradiation Chamber Performance. The custom irradiation chamber simulated the transmission of aerosolized viruses produced via human coughing and breathing. The chamber operated at an average relative humidity of 66% and an average temperature of 24° C across all runs. The average particle size distribution was 83% between 0.3 µm and 0.5 µm, 12% between 0.5 µm and 0.7 µm, and 5% > 0.7 µm (Table 3). Aerosolized viruses were efficiently transmitted through the system as The distance between the lamp and the irradiation chamber permitted a single lamp to uniformly irradiate the entire exposure window area. Measurements using the silicon photodetector indicated an exposure intensity of approximately 90 µW/cm 2 across the exposure area. The chamber is equipped with a reflective aluminum surface opposite of the exposure window. As in our previous work with this chamber 23 , the reflectivity of this surface was approximately 15%. We have therefore conservatively estimated the intensity across the entire exposure area to be 100 µW/cm 2 . With the lamp positioned exposure window, we calculated the total exposure dose to a particle to be 2 mJ/cm 2 . We used additional sheets of UV transmitting plastic windows to uniformly reduce the intensity across the exposure region to create different exposure conditions. While in our previous work with these sheets we measured a transmission closer to 65% 23 , for these tests we measured the 222-nm transmission of each sheet to be approximately 50%. This decrease in transmission is likely due to the photodegradation of the plastic over time 4 . The addition of one or two sheets of the plastic covering the exposure window decreases the exposure dose to 1 and 0.5 mJ/cm 2 , respectively.
Experimental Protocol. As previously described 23  Virus Infectivity Assays.
TCID 50. We used the 50% tissue culture infectious dose assay to determine virus infectivity 28 . Briefly, 10 5 host cells were plated in each well of 96-well plates the day prior the experiment. Cells were washed twice in HBSS ++ and serial 1:10 dilutions in infection medium of the exposed virus from the BioSampler was overlaid on cells for two hours. The cells were then washed twice in HBSS ++ , covered with fresh infection medium, and incubated for three or four days at 34ºC. Cytopathic effects (CPE) were scored at a bright field microscope (10x) as vacuolization of cytoplasm, cell rounding and sloughing. The TCID 50 was calculated with the Reed and Muench method 28,35 .      Table 1).

Figure 2
Infection of human lung cells from irradiated aerosolized alpha HCoV-229E as function of dose of far-UVC light. Representative fluorescent images of MRC-5 normal human lung fibroblasts infected with human alphacoronavirus 229E exposed in aerosolized form. The viral solution was collected from the BioSampler after running through the aerosol chamber while being exposed to 0, 0.5, 1 or 2 mJ/cm2 of 222-nm light. Green fluorescence qualitatively indicates infected cells (Green= Alexa Fluor®-488 used as secondary antibody against anti-human coronavirus spike glycoprotein antibody; Blue = nuclear stain DAPI).
Images were acquired with a 10x objective; the scale bar applies to all the panels in the figure. Images were acquired with a 10x objective; the scale bar applies to all the panels in the figure.