Far-UVC light: A new tool to control the spread of airborne-mediated microbial diseases

Airborne-mediated microbial diseases such as influenza and tuberculosis represent major public health challenges. A direct approach to prevent airborne transmission is inactivation of airborne pathogens, and the airborne antimicrobial potential of UVC ultraviolet light has long been established; however, its widespread use in public settings is limited because conventional UVC light sources are both carcinogenic and cataractogenic. By contrast, we have previously shown that far-UVC light (207–222 nm) efficiently inactivates bacteria without harm to exposed mammalian skin. This is because, due to its strong absorbance in biological materials, far-UVC light cannot penetrate even the outer (non living) layers of human skin or eye; however, because bacteria and viruses are of micrometer or smaller dimensions, far-UVC can penetrate and inactivate them. We show for the first time that far-UVC efficiently inactivates airborne aerosolized viruses, with a very low dose of 2 mJ/cm2 of 222-nm light inactivating >95% of aerosolized H1N1 influenza virus. Continuous very low dose-rate far-UVC light in indoor public locations is a promising, safe and inexpensive tool to reduce the spread of airborne-mediated microbial diseases.

cells in a particular field of view, while green fluorescence indicated the integration of live influenza A (H1N1) viruses into the cells. Results from the zero-dose control studies (Fig. 1, top left) confirmed that the aerosol irradiation chamber efficiently transmitted the aerosolized viruses through the system, after which the live virus efficiently infected the test mammalian epithelial cells. Figure 2 shows the surviving fraction, as a function of the incident 222-nm far-UVC dose, of exposed H1N1 aerosolized viruses, as measured by the number of focus forming units in incubated epithelial cells relative to unexposed controls. Linear regressions (see below) showed that the survival results were consistent with a classical exponential UV disinfection model with rate constant k = 1.8 cm 2 /mJ (95% confidence intervals 1.5-2.1 cm 2 / mJ). The overall model fit was good, with a coefficient of determination, R 2 = 0.95, which suggests that most of the  variability in virus survival was explained by the exponential model. The rate constant of 1.8 cm 2 /mJ corresponds to an inactivation cross-section (dose required to inactivate 95% of the exposed viruses) of D 95 = 1.6 mJ/cm 2 (95% confidence intervals 1.4-1.9 mJ/cm 2 ).

Discussion
We have developed an approach to UV-based sterilization using single-wavelength far-UVC light generated by filtered excilamps, which selectively inactivate microorganisms, but does not produce biological damage to exposed mammalian cells and tissues [13][14][15] . The approach is based on biophysical principles in that far-UVC light can traverse and therefore inactivate bacteria and viruses which are typically micrometer dimensions or smaller, whereas due to its strong absorbance in biological materials, far-UVC light cannot penetrate even the outer dead-cell layers of human skin, nor the outer tear layer on the surface of the eye.
Here we applied this approach to test the efficacy of the 222-nm far-UVC light to inactivate influenza A virus (H1N1) carried by aerosols in a benchtop aerosol UV irradiation chamber, which generated aerosol droplets of sizes similar to those generated by human coughing and breathing. Aerosolized viruses flowing through the irradiation chamber were exposed to UVC emitting lamps placed in front of the chamber window.
As shown in Fig. 2, inactivation of influenza A virus (H1N1) by 222-nm far-UVC light follows a typical exponential disinfection model, with an inactivation cross-section of D 95 = 1.6 mJ/cm 2 (95% CI: 1.4-1.9). For comparison, using a similar experimental arrangement, but using a conventional 254 nm germicidal UVC lamp, McDevitt et al. 19 found a D 95 value of 1.1 mJ/cm 2 (95% CI: 1.0-1.2) for H1N1 virus. Thus as we 13,15 and others [16][17][18] reported in earlier studies for bacterial inactivation, 222-nm far-UVC light and 254-nm broad-spectrum germicidal light are also comparable in their efficiencies for aerosolized viral inactivation. Other recent work comparing viral inactivation across the UVC spectrum has shown variations in efficiency are expected, but in general both regions of the spectrum are effective in inactivation, though the precise cause of inactivation may differ 20,21 . However as discussed above, based on biophysical considerations and in contrast to the known human health safety issues associated with conventional germicidal 254-nm broad-spectrum UVC light, far-UVC light does not appear to be cytotoxic to exposed human cells and tissues in vitro or in vivo [13][14][15] .
If these results are confirmed in other scenarios, it follows that the use of overhead low-level far-UVC light in public locations may represent a safe and efficient methodology for limiting the transmission and spread of airborne-mediated microbial diseases such as influenza and tuberculosis. In fact the potential use of ultraviolet light for airborne disinfection is by no means new, and was first demonstrated more than 80 years ago 8,22 . As applied more recently, airborne ultraviolet germicidal irradiation (UVGI) utilizes conventional germicidal UVC light in the upper part of the room, with louvers to prevent direct exposure of potentially occupied room areas 23 . This results in blocking more than 95% of the UV radiation exiting the UVGI fixture, with substantial decrease in effectiveness 24 . By contrast, use of low-level far-UVC fixtures, which are potentially safe for human exposure, could provide the desired antimicrobial benefits without the accompanying human health concerns of conventional germicidal lamp UVGI.
A key advantage of the UVC based approach, which is in clear contrast to vaccination approaches, is that UVC light is likely to be effective against all airborne microbes. For example, while there will almost certainly be variations in UVC inactivation efficiency as different influenza strains appear, they are unlikely to be large 7,10 . Likewise, as multi-drug-resistant variants of bacteria emerge, their UVC inactivation efficiencies are also unlikely to change greatly 9 .
In conclusion, we have shown for the first time that very low doses of far-UVC light efficiently inactivate airborne viruses carried by aerosols. For example, a very low dose of 2 mJ/cm 2 of 222-nm light inactivates >95% of airborne H1N1 virus. Our results indicate that far-UVC light is a powerful and inexpensive approach for prevention and reduction of airborne viral infections without the human health hazards inherent with conventional germicidal UVC lamps. If these results are confirmed in other scenarios, it follows that the use of overhead very low level far-UVC light in public locations may represent a safe and efficient methodology for limiting the transmission and spread of airborne-mediated microbial diseases. Public locations such as hospitals, doctors' offices, schools, airports and airplanes might be considered here. This approach may help limit seasonal influenza epidemics, transmission of tuberculosis, as well as major pandemics.

Methods
Far-UVC lamps. We used a bank of three excimer lamps containing a Kr-Cl gas mixture that predominantly emits at 222 nm 25,26 . The exit window of each lamp was covered with a custom bandpass filter designed to remove all but the dominant emission wavelength as previously described 15 . Each bandpass filter (Omega Optical, Brattleboro, VT) had a center wavelength of 222 nm and a full width at half maximum (FWHM) of 25 nm and enables >20% transmission at 222 nm. A UV spectrometer (SPM-002-BT64, Photon Control, BC, Canada) with a sensitivity range between 190 nm and 400 nm was utilized to verify the 222 nm emission spectrum. A deuterium lamp standard with a NIST-traceable spectral irradiance (Newport Model 63945, Irvine, CA) was used to radiometrically calibrate the UV spectrometer. An SM-70 Ozone Monitor (Aeroqual, Avondale, Auckland, New Zealand) measured the ozone generation from the lamps to be <0.005 ppm, which is not a significant level to provide an antimicrobial effect to aerosolized viruses 27 .
Far-UVC dosimetry. Optical power measurements were performed using an 818-UV/DB low-power UV enhanced silicon photodetector with an 843-R optical power meter (Newport, Irvine, CA). Additional dosimetry to determine the uniformity of the UV exposure was performed using far-UVC sensitive film as described in our previous work 28,29 . This film has a high spatial resolution with the ability to resolve features to at least 25 µm, and exhibits a nearly ideal cosine response 30,31 . Measurements were taken between experiments therefore allowing placement of sensors inside the chamber.
Scientific RePoRtS | (2018) 8:2752 | DOI:10.1038/s41598-018-21058-w A range of far-UVC exposures, from 3.6 µJ/cm 2 up to 281.6 mJ/cm 2 , were used to define a response calibration curve. Films were scanned as 48 bit RGB TIFF images at 150 dpi using an Epson Perfection V700 Photo flatbed scanner (Epson, Japan) and analyzed with radiochromic film analysis software 32 to calculate the total exposure based on measured changes in optical density.
Measurements using both a silicon detector and UV sensitive films were combined to compute the total dose received by a particle traversing the exposure window. The three vertically stacked lamps produced a nearly uniform dose distribution along the vertical axis thus every particle passing horizontally through the irradiation chamber received an identical dose. The lamp width (100 mm) was smaller than the width of the irradiation chamber window (260 mm) so the lamp power was higher near the center of the irradiation chamber window compared to the edge. The UV sensitive film indicated a power of approximately 120 µW/cm 2 in the center third of the window and 70 µW/cm 2 for the outer thirds. The silicon detector was used to quantify the reflectivity of the aluminum sheet at approximately 15% of the incident power. Combining this data allowed the calculation of the average total dose of 2.0 mJ/cm 2 to a particle traversing the window in 20 seconds. Additionally, the silicon detector was used to confirm the attenuation of 222-nm light through a single sheet of plastic film was 65%. The addition of one or two sheets of plastic film between the lamps and the irradiation chamber window yielded average doses of 1.3 mJ/cm 2 and 0.8 mJ/cm 2 , respectively.
Benchtop aerosol irradiation chamber. A one-pass, dynamic aerosol / virus irradiation chamber was constructed in a similar configuration to that used by Ko et al. 33 , Lai et al. 34 , and McDevitt et al. 19,35 . A schematic overview of the system is shown in Fig. 3 and is pictured in Fig. 4. Aerosolized viruses were generated by adding a virus solution into a high-output extended aerosol respiratory therapy (HEART) nebulizer (Westmed, Tucson, AZ) and operated using a dual-head pump (Thermo Fisher 420-2901-00FK, Waltham, MA) with an input flow rate of 11 L/min. The aerosolized virus flowed into the irradiation chamber where it was mixed with independently controlled inputs of humidified and dried air. Humidified air was produced by bubbling air through water, while dry air was provided by passing air through a desiccant air dryer (X06-02-00, Wilkerson Corp, Richland, MI). Adjusting the ratio of humid and dry air enabled control of the relative humidity (RH) within the irradiation chamber which, along with the nebulizer settings, determined the aerosol particle size distribution. An optimal RH value of 55% resulted in a distribution of aerosol particle sizes similar to the natural distribution from human coughing and breathing, which has been shown to be distributed around approximately 1 µm, with a significant tail of particles less than 1 µm 36-38 .
After combining the humidity control inputs with the aerosolized virus, input flow was directed through a series of baffles that promoted droplet drying and mixing to produce an even particle distribution and stable humidity 34 . The RH and temperature inside the irradiation chamber were monitored using an Omega RH32 meter (Omega Engineering Inc., Stamford, CT) immediately following the baffles. A Hal Technologies HAL-HPC300 particle sizer (Fontana, CA) was adjoined to the irradiation chamber to allow for sampling of particle sizes throughout operation.
During UV exposure, the 222-nm lamps were placed 11 cm from the irradiation chamber window. The lamps were directed at the 26 cm × 25.6 cm chamber window which was constructed of 254-µm thick UV transparent plastic film (Topas 8007x10, Topas Advanced Polymers, Florence, KY), and which had a transmission of ~65% at 222 nm. The wall of the irradiation chamber opposite the transparent window was constructed with polished aluminum in order to reflect a portion of the UVC light back through the exposure region, therefore increasing the overall exposure dose by having photons pass in both directions. The depth of the irradiation chamber between the window and the aluminum panel was 6.3 cm, creating a total exposure volume of 4.2 L. Flow of the aerosols continues out of the irradiation chamber to a set of three way valves that could be configured to either pass through a bypass channel (used when no sampling was required), or a BioSampler (SKC Inc, Eighty Four, PA) used to collect the virus. The BioSampler uses sonic flow impingement upon a liquid surface to collect aerosols when operated at an air flow of 12.5 L/min. Finally, flow continued out of the system through a final HEPA filter and to a vacuum pump (WP6111560, EMD Millipore, Billerica, MA). The vacuum pump at the end of the system powered flow through the irradiation chamber. The flow rate through the system was governed by the BioSampler. Given the flow rate and the total exposure volume of the irradiation chamber, 4.2 L, a single aerosol droplet passed through the exposure volume in approximately 20 seconds.
The entire irradiation chamber was set up inside a certified class II type A2 biosafety cabinet (Labconco, Kansas City, MO). All air inputs and outputs were equipped with HEPA filters (GE Healthcare Bio-Sciences, Pittsburgh, PA) to prevent unwanted contamination from entering the chamber as well as to block any of the virus from releasing into the environment.
Irradiation chamber performance. The custom irradiation chamber simulated the transmission of aerosolized viruses produced via human coughing and breathing. The chamber operated at a relative humidity of 55% which resulted in a particle size distribution of 87% between 0.3 µm and 0.5 µm, 11% between 0.5 µm and 0.7 µm, and 2% > 0.7 µm. A comparison to published ranges of particle size distributions is shown in Table 1. Aerosolized viruses were efficiently transmitted through the system as evidenced from the control (zero exposure) showing clear virus integration (Fig. 1, top left).    , (1) where k is the UV inactivation rate constant or susceptibility factor (cm 2 /mJ). The regression was performed with the intercept term set to zero, which represents the definition of 100% relative survival at zero UV dose. Bootstrap 95% confidence intervals for the parameter k were calculated using R 3.2.3 software 41 . The virus inactivation cross section, D 95 , which is the UV dose that inactivates 95% of the exposed virus, was calculated as D 95 = −ln[1 − 0.95]/k.