key: cord-0986497-0bbw64p5
authors: Schuit, Michael; Ratnesar-Shumate, Shanna; Yolitz, Jason; Williams, Gregory; Weaver, Wade; Green, Brian; Miller, David; Krause, Melissa; Beck, Katie; Wood, Stewart; Holland, Brian; Bohannon, Jordan; Freeburger, Denise; Hooper, Idris; Biryukov, Jennifer; Altamura, Louis A; Wahl, Victoria; Hevey, Michael; Dabisch, Paul
title: Airborne SARS-CoV-2 is Rapidly Inactivated by Simulated Sunlight
date: 2020-06-11
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa334
sha: 9f0f37608fa07be85c0da57fc3dc1357b894f41c
doc_id: 986497
cord_uid: 0bbw64p5

Aerosols represent a potential route of transmission of COVID-19. This study examined the effect of simulated sunlight, relative humidity, and suspension matrix on the stability of SARS-CoV-2 in aerosols. Both simulated sunlight and matrix significantly affected the decay rate of the virus. Relative humidity alone did not affect the decay rate; however, minor interactions between relative humidity and the other factors were observed. Decay rates in simulated saliva, under simulated sunlight levels representative of late winter/early fall and summer were 0.121±0.017 min(-1) (90% loss: 19 minutes) and 0.379±0.072 min(-1) (90% loss: 6 minutes), respectively. The mean decay rate without simulated sunlight across all relative humidity levels was 0.008±0.011 min(-1) (90% loss: 125 minutes). These results suggest that the potential for aerosol transmission of SARS-CoV-2 may be dependent on environmental conditions, particularly sunlight. These data may be useful to inform mitigation strategies to minimize the potential for aerosol transmission.

A c c e p t e d M a n u s c r i p t For aerosol transmission to occur, viruses within aerosol particles must remain infectious between generation and inhalation by a susceptible host. Loss of infectivity during this period will decrease the likelihood of aerosol transmission. van Doremalen et al. [15] have reported that SARS-CoV-2 is detectable in aerosols for several hours in darkness at room temperature. Similar results have been reported previously for other coronaviruses under similar conditions [16, 17] . Environmental conditions, including relative humidity and sunlight, have been shown to influence the decay rate of infectious viruses in aerosols [16, [18] [19] [20] [21] [22] . However, no such data on the influence of these factors on the aerosol persistence of SARS-CoV-2 exist. Therefore, the present study examined the influence of both simulated sunlight and relative humidity on the stability of SARS-CoV-2 in aerosols generated from virus suspended in different liquid matrices. The data generated will further our understanding of factors which have the potential to influence aerosol transmission of SARS-CoV-2 and could be utilized to inform mitigation strategies for aerosol transmission of virus during the current pandemic.

Vero Cells (ATCC CCL-81) were grown at 37 °C and 5% CO 2 in culture medium, consisting of Minimum Essential Medium (MEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone or Atlanta Biologicals), 2mM GlutaMAX (Gibco), 0.1 mM nonessential amino acids solution (Gibco), 1mM sodium pyruvate (Gibco), and 1% antibiotic-antimycotic solution (Gibco).

A passage four isolate of SARS-CoV-2 (BetaCoV/USA/WA1/2020) was obtained from BEI resources and passaged twice in Vero cells to produce a stock of virus that was concentrated by tangential flow filtration and frozen at -80°C until use. For aerosol tests, aliquots of the concentrated virus were thawed and diluted 1:10 in either fresh culture medium or simulated saliva, formulated as described in the ASTM standard for measuring virus decontamination efficacy [23] , but prepared with KH 2 PO 4 A c c e p t e d M a n u s c r i p t and K 2 HPO 4 at final concentrations of 15.4 mM and 24.6 mM, respectively. Diluted virus aliquots were prepared daily from frozen stocks and kept on ice between tests. Titers of infectious virus in aerosol samples were determined by microtitration assay on confluent monolayers of Vero cells in 96-well plates. Plates were incubated at 37°C and 5% CO 2 , with cytopathic effect read four days postinfection and viral titers calculated according to the method of Kärber and Spearman [24, 25] . The pH and solids content of viral suspensions diluted in each matrix was measured in triplicate using a SevenExcellence pH meter (Mettler-Toledo) and MA35 Moisture Analyzer (Sartorius AG), respectively. Protein content was quantified using a Pierce BCA Protein Assay Kit (23225, Thermo Fisher Scientific) with an albumin standard curve. The assay was read on a SpectraMax M5 plate reader (Molecular Devices).

Two different environmentally controlled rotating drum aerosol chambers, with volumes of 16-L and 208-L, were used in the present study to expose aerosols containing SARS-CoV-2 to controlled levels of temperature, relative humidity, and simulated sunlight. The environmental control systems were similar for both drums, and have been described previously for one of these drums [19] . Briefly, the temperature of the air inside the drum was regulated by a temperature-controlled glycol solution circulated through channels in the walls of the drums. Relative humidity was controlled by adjusting the balance of dry and humid air entering the drum prior to tests, during filling, and as makeup air when aerosol samples were collected from the drums. Temperature and relative humidity probes in the interior of each drum were used to record the values of these parameters in ten-second intervals over the course of each test. For each test, the mean and standard deviation were determined for these parameters using data from the beginning of the first aerosol sample to the end of the final sample.

For a subset of tests, SARS-CoV-2 aerosols were exposed to simulated sunlight generated by a solar simulator (Newport Oriel) equipped with a 320-nm highpass filter (WG320 filter PN SL07614, Solar A c c e p t e d M a n u s c r i p t Light Co.) through a fused-silica window on one face of the chambers. Tests were conducted at one of two intensity levels, with spectra designed to represent the ultraviolet (UV) range (280-400 nm) of natural sunlight. The two spectra used in the present study, referred to hereafter as high-intensity and mid-intensity, have similar UV irradiances to model spectra from the National Center for A c c e p t e d M a n u s c r i p t

Virus-containing aerosol particles of respiratory origin have been found in a range of particle sizes from sub-micron to several microns in diameter [8, [26] [27] [28] . In the present study, the target mass median aerodynamic diameter (MMAD) was 2 µm, an approximate mid-point of the range of relevant possible sizes. Aerosols were generated into an external stainless steel plenum attached to each drum using an air assist nozzle (IAZA5200415K; Lee Company). The nozzle was supplied with dry, compressed air at 45 psig and supplied with the viral suspension at 200 to 300 µL/min using a syringe pump.

Aerosol was drawn from the external plenum into the drums. The filling time differed for the two drums due to the difference in volume and was 30 seconds for the smaller drum and 60 seconds for the larger drum. Following filling, aerosols were allowed to mix in the drum for thirty seconds prior to collection of the first sample. Aerosols were then aged in the drums for up to 60 minutes. Five samples of the aerosol present in a drum were collected over the course of each test. The test duration and sample intervals were determined based on the anticipated decay rate for a given set of environmental conditions. At each sampling time point, a ten second sample was collected using an Aerodynamic Particle Sizer (APS; Model 3321, TSI Inc.) to measure the mass concentration and size distribution of the aerosol in the drum. Immediately following the APS sample, a 20 to 60 second sample was collected onto a 25 mm gelatin filter (PN 225-9551; SKC, Inc.) in a Delrin filter holder (PN 1109; Pall Corp.) operated at 5 L/min. The gelatin filter was immediately removed from the holder and dissolved in 10 mL of culture medium to re-suspend the collected virus. Relative humidity-conditioned makeup air entered the drum during both APS and gelatin filter sampling to maintain the relative humidity and neutral pressure in the chamber.

Tests were conducted in both suspension matrices across a range of relative humidity levels (20, 45, and 70%) and simulated sunlight intensities (darkness, mid-intensity, and high-intensity). A 2x2 full factorial design with a center-point was utilized to examine the effect of each parameter, as well as A c c e p t e d M a n u s c r i p t interactions between parameters, on the decay rate of aerosolized SARS-CoV-2. Tests were conducted at all combinations of the low and high levels of both factors, as well as at the mid-point levels of both factors. This experimental design is an efficient approach that allows examination of the impact of relative humidity and simulated sunlight, as well as potential interactions of these factors, while minimizing the total number of tests required. Additional tests were conducted without simulated sunlight at target relative humidity values of 37 and 53% to examine the effect of relative humidity under temperature and light conditions relevant to indoor environments in greater detail. Three to six replicate tests were performed for each combination of suspension matrix and environmental condition. All tests were conducted at a target temperature of 20°C.

The aerosol concentration of infectious SARS-CoV-2 within the drum at each time point, in TCID 50 /Lair, was calculated as the total amount of virus collected by the gelatin filter divided by the amount of air sampled. The aerosol mass concentration within the drum at each time point, in mg/m 3 , was calculated from the data collected by the APS. For each test, time-series log 10 transformed viral and mass aerosol concentration data were fit using linear regression in Microsoft Excel (v. 2016). The slopes of these regression lines represent the decay rates of infectious virus and total aerosol mass in the chamber, respectively. In the published literature, decay is often reported as the decay constant from a one-phase exponential fit [16, 19, [29] [30] [31] [32] . To allow a direct comparison to these values, the slope was converted from log base 10 to log base e, as this value is equivalent to the decay constant from a one-phase exponential decay fit of the data. The mean MMAD and geometric standard deviation (GSD) at the first sample collected across all tests in simulated saliva were 1.96 ± 0.05 µm and 1.62 ± 0.04, respectively. For tests in culture medium, these values were 1.98 ± 0.08 and 1.60 ± 0.04, respectively. A small downward shift in the MMAD occurred over the course of each test due to a more rapid physical loss of larger particles in the size distribution. As a result, the mean MMAD of aerosols generated from simulated saliva and culture medium at the final sample were 1.78 ± 0.14 and 1.88 ± 0.13, respectively.

Decay data for SARS-CoV-2 in aerosols are shown in Figure 2 , Figure 3 , and Table 1 . Average decay constants for infectivity ranged from near zero for tests without simulated sunlight to 0.48 min -1 , or 38%/min, for tests with high-intensity simulated sunlight at 70% relative humidity. Stepwise regression analysis demonstrated that k Infectivity was dependent on the simulated sunlight intensity and the suspension matrix (P<0.0001 and P=.0004, respectively), but not relative humidity (P=0.0946). Interactions between suspension matrix and simulated sunlight intensity (P<0.0001), suspension matrix and relative humidity (P=0.0017), and simulated sunlight intensity and relative A c c e p t e d M a n u s c r i p t humidity (P=0.0463), were also significant. While the effect of suspension matrix was statistically significant, the magnitude of the effect of simulated sunlight was much greater, as suggested by a greater standardized regression coefficient (-0.117 for simulated sunlight vs. 0.022 for matrix). The overall adjusted r 2 for the model was 0.88.

The present study examined the influence of simulated sunlight and relative humidity on the stability of SARS-CoV-2 in aerosols generated from virus suspended in either simulated saliva or culture medium at 20°C. Simulated sunlight rapidly inactivated the virus in aerosols in either suspension matrix , with half-lives of less than 6 minutes and 90% of the virus inactivated in less than 20 minutes for all simulated sunlight levels tested. There was a small but statistically significant reduction in decay rate under high-intensity sunlight when the virus was suspended in culture medium compared to simulated saliva, suggesting that the matrix in which the virus is suspended may also be an important factor to consider when examining the persistence of SARS-CoV-2 in an aerosol. While it has been reported previously that UVC can inactivate aerosolized coronaviruses [33] , the present study is the first to demonstrate that simulated sunlight, with UVA and UVB levels similar to natural sunlight, is also able to inactivate airborne coronaviruses. It should be noted that many additional factors beyond the relative stability of the virus in an aerosol contribute to the potential for aerosol transmission of disease. These include the amount of virus present in an aerosol, the size and infectious dose of aerosol particles, the distance and airflow dynamics between infected and uninfected individuals, the presence of mitigation measures such as personal protective equipment. Therefore, while the results of the present study provide novel data regarding the stability of SARS-CoV-2 aerosols in the environment, additional data are needed to provide a comprehensive assessment of the potential for aerosol transmission.

A c c e p t e d M a n u s c r i p t Relative humidity alone did not significantly affect decay of the virus, although there were interactions identified between relative humidity and the other factors. However, the magnitude of these interactions was minor compared to the magnitude of the effect of simulated sunlight. The half-lives estimated from the mean decay constants across all relative humidity levels without simulated sunlight present were 55 and 86 minutes for aerosols generated from virus suspended in culture medium and simulated saliva, respectively. The half-life from the present study for culture medium is similar to the value of 1.1 hours reported recently for SARS-CoV-2 in darkness and 65% relative humidity by van Doremalen et al. [20] . The prolonged persistence of SARS-CoV-2 under conditions representative of indoor environments highlights the need for additional studies to better understand the potential sources of aerosols and viral load present in these settings.

It has been previously reported that other coronaviruses were significantly less stable at higher relative humidities, with the half-life for human coronavirus 229E decreasing from 67.3 ± 8.2 hours to 3.3 ± 0.2 hours for relative humidity levels of 50 and 80%, respectively [16] . While a similar effect was not observed for SARS-CoV-2 in the present study, it is possible that the shorter test durations used in the present study precluded detection of this effect of relative humidity. It is possible that additional tests of longer duration without simulated sunlight would allow a better assessment of the effect of relative humidity on SARS-CoV-2 in aerosols, but the results of the present study suggest that any such effect would be relatively minor in comparison to the effect of sunlight.

Previous studies have also demonstrated that numerous other factors can influence the survival of microorganisms in aerosols. In particular, temperature has been shown previously to affect the survival of coronaviruses, including MERS, in aerosols [16, 17] . Furthermore, while the stability SARS-CoV-1 and SARS-CoV-2 in aerosols were shown to be similar under a single set of conditions [15] , other studies have demonstrated that the aerosol stability can vary between related viruses [34] [35] [36] [37] .

Therefore, additional testing incorporating a range of relevant temperatures and additional isolates A c c e p t e d M a n u s c r i p t of SARS-CoV-2 should be conducted to better estimate the range of potential decay rates associated with SARS-CoV-2.

It was necessary to concentrate the viral stock used in the present study to ensure that quantifiable concentrations of virus were present in aerosols. However, the addition of the concentrated viral stock to the simulated saliva significantly altered the properties of the simulated saliva, specifically the fractional solids and protein content. Thus, while a small difference in decay was observed between the simulated saliva and culture medium in the presence of simulated sunlight, it is possible that the viral suspension diluted in simulated saliva is not representative of the composition of expelled particles in infected individuals. Previous studies have shown that particle composition can affect the decay rate of infectious viruses in aerosols [38] [39] [40] . Therefore, additional studies 

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A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Table 1 . Summary of SARS-CoV-2 decay at 20°C in aerosols. Decay constants (k Infectivity ), decay rate, and half-life calculated from the mean k Infectivity values are summarized as a function of matrix and simulated sunlight level. Decay constants and rates are presented as the arithmetic mean ± standard deviation of each data set. Results across different relative humidity levels were pooled since relative humidity was determined not to be a significant factor affecting decay. Data from fifty-six tests are included; three tests were not included due to poor linear regression fits of the time-series viral aerosol concentration data. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t