key: cord-0976078-zx5uhe61
authors: Schilling-Loeffler, Katja; Falkenhagen, Alexander; Johne, Reimar
title: Coronaviruses are stable on glass, but are eliminated by manual dishwashing procedures
date: 2022-04-06
journal: Food Microbiol
DOI: 10.1016/j.fm.2022.104036
sha: 964dd29e431d5dce32648106bb0f2e3352debdc4
doc_id: 976078
cord_uid: zx5uhe61

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is primarily transmitted from human to human via droplets and aerosols. While transmission via contaminated surfaces is also considered possible, the overall risk of this transmission route is assumed to be low. Nevertheless, transmission through contaminated drinking glasses may pose an increased risk as the glass is in direct contact with the mouth and oral cavity. Using human coronavirus 229E (HCoV-229E) as surrogate for SARS-CoV-2, this study examined coronavirus stability on glass, inactivation by dishwashing detergents, and virus elimination by a manual glass scrubbing device. Infectious HCoV-229E was recovered from glass up to 7 and 21 days storage under daylight and dark conditions, respectively. Near complete inactivation of HCoV-229E (>4 log(10) reduction) was observed after incubation with two common dishwashing detergents at room temperature for 15 s, whereas incubation at 43 °C for 60 s was necessary for a third detergent to achieve a similar titer reduction. The virus was efficiently removed from contaminated drinking glasses using a manual glass scrubbing device in accordance with German standard DIN 6653-3. The results confirm that coronaviruses are relatively stable on glass, but indicate that common manual dishwashing procedures can efficiently eliminate coronaviruses from drinking glasses.

The Orthocoronavirinae, a subfamily of the Coronaviridae family of the order Nidovirales, comprises a 36 large group of coronaviruses, which represent enveloped viruses with a large single-stranded RNA 37 genome (Walker et al., 2020) . Within this subfamily, the human coronaviruses (HCoVs) belong to the 38 genera Alphacoronavirus and Betacoronavirus. The two Alphacoronavirus members HCoV-229E 39 (Tyrrell and Bynoe, 1965) and HCoV-NL-63 (van der Hoek et al., 2004) as well as the two 40

Betacoronavirus members HCoV-OC43 (Hamre and Procknow, 1966) and HCoV-HKU-1 (Woo et al., 41 2005) are globally endemic and mostly cause mild respiratory symptoms of the common cold. The predominant transmission route of SARS-CoV-2 is from human to human through respiratory 54 droplets and aerosols and the virus remains infectious in aerosols for hours (van Doremalen et al., 55 2020; Zhang et al., 2020) . However, fomite transmission was also considered possible, as the virus can 56 remain infectious on surfaces for several days (van Doremalen et al., 2020) and has been recovered 57 from household surfaces of SARS-CoV-2-infected individuals (Marcenac et al., 2021) . The stability of 58

HCoV on a variety of surfaces depends on the inoculation medium, inoculation substrate, relative 59 humidity, UV exposure, virus titer, and temperature. HCoV stability on surfaces ranges from several 60 hours to several days and has recently been extensively reviewed (Bueckert et al., 2020) . The longest 61 time-period after which infectious HCoV was still extracted from a surface was 28 days in the dark at 62 removed. Thereafter, the fixed cells were covered with 500 µL crystal violet solution (Merck, 135 Darmstadt, Germany) and incubated for 15 min while rocking the plate. Excess crystal violet was then 136 removed (crystal violet solution was reused for up to 3 times), stained cells were washed with water  137   and plates dried and stored in the dark until plaques were counted on a light table (Kaiser Fototechnik,  138 Buchen, Germany). Throughout all plaque assays each virus sample was analyzed in duplicate. Virus 139 titer in PFU/mL was calculated by multiplying the counted plaque numbers (mean value of the assayed 140 duplicate) with the dilution factor and taking into account the volume of virus sample inoculum. 141

Sterile glass slides (Menzel GmbH&Co KG, Braunschweig, Germany) were inoculated with 100 µL virus 144 stock (corresponding to 2.5x10 5 PFU/glass slide), the aliquot left to dry within 1 h in a biosafety cabinet 145 and transferred into a container for storage. At the indicated time-points, the remaining virus was 146 removed from the slides using a sterile cotton swab previously moistened with DMEM (Paul Boettger  147 GmbH&Co KG, Bodenmais, Germany). The cotton tip of the swab was transferred into 700 µL DMEM 148 in a 1.5 mL tube, mixed by vortexing for 15 s, and the tip removed with sterile forceps. Samples were 149 stored at -80°C until concentration of infectious virus was determined by plaque assay (2.2). In a first 150 experiment, virus titer was determined before and after drying on glass (within 1 h after virus sample 151 was added to the glass slide) and at the following time points: 0h, 1h, 2h, 4h, 6h, 8h, 16h, and 24h in 152 triplicate after incubation at room temperature in the dark (glass slides were stored in a light-sealed 153 box). In a second experiment, infectious virus titer extracted from glass after drying was determined 154 at the time points: 0h, 3d, 7d, 14 d, 21 d and 28 d at room temperature, in the dark (glass slides were 155 stored in a light-sealed box) or in presence of indirect sunlight (glass slides were stored in a transparent 156 glass box on a laboratory board in about 2 m distance from an east-facing window) in duplicates. Both 157 experiments were performed in parallel and samples were collected simultaneously for both lighting 158 conditions. 159

The virus stock was serially diluted in complete DMEM with 5% FBS and 3 g/L BSA, and 100 µL 161 aliquots were transferred to sterile glass slides. To exclude infectivity reduction by the drying process 162 itself, the sample was not dried but taken 10 min after application with a cotton swab as described in 163 2.3.1. Subsequently, virus was removed and analyzed by plaque assay as described above (2.2). The 164 amount of virus in the highest dilution detected by the plaque assay in at least one of three 165 replicates was defined as the limit of detection, as previously described (Bartsch et al., 2016) . The 166 detection limit was determined in triplicate. 167 J o u r n a l P r e -p r o o f

To determine the recovery rate, the ratio of the virus titer extracted from sterile slides (without drying) 169 and the virus titer of the stock solution was multiplied by 100 %, as previously described ( 

The virus stock was serially diluted in complete DMEM with 5% FBS and 3 g/L BSA and purified using 206 Detergent Removal Spin Columns (Pierce TM , ThermoScientific). Virus concentration in the eluate was 207 determined by plaque assay. The amount of virus in the highest dilution detected by plaque assay in 208 at least one of three replicates was defined as the detection limit, as previously described (Bartsch et 209 al., 2016) . 210

To determine the recovery rate, the ratio of the virus titer of the eluate after purification using 212 Detergent Removal Spin Columns (Pierce TM , ThermoScientific) and the virus titer of the stock solution 213 was multiplied by 100 %, as previously described (Blondin-Brosseau et al., 2021). 214

An area of approximately 1.5 cm 2 was marked on the drinking rim of the outside of drinking glasses 216 (ARCOROC, Arques, France) using a diamond pen. The marked glasses were placed on the side and an 217 aliquot of 100 µL virus stock (corresponding to 2.5x10 5 PFU/marked area) was added to the inside of 218 each glass within the marked area and allowed to dry for two hours. The glasses were then washed 219 with a commercially available manual glass scrubbing and washing system, which is in accordance with 220 the requirements of the German standard for manual glass scrubbing devices with physically separated 221 pre-rinsing and post-rinsing canisters (DIN 6653-3:2011-01), which will hereafter be referred to as 222 'manual glass scrubbing device'. This device consisted of two physically separated pre-rinsing and post-223 rinsing canisters (Supplementary Material 1), with the pre-rinsing canister containing a detergent 224 solution and the post-rinsing canister comprising horizontal freshwater jets. Six drinking glasses were 225 washed using tap water at room temperature (23°C) and six additional drinking glasses were washed 226 using a water temperature of 18°C (corresponding to the lowest tap water temperature reached in the 227 laboratory). A detergent tablet provided by the manufacturer of the device was added to the pre-228 rinsing canister prior to the experiment. Additionally, the water pressure was adjusted, resulting in 229 horizontal water jets in the post-rinse canister. For the washing process, the manufacturer's 230 instructions were followed by cleaning the glass with four pumps on the brush in the pre-rinse canister 231 without rotation and rinsing the glass in the second (post-rinse) canister by pressing the glass on the 232 telescope rinse bar for 3 s. The glasses were thereafter air-dried and virus was recovered from the 233 marked 1.5 cm 2 area as described for the glass slides in 2.3.1. A positive control was included in 234 triplicate, were the same amount of virus stock was dried on the glass rim and extracted without the 235 washing process. The detection limit and recovery rate as determined in 2.3.2 and 2.3.3 also apply 236 here, as the mechanism of virus extraction from drinking glasses and from glass slides as well as the 237 plaque assay were the same. Photos of the experimental setup are shown in Supplemental Material 1. 238 indicated that the HCoV-229E mean titer did not decrease by more than 0.5 log10 immediately after 251 drying and within 24 h in the dark (Supplemental Material 2 Figure 1) . Therefore, the change of the 252 HCoV-229E titer was monitored for several days to weeks (Figure 1) . Under daylight conditions, the 253 mean titer of infectious HCoV-229E decreased by 1.5 log10 after three days, but then remained 254 relatively stable until day 7. After 14 days, no remaining infectious virus was detected on the glass 255 slides under these conditions. In the dark, the mean virus titer decreased by 0.5 log10 after three days 256 and remained stable until day 7 (Figure 1) . After 14 days, the mean virus titer was decreased by about 257 2 log10 compared to day 0, and after 21 days, a HCoV-229E reduction of > 4 log10 was observed. On day 258 28, no infectious HCoV-229E was detected on the glass slides under dark storage conditions. 259

Considering the LOD of 75 (1.87 log10) PFU/glass slide determined for virus recovery from glass slides, 260 J o u r n a l P r e -p r o o f exposure of dried HCoV-229E on glass slides for 14 days at daylight condition and for 21 days at dark 261 condition resulted in mean titer reductions of >2.8 log10. 262

The reduction of the HCoV-229E titer was analyzed after exposure to three different dishwashing 264 detergents as well as S/D solution in aqueous suspensions at room temperature for 15 s (Figure 2A ) 265 and 60 s ( Figure 2B ) and at 43°C for 15 s ( Figure 2C ) and 60 s ( Figure 2D ). The LOD for the whole 266 experimental process was 7.5 PFU/inoculum; the recovery rate was 82.67±21.00 %. Using detergent 1, 267 detergent 3 or S/D, the HCoV-229E mean titer decreased by > 4 log10 at all tested conditions (Table 2) . 

The decrease of the HCoV-229E titer by the use of a commercial manual glass scrubbing device was 282 analyzed using virus-contaminated drinking glasses and water temperatures of 18 °C or room 283 temperature. After completion of the washing procedure, no remaining HCoV-229E was detected on 284 the glasses at either temperature (Table 3) . CoV-2 was described as most resilient, being stable on glass surfaces for days to weeks (Pastorino et 307 al., 2020; Riddell et al., 2020) . However, in two studies HCoV-229E has also been shown to stay 308 infectious for several days after drying on glass surfaces (Bonny et al., 2018; Warnes et al., 2015) . In 309 both cases, the experiments were stopped after 5 or 7 days, leaving the time necessary for complete 310 inactivation unknown. Besides possible differences between the distinct inactivation times, the 311 relative contributions of environmental factors on stability seem to be similar for all HCoVs, e.g. low 312 temperatures and/or low relative humidity usually increased their persistence (Bueckert et al., 2020) . 313 Therefore, although slight differences in inactivation of HCoV-229E and SARS-CoV-2 have to be 314 expected, the general conclusions drawn from our study regarding inactivation by detergents and 315 during manual washing procedures should apply to both viruses. 316

For the method applied in our stability study on glass, an approximate LOD of 75 PFU (1.87 log10) was 317 determined, which is comparable to LODs of about of 50-125 PFU and 1.8 log10 TCID50 /mL described 318 in similar previous studies (Blondin-Brosseau et al., 2021; . Using this 319 method, we detected infectious virus for up to 21 days after drying on glass, albeit with a very low 320 titer. Previous studies investigated HCoV reduction times after drying on glass found a 3.5 log10 321 reduction for SARS-CoV-2 in seven days or 2 log10 reduction for HCoV-229E in five days (Liu et The results of our study obtained for HCoV-229E with the control S/D are similar to those described 356 for SARS-CoV, which resulted in a > 4.5 log10 reduction after 1 min (Rabenau, Biesert, et al., 2005) . 357

Another recent study confirmed the inactivation of SARS-CoV-2 using a 0.5 % Triton X-100 solution 358 achieving a 5.9 log10 reduction after exposure of less than 2 minutes (Welch et al., 2020) . Only a few 359 studies have assessed the efficiency of household detergents for HCoV inactivation. Lai et al. (2005) 360 reported a reduction of >3 log10 for SARS-CoV after exposure of to a 1:100 dilution of a household 361 detergent for 5 min. From our study and the published data, it can generally be concluded that 362 household detergents inactivate HCoVs in solution. However, differences in efficiency exist between 363 different products, and inactivation efficiency is affected by both temperature and contact time. 364

In addition to the detergent, mechanical removal of contaminants from the surface and dilution in the 365 washing water represent important factors during a complete washing procedure of dishes and 366 drinking glasses. In order to test the efficiency of a whole washing procedure for drinking glasses under 367 more realistic conditions, a commercially available manual glass scrubbing device was used in our 368 study. This device was in accordance with DIN 6653-3 (2011) performance requirements, which 369 guarantees high cleaning efficiencies due to its construction. In the device, virus is mechanically eluted 370 from the glass by scrubbing in the pre-rinse canister, where eluted virus is exposed to detergent. The 371 glass is subsequently transferred to the post-rinse canister, where the glass is rinsed with fresh tap 372 water to remove detergent and loosened dirt. We did not detect any remaining virus on the initially 373

HCoV-229E-inoculated glasses after completion of the washing process. However, the variation of the 374 applied parameters such as shorter washing times and lower flow-through of fresh water, but also the 375 presence of higher concentrations of protein or fat, may reduce the elimination efficiency of viruses. 376

Our study has some limitations. First, as discussed above, it was conducted with HCoV-299E as 377 surrogate virus for SARS CoV-2, and confirmation with SARS-CoV-2 would be desirable in future 378 investigations. Second, the virus was only tested in culture medium with BSA, but not in other matrices. 379

Although the use of this standardized solution enables comparison with data from similar studies, it 380 does not fully reflect conditions in real-life, and negative effects on virus inactivation in complex 381 matrices have been described (Bertrand et al., 2012) . Therefore, studies investigating virus reduction 382 in complex matrices such as saliva or specific beverages would be useful, although a wide variety of 383 other matrices may be considered. Third, only a small spectrum of detergents and only one glass 384 scrubbing device was tested here, which should be extended in further studies. 385

In summary, the results of our study indicate that HCoV-229E can stay infectious for days to weeks 386 after drying on glass, where daylight exposure contributes to a more rapid inactivation. Sufficient 387 cleaning of dishes and drinking glasses is therefore crucial. Common household detergent solutions 388 can inactivate the virus, some of them with high efficiency even at room temperature and with low 389 contact times. However, as one tested dishwashing detergent with lower overall surfactant content 390 showed lower efficiency, the use of higher temperatures and longer contact times should generally be 391 suggested for manual dish washing procedures. Finally, it was shown that a commercial glass scrubbing Where data points are on the x-axis, zero plaques were detected (zpd). Overlapping data points are 418 indicated in bold. 419 420 Literature 421

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