key: cord-0291274-qxmhxm3p authors: Newey, Colleen; Olausson, Abigail T.; Applegate, Alyssa; Reid, Ann Aubrey; Robison, Richard A.; Grose, Julianne H. title: Presence and Stability of SARS-CoV-2 on Environmental Currency and Money Cards date: 2021-08-23 journal: bioRxiv DOI: 10.1101/2021.08.23.457328 sha: f5136ce5f032732b2167408947c714c5b48c4498 doc_id: 291274 cord_uid: qxmhxm3p The highly contagious nature of SARS-CoV-2 has led to several studies on the transmission of the virus. A little studied potential fomite of great concern in the community is currency, which has been shown to harbor microbial pathogens in several studies. Since the onset of the COVID-19 pandemic, many businesses in the United States have limited the use of banknotes in favor of credit cards. However, SARS-CoV-2 has shown greater stability on plastic in several studies. Herein, the stability of SARS-CoV-2 at room temperature on banknotes, money cards and coins was investigated. In vitro studies with live virus suggested SARS-CoV-2 was highly unstable on banknotes, showing an initial rapid reduction in viable virus and no viral detection by 24 hours. In contrast, SARS-CoV-2 was far more stable on money cards with live virus detected after 48 hours. Environmental swabbing of currency and money cards on and near the campus of Brigham Young University supported these results, with no detection of SARS-CoV-2 RNA on banknotes, and a low level on money cards. No viable virus was detected on either. These preliminary results suggest that the use of money cards over banknotes in order to slow the spread of this virus may be ill-advised. These findings should be investigated further through larger environmental studies involving more locations. 252 time 0.5 hours onward, Table 1 ). The initial apparent half-life of SARS-CoV-2 was shortest on 253 banknotes (~1.7 minute half-life), quarters (~ 4.0 minutes) and pennies (~ 4.0 minutes) compared 254 to the money card (~ 9 minutes) (Table 1 and Fig. 1 ). Second step decay rates were again highly 255 variable, with quarters displaying slower decay than money cards or pennies, however money 256 cards decay appeared to level out at 24 hours while no or extremely low levels of SARS-CoV-2 257 were detected on pennies after 24 hours. The $1 U.S.A. banknote data for second step decay 258 calculations is unreliable due to variability the detection of extremely low levels of virus at the 259 four-hour time point (0 versus 10 or 15 PFU/mL), suggesting an almost complete loss of titer at 260 the 30 minute time point (see Fig. 1 ). A $1 banknotes, coin and money cards in the laboratory is studied herein. In addition, the 116 frequency of detection of SARS-CoV-2 on environmental paper money and money cards was 117 assessed in the Brigham Young University (BYU), Provo, Utah area. live SARS-CoV-2 was performed in the biosafety level-3 facility at BYU, Provo, 124 UT. All work was conducted under proper safety handling procedures approved by the BYU 125 Cell Culture: Viral cultivation and plaque assays were performed using African green monkey 128 kidney (VERO 76, C1008) cells obtained from American Type Culture Collection (ATCC) VERO cells were maintained in T-75 flasks containing Dulbecco's Modified Eagle's Medium 130 (DMEM; Corning, 10-017-CV) supplemented with 10% 010-CV) at 37°C, and 5% CO 2 . For 24-well plate preparation, cells were seeded at 200,000 cells 132 per well in DMEM with 10% FBS and used for assays 18-24 hours later Viral Strain: SARS-CoV-2 (2019-nCoV/USA-WA1/2020) was obtained from the ATCC. Viral 135 titer was obtained by plaque assay in VERO cells. Briefly, stock virus was diluted 1:2 and from 136 10 -1 through 10 -10 by 10-fold serial dilutions in DMEM. VERO cells in 24-well plates were then 7 Plates were placed at 37°C and 5% CO 2 for 1 hour and 138 manually rocked every 10 minutes. A 1ml-overlay of 1:1 mixture of 2X Minimal Essential Agarose plugs were allowed to 141 solidify at room temperature for 30 minutes. Plates were then incubated for 72 hours at 37°C and 142 5% CO 2 . Post incubation, wells were fixed with 10% of SARS-CoV-2 RNA (positive control for LAMP assays): SARS-CoV-2 149 7 cells directly in the culture dish and pipetting up and 151 down several times to homogenize. Samples were allowed to incubate for 5 minutes, and then 152 centrifuged for 5 minutes at 12,000 ×g at 4-10°C. The clear supernatant was Experiments: Due to the reduction in usage of cash in and 156 around campus, environmental samples of cash were primarily obtained from the Brigham 157 Young University vault, a daily collection of currency and coin from university-based stores, 158 restaurants, dormitories and vending machines. In addition, fresh samples were obtained from around the edges of the bill and then back and forth across the surface for ~10 seconds assayed for viral signatures within one hour after collection using a loop-mediated 165 isothermal amplification (LAMP) assay England BioLabs) was utilized using SARS-CoV-2 specific primers[43] or SARS-CoV-2 Rapid were diluted to a final 169 concentration of: 16 uM FIP Lab Inoculation of U.S Currency Experiments: Viable SARS-CoV-2 virus (20ul of stock at 178 1.5x10 7 pfu/mL) was inoculated onto designated Negative controls were inoculated with 20ul of sterile DMEM. The initial dilution; Undiluted, 10 -1 , 10 -2 , 10 -3 , 10 -4 , and DMEM negative control. Plates were placed at 188 37°C and 5% CO 2 for 1 hour and manually rocked every 10 minutes 4%) were placed in each well for 3 minutes at 195 room temperature, followed by rinsing with distilled water twice and air drying Local Coronavirus Case Counts: Local coronavirus case counts were San Diego California) and ANOVA with post hoc Tukey HSD was 205 performed using JMP pro version 15 (JMP®, Version 15 Viable SARS-CoV-2 was spotted on $1 banknotes, quarters, pennies and credit cards and then 214 extracted by swabbing with a MEM-moistened sterile swab at time points 30 minutes (allowing 215 time for drying) as well as 4, 24, and 48 hours post inoculation. Extracted samples were 216 immediately assayed for viable SARS-CoV-2 via plaque counts in VERO cells In contrast, money cards displayed only a 10-fold or 90% reduction in titer at 222 30 minutes (2.5x10 6 to 2.5x10 5 pfu/mL, p = 1.51 x10 -3 ) which may in part be due to a difference 223 in the ability of the viral suspension to soak into or bind plastic money cards versus paper 224 banknotes. Further significant reductions in titer occurred at 4 hours (99.6% reduction compared 225 with 30 minute values, p = 1.7 x10 -7 ), however live virus was still detectable at 24 and 48 hours 226 post inoculation, displaying a 99.96% and 99.97% reduction, respectively, when compared with 227 time 30 minutes (p = 1.2 x10 -7 and p = 7.9 x10 -7 , respectively). Quarters and pennies were similar 228 to the money card, with a stronger initial reduction in viral titer (99.4% and 99.6%, respectively, 229 at time 30 minutes A. banknotes, money cards, quarters, 234 and pennies. A) Representative plaques of a SARS-CoV-2 on VERO cells from this study at the 235 four hour time point. B) Recovery of virus after inoculation of $1 U.S.A. banknotes after 0.5, 4, 236 24 and 48 hours. C) Recovery of virus after inoculation of money cards Log 2 of the virus 239 concentration was represented on the y-axis of all charts to better display differences in numbers At zero titer, log 2 is undefined and is therefore shown at 2 0 . Time 0.5 was used as an initial time ) was 243 used to detect differences between time points, and all significant differences (p< 0.10) between 244 the time 0.5 hours and other time points as indicated by "*". For every surface, the starting point 245 (just prior to inoculation) was significantly different (p<0.05) from all other time points All samples displayed a rapid, initial drop in viable virus (PFU/mL), followed by a different rate 249 of decay. This may be due to surface drying times CoV-2 on these surfaces was conducted with and without inclusion of the starting titer to obtain 251 two-step decay rates Due to the apparent instability of SARS-CoV-2 on U.S. banknotes and the improved stability on 278 money cards as assayed in the laboratory, a large-scale screening was performed to detect SARS Two hundred and seventy-nine money cards, including 280 credit cards and Brigham Young University (BYU) ID money cards, were swabbed and assayed 281 via SARS-CoV-2 LAMP assay (Fig. 2, Table 2). A total of 17 money cards tested positive in 282 duplicate LAMP assays To determine if there was live virus present, a total of 14 samples 284 from the 2/19/2021 assays (including all 6 samples that were positive) were interrogated in the 285 BSL-3 laboratory for live virus using a standard plague assay. No plaques were seen for any of 286 the samples. Dilutions of a viable suspension of SARS-CoV-2 with a titer of 7 LAMP assay-based detection of SARS-CoV-2. A) Representative colorimetric LAMP 291 SARS-CoV-2 assay using the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit Biolabs) and NEB positive control or purified SARS-CoV-2 RNA. B) Associated gel 293 electrophoresis of samples from (A). NEB positive control and SARS-CoV-2 RNA were used 294 straight (sample number 1), and then serially diluted 1:10, four times 6% reduction of the virus after four hours, compared with a 99.9993% reduction of the virus 337 on $1 U.S.A. banknotes immediately after inoculation of U.V.-sterilized CoV-2 at 24 hours post inoculation of uncirculated $1 U.S.A. banknotes at room 340 temperature.[41] In addition, we were unable to detect some SARS-CoV-2 RNA on U.S.A. 341 banknotes in circulation (Table 3), consistent with the instability of SARS-CoV-2 on banknotes 342 in vitro. 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