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      End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments

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          Abstract

          The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus responsible for coronavirus disease 2019 [COVID-19]) pandemic has led to a global slowdown with far-reaching financial and social impacts. The SARS-CoV-2 respiratory virus is primarily transmitted from person to person through inhalation of infected droplets or aerosols. However, some studies have shown that virions can remain infectious on surfaces for days and can lead to human infection from contact with infected surfaces. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end study showed that the effective combination for monitoring SARS-CoV-2 on surfaces required a minimum of 1,000 viral particles per 25 cm 2 to successfully detect virus from surfaces. This comprehensive study can provide valuable information regarding surface monitoring of various materials as well as the capacity to retain viral RNA and allow for effective disinfection.

          ABSTRACT

          Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted person to person through inhalation of droplets or aerosols, laden with viral particles. However, as recent studies have shown, virions can remain infectious for up to 72 h on surfaces, which can lead to transmission through contact. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination for monitoring SARS-CoV-2 on surfaces includes using an Isohelix swab collection tool, DNA/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative PCR (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, noninfectious viral fragments of SARS-CoV-2 persisted on surfaces for as long as 8 days even after bleach treatment. Additionally, debris associated with specific built environment surfaces appeared to inhibit and negatively impact the recovery of RNA; Amerstat demonstrated the highest inhibition (>90%) when challenged with an inactivated viral control. Overall, it was determined that this E2E protocol required a minimum of 1,000 viral particles per 25 cm 2 to successfully detect virus from test surfaces. Despite our findings of viral fragment longevity on surfaces, when this method was employed to evaluate 368 samples collected from various built environmental surfaces, all samples tested negative, indicating that the surfaces were either void of virus or below the detection limit of the assay.

          IMPORTANCE The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus responsible for coronavirus disease 2019 [COVID-19]) pandemic has led to a global slowdown with far-reaching financial and social impacts. The SARS-CoV-2 respiratory virus is primarily transmitted from person to person through inhalation of infected droplets or aerosols. However, some studies have shown that virions can remain infectious on surfaces for days and can lead to human infection from contact with infected surfaces. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end study showed that the effective combination for monitoring SARS-CoV-2 on surfaces required a minimum of 1,000 viral particles per 25 cm 2 to successfully detect virus from surfaces. This comprehensive study can provide valuable information regarding surface monitoring of various materials as well as the capacity to retain viral RNA and allow for effective disinfection.

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          Most cited references 44

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          Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1

          To the Editor: A novel human coronavirus that is now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (formerly called HCoV-19) emerged in Wuhan, China, in late 2019 and is now causing a pandemic. 1 We analyzed the aerosol and surface stability of SARS-CoV-2 and compared it with SARS-CoV-1, the most closely related human coronavirus. 2 We evaluated the stability of SARS-CoV-2 and SARS-CoV-1 in aerosols and on various surfaces and estimated their decay rates using a Bayesian regression model (see the Methods section in the Supplementary Appendix, available with the full text of this letter at NEJM.org). SARS-CoV-2 nCoV-WA1-2020 (MN985325.1) and SARS-CoV-1 Tor2 (AY274119.3) were the strains used. Aerosols (<5 μm) containing SARS-CoV-2 (105.25 50% tissue-culture infectious dose [TCID50] per milliliter) or SARS-CoV-1 (106.75-7.00 TCID50 per milliliter) were generated with the use of a three-jet Collison nebulizer and fed into a Goldberg drum to create an aerosolized environment. The inoculum resulted in cycle-threshold values between 20 and 22, similar to those observed in samples obtained from the upper and lower respiratory tract in humans. Our data consisted of 10 experimental conditions involving two viruses (SARS-CoV-2 and SARS-CoV-1) in five environmental conditions (aerosols, plastic, stainless steel, copper, and cardboard). All experimental measurements are reported as means across three replicates. SARS-CoV-2 remained viable in aerosols throughout the duration of our experiment (3 hours), with a reduction in infectious titer from 103.5 to 102.7 TCID50 per liter of air. This reduction was similar to that observed with SARS-CoV-1, from 104.3 to 103.5 TCID50 per milliliter (Figure 1A). SARS-CoV-2 was more stable on plastic and stainless steel than on copper and cardboard, and viable virus was detected up to 72 hours after application to these surfaces (Figure 1A), although the virus titer was greatly reduced (from 103.7 to 100.6 TCID50 per milliliter of medium after 72 hours on plastic and from 103.7 to 100.6 TCID50 per milliliter after 48 hours on stainless steel). The stability kinetics of SARS-CoV-1 were similar (from 103.4 to 100.7 TCID50 per milliliter after 72 hours on plastic and from 103.6 to 100.6 TCID50 per milliliter after 48 hours on stainless steel). On copper, no viable SARS-CoV-2 was measured after 4 hours and no viable SARS-CoV-1 was measured after 8 hours. On cardboard, no viable SARS-CoV-2 was measured after 24 hours and no viable SARS-CoV-1 was measured after 8 hours (Figure 1A). Both viruses had an exponential decay in virus titer across all experimental conditions, as indicated by a linear decrease in the log10TCID50 per liter of air or milliliter of medium over time (Figure 1B). The half-lives of SARS-CoV-2 and SARS-CoV-1 were similar in aerosols, with median estimates of approximately 1.1 to 1.2 hours and 95% credible intervals of 0.64 to 2.64 for SARS-CoV-2 and 0.78 to 2.43 for SARS-CoV-1 (Figure 1C, and Table S1 in the Supplementary Appendix). The half-lives of the two viruses were also similar on copper. On cardboard, the half-life of SARS-CoV-2 was longer than that of SARS-CoV-1. The longest viability of both viruses was on stainless steel and plastic; the estimated median half-life of SARS-CoV-2 was approximately 5.6 hours on stainless steel and 6.8 hours on plastic (Figure 1C). Estimated differences in the half-lives of the two viruses were small except for those on cardboard (Figure 1C). Individual replicate data were noticeably “noisier” (i.e., there was more variation in the experiment, resulting in a larger standard error) for cardboard than for other surfaces (Fig. S1 through S5), so we advise caution in interpreting this result. We found that the stability of SARS-CoV-2 was similar to that of SARS-CoV-1 under the experimental circumstances tested. This indicates that differences in the epidemiologic characteristics of these viruses probably arise from other factors, including high viral loads in the upper respiratory tract and the potential for persons infected with SARS-CoV-2 to shed and transmit the virus while asymptomatic. 3,4 Our results indicate that aerosol and fomite transmission of SARS-CoV-2 is plausible, since the virus can remain viable and infectious in aerosols for hours and on surfaces up to days (depending on the inoculum shed). These findings echo those with SARS-CoV-1, in which these forms of transmission were associated with nosocomial spread and super-spreading events, 5 and they provide information for pandemic mitigation efforts.
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            Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

            Background The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project. Conclusion The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
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              The Architecture of SARS-CoV-2 Transcriptome

              Summary SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3′ tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.
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                Author and article information

                Contributors
                Role: Editor
                Role: ad hoc peer reviewer
                Journal
                mSystems
                mSystems
                msys
                msys
                mSystems
                mSystems
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                2379-5077
                6 October 2020
                Sep-Oct 2020
                6 October 2020
                : 5
                : 5
                Affiliations
                [a ]NASA Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California, USA
                [b ]Vermont Integrative Genomics Resource, Larner College of Medicine, The University of Vermont, Burlington, Vermont, USA
                [c ]ZIN Technologies Inc., Middleburg Heights, Ohio, USA
                [d ]Biotia, New York, New York, USA
                [e ]SUNY Downstate Health Sciences University, Brooklyn, New York, USA
                [f ]Weill Medical College of Cornell University, New York, New York, USA
                [g ]Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital, Tubingen, Germany
                Institute for Systems Biology
                MIT
                Author notes
                Address correspondence to Kasthuri Venkateswaran, kasthuri.j.venkateswaran@ 123456jpl.nasa.gov .

                Ceth W. Parker, Nitin Singh, and Scott Tighe contributed equally. The placement is based on alphabetical order.

                Citation Parker CW, Singh N, Tighe S, Blachowicz A, Wood JM, Seuylemezian A, Vaishampayan P, Urbaniak C, Hendrickson R, Laaguiby P, Clark K, Clement BG, O’Hara NB, Couto-Rodriguez M, Bezdan D, Mason CE, Venkateswaran K. 2020. End-to-end protocol for the detection of SARS-CoV-2 from built environments. mSystems 5:e00771-20. https://doi.org/10.1128/mSystems.00771-20.

                Article
                mSystems00771-20
                10.1128/mSystems.00771-20
                7542562
                33024053
                Copyright © 2020 Parker et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                Page count
                supplementary-material: 9, Figures: 6, Tables: 0, Equations: 0, References: 44, Pages: 18, Words: 13327
                Product
                Funding
                Funded by: NASA | Jet Propulsion Laboratory (JPL), https://doi.org/10.13039/100006196;
                Award ID: Directors Discretionary Fund
                Award Recipient :
                Funded by: Testing for America (501c3);
                Award Recipient :
                Funded by: OpenCovidScreen Foundation;
                Award Recipient :
                Funded by: Bert L and N Kuggie Vallee Foundation;
                Award Recipient :
                Funded by: Alfred P. Sloan Foundation, https://doi.org/10.13039/100000879;
                Award ID: G-2015-13964
                Award Recipient :
                Funded by: Citadel Foundation (TCF), https://doi.org/10.13039/100014431;
                Award Recipient :
                Funded by: HHS | National Institutes of Health (NIH), https://doi.org/10.13039/100000002;
                Award ID: R01AI125416
                Award ID: R21AI129851
                Award ID: R01AI151059
                Award Recipient :
                Funded by: WorldQuant Foundation, https://doi.org/10.13039/100015169;
                Award Recipient :
                Funded by: Pershing Square Foundation, https://doi.org/10.13039/100010304;
                Award Recipient :
                Categories
                Research Article
                Clinical Science and Epidemiology
                Custom metadata
                September/October 2020

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