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      Stability of SARS-CoV-2 in different environmental conditions

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          Abstract

          We previously reported the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different clinical samples. 1 This virus can be detected on different surfaces in a contaminated site. 2 Here, we report the stability of SARS-CoV-2 in different environmental conditions. We first measured the stability of SARS-CoV-2 at different temperatures. SARS-CoV-2 in virus transport medium (final concentration ∼6·8 log unit of 50% tissue culture infectious dose [TCID50] per mL) was incubated for up to 14 days and then tested for its infectivity (appendix p 1). The virus is highly stable at 4°C, but sensitive to heat. At 4°C, there was only around a 0·7 log-unit reduction of infectious titre on day 14. With the incubation temperature increased to 70°C, the time for virus inactivation was reduced to 5 mins. We further investigated the stability of this virus on different surfaces. Briefly, a 5 μL droplet of virus culture (∼7·8 log unit of TCID50 per mL) was pipetted on a surface (appendix p 1; ∼cm2 per piece) and left at room temperature (22°C) with a relative humidity of around 65%. The inoculated objects retrieved at desired time-points were immediately soaked with 200 μL of virus transport medium for 30 mins to elute the virus. Therefore, this recovery of virus does not necessarily reflect the potential to pick up the virus from casual contact. No infectious virus could be recovered from printing and tissue papers after a 3-hour incubation, whereas no infectious virus could be detected from treated wood and cloth on day 2. By contrast, SARS-CoV-2 was more stable on smooth surfaces. No infectious virus could be detected from treated smooth surfaces on day 4 (glass and banknote) or day 7 (stainless steel and plastic). Strikingly, a detectable level of infectious virus could still be present on the outer layer of a surgical mask on day 7 (∼0·1% of the original inoculum). Interestingly, a biphasic decay of infectious SARS-CoV-2 could be found in samples recovered from these smooth surfaces (appendix pp 2–7). 39 representative non-infectious samples tested positive by RT-PCR 3 (data not shown), showing that non-infectious viruses could still be recovered by the eluents. We also tested the virucidal effects of disinfectants by adding 15 μL of SARS-CoV-2 culture (∼7·8 log unit of TCID50 per mL) to 135 μL of various disinfectants at working concentration (appendix p 1). With the exception of a 5-min incubation with hand soap, no infectious virus could be detected after a 5-min incubation at room temperature (22°C). Additionally, we also found that SARS-CoV-2 is extremely stable in a wide range of pH values at room temperature (pH 3–10; appendix p 1). Overall, SARS-CoV-2 can be highly stable in a favourable environment, 4 but it is also susceptible to standard disinfection methods.

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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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            Viral load of SARS-CoV-2 in clinical samples

            An outbreak caused by a novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in December 2019, 1 and has since spread within China and to other countries. Real-time RT-PCR assays are recommended for diagnosis of SARS-CoV-2 infection. 2 However, viral dynamics in infected patients are still yet to be fully determined. Here, we report our findings from different types of clinical specimens collected from 82 infected individuals. Serial samples (throat swabs, sputum, urine, and stool) from two patients in Beijing were collected daily after their hospitalisation (patient 1, days 3–12 post-onset; patient 2, days 4–15 post-onset). These samples were examined by an N-gene-specific quantitative RT-PCR assay, as described elsewhere. 3 The viral loads in throat swab and sputum samples peaked at around 5–6 days after symptom onset, ranging from around 104 to 107 copies per mL during this time (figure A, B ). This pattern of changes in viral load is distinct from the one observed in patients with SARS, which normally peaked at around 10 days after onset. 4 Sputum samples generally showed higher viral loads than throat swab samples. No viral RNA was detected in urine or stool samples from these two patients. Figure Viral dynamics of SARS-CoV-2 in infected patients Viral load (mean [SD]) from serial throat swab and sputum samples in patient 1 (A) and patient 2 (B). (C) Viral load (median [IQR]) in throat and sputum samples collected from 80 patients at different stages after disease onset. (D) Correlation between viral load in throat swab samples and viral load in sputum samples. We also studied respiratory samples (nasal [n=1] and throat swabs [n=67], and sputum [n=42]) collected from 80 individuals at different stages of infection. The viral loads ranged from 641 copies per mL to 1·34 × 1011 copies per mL, with a median of 7·99 × 104 in throat samples and 7·52 × 105 in sputum samples (figure C). The only nasal swab tested in this study (taken on day 3 post-onset) showed a viral load of 1·69 × 105 copies per mL. Overall, the viral load early after onset was high (>1 × 106 copies per mL). However, a sputum sample collected on day 8 post-onset from a patient who died had a very high viral load (1·34 × 1011 copies per mL). Notably, two individuals, who were under active surveillance because of a history of exposure to SARS-CoV-2-infected patients showed positive results on RT-PCR a day before onset, suggesting that infected individuals can be infectious before them become symptomatic. Among the 30 pairs of throat swab and sputum samples available, viral loads were significantly correlated between the two sample types for days 1–3 (R2=0·50, p=0·022), days 4–7 (R2=0·93, p<0·001), and days 7–14 (R2=0·95, p=0·028). From 17 confirmed cases of SARS-CoV-2 infection with available data (representing days 0–13 after onset), stool samples from nine (53%; days 0–11 after onset) were positive on RT-PCR analysis. Although the viral loads were less than those of respiratory samples (range 550 copies per mL to 1·21 × 105 copies per mL), precautionary measures should be considered when handling faecal samples.
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              Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia

              Abstract Background A novel coronavirus of zoonotic origin (2019-nCoV) has recently been identified in patients with acute respiratory disease. This virus is genetically similar to SARS coronavirus and bat SARS-like coronaviruses. The outbreak was initially detected in Wuhan, a major city of China, but has subsequently been detected in other provinces of China. Travel-associated cases have also been reported in a few other countries. Outbreaks in health care workers indicate human-to-human transmission. Molecular tests for rapid detection of this virus are urgently needed for early identification of infected patients. Methods We developed two 1-step quantitative real-time reverse-transcription PCR assays to detect two different regions (ORF1b and N) of the viral genome. The primer and probe sets were designed to react with this novel coronavirus and its closely related viruses, such as SARS coronavirus. These assays were evaluated using a panel of positive and negative controls. In addition, respiratory specimens from two 2019-nCoV-infected patients were tested. Results Using RNA extracted from cells infected by SARS coronavirus as a positive control, these assays were shown to have a dynamic range of at least seven orders of magnitude (2x10−4-2000 TCID50/reaction). Using DNA plasmids as positive standards, the detection limits of these assays were found to be below 10 copies per reaction. All negative control samples were negative in the assays. Samples from two 2019-nCoV-infected patients were positive in the tests. Conclusions The established assays can achieve a rapid detection of 2019n-CoV in human samples, thereby allowing early identification of patients.
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                Author and article information

                Contributors
                Journal
                The Lancet Microbe
                The Author(s). Published by Elsevier Ltd.
                2666-5247
                2666-5247
                2 April 2020
                May 2020
                2 April 2020
                : 1
                : 1
                : e10
                Affiliations
                [a ]School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China
                Article
                S2666-5247(20)30003-3
                10.1016/S2666-5247(20)30003-3
                7214863
                32835322
                e27a916c-d648-4c52-8f96-9c0f462100b3
                © 2020 The Author(s). Published by Elsevier Ltd.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

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