Prolonged Infectivity of SARS-CoV-2 in Fomites

We spotted severe acute respiratory syndrome coronavirus 2 on polystyrene plastic, aluminum, and glass for 96 hours with and without bovine serum albumin (3 g/L). We observed a steady infectivity (<1 log10 drop) on plastic, a 3.5 log10 decrease on glass, and a 6 log10 drop on aluminum. The presence of proteins noticeably prolonged infectivity.

S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide, demonstrating a great potential for direct and indirect transmission between humans. Coronaviruses can keep their infectivity in fomites and thus can remain infectious on dry surfaces for hours (1,2). However, limited data are available for SARS-CoV-2 (1). Specifically, there are no data about the role of interfering substances such as proteins on SARS-CoV-2 infectivity in the environment. We evaluated the stability and infectivity of SARS-CoV-2 deposited on polystyrene plastic, aluminum, and glass for 96 hours at 45%-55% relative humidity (recommended for indoor living spaces by the American Society of Heating, Refrigeration and Air Conditioning Engineers) and 19°C -21°C temperature range using a 10 6 50% tissue culture infectivity dose (TCID 50 )/mL inoculum.
We inoculated SARS-CoV-2 at a multiplicity of infection of 0.001 onto Vero E6 cells incubated at 37°C in 5% CO 2 for 72 h (Appendix, https://wwwnc.cdc.gov/ EID/article/26/10/20-1788-App1.pdf). We collected the supernatant and clarified it by spinning at 1500 × g for 10 min. We prepared aliquots and stored them at -80°C before titration. We measured virus infectivity using TCID 50 . We diluted the inoculum in cell culture medium containing 5% fetal bovine serum (FBS; final protein concentration 1.8 g/L) to 10 6 TCID 50 /mL. For experiments with a higher protein concentration, we used a concentrated bovine serum albumin (BSA) solution (40 g/L) to result in a final protein concentration of 11.4 g/L. We measured virus infectivity sequentially on polypropylene plastic, aluminum, and glass slides. We deposited a 50-μL drop in triplicate on the various surfaces (≈1 cm 2 per piece) and recovered them sequentially to quantify viable infectious virions by endpoint titration on Vero E6 cells. The limit of detection for the assays was 10 0.5 TCID 50 /mL.
We conducted our experiments with and without BSA to mimic the protein content within body fluids of the respiratory system such as cough droplets, sputum, and airway mucosal secretions (3). Final protein concentration was 1.8 g/L without BSA conditions and 11.4 g/L with BSA conditions. We observed 3 different profiles, depending on surface type: a 3.5 log 10 decrease over 44 h on glass ( Figure 1, panel A), a steady infectivity with a <1 log 10 drop over 92 h on polystyrene plastic ( Figure 1, panel B), and a sharp 6 log 10 drop in <4 h on aluminum (Figure 1 panel C). The probable adsorption of viral particles onto a plastic polystyrene surface was associated with prolonged infectivity, whereas a high drop on aluminum was observed as in previously published data on SARS-CoV, adenovirus, or poliovirus (4,5). Our results have also shown higher stability for SARS-CoV-2 on polystyrene plastic, with or without BSA, in comparison with a recent study (1); this variation could be explained by a different type of plastic used in the 2 studies. Regardless of the type of surface, virus infectivity decreased ≈1 log 10 within 2 h (Table).
To study SARS-CoV-2 stability in solution, we titrated cell culture supernatants containing 10 6 TCID 50 /mL every 24 h for 96 h. We found that SARS-CoV-2 was very stable, showing an overall decreased infectivity <1.4 log 10 reduction, results similar to those described for SARS-CoV (Appendix Figure) (4).

RESEARCH LETTERS
We spotted severe acute respiratory syndrome coronavirus 2 on polystyrene plastic, aluminum, and glass for 96 hours with and without bovine serum albumin (3 g/L). We observed a steady infectivity (<1 log 10 drop) on plastic, a 3.5 log 10 decrease on glass, and a 6 log 10 drop on aluminum.
The presence of proteins noticeably prolonged infectivity.

Figure.
Viability of severe acute respiratory syndrome coronavirus 2 on various surfaces and in suspension. Viruses were applied to glass (A), polystyrene plastic (B), and aluminum (C) at 45%-55% relative humidity at 19°C-21°C for 96 h. The titer of viable virus is expressed as TCID 50 /mL of collection medium. All samples were quantified by endpoint titration on Vero E6 cells with a limit of detection of ≈10 0.5 TCID 50 /mL. TCID 50 , 50% tissue culture infectivity dose.
Our data showed that SARS-CoV-2 infectivity was remarkably preserved in the presence of proteins, regardless of the type of surface. A final concentration of 11.4 g/L of proteins, as used in our study, closely mimics that of respiratory fluids, which possess protein concentrations of a similar order of magnitude. However, the respiratory body fluids are complex media including not only proteins, but also enzymes and mucins (present in mucus) that may have a negative effect on virus infectivity. Regarding viral load measurement, the reason for avoiding the use of molecular techniques such as reverse transcription PCR is that despite that they allow quantification of RNA copies and determination of RNA decay, they cannot measure residual infectivity on various surfaces.
The protective effect of proteins had already been described for pandemic SARS-CoV or suggested for influenza A(H1N1) virus, but with less notable effects (4,6). As illustrated in other virus models (7), interfering substances such as proteins influenced the resistance of SARS-CoV-2 to drying and thus its persistence in the environment.
In conclusion, we showed that a moderate protein concentration in droplets markedly increased the infectivity of SARS-CoV-2, suggesting that a proteinrich medium like airway secretions could protect the virus when it is expelled and may enhance its persistence and transmission by contaminated fomites. Accordingly, it is plausible that fomites infected with SARS-CoV-2 play a key role in the indirect transmission of coronavirus disease (COVID-19). This finding supports surface cleaning as a necessary action that should be enforced and repeated becuase it may play a key role in halting SARS-CoV-2 transmission and mitigating the COVID-19 pandemic.

About the Author
Dr. Pastorino is research engineer with a PhD in virology. His primary research interests are biosafety aspects of Biosafety Level 3 viruses in the context of preparedness and response against epidemics.