Cells, viruses, and quantification of infectious titers. Propagation and quantification of virus stocks and samples were performed using VeroE6 cells12. All cultures were maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 10% tryptose phosphate broth, penicillin (100 unit/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM). SARS-CoV-2 USA-WA-1/2020 was originally acquired from BEI Resources (Lot number 70033175) and passaged twice to generate the stocks used in this study. Infectious titers of all samples were quantified using median tissue culture infectious dose (TCID50) method as previously described13.
Generation of Contaminated Surface and treatment with Dry Hydrogen Peroxide. Dried contaminated surfaces were created using 25 mm (W) x 75 mm (L) x 1 mm (D) microscope glass slides spiked with SARS-CoV-2. Standard glass microscope slides were selected for testing based on the published report on the recovery of infectious viruses up to 48 hours after the inoculation of SARS-CoV-2 and desiccation14. Contaminated glass slides were used to determine the decay of residual infectious titers after the initiation of DHP treatment and compared to slides in the control group, which did not receive DHP treatment.
Each glass slide was spiked with 250 μl of SARS-CoV-2 stocks at approximately 6 log10TCID50/ml followed by drying inside a class II A2 biosafety cabinet for one hour. Three contaminated glass slides were randomly assigned as a set, placed inside a 100 mm x 100 mm square petri dishes, sealed with parafilm and transported to two biosafety level-3 large animal (BSL-3Ag) laboratories that are each 50 m2 in size. The first laboratory was charged with DHP by allowing the continuous operation of a Sentry DHP generator at full output of 38 cubic feet per minute (Synexis LLC, Lenexa, KS) for 24 hours prior to the experiment. This setting generates DHP at a concentration no greater than 25 parts per billion (ppb)15.The second laboratory was used as a control environment which maintained the negative air pressure required for BSL-3 laboratories. To simulate the air exchange conditions in public spaces, the rate of air exchange in both BSL-3Ag laboratories was reduced to seven air exchanges per hour, the minimal frequency required for the maintenance of negative air pressure. Ambient temperature was maintained at 22°C throughout the experiment. Relative humidity was set at 55% for the entire time course. A total of nine sets of triplicate samples were used in each group to quantify residual infectious viruses present on contaminated glass slides incubated in each room at 0, 15, 30, 45, 60, 90, 120, 240, and 1440 minutes after the exposure to DHP and ambient air. The time course was designed to reflect the stability of SARS-CoV-2 on a variety of contaminated surfaces as residual infectious titers can maintain above the limit of detection of our assay at 1.06 log10TCID50/ml for up to 24 hours16. At each designated timepoint, the lid of each petri dish was returned, sealed with clear adhesive tape and kept on ice until the elution of residual viruses.
Elution of residual infectious viruses from contaminated glass slides. Residual infectious viruses were eluted by gently wiping the surface of each contaminated glass slide with a sterile polyester applicator (Fisher Scientific) saturated with Dulbecco’s phosphate buffer saline for 20 times. The tip of each applicator was submerged in 1 ml of DMEM media followed by vortexing at 3,000 rpm for 15 seconds to dislodge infectious viruses. All DMEM media containing infectious viruses were filtered using 0.22 μm disc filters to eliminate environmental contaminants and titrated using TCID50 method.
Statistical analysis. Residual infectious titers of individual samples collected at designated timepoints were calculated using the Reed-Munich method. Whilst the triplicate samples are a standard practice for environmental stability, the numbers of replicates do not warrant the analysis using repeated measures t-test17. Therefore, the kinetics in the decay of infectious titers between two groups was compared using mixed effects model.