Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 2019 (COVID-19) pandemic.1–3 There is a high demand for safe sample handling to perform tests on in vitro and in vivo material in research and diagnostic laboratories worldwide. SARS-CoV-2 is considered a risk group 3 pathogen, and safety at workplaces is of the highest priority.4 SARS-CoV-2 is an enveloped, single-strand, positive-sense RNA virus with a genome size of approximately 30 kb.2,3,5 Its biological, biochemical, and physical features make the virus sensitive to chemical and physical inactivation procedures. We evaluated commonly used inactivation procedures to generate safe material for downstream genome, protein, immune response, and histopathology analyses.
All infectious work was performed under high biocontainment conditions at the Rocky Mountain Laboratories of the National Institute of Allergy and Infectious Diseases (NIAID) according to standard operating protocols (SOPs) approved by the Institutional Biosafety Committee (IBC). For our studies, we used the SARS-CoV-2 isolate nCoV-WA1-2020 (MN985325.1; kindly provided by the Centers for Disease Control and Prevention).6 SARS-CoV-2 replication in cell culture causes a cytopathic effect (CPE), thus allowing for a simple readout parameter. SARS-CoV-2 stocks were grown in VeroE6 cells and titrated using a tissue culture infectious dose 50% (TCID50) assay.7 The TCID50 was calculated via the Reed-Muench formula to a concentration of 4 × 106 TCID50/mL.8 Cells were produced by infecting VeroE6 cells at a multiplicity of infection of 0.01 SARS-CoV-2. Cells were harvested in Dulbecco's phosphate-buffered saline (DPBS) at CPE of approximately 75%, counted, and frozen (−80°C) in aliquots of 2 × 106, 5 × 106, and 2 × 107 cells/mL. SARS-CoV-2-infected lung tissue (≈1 × 1010 TCID50/g) was obtained from a previous Syrian hamster study approved by the Institutional Animal Care and Use Committee.9
We tested the physical and chemical inactivation of virus stocks as well as chemical inactivation of infected cells and tissue. Triplicate samples were dialyzed with an 8- to 10-kDa molecular weight cutoff (Repligen Corporation, Waltham, MA) using DPBS over a stir plate at 4°C (> 500-fold exchange volumes, five changes during 32–48 hours) or run over a detergent removal column (DetergentOUT GBS10-5000 columns; G-Biosciences, St. Louis, MO). DPBS and noninfected VeroE6 cells and hamster lung tissues served as negative controls. Untreated virus stocks and SARS-CoV-2-infected VeroE6 cells and hamster lung tissue were used as positive controls. All samples were brought to a final volume of 3 ml and equally divided to infect VeroE6 cells (80% confluency) in triplicate for a total of 9 flasks per sample type. Cells were incubated at 37°C for 7 days and monitored regularly for CPE. Three days after initial infection, samples were passaged by transferring 1 mL supernatant each to a new flask of VeroE6 cells (80% confluency). Additionally, we tested extracted RNA with or without transfection reagents to assess RNA infectivity. The results are summarized in Table 1, and detailed protocols are provided as Supplementary Material.
Summary of inactivation methods and results
|Inactivation method||Reagent volume||Sample type*||Inactivated sample (final viral load)||Contact time||Temp.||Reagent removal process||Result (initial infection)||Result (passage)|
|Irradiation||1 mL||Liquid virus stock||1 × 106 TCID50||0, 0.2, 0.4, 0.6, or 0.8 Mrd dose||Dry ice||Positive (9/9)||Positive (9/9)|
|1 mL||Liquid virus stock||1 × 106 TCID50||1.0 Mrd dose||Dry ice||Negative (0/9)||Negative (0/9)|
|Buffer AVL + ethanol†||560 μL||Liquid virus stock||140 μL (5.6 × 105 TCID50)||10 min + 20 min||20°C||Dialysis||Negative (0/9)||Negative (0/9)|
|Buffer AVL RNA extract transfection||560 μL||Liquid virus stock RNA extract||30 μL RNA extract (2.8 × 105 TCID50 equivalent)||10 min + 20 min||20°C||Extraction||Negative (0/6)||Negative (0/6)|
|560 μL||Liquid virus stock RNA extract with TransIT LT1||30 μL RNA extract (2.8 × 105 TCID50 equivalent)||10 min + 20 min||20°C||Extraction||Negative (0/6)||Negative (0/6)|
|560 μL||Liquid virus stock RNA extract with Lipofectamine LTX||30 μL RNA extract (2.8 × 105 TCID50 equivalent)||10 min + 20 min||20°C||Extraction||Negative (0/6)||Negative (0/6)|
|560 μL||Liquid virus stock RNA extract with TransIT mRNA||30 μL RNA extract (2.8 × 105 TCID50 equivalent)||10 min + 20 min||20°C||Extraction||Positive (6/6)||Positive (6/6)|
|Buffer RLT + ethanol†||600 μL||Cell pellet||5 × 106 infected cells (∼5 × 106 TCID50)||10 min + 20 min||20°C||Dialysis||Negative (0/9)||Negative (0/9)|
|600 μL||Tissue||30 mg (∼3 × 108 TCID50)||10 min + 20 min||20°C||Dialysis||Negative (0/9)||Negative (0/9)|
|Trizol||275 μL||Liquid virus stock||125 μL (5 × 105 TCID50)||10 min||20°C||Dialysis||Negative (0/9)||Negative (0/9)|
|300 μL||Cells in 150 µL||5 × 106 infected cells (∼5 × 106 TCID50)||10 min||20°C||Dialysis||Negative (0/9)||Negative (0/9)|
|600 μL||Tissue||50 mg (∼5 × 108 TCID50)||10 min||20°C||Dialysis||Negative (0/9)||Negative (0/9)|
|Formalin||1 mL||Cells||2 × 106 infected cells (∼2 × 106 TCID50)||Overnight||4°C||Dialysis||Negative (0/9)||Negative (0/9)|
|10 mL, 10% final||Tissue||1.4 g (∼1.4×10 TCID50)||7 days||4°C||Dialysis||Negative (0/9)||Negative (0/9)|
|Paraformaldehyde||1 mL, 2% final||Cells||2 × 107 infected cells (∼2 × 107 TCID50)||Overnight||4°C||Dialysis||Negative (0/9)||Negative (0/9)|
|10 mL, 2% final||Tissue||0.7 g (∼7 × 109 TCID50)||7 days||4°C||Dialysis||Negative (0/9)||Negative (0/9)|
|1% SDS‡||100 μL, 4×||Liquid virus stock||300 µL (1.2 × 106 TCID50)||10 min||100°C§||Detergent column||Negative (0/9)||Negative (0/9)|
|100 μL, 4×||Cells in 300 μL||5 × 106 infected cells (∼5 × 106 TCID50)||10 min||100°C§||Detergent column||Negative (0/9)||Negative (0/9)|
Mrd = Megarad.
Initial sample viral titers were as follows: liquid SARS-CoV-2 virus stock (4 × 106 tissue culture infectious dose 50% [TCID50]/mL); infected cells (∼1 × 107 TCID50/mL, 1 × 107 cells/mL); and tissue (∼1010 TCID50/g). Infected cells were pelleted by centrifugation and lysed directly in Buffer RLT or pelleted and resuspended in Dulbecco's phosphate-buffered saline at the concentration and volumes described in the Table before addition of other test reagents). Lung tissue was collected from Syrian hamsters at the height of infection with SARS-CoV-2 or from mock-infected hamsters on a corresponding day.
Addition of ethanol: after contact time with buffer AVL or buffer RLT, samples were transferred to a clean tube with 560 μL 100% ethanol (for AVL inactivation) or 600 μL 70% ethanol (for RLT inactivation) and allowed an additional 20 minutes of contact time at 20°C.
4 × SDS loading buffer contains 200 mM Tris (pH 6.8), 4% SDS, 35% glycerol, 0.05% bromophenol blue, and 20% 2-ME (added at the time of use).
Samples were boiled in an AccuBlock Digital Dry Bath.
The authors acknowledge the Viral Special Pathogens Branch of the Centers for Disease Control and Prevention, Atlanta, GA, USA, for providing SARS-CoV-2. The American Society of Tropical Medicine and Hygiene has waived the Open Access fee for this article due to the ongoing COVID-19 pandemic.
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