Table 1.

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.