Volume 22, Number 7—July 2016
Dispatch
Effective Chemical Inactivation of Ebola Virus
Table
Summary of methods and results for chemical inactivation of Ebola virus*
Inactivation method | Reagent volume | Sample type† | Inactivated sample (final viral load) | Contact time | Temp. | Reagent removal process | Result (in vitro) | Result (in vivo) |
---|---|---|---|---|---|---|---|---|
Buffer AVL |
560 µL | Liquid virus stock | 140 µL (1.4 × 106 TCID50) | 10 min | 20°C | Dialysis | Pos (1/9) | Neg (0/15) |
600 µL | Liquid virus stock | 100 µL (106 TCID50) | 10 min | 20°C | Dialysis | Pos (1/9) | Neg (0/15) | |
560 µL | Liquid virus stock | 140 µL (1.4 × 106 TCID50) | Overnight | 4°C | Dialysis | Neg (0/9) | ND | |
560 µL |
Liquid virus
stock |
140 µL
(1.4 × 106 TCID50) |
7 d |
–80°C |
Dialysis |
Neg
(0/9) |
ND |
|
Buffer AVL
+ ethanol‡ |
560 µL | Liquid virus stock | 140 µL (1.4 × 106 TCID50) | 10 min + 20 min | 20°C | Dialysis | Neg (0/9) | Neg (0/15) |
600 µL |
Liquid virus
stock |
100 µL
(106 TCID50) |
10 min +
20 min |
20°C |
Dialysis |
Neg
(0/9) |
Neg
(0/15) |
|
Buffer RLT |
600 µL | Cell pellet | 5 × 106 infected cells (≈5 × 106 TCID50) | 10 min | 20°C | Dialysis | Pos (4/9) | Pos (15/15) |
800 µL |
Cell pellet |
5 × 106 infected cells
(≈5 × 106 TCID50) |
10 min |
20°C |
Dialysis |
Pos (6/9) |
Pos
(14/15) |
|
Buffer RLT + ethanol |
600 µL | Cell pellet | 5 × 106 infected cells (≈5 × 106 TCID50) | 10 min + 20 min | 20°C | Dialysis | Neg (0/9) | Neg (0/15) |
800 µL | Cell pellet | 5 × 106 infected cells (≈5 × 106 TCID50) | 10 min + 20 min | 20°C | Dialysis | Neg (0/9) | Neg (0/15) | |
600 µL |
Tissue |
30 mg
(≈3 × 105 TCID50) |
10 min +
20 min |
20°C |
Dialysis |
Neg
(0/9) |
ND |
|
TRIzol§ |
750 µL, 75% final | Cell pellet in 250 µL | 5 × 106 infected cells (≈5 × 106 TCID50) | 10 min | 20°C | Dialysis | Neg (0/9) | ND |
750 µL, 75% final | Blood | 250 µL (≈2.5 × 105 TCID50) | 10 min | 20°C | Dialysis | Neg (0/9) | ND | |
1 mL |
Tissue |
50 mg
(≈5 × 105 TCID50) |
10 min |
20°C |
Dialysis |
Neg
(0/9) |
ND |
|
Formalin |
750 µL, 7.5% final | Cells, 250 µL | 2.5 × 106 infected cells (≈2.5 × 106 TCID50) | Overnight | 4°C | Dialysis | Neg (0/9) | Neg (0/15) |
10 mL, 10% final |
Tissue |
150 mg
(≈1.5x6 TCID50) |
7 d or 30 d¶ |
4°C |
Dialysis |
Neg
(0/9) |
ND |
|
Glutaraldehyde |
1.3 mL, 2% final | Cells, 330 µL | 3.3 × 106 infected cells (≈3.3 × 106 TCID50) | Overnight | 4°C | Dialysis | Neg (0/9) | Neg (0/15) |
10 mL, 2% final |
Tissue |
150 mg
(≈1.5 × 106 TCID50) |
7 d |
4°C |
Dialysis |
Neg
(0/9) |
ND |
|
Paraformaldehyde |
1.3 mL, 2% final | Cells, 330 µL | 3.3 × 106 infected cells (≈3.3 × 106 TCID50) | Overnight | 4°C | Dialysis | Neg (0/9) | Neg (0/15) |
10 mL, 2% final |
Tissue |
150 mg
(≈1.5x6 TCID50) |
7 d |
4°C |
Dialysis |
Neg
(0/9) |
ND |
|
Heat# |
NA | Cells | 1 mL, 1:10 dilution (≈106 TCID50) | 5 min | 100°C | NA | Pos (3/3) | ND |
NA | Cells | 1 mL, 1:10 dilution (≈106 TCID50) | 10 min | 100°C | NA | Neg (0/9) | ND | |
NA | Cells | 1 mL, 1:10 dilution (≈106 TCID50) | 5 min | 120°C | NA | Neg (0/9) | ND | |
NA | Cells | 1 mL, 1:10 dilution (≈106 TCID50) | 10 min | 120°C | NA | Neg (0/9) | ND | |
NA | Liquid virus stock | 1 mL, 1:10 dilution (106 TCID50) | 15 min | 65°C or 70°C | NA | Pos (6/6) | ND | |
NA |
Liquid virus stock |
1 mL, 1:10 dilution
(106 TCID50) |
30 min |
60°C or 65°C |
NA |
Pos (6/6) |
ND |
|
ELISA buffer |
960 µL |
Liquid virus stock |
40 µL
(4 × 105 TCID50) |
10 min |
20°C |
Detergent
column |
Pos (2/9) |
ND |
Heat + ELISA buffer |
960 µL | Liquid virus stock | 40 µL (4 × 105 TCID50) | 30 min | 60°C | Detergent column | Neg (0/9) | Neg (0/15) |
960 µL | Liquid virus stock | 40 µL (4 × 105 TCID50) | 15 min | 65°C | Detergent column | Neg (0/9) | ND | |
960 µL | Liquid virus stock | 40 µL (4 × 105 TCID50) | 30 min | 65°C | Detergent column | Neg (0/9) | ND | |
960 µL |
Liquid virus stock |
40 µL
(4 × 105 TCID50) |
15 min |
70°C |
Detergent
column |
Neg
(0/9) |
ND |
|
1% SDS, 5% 2-ME** |
250 µL, 4× | Cells, 250 µL | 2.5 × 106 infected cells (≈2.5 × 106 TCID50) | 10 min | 20°C | Detergent column | Neg (0/9) | ND |
250 µL, 4× |
Tissue |
150 mg
(≈1.5 × 106 TCID50) |
10 min |
20°C |
Detergent
column |
Neg
(0/9) |
ND |
|
1% SDS | 250 µL, 4× | Cells, 250 µL | 2.5 × 106 infected cells (≈2.5 × 106 TCID50) | 10 min | 20°C | Detergent column | Neg (0/9) | ND |
*DPBS, Dulbecco’s phosphate-buffered saline; NA, non-applicable; ND, not done; SDS, sodium dodecyl sulfate; TCID50, tissue culture infectious dose 50.
†Initial sample virus titers were as follows: liquid virus stock (1 × 107 TCID50/mL), infected cells (≈1 × 107 TCID50/mL, 1 × 107 cells/mL), blood (≈106 TCID50/mL), tissue (≈104 TCID50/mg). Infected cells (5 × 106 cells) were pelleted by centrifugation and lysed in test reagent (Buffer RLT; QIAGEN, Valencia, CA, USA), pelleted and resuspended in 250 μL DPBS before addition of test reagent (TRIzol; Life Technologies, Grand Island, NY, USA), or used directly in solution at the concentration and volume described in the table (fixative, heat, and SDS testing). Ebola virus–infected blood and liver tissue were collected from BALB/c mice infected with mouse-adapted Ebola virus at the height of infection, or from mock infected mice at a corresponding day.
‡Addition of ethanol: after contact time with Buffer AVL (QIAGEN, Valencia, CA, USA) 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 contact time at 20°C.
§Although Spectra/Por Float-A-Lyzer G2 dialysis tubes (Spectrum Laboratories, Lawrenceville, GA, USA) were used for all other reagent removal by dialysis, Slide-A-Lyzer cassettes (Fisher Scientific, Pittsburgh, PA, USA) were used with TRIzol because the cassettes are more resistant to direct degradation by the reagent.
¶Tissue samples <1 cm3 were treated with a 7-day contact time, whereas samples >1 cm3 were treated with a 30-day contact time.
#Samples were boiled in an AccuBlock Digital Dry Bath (Sigma-Aldrich, St. Louis, MO, USA). All other heat treatments were carried out in a nonshaking water bath.
**4× SDS loading buffer contains 200 mmol/L Tris (pH 6.8), 4% SDS, 35% glycerol, 0.05% bromophenol blue and 20% 2-ME (added at the time of use). A combination of 250 μL 4× buffer, sample, and DPBS for a combined volume of 1 mL was tested; an appropriate volume of DPBS was substituted for 2-ME as necessary for testing of SDS as the single inactivating reagent.