Effective Chemical Inactivation of Ebola Virus

Reliable inactivation of specimens before removal from high-level biocontainment is crucial for safe operation. To evaluate efficacy of methods of chemical inactivation, we compared in vitro and in vivo approaches using Ebola virus as a surrogate pathogen. Consequently, we have established parameters and protocols leading to reliable and effective inactivation.

the Institutional Animal Care and Use Committee (IACUC) of RML and performed following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC), by certified staff in an AAALAC-approved facility.

Viruses and Infected Cells
Testing involved 10 7 FFU/ mL stock virus of wild-type EBOV expressing enhanced green fluorescent protein (EBOV-eGFP) or 10 7 FFU/ mL stock virus of mouse-adapted EBOV (MA-EBOV). Virus stocks were grown in VERO E6 cells and titrated using a focus forming unit (FFU) assay. Infected cells were produced by infecting VERO E6 cells at a multiplicity of infection of 0.01. Cells were harvested at CPE of 75%, pelleted, resuspended in 6 mL Dulbecco's phosphate-buffered saline (DPBS) and 1 mL aliquots were stored at -80 o C until use (1 × 10 7 cells/ mL). Negative control cells were made of similarly treated, uninfected cell monolayers. For those tests involving blood or tissues, BALB/c mice were infected with MA-EBOV and blood and liver were collected at the height of disease. Blood and liver from uninfected control animals were used as negative control samples.

Dialysis
For those tests requiring dialysis, samples were dialyzed with a 10kDa molecular weight cutoff. DPBS was used as a dialysis buffer at >500-fold exchange volumes over five changes and 32-48 hours, at 4 o C over a stir plate, before samples were collected and used to infect for testing.
All samples were dialyzed using Spectra/Por Float-A-Lyzer G2 tubing (Spectrum Laboratories) with the exception of those involving TRIzol, which was dialyzed using Slide-A-Lyzer cassettes (Fisher Scientific).

Detergent Removal
DetergentOUT GBS10-5000 columns (G-Biosciences) were utilized to remove detergent from samples, per manufacturer's recommended protocol. Columns were equilibrated twice with DPBS before the detergent-containing sample was incubated on the column for two minutes, spun through the column, collected and used to infect for testing.

Validation Protocol, Cell Culture Model
Virus-infected samples (in triplicate unless otherwise noted) were treated according to the specific testing parameters and dialyzed or run over detergent-removal columns to remove inactivating reagents. Each of the treated samples was then increased in volume to 3 mL as necessary and equally divided to infect triplicate 25 cm 2 flasks of fresh VERO E6 cells at 80% confluency. Following an infection time of 60 minutes at 37 o C, inoculum was removed and 6 mL/flask DMEM with 2% FBS was added. Unless otherwise noted, cells were not washed before addition of fresh medium. Cells were incubated at 37 o C for an additional 14 days and monitored regularly for CPE (MA-EBOV) or CPE and fluorescence (EBOV-eGFP). Positive and negative samples were included in every validation, subjected to the same mechanical treatments (e.g. dialysis, spin columns) as the test samples, and tested on 3 flasks of fresh cells each. Negative control samples were DPBS or uninfected cells /tissue homogenates from animals which were not infected; positive control samples were virus stock/infected cells/infected tissue homogenates Page 3 of 10 that were not treated. A DPBS mock infection of 3 flasks was included in each experiment to control for residual inactivating reagent.

Validation Protocol, Animal Model
Six-to eight-week old female BALB/c mice (Charles River Laboratories) were housed in microisolator cages and allowed to acclimatize prior to use in experiments. Three virus-infected samples were treated according to the specific testing parameters and dialyzed or run over detergent-removal columns to remove inactivating reagents. Each of the treated samples was then increased in volume to 1 mL as necessary and equally divided to infect 5 mice Or 50 mg infected liver (5 × 10 5 TCID50) + 1 mL TRIzol A larger piece (100 mg) was homogenized in 1 mL TRIzol at 30Hz with a stainless steel bead for 10 minutes in a 2 mL tube at 20 o C. The equivalent volume of 50 mg (0.5 mL) was transferred to a clean 2 mL tube and a volume of TRIzol (0.5 mL) added to bring volume back to 1 mL total. This was followed by a 10 minute contact time in 2 mL tube at 20 o C. Dialyzed in Slide-A-Lyzer tubes.
All samples were removed from dialysis tubing, raised to 3 mL final volume with DPBS, and split evenly to infect three flasks of VERO E6 cells. Infection contact time was 1 hour; inoculum was removed, cells were washed to remove any traces of TRIzol and medium (DMEM) was added. TRIzol samples were not tested in mice.

Or
One-half infected liver lobe + 10 mL 10% formalin 7 day contact time in 15 mL tube at 4 o C This larger piece was dissected following contact time for a smaller internal piece (150 mg, 1.5 × 10 6 TCID50) which was homogenized in 1 mL DPBS at 30Hz with a stainless steel bead for 10 minutes in a 2 mL tube at 20 o C. Dialyzed in Spectra/Por Float-A-Lyzer tubes.

Or
One infected liver + 10 mL 10% formalin 30 day contact time in 15 mL tube at 4 o C This larger piece was dissected following contact time for a smaller internal piece (150 mg, 1.5 × 10 6 TCID50) which was homogenized in 1 mL DPBS at 30Hz with a stainless steel bead for 10 minutes in a 2 mL tube at 20 o C. Dialyzed in Spectra/Por Float-A-Lyzer tubes.
All samples were removed from dialysis tubing, raised to 3 mL final volume with DPBS, and split evenly to infect three flasks of VERO E6 cells. Infection contact time was 1 hour; inoculum was removed and replaced with medium (DMEM). Cells were not washed. For those methods tested in mice, samples were split equally to infect 5 mice. All samples were removed from dialysis tubing, raised to 3 mL final volume with DPBS, and split evenly to infect three flasks of VERO E6 cells. Infection contact time was 1 hour;

Glutaraldehyde and Paraformaldehyde Testing
inoculum was removed and replaced with medium (DMEM). Cells were not washed. For those methods tested in mice, samples were split equally to infect 5 mice.
All samples were raised to 3 mL final volume with DPBS, and split evenly to infect three flasks of VERO E6 cells. Infection contact time was 1 hour; inoculum was removed, cells washed and medium (DMEM) was added. After incubation for 24 hours, half the medium was removed and replaced with and additional full volume to avoid a pH drop occasionally seen after use of detergent removal columns. For those methods tested in mice, samples were split equally to infect 5 mice.