Protocol for SARS-CoV-2 Coronavirus Inactivation with Sonication
Inactivation of the SARS-CoV-2 Coronavirus with the VialTweeter
After removing the fixative, monolayers were washed three times with phosphate-buffered saline (PBS) before scraping cells into 1mL MEM/5% FBS and sonicated (3 × 10 second on, 10 seconds off at 100% power and amplitude) using the Hielscher ultrasonic processor UP200St with VialTweeter attachment. Supernatants were clarified by centrifuging at 3000 × g for 10 mins. Read the full protocol here for the SARS-CoV-2 coronavirus inactivation by Welch et al. (2020) below:
Cells and Virus
Vero E6 cells (Vero C1008; ATCC CRL-1586) were cultured in modified Eagle’s minimum essential medium (MEM) supplemented with 10% (v/v) fetal calf serum (FCS). Virus used was SARS-CoV-2 strain hCOV-19/England/2/2020, isolated by PHE from the first patient cluster in the UK on 29/01/2020. This virus was obtained at passage 1 and used for inactivation studies at passage 2 or 3.
For reagents and chemicals used for SARS-CoV-2 inactivation as well as the removal of reagent cytotoxicity please see the scientific report of Welch et al. (2020).
For commercial products, virus preparations (tissue culture fluid, titers ranging from 1 × 106 to 1 × 108 PFU/ml) were treated in triplicate with reagents at concentrations and for contact times recommended in the manufacturers’ instructions for use, where available, or for concentrations and times specifically requested by testing laboratories. Where a range of concentrations was given by the manufacturer, the lowest ratio of product to sample was tested (i.e. lowest recommended concentration of test product). Specimen transport tube reagents were tested using a ratio of one volume of tissue culture fluid to ten volumes of reagent, unless a volume ratio of sample fluid to reagent was specified by the manufacturer. Detergents, fixatives and solvents were tested at the indicated concentrations for the indicated times. All inactivation steps were performed at ambient room temperature (18 – 25°C). For testing of alternative sample types, virus was spiked into the indicated sample matrix at a ratio of 1:9, then treated with test reagents as above. All experiments included triplicate control mock-treated samples with an equivalent volume of PBS in place of test reagent. Immediately following the required contact time, 1mL of treated sample was processed using the appropriately selected filtration matrix. Reagent removal for inactivation testing was carried out in a larger spin column format using Pierce 4mL Detergent Removal Spin Columns (Thermo Fisher), or by filling empty Pierce 10mL capacity centrifuge columns (Thermo Fisher) with SM2 Bio-Beads, Sephacryl S-400HR or Sephadex LH-20 to give 4mL packed beads/resin. For purification using Amicon filters, 2 × 500μl samples were purified using two centrifugal filters by the method previously described, then pooled together. For formaldehyde and formaldehyde with glutaraldehyde removal, one filter was used with 1× 500μl sample volume, resuspended after processing in 500μl PBS, and added to 400ul MEM/5% FBS. For inactivation of infected monolayers, 12.5 cm2 flasks of Vero E6 cells (2.5 × 106 cells/flask in 2.5mL MEM/5% FBS) were infected at MOI 0.001 and incubated at 37°C/5% CO2 for 24 hours. Supernatant was removed, and cells fixed using 5mL of formaldehyde, or formaldehyde and glutaraldehyde at room temperature for 15 or 60 mins. The fixative was removed, and monolayers washed three times with PBS before scraping cells into 1mL MEM/5% FBS and sonicated (3 × 10 second on, 10 seconds off at 100% power and amplitude) using a UP200St with VialTweeter attachment (Hielscher Ultrasound Technology). Supernatants were clarified by centrifuging at 3000 × g for 10 mins.
The full protocol including the use of the Hielscher VialTweeter can be found here:
Welch, Stephen R.; Davies, Katherine A.; Buczkowski, Hubert; Hettiarachchi, Nipunadi; Green, Nicole; Arnold, Ulrike; Jones, Matthew; Hannah, Matthew J.; Evans, Reah; Burton, Christopher; Burton, Jane E.; Guiver, Malcolm; Cane, Patricia A.; Woodford, Neil; Bruce, Christine B.; Roberts, Allen D. G.; Killip, Marian J. (2020): Inactivation analysis of SARS-CoV-2 by specimen transport media, nucleic acid extraction reagents, detergents and fixatives. Journal of Clinical Microbiology. Accepted Manuscript Posted Online 24 August 2020.
- Sonication of up to 10 vials simultaneously
- No cross-contamination
- No sample loss
- Automatic data recording
- Easy and safe to operate
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The multi-sample ultrasonicator VialTweeter is just one of many ultrasonic solutions for sample preparation in biological, biochemical and clinical laboratories. Hielscher Ultrasonics offers the ideal ultrasonic dismembrator for your application, e.g. cell lysis, cell extraction, tissue homogenization, lysate solubilization, dissolving, sample degassing etc.
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Literature / References
- Welch, Stephen R.; Davies, Katherine A.; Buczkowski, Hubert; Hettiarachchi, Nipunadi; Green, Nicole; Arnold, Ulrike; Jones, Matthew; Hannah, Matthew J.; Evans, Reah; Burton, Christopher; Burton, Jane E.; Guiver, Malcolm; Cane, Patricia A.; Woodford, Neil; Bruce, Christine B.; Roberts, Allen D. G.; Killip, Marian J. (2020): Inactivation analysis of SARS-CoV-2 by specimen transport media, nucleic acid extraction reagents, detergents and fixatives. Journal of Clinical Microbiology. Accepted Manuscript Posted Online 24 August 2020.
- Natacha S. Ogando; Tim J. Dalebout; Jessika C. Zevenhoven-Dobbe; Ronald W. Limpens; Yvonne van der Meer; Leon Caly; Julian Druce; Jutte J. C. de Vries; Marjolein Kikkert; Montserrat Bárcena; Igor Sidorov; Eric J. Snijder (2020): SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology. bioRxiv posted 20 April 2020.
Facts Worth Knowing
What are Vero Cells?
Vero E6, also known as Vero C1008 (ATCC No. CRL-1586) is clone cell line from Vero 76 and is used in the research of SARS-CoV and SARS-CoV-2 coronaviruses. Vero cells are a lineage of cells used in cell cultures. The ‘Vero’ lineage was isolated from kidney epithelial cells extracted from an African green monkey (Chlorocebus sp.).
Vero E6 cells show some contact inhibition, so are suitable for propagating viruses that replicate slowly. Vero E6 cell lines are commonly used to investigate the cytopathology of coronaviruses SARS-CoV and SARS-CoV-2 as Vero cells (African green monkey kidney cells) exhibit an abundant expression of angiotensin-converting enzyme 2 (ACE2) receptors. ACE2 receptors are a major docking site for the SARS-CoV-2 coronavirus.
For instance, Ogando et al. (2020) found that SARS-CoV-2 – in comparison to SARS-CoV – generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Furthermore, they determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (Remdesivir, Alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that – upon passaging in Vero E6 cells – SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative ‘furin-like cleavage site’ in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.