Inactivation of SARS-CoV-2 and COVID-19 Patient Samples for Contemporary Immunology and Metabolomics Studies

  1. Devon J Eddins
  2. Leda C Bassit
  3. Joshua D Chandler
  4. Natalie S Haddad
  5. Kathryn L Musall
  6. Junkai Yang
  7. Astrid Kosters
  8. Brian S Dobosh
  9. Mindy R Hernández
  10. Richard P Ramonell
  11. Rabindra M Tirouvanziam
  12. F Eun-Hyung Lee
  13. Keivan Zandi
  14. Raymond F Schinazi
  15. Eliver E B Ghosn

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Due to the severity of COVID-19 disease, the U.S. Centers for Disease Control and Prevention and World Health Organization recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level (BSL)3 laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred to lower containment levels (BSL2), while maintaining the fidelity of complex downstream assays to expedite the development of medical countermeasures. In this study, we demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with commonly used reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and Ab detection, heat inactivation following cDNA amplification for droplet-based single-cell mRNA sequencing, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of viral inactivation protocols for downstream contemporary assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Article activity feed

  1. Version published to 10.4049/immunohorizons.2200005
  2. ScreenIT

    SciScore for 10.1101/2021.10.22.465481: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIRB: All studies were approved by the Emory Institutional Review Board (IRB) under protocol numbers IRB00058507, IRB00057983 and IRB00058271.
    Consent: Informed consent was obtained from the patients when they had decision making ability or from a legal authorized representative (LAR) if the patient was unable to provide consent.
    Field Sample Permit: All work with infectious virus and respiratory samples from COVID-19 patients was conducted inside a biosafety cabinet within the Emory Health and Safety Office (EHSO)- and United States Department of Agriculture (USDA)-approved BSL3 containment facility in the Health Sciences Research Building at Emory University following protocols approved by the Institutional Biosafety Committee (IBC) and Biosafety Officer. 2.2 Virus and cells: African green monkey (Cercopithecus aethiops) kidney epithelial cells (Vero E6 cells; ATCC® CRL-1586™) were maintained in complete (c)DMEM containing: 1X DMEM supplemented with 25 mM HEPES, 2 mM L-glutamine,1 mM sodium pyruvate, 1X non-essential amino acids (NEAA), 1X antibiotic/antimycotic solution (all from Corning) and 10% heat-inactivated FBS (Gibco), unless indicated otherwise.
    IACUC: All work with infectious virus and respiratory samples from COVID-19 patients was conducted inside a biosafety cabinet within the Emory Health and Safety Office (EHSO)- and United States Department of Agriculture (USDA)-approved BSL3 containment facility in the Health Sciences Research Building at Emory University following protocols approved by the Institutional Biosafety Committee (IBC) and Biosafety Officer. 2.2 Virus and cells: African green monkey (Cercopithecus aethiops) kidney epithelial cells (Vero E6 cells; ATCC® CRL-1586™) were maintained in complete (c)DMEM containing: 1X DMEM supplemented with 25 mM HEPES, 2 mM L-glutamine,1 mM sodium pyruvate, 1X non-essential amino acids (NEAA), 1X antibiotic/antimycotic solution (all from Corning) and 10% heat-inactivated FBS (Gibco), unless indicated otherwise.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were incubated with an anti-SARS-CoV-2 spike RBD polyclonal antibody (Gentaur) at 1:3000 overnight at 37°C.
    anti-SARS-CoV-2
    suggested: (Leinco Technologies Cat# LT3000, RRID:AB_2893948)
    Cells were washed to remove excess antibody, then incubated with a secondary HRP-conjugated anti-human IgG for 1 h at 37°C.
    anti-human IgG
    suggested: None
    Sera were assayed at 1:500 dilutions (in assay buffer) and surveyed for anti-SARS-CoV-2 N or RBD antibodies by 1 h incubation on a plate shaker at 800 rpm in the dark.
    anti-SARS-CoV-2 N
    suggested: None
    Median fluorescent intensity (MFI) using combined or individual detection antibodies (i.e., anti-IgA, anti-IgG, or anti-IgM) was measured and the background value of assay buffer was subtracted from each serum sample result to obtain MFI minus background values (net MFI).
    anti-IgA
    suggested: None
    anti-IgG
    suggested: None
    anti-IgM
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    2.3.3 TCID50 assays: Vero E6 cells were seeded in 96-well plates with 2 × 104 cells/well in 5% MEM 24 h prior to infection and checked to verify ≥80% confluency. 10-fold dilutions of stock SCV2-WA1 virus in serum-free MEM (100 μL) were incubated on Vero E6 monolayers in quadruplicates for 2 h absorption at 37°C without rocking.
    Vero E6
    suggested: None
    Supernatants were collected and diluted in Opti-MEM™ to final concentrations of 1:100 the extraction solvent or 1% Triton X-100, and >100 FFU/mL SARS-CoV-2 per well for analysis by FRNA. 2.7 Inactivation for scRNA-seq (10X Genomics): SARS-CoV-2-infected Vero E6 cells (MOI 0.04 for 72 h) or Calu-3 cells (MOI 0.04 for 48 h) were encapsulated for scRNA-seq following the manufacturer’s protocol “Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 User Guide with Feature Barcode technology for Cell Surface Protein” (document number CG000208; 10X Genomics) targeting 20,000 and 10,000 cells, respectively.
    Calu-3
    suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
    Software and Algorithms
    SentencesResources
    Compound Discoverer 3.2 (ThermoFisher) was used to quantify peak areas and assign annotations based on a local library of reference standards or via matching metabolites to reference spectra in mzCloud (mzcloud.org).
    mzCloud
    suggested: (mzCloud, RRID:SCR_014669)
    Splicing-aware aligner STAR (40) was used in FASTQ alignments.
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Complimentary (c)DNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™) per manufacturer’s instructions and diluted 1:5 in nuclease-free water, then 10 μL of diluted cDNA was used with the NEB Luna Universal Probe qPCR Mastermix (New England BioLabs®) following the manufacturer’s protocol and RT-qPCR performed in 384-well plates using a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems™)
    New England BioLabs®
    suggested: (New England Biolabs, RRID:SCR_013517)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.10.22.465481v1 on bioRxiv