Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings

  1. Marek Widera
  2. Sandra Westhaus
  3. Holger F. Rabenau
  4. Sebastian Hoehl
  5. Denisa Bojkova
  6. Jindrich Cinatl
  7. Sandra Ciesek

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Abstract

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In this study, we evaluated lysis buffers that are commonly used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. In addition, viral stability in cell culture media at 4 °C and on display glass and plastic surfaces used in laboratory environment was analyzed. Furthermore, we evaluated chemical and non-chemical inactivation methods including heat inactivation, UV-C light, addition of ethanol, acetone-methanol, and PFA, which might be used as a subsequent inactivation step in the case of insufficient inactivation. We infected susceptible Caco-2 and Vero cells with pre-treated SARS-CoV-2 and determined the tissue culture infection dose 50 (TCID 50 ) using crystal violet staining and microscopy. In addition, lysates of infected cells and virus containing supernatant were subjected to RT-qPCR analysis. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate the virus. In conclusion, careful evaluation of the used inactivation methods is required especially for non-denaturing buffers. Additional inactivation steps might be necessary before removal of lysed viral samples from BSL-3.

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  1. Version published to 10.1007/s00430-021-00716-3
  2. Version published to 10.1101/2020.09.11.292581v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.09.11.292581: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: All infectious work was performed under biosafety level 3 (BSL-3) conditions in a BSL-3 facility according to the Committee on Biological Agents (ABAS) and Central Committee for Biological Safety (ZKBS). 2.2 Determination of SARS-CoV-2 infectivity by TCID50: Infectious titer of SARS-CoV-2 supernatant were determined by an end-point limiting dilution assay as 50% tissue culture infectious dose (TCID50/ml) in confluent cells in 96-well microtiter plates.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Influenza isolates PR8 (H1N1) [ATCC-VR-1469; p1; Titer: 1.1× 107] and Victoria (H3N2) [ATCC-VR-822; p5; Titer: 1.4× 107] were prepared by infecting MDCK cells.
    MDCK
    suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
    Briefly, Caco-2 were seeded onto a 96-well plate and infected with untreated or treated SARS-CoV-2 containing supernatant in an initial 1:10 dilution (quadruplicates).
    Caco-2
    suggested: None
    Analyzing different fixation solutions, compound-virus mixture was further diluted 1:100 before added to the seeded Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    Data analysis was performed in Microsoft Excel and GraphPad Prism 6 (GraphPad Software, USA).
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: We detected the following sentences addressing limitations in the study:
    This study has limitations that need to be considered. To maintain real-lab-settings, we performed the stability evaluation with daily used virus stocks thawed for routine infectious experiments. Since the stocks in use had different lifetimes, longer standing times at ambient temperature could possibly have influenced viral stability. Hence we quantitatively cannot compare the samples with each other and also statistical evaluation was not applicable. Far more replicates under more controlled conditions would be necessary here. Nonetheless, even after 23 weeks we still found high-titer virus that has to be adequately inactivated by appropriate lysis buffer. These data are therefore important as a derivation for the inactivation tests. A further limitation of this study is based on the fact that inactivation efficiency largely depends on the initial virus load and the reaction volume. Especially in the case of heat inactivation, the heating time of the respective medium must be considered (Supplementary Figure 1, Supplementary Tables 1–2). In addition, the higher the virus titer, the longer the inactivation time is necessary for complete inactivation. SARS-CoV-2 is able to replicate in susceptible cells lines like Caco2 and Vero cells both yielding high viral titers in the cell culture supernatants. The main difference is the ability to form a CPE, which is definitely more pronounced in Caco2 cells (Hoehl et al., 2020). However, in this study we did not match results obtained...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

    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.

  4. Version published to 10.1101/2020.09.11.292581v1 on bioRxiv