The SARS-CoV2 envelope differs from host cells, exposes procoagulant lipids, and is disrupted in vivo by oral rinses

  1. Zack Saud
  2. Victoria J. Tyrrell
  3. Andreas Zaragkoulias
  4. Majd B. Protty
  5. Evelina Statkute
  6. Anzelika Rubina
  7. Kirsten Bentley
  8. Daniel A. White
  9. Patricia Dos Santos Rodrigues
  10. Robert C. Murphy
  11. Harald Köfeler
  12. William J. Griffiths
  13. Jorge Alvarez-Jarreta
  14. Richard William Brown
  15. Robert G. Newcombe
  16. James Heyman
  17. Manon Pritchard
  18. Robert WJ. Mcleod
  19. Arvind Arya
  20. Ceri-Ann Lynch
  21. David Owens
  22. P Vince Jenkins
  23. Niklaas J. Buurma
  24. Valerie B. O’Donnell
  25. David W. Thomas
  26. Richard J. Stanton

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Abstract

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  1. Version published to 10.1016/j.jlr.2022.100208
  2. ScreenIT

    SciScore for 10.1101/2022.02.16.22270842: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Briefly, following informed consent, in-patients with PCR-confirmed COVID-19 infection within the last 14 days, were recruited at the University Hospital of Wales, the Royal Glamorgan Hospital and Betsi Cadwalader University Health Board in Wales UK.
    Sex as a biological variablenot detected.
    RandomizationClinical study design: A four-arm, randomised controlled trial was conducted to study the effectiveness of anti-microbial mouthwashes in vivo.
    BlindingClinical and research staff involved in sample collection and laboratory analysis were blinded as to which product was which.
    Power AnalysisOriginal sample size was calculated based on reported mouthwash activity against enveloped herpes virus; designed with a >80% power to detect a 2-fold reduction in viral load(58).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies were all diluted in blocking buffer, and were rabbit anti-actin (A2066, Sigma-aldrich 1:2,000) and mouse anti-SARS-CoV2 Spike (Clone 1A9, Insight; 1:2,000), as well as anti-mouse HRP or anti-rabbit HRP (GE Healthcare; 1:2,000).
    anti-actin
    suggested: (Sigma-Aldrich Cat# A2066, RRID:AB_476693)
    anti-SARS-CoV2
    suggested: None
    anti-mouse HRP
    suggested: None
    anti-rabbit HRP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and viruses: Virucidal assays utilised VeroE6 or A549 cells, a gift from the University of Glasgow/MRC Centre for Virology, UK.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    The England2 strain of SARS-CoV2 was provided by Public Health England, and amplified in VeroE6 cells before being harvested from the supernatant.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Virus was titrated by plaque assay; serial dilutions were used to infect VeroE6/ACE2/TMPRSS2 cells for 1 h.
    VeroE6/ACE2/TMPRSS2
    suggested: None
    Harvest of virus particles and lipid extraction for lipidomics profiling: Cells were infected with SARS-CoV2 at MOI=0.01, when cells were 70% confluent, in either serum-free media (Vero cells) or at 2% FCS (A549).
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    Prior to feature analysis the data was processed using the Waters compression tool to reduce the noise, changed to centroid using MassLynx and converted to .
    MassLynx
    suggested: (MassLynx , RRID:SCR_014271)
    MZxml by the MSconvert module in Proteowizard.
    Proteowizard
    suggested: (ProteoWizard, RRID:SCR_012056)
    Feature analysis was carried out using the HPLC/QTOF parameters in XCMS online(37).
    XCMS
    suggested: (XCMS, RRID:SCR_015538)
    The two resulting feature lists (positive and negative) were further processed using the Python program LipidFinder 2.0 in its default configuration(36).
    Python
    suggested: (IPython, RRID:SCR_001658)
    LipidFinder
    suggested: None
    Membranes were washed, incubated with secondary antibody for 1 h at room temperature, washed, developed with Supersignal West Pico (Thermo), and imaged using a G:Box Chemi XX6 (Syngene).
    Thermo
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    Antibodies were all diluted in blocking buffer, and were rabbit anti-actin (A2066, Sigma-aldrich 1:2,000) and mouse anti-SARS-CoV2 Spike (Clone 1A9, Insight; 1:2,000), as well as anti-mouse HRP or anti-rabbit HRP (GE Healthcare; 1:2,000).
    GE Healthcare
    suggested: (GE Healthcare, RRID:SCR_000004)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    As a caveat of the method, membrane proteins or sugars could in theory hinder derivatisation of aPL, and so this value may be a lower-level estimate. In primary cells, energy-dependent processes maintain asymmetry of plasma membranes. This ensures that very low mol% of PE and PS are exposed on the surface, for example, only 3 - 4 % is present on platelets following thrombin activation (40). This is because PE and PS promote coagulation, complement binding and uptake of apoptotic cells through their electronegative interactions with Ca2+ ions and various proteins (3, 20, 40). Although the overall amounts of PS appear to be rather low in virions, PE levels are similar to plasma membrane (Table 1). Thus, exposure of 50 % of aPL on the surface will result in levels of external PE that are around 12-fold-higher than activated platelets(40). Indeed, in line with this, we found that purified virions significantly accelerate plasma coagulation in vitro (Figure 4 B). In addition, a recent study showed (using a less specific ELISA) that levels of PS on the surface of SARS-CoV2 are sufficient to support PS-receptor dependent viral entry (17). Our work extends this significantly by reporting on the ng amounts of PS and PE present, the proportions of PE and PS that are externalised, and the specific molecular species of PE and PS in the membrane. In addition to SARS-CoV2, PS has been implicated in the cellular uptake of several other viruses, thus knowing how much and which molecular spec...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    ISRCTN25647404NANA

    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.
    • Thank you for including a protocol registration statement.

    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 10.1101/2022.02.16.22270842v1 on medRxiv
  4. ScreenIT

    SciScore for 10.1101/2020.11.13.381079: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    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
    In a further modification to the methods of Meister et al [3], we titrated virus onto VeroE6 cells transduced with Lentivirus vectors expressing ACE2 and TMPRSS2 and drug selected, to enhance virus entry (>1-log), generating a more sensitive test for virucidal activity.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Titrations were performed by plaque assay; serial dilutions were used to infect VeroE6/ACE2/TMPRSS2 cells for 1 h.
    VeroE6/ACE2/TMPRSS2
    suggested: None

    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.
    • No funding statement was detected.
    • 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.

  5. Version published to 10.1101/2020.11.13.381079v2 on bioRxiv
  6. ScreenIT

    SciScore for 10.1101/2020.11.13.381079: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.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
    The England2 strain of SARS-CoV2 was provided by Public Health England, and amplified in VeroE6 cells before being harvested from the supernatant.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Titrations were performed by plaque assay; serial dilutions were used to infect VeroE6/ACE2/TMPRSS2 cells for 1 h.
    VeroE6/ACE2/TMPRSS2
    suggested: None

    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 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.

    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.

  7. Version published to 10.1101/2020.11.13.381079v1 on bioRxiv