SARS-CoV-2 Bearing a Mutation at the S1/S2 Cleavage Site Exhibits Attenuated Virulence and Confers Protective Immunity

  1. Michihito Sasaki
  2. Shinsuke Toba
  3. Yukari Itakura
  4. Herman M. Chambaro
  5. Mai Kishimoto
  6. Koshiro Tabata
  7. Kittiya Intaruck
  8. Kentaro Uemura
  9. Takao Sanaki
  10. Akihiko Sato
  11. William W. Hall
  12. Yasuko Orba
  13. Hirofumi Sawa

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Abstract

SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism.

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  1. Version published to 10.1128/mbio.01415-21
  2. ScreenIT

    SciScore for 10.1101/2021.04.29.442060: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: The protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Hokkaido University (approval no. 20-0060).
    Euthanasia Agents: At 2, 4 or 19 dpi, a subset of the infected hamsters were euthanized under deep anesthesia by isoflurane inhalation, and tissue samples (nasal turbinate, lung and blood) were harvested.
    Sex as a biological variableHamster infection: For virological and histopathological analyses in single infection, 4-to 6-week-old male Syrian hamsters (Japan SLC, Shizuoka, Japan) were inoculated intranasally with 1.5×104 plaque forming units (pfu) of wild-type WK-521 (WT), del2 or R685H viruses in 200 μl of PBS.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For immunohistochemical analysis, slides were heated in citrate buffer for 5 min using a pressure cooker for antigen retrieval and blocking with Block Ace (KAC, Kyoto, Japan), followed by staining with anti-SARS-CoV-2 spike antibody (GTX632604, GeneTex, Hsinchu, Taiwan),
    anti-SARS-CoV-2
    suggested: None
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    anti-SARS-CoV-2 nucleocapsid antibody (GTX635679, GeneTex), anti-CD3 (ab16669, Abcam, Cambridge, UK), anti-MPO (A039829-2
    anti-SARS-CoV-2 nucleocapsid antibody ( GTX635679
    suggested: None
    anti-CD3
    suggested: (Abcam Cat# ab16669, RRID:AB_443425)
    anti-MPO
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells: Vero (Vero E6, ATCC, Manassas, VA) and Vero-TMPRSS2 [18] cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (
    Vero E6
    suggested: None
    Saijyo, Shimojima and Ito (National Institute of Infectious Diseases, Japan); the original stock of these virus strains was prepared by inoculation of Vero-TMPRSS2 cells.
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    Vero cells were infected with either WT, del2 or R685H viruses at an MOI of 0.01.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical analysis was performed by One-way analysis of variance (ANOVA) with Tukey’s test using GraphPad Prism 8 (GraphPad Prism Software, San Diego, CA).
    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: 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 10.1101/2021.04.29.442060v1 on bioRxiv