Spike-Independent Infection of Human Coronavirus 229E in Bat Cells

  1. Marcus G. Mah
  2. Martin Linster
  3. Dolyce H. W. Low
  4. Yan Zhuang
  5. Jayanthi Jayakumar
  6. Firdaus Samsudin
  7. Foong Ying Wong
  8. Peter J. Bond
  9. Ian H. Mendenhall
  10. Yvonne C. F. Su
  11. Gavin J. D. Smith

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Abstract

Many viruses, including coronaviruses, originated from bats. Yet, we know little about how these viruses switch between hosts and enter human populations.

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  1. Version published to 10.1128/spectrum.03483-22
  2. Version published to 10.1101/2021.09.18.460924v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.09.18.460924: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Viruses were titrated for 5 days in a 96-well format, plates were fixed with 100% methanol, blocked with 3% bovine serum albumin (BSA), stained with 1E7 primary monoclonal antibody (Eurofins Technologies Ingenasa) binding to the 229E nucleocapsid protein and a goat anti-mouse secondary antibody conjugated with fluorescein isothiocyanate (Abcam, ab6785).
    anti-mouse
    suggested: (Abcam Cat# ab6785, RRID:AB_955241)
    This was followed by overnight probing with primary antibody at 4 °C and 1 hour with either anti-mouse or-rabbit horseradish peroxidase-linked IgG secondary antibodies.
    horseradish peroxidase-linked IgG
    suggested: None
    The primary antibodies include mouse monoclonal antibody 1E7 which is anti-229E nucleocapsid protein and polyclonal rabbit sera derived from immunization of rabbits with 229E spike protein (GenScript).
    anti-229E nucleocapsid protein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Caco2 and Rhileki cells were infected with C2 or C2R10 viruses respectively for 2 hours at 33 °C.
    Caco2
    suggested: None
    Software and Algorithms
    SentencesResources
    Raw NGS reads were trimmed by Trimmomatic v0.39 to remove adaptors and low-quality reads41.
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    In order to obtain consensus sequences, C2 virus consensus of each isolate was used as reference to assemble trimmed and raw reads using the BWA-MEM algorithm in UGENE v.3442.
    UGENE
    suggested: (Unipro UGENE, RRID:SCR_005579)
    Coverage, depth and statistics of the assembled reads were visualized in Geneious R9.1.8 and Integrative Genomics Viewer43.
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    Consensus sequences were aligned using MAFFT v7.222 and annotated in Geneious R9.1.8.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    PCR products were visualized using a 1.5% gel and sequences confirmed by Sanger sequencing (Macrogen)
    Macrogen
    suggested: (Macrogen, RRID:SCR_014454)
    Blots were visualized using a CCD imager detector (Bio-Rad Laboratories) after ECL detection (Amersham Biosciences).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    These amino acid sequences were then aligned to the full-length sequences of 229E S and ORF4 proteins obtained from GenBank (accession number: NC_002645) using Clustal Omega webserver (https://www.ebi.ac.uk/Tools/msa/clustalo/)50 to determine reading frames corresponding to each protein and deleted residues.
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)

    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.

  4. Version published to 10.1101/2021.09.18.460924v1 on bioRxiv