Many bat species are not potential hosts of SARS-CoV and SARS-CoV-2: Evidence from ACE2 receptor usage

  1. Huan Yan
  2. Hengwu Jiao
  3. Qianyun Liu
  4. Zhen Zhang
  5. Xin Wang
  6. Ming Guo
  7. Bing-Jun Wang
  8. Ke Lan
  9. Yu Chen
  10. Huabin Zhao

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Abstract

Bats are the suggested natural hosts for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2, the latter of which caused the coronavirus disease 2019 (COVID-19) pandemic. The interaction of viral Spike proteins with their host receptor angiotensin-converting enzyme 2 (ACE2) is a critical determinant of potential hosts and cross-species transmission. Here we use virus-host receptor binding and infection assays to show that ACE2 orthologs from 24, 21, and 16 of 46 phylogenetically diverse bat species – including those in close and distant contact with humans – do not support entry of SARS-CoV, SARS-CoV-2, and both of these coronaviruses, respectively. Furthermore, we used genetic and functional analyses to identify genetic changes in bat ACE2 receptors associated with viral entry restrictions. Our study demonstrates that many – if not most – bat species are not potential hosts of SARS-CoV and SARS-CoV-2, and provides important insights into pandemic control and wildlife conservation.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.08.284737: (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

    Antibodies
    SentencesResources
    Cell culture: HEK293T cells (293T, ATCC, CRL-3216) and VERO-E6 cells (ATCC, CRL-1586) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS), 2.0 mM L-Glutamine, 110 mg/L sodium pyruvate, and 4.5 g/L D-glucose. l1-Hybridoma (CRL-2700) secreting a monoclonal antibody targeting against VSV glycoprotein was cultured in Minimum Essential Medium with Earle’s salts and 2.0 mM L-Glutamine (MEM; Gibco).
    CRL-3216
    suggested: None
    l1-Hybridoma
    suggested: None
    VSV
    suggested: None
    Next they were incubated with the mouse monoclonal antibody targeting Flag tag (9A3, #8146S,
    antibody targeting Flag tag ( 9A3
    suggested: (Cell Signaling Technology Cat# 8146, RRID:AB_10950495)
    After three rounds of PBS washing, cells were subsequently incubated with 2 μg/ml of the secondary goat anti-rabbit antibody conjugated with Alexa Fluor 594 (A11032, Thermo Fisher Scientific, United States) diluted in 1% BSA /PBS at room temperature for 30 mins.
    anti-rabbit
    suggested: None
    A11032
    suggested: (Molecular Probes Cat# A-11032, RRID:AB_2534091)
    Cells were subsequently incubated with a mouse monoclonal antibody SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (40143-MM05, Sino Biological, China) at 1:500 at 37°C for 1 hour, and then incubated with 2μg/ml of goat anti-mouse secondary antibody, Alexa Fluor 594 (A-11032, Thermo Fisher Scientific) at 37°C for 1 hour.
    anti-mouse
    suggested: (Molecular Probes Cat# A-11032, RRID:AB_2534091)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: HEK293T cells (293T, ATCC, CRL-3216) and VERO-E6 cells (ATCC, CRL-1586) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS), 2.0 mM L-Glutamine, 110 mg/L sodium pyruvate, and 4.5 g/L D-glucose. l1-Hybridoma (CRL-2700) secreting a monoclonal antibody targeting against VSV glycoprotein was cultured in Minimum Essential Medium with Earle’s salts and 2.0 mM L-Glutamine (MEM; Gibco).
    HEK293T
    suggested: None
    Coronavirus RBD-hFc binding assay: Recombinant SARS-CoV-RBD-hFc and SARS-CoV-2-RBD-hFc proteins were produced by transient transfection of 293T cells with Lipofectamine 3000.
    293T
    suggested: None
    SARS-CoV-2 was amplified on Vero-E6 cells and stored at −150°C, and the titer was determined on Vero-E6 cells through a plaque assay.
    Vero-E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Next, we aligned the deduced ACE2 protein sequences using the MUSCLE program (
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)
    The sequence logo was generated with WebLogo (https://weblogo.berkeley.edu/logo.cgi).
    WebLogo
    suggested: (WEBLOGO, RRID:SCR_010236)
    We performed selective pressure analysis on bat ACE2 using CodeML implemented in PAML (24).
    PAML
    suggested: (PAML, RRID:SCR_014932)
    Homology-based structural modeling: Molecular models of different bat ACE2 were predicted by I-TASSER (Iterative Threading ASSEmbly Refinement) version 5.1 (31).
    I-TASSER
    suggested: (I-TASSER, RRID:SCR_014627)
    The structural alignment and visualization were implemented in PyMOL (33).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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.

    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.

  2. Version published to 10.1101/2020.09.08.284737 on bioRxiv