Isolation of bat sarbecoviruses of SARS-CoV-2 clade, Japan

  1. Shin Murakami
  2. Tomoya Kitamura
  3. Hiromichi Matsugo
  4. Haruhiko Kamiki
  5. Ken Oyabu
  6. Wataru Sekine
  7. Akiko Takenaka-Uema
  8. Yuko Sakai-Tagawa
  9. Yoshihiro Kawaoka
  10. Taisuke Horimoto

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Betacoronaviruses have caused 3 outbreaks in the past 2 decades. SARS-CoV-2, in particular, has caused a serious pandemic. As the betacoronaviruses are considered to originate from bats, surveillance of bat betacoronaviruses is crucial for understanding the mechanism of cross-species transition and potential for future outbreaks. We previously detected and characterized a SARS-CoV-2-related sarbecovirus, Rc-o319, from Rhinolophus cornutus in Japan. Here, we detected several bat sarbecoviruses of the SARS-CoV-2 clade from R. cornutus in multiple locations in Japan, and successfully isolated them using Vero/TMPRSS2 cells stably expressing R. cornutus ACE2 (Vero-RcACE2). The coding sequences of S1 region varied among isolates, whereas other genetic regions were highly conserved. Isolates were efficiently grown in Vero-RcACE2 cells, but did not replicate in Vero/TMPRSS2 cells stably expressing human ACE2, suggesting a narrow host range. Further long-term epidemiological studies of sarbecoviruses in wildlife are expected to facilitate the assessment of the risk of their spillover potential.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2022.05.16.492045: (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.
    RandomizationDrug-resistant clones were randomly selected and their genomic DNA was sequenced.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cells and virus: Vero/TMPRSS2 cells (19) were kindly provided by Dr. Makoto Takeda, National Institute of Infectious Diseases, Japan, and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque, Kyoto, Japan) supplemented with 10 % fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 μg/mL of streptomycin.
    Vero/TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    The successful isolation of viruses was confirmed by rRT-PCR, and isolates were propagated in Vero -RcACE2 cells, with aliquots being stored at −80 °C.
    Vero -RcACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Libraries of Rc-mk2 and Rc-kw8 strains were sequenced using a Novaseq 6000 sequencer (Illumina), whereas those of Rc-os20 were sequenced using a DNBSEQ-G400RS (MGI) sequencer.
    Rc-kw8
    suggested: None
    Recombinant DNA
    SentencesResources
    Establishment of ACE2-stably expressing cells: We constructed a plasmid, pCAGGS-blast, by inserting the XhoI-EcoRV fragment of pMXs-IRES-Bsd (20), which contains the encephalomyocarditis virus internal ribosomal entry site and blasticidin-resistant gene, into the XhoI and StuI sites of the pCAGGS-MCS vector.
    pMXs-IRES-Bsd
    suggested: None
    pCAGGS-MCS
    suggested: None
    Open reading frame (ORF) sequences of RcACE2 or hACE2 were PCR-amplified from pCAGGS-RcACE2- or pCAGGS-hACE2-expressing plasmids (10) and cloned into EcoRI- and XhoI-digested pCAGGS-blast plasmids using NEBuilder (New England Biolabs, Ipswich, MA, USA).
    pCAGGS-RcACE2-
    suggested: None
    pCAGGS-hACE2-expressing
    suggested: None
    pCAGGS-blast
    suggested: None
    Vero/TMPRSS2 cells were transfected with pCAGGS-blast-RcACE2 or pCAGGS-blast-hACE2 plasmids using the PEI MAX transfection reagent (Polysciences, Warrington, PA, USA)
    pCAGGS-blast-RcACE2
    suggested: None
    pCAGGS-blast-hACE2
    suggested: None
    The target sequence for the ACE2 gene (5′-TGCTGCTCAGTCCACCATTG-3′) was designed using CRISPR direct (https://crispr.dbcls.jp) and cloned into plentiCRISPR plasmids (21) (Addgene plasmid #52961, a gift from Dr. Feng Zhang) using NEBuilder (NEB).
    plentiCRISPR
    suggested: RRID:Addgene_102315)
    Vero/TMPRSS2 cells were transfected with an ACE2-targeting plasmid using PEI MAX (Polysciences).
    ACE2-targeting
    suggested: None
    Software and Algorithms
    SentencesResources
    Read sequences were mapped to the Rc-o319 genome sequence (GenBank accession No. LC556375) and sarbecoviral sequences were determined using the CLC genomic workbench version 8.0.1 (Qiagen, https://www.qiagen.com) software.
    https://www.qiagen.com
    suggested: (QIAGEN, RRID:SCR_008539)
    Phylogenetic analysis: The nucleotide sequences of sarbecoviruses were aligned using ClustalW version 2.1 (Clustal, https://www.clustal.org).
    ClustalW
    suggested: (ClustalW, RRID:SCR_017277)
    Phylogenetic trees were then constructed by performing a maximum-likelihood analysis using MEGA version X (22) in combination with 500 bootstrap replicates.
    MEGA
    suggested: (Mega BLAST, RRID:SCR_011920)

    Results from OddPub: Thank you for sharing your data.

    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.

  2. Version published to 10.1101/2022.05.16.492045 on bioRxiv