Isolation, sequence, infectivity and replication kinetics of SARS-CoV-2

  1. Arinjay Banerjee
  2. Jalees A. Nasir
  3. Patrick Budylowski
  4. Lily Yip
  5. Patryk Aftanas
  6. Natasha Christie
  7. Ayoob Ghalami
  8. Kaushal Baid
  9. Amogelang R. Raphenya
  10. Jeremy A. Hirota
  11. Matthew S. Miller
  12. Allison J. McGeer
  13. Mario Ostrowski
  14. Robert A. Kozak
  15. Andrew G. McArthur
  16. Karen Mossman
  17. Samira Mubareka

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Abstract

SARS-CoV-2 emerged in December 2019 in Wuhan, China and has since infected over 1.5 million people, of which over 107,000 have died. As SARS-CoV-2 spreads across the planet, speculations remain about the range of human cells that can be infected by SARS-CoV-2. In this study, we report the isolation of SARS-CoV-2 from two cases of COVID-19 in Toronto, Canada. We determined the genomic sequences of the two isolates and identified single nucleotide changes in representative populations of our virus stocks. More importantly, we tested a wide range of human immune cells for productive infection with SARS-CoV-2. Here we confirm that human primary peripheral blood mononuclear cells (PBMCs) are not permissive for SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor single nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine viral genotype and phenotype using in vitro and in vivo infection models.

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    SciScore for 10.1101/2020.04.11.037382: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This work was approved by the Sunnybrook Research Institute Research Ethics Board (149-1994) and the Research Ethics Boards of St.
    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
    Cells: Vero E6 cells (African green monkey cells; ATCC, https://www.atcc.org) were maintained in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, https://www.sigmaaldrich.com), 1x L-Glutamine and Penicillin/Streptomycin (Pen/Strep; Corning, https://ca.vwr.com).
    Vero E6
    suggested: None
    Calu-3 cells (human lung adenocarcinoma derived; ATCC, https://www.atcc.org) were cultured as previously mentioned (4).
    Calu-3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    THP-1 cells were differentiated into macrophages and dendritic cells using 50 ng/ml GM-CSF (R&D Systems, https://www.rndsystems.com) + 50 ng/ml M-CSF (R&D Systems, https://www.rndsystems.com) and 50 ng/ml GM-CSF + 500 U/ml IL-4 (Biolegend, https://www.biolegend.com), respectively.
    THP-1
    suggested: None
    Software and Algorithms
    SentencesResources
    Calu-3 cells (human lung adenocarcinoma derived; ATCC, https://www.atcc.org) were cultured as previously mentioned (4).
    https://www.atcc.org
    suggested: (ATCC, RRID:SCR_001672)
    THP-1 cells were differentiated into macrophages and dendritic cells using 50 ng/ml GM-CSF (R&D Systems, https://www.rndsystems.com) + 50 ng/ml M-CSF (R&D Systems, https://www.rndsystems.com) and 50 ng/ml GM-CSF + 500 U/ml IL-4 (Biolegend, https://www.biolegend.com), respectively.
    https://www.rndsystems.com
    suggested: (R and D Systems, RRID:SCR_006140)
    https://www.biolegend.com
    suggested: (BioLegend, RRID:SCR_001134)
    , CD8 positive selection kit (STEMCELL Technologies, https://www.stemcell.com), PE positive selection kit (STEMCELL Technologies, https://www.stemcell.com) and Monocyte negative selection kit (STEMCELL Technologies, https://www.stemcell.com), respectively.
    https://www.stemcell.com
    suggested: (STEMCELL Technologies, RRID:SCR_013642)
    Briefly, viral RNA was extracted from infected cells using QIAamp viral RNA kit (Qiagen, https://www.qiagen.com) according to manufacturer’s instructions.
    https://www.qiagen.com
    suggested: (QIAGEN, RRID:SCR_008539)
    Electron Microscopy: Samples were fixed in 10% neutral buffered formalin (Sigma-Aldrich, https://www.sigmaaldrich.com/) for 1 hr.
    https://www.sigmaaldrich.com/
    suggested: (Sigma-Aldrich, RRID:SCR_008988)
    All PCR reactions were purified using RNAClean XP (Beckman Coulter, https://www.beckmancoulter.com) at 1.8x bead to amplicon ratio and eluted in 30 µL.
    https://www.beckmancoulter.com
    suggested: (Beckman Coulter, RRID:SCR_008940)
    Paired-end 150 bp sequencing was performed for each library on a MiniSeq with the 300-cycle mid-output reagent kit (Illumina, https://www.illumina.com), multiplexed with targeted sampling of ∼40,000 clusters per library.
    MiniSeq
    suggested: None
    https://www.illumina.com
    suggested: (Illumina, RRID:SCR_010233)
    Sequencing reads from pool #1 and pool #2 were combined (as R1 and R2), amplification primer sequences removed using cutadapt (version 1.18) (11), and Illumina adapter sequences were removed and low quality sequences trimmed or removed using Trimmomatic (version 0.36) (12).
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    Final sequence quality and confirmation of adapter/primer trimming were confirmed by FASTQC (version 0.11.5) (13).
    FASTQC
    suggested: (FastQC, RRID:SCR_014583)
    SARS-CoV-2 genome sequences were assembled using UniCycler (version 0.4.8; default settings, except for --mode conservative) (14) and assembly statistics generated by QUAST (version 5.0.2) (15)
    QUAST
    suggested: (QUAST, RRID:SCR_001228)
    Sequencing depth and completeness of coverage of the assembled genomes was additionally assessed by Bowtie2 (version 2.3.4.1) (16) alignment of the sequencing reads against the assembled contigs and statistics generated by ngsCAT (version 0.1) (17).
    Bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    Sequence variation in the assembled genomes was assessed by BLASTN against SARS-CoV-2 genome sequences available in GenBank as well as BreSeq (version 0.35.0) (18) analysis relative to GenBank entry MN908947.3 (first genome sequence reported from the original Wuhan outbreak, China).
    BLASTN
    suggested: (BLASTN, RRID:SCR_001598)
    BreSeq
    suggested: (breseq, RRID:SCR_010810)

    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.

    About SciScore

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  2. Version published to 10.1101/2020.04.11.037382v1 on bioRxiv
  3. Version published to 10.1101/2020.04.11.037382v2 on bioRxiv