SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients

  1. William B. Klimstra
  2. Natasha L. Tilston-Lunel
  3. Sham Nambulli
  4. James Boslett
  5. Cynthia M. McMillen
  6. Theron Gilliland
  7. Matthew D. Dunn
  8. Chengun Sun
  9. Sarah E. Wheeler
  10. Alan Wells
  11. Amy L. Hartman
  12. Anita K. McElroy
  13. Douglas S. Reed
  14. Linda J. Rennick
  15. W. Paul Duprex

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

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  1. Version published to 10.1099/jgv.0.001481 on Access Microbiology
  2. Version published to 10.1101/2020.06.19.154930v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.06.19.154930: (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
    Primary antibody (rabbit anti-SARS-CoV-2 S monoclonal antibody, 40150- R007, Sino Biological, rabbit anti-SARS-CoV-2 N monoclonal antibody, 40143-R019, Sino Biological, or the day 14 serum from patient 3 (Table 1), diluted 1:500 in PBS containing 0.1% (v/v) Triton X was added and incubated at 37 °C for 1 hour.
    anti-SARS-CoV-2 S
    suggested: None
    After washing the cells three times with PBS, secondary antibody (chicken anti-rabbit Alexa Fluor-488, Invitrogen, or goat anti-human Alexa Fluor-488, Invitrogen) diluted 1:400 in PBS containing 0.1% (v/v) Triton X was added and incubated at 37 °C for 1 hour.
    anti-rabbit
    suggested: None
    anti-human Alexa Fluor-488 , Invitrogen
    suggested: None
    Cells were incubated with 200 µl primary antibody (rabbit anti-SARS-CoV-2 N, Sino Biological, 40143-R019) diluted 1:1000 in blocking buffer (4% dried milk/0.1% Tween-20 in PBS) for 1 hour at room temperature.
    anti-SARS-CoV-2 N , Sino Biological , 40143-R019
    suggested: None
    Cells were incubated with 200 µl secondary antibody (goat anti-rabbit HRP, Abcam, ab6721) diluted 1:1000 in blocking buffer for 1 hour at room temperature.
    anti-rabbit HRP
    suggested: (Abcam Cat# ab6721, RRID:AB_955447)
    The membranes were incubated with rabbit anti-SARS coronavirus spike protein polyclonal antibody (Invitrogen, PA1-41513) diluted 1:10,000, or rabbit anti-SARS-CoV-2 N monoclonal antibody (Sino Biological, 40143-R019) diluted 1:15,000, in TBST buffer containing 1% (w/v) non-fat milk overnight at 4 °C.
    anti-SARS
    suggested: (Thermo Fisher Scientific Cat# PA1-41513, RRID:AB_1087211)
    anti-SARS-CoV-2 N
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells: Vero 76 cells, Clone E6, were purchased from ATCC and were grown in DMEM (Corning) supplemented with 10% (v/v) FBS (Atlanta Biologicals), 1% (v/v) L-glutamine (Corning) and 1% (v/v) penicillin-streptomycin (pen-strep; Corning).
    Vero 76
    suggested: None
    Western blot: Confluent monolayers of Vero-E6 cells in 6-well plates were infected with Wash1 (P5 or P6) or Munich (P2 or P3) virus at an MOI of 0.01 or transfected with a pCAGGS vector expressing a codon-optimized (GeneScript) S protein (synthesized by GeneScript) possessing the amino acid sequence of the SARS-CoV-2 Wuhan-Hu-1 isolate (accession number MN908947).
    Vero-E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Oxford Nanopore (MinION) cDNA-PCR sequencing: SARS-CoV-2 transcripts from Wash1: P6 and Munich: P3 virus stocks were sequenced on the MinION device (MinION Mk1B, MIN-101B) using ONT Ligation Sequencing Kit 1D (SQK- LSK108).
    MinION
    suggested: (MinION, RRID:SCR_017985)
    Each cDNA library was loaded individually onto separate SpotON Flow Cells (Version R9; FLO-MIN 106) and run using the default script for sequencing Kit SQK-LSK108 via MinKNOW
    MinKNOW
    suggested: None

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 30. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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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  4. Version published to 10.1101/2020.06.19.154930v1 on bioRxiv