Evolutionary and Phenotypic Characterization of Two Spike Mutations in European Lineage 20E of SARS-CoV-2

  1. Paula Ruiz-Rodriguez
  2. Clara Francés-Gómez
  3. Álvaro Chiner-Oms
  4. Mariana G. López
  5. Santiago Jiménez-Serrano
  6. Irving Cancino-Muñoz
  7. Paula Ruiz-Hueso
  8. Manuela Torres-Puente
  9. Maria Alma Bracho
  10. Giuseppe D’Auria
  11. Llúcia Martinez-Priego
  12. Manuel Guerreiro
  13. Marta Montero-Alonso
  14. María Dolores Gómez
  15. José Luis Piñana
  16. SeqCOVID-SPAIN Consortium
  17. Fernando González-Candelas
  18. Iñaki Comas
  19. Alberto Marina
  20. Ron Geller
  21. Mireia Coscolla

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Abstract

We observed repeated, independent emergence of mutations in the SARS-CoV-2 spike involving amino acids 1163 and 1167, within the HR2 functional motif. Conclusions derived from evolutionary and genomic diversity analysis suggest that the co-occurrence of both mutations might pose an advantage for the virus and therefore a threat to effective control of the epidemic.

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  1. Version published to 10.1128/mbio.02315-21
  2. ScreenIT

    SciScore for 10.1101/2021.03.08.21253075: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Sequencing of the samples has been approved by the ethics committee: Comité Ético de Investigación de Salud Pública y Centro Superior de Investigación en Salud Pública (CEI DGSP-CSISP) N° 20200414/05.
    IACUC: For the first wave, serum samples and data from patients included in this study were provided by the Consorcio Hospital General de Valencia Biobank, integrated in the Valencian Biobanking Network, and they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees.
    Consent: All samples from Hospital Universitario y Politécnico La Fe de Valencia were collected after informed written consent and the project has been approved by the ethical committee and institutional review board (registration number 2020-123-1).
    RandomizationThe dataset to represent 20I/501Y.V1 phylogenetic relationships include 3,067 randomly selected samples identified by Pangolin typing system (https://github.com/cov-lineages/pangolin) as lineage B.1.1.7 plus the 33 sequences with amino acid replacements in D1163 and/or G1167 (Supplementary Table 7).
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    To evaluate the efficiency of virus production, three transfections in HEK293 cells (CRL-1573 from ATCC) were performed for each plasmid to generate pseudotyped VSV harbouring the indicated S protein83.
    HEK293
    suggested: None
    The titers of the virus produced were then assayed by serial dilution, followed by infection of either Vero cells (CCL-81 from ATCC) or A549 cells expressing ACE2 and TMPRSS2 (InvivoGen catalog code a549-hace2tpsa), and counting of GFP positive cells (focus forming units; FFU) at 16 hours post infection.
    A549
    suggested: None
    For assessing thermal stability, 1000 FFU (as measured on Vero cells) were incubated for 15 minutes at 30.4, 31.4, 33, 35.2, 38.2, 44.8, 47, 48.6 or 49.6°C before addition to Vero cells previously seeded in a 96 well plate (10,000 cells/well).
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    Whole-genome sequencing was performed in the MiSeq platform (2×200 cycles paired-end run;
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)
    The 14 sequences harbouring the spike gene were concatenated and aligned with MEGA-X71 using amino acids with ClustalW algorithm with default options (alignment is available at https://github.com/PathoGenOmics/B.1.177.637_SARS-CoV-2) Sampling SARS-CoV-2 from non-Spanish consortium sequences: To build the global alignment, sequences were downloaded from GISAID1 including all the pandemic periods since the first known case sequenced (from 24 December 2019) until the last sample on 22 December 2020.
    ClustalW
    suggested: (ClustalW, RRID:SCR_017277)
    Genotypes detected that involved mutations in 1163 and 1167 such as B.1.177.637 and cluster 163.654 were represented in a circos plot with the R package circlize76 v 0.4.12.1004
    circos
    suggested: (Circos, RRID:SCR_011798)
    circlize76
    suggested: None
    Duplicated sequences were removed with seqkit v 0.13.2 (arguments employed: rmdup -s). 8,397 sequences were selected at random with the same temporal distribution by month as the initial dataset by Python scripting (available at https://github.com/PathoGenOmics/B.1.177.637_SARS-CoV-2).
    Python
    suggested: (IPython, RRID:SCR_001658)
    Mutations were introduced using single mutation tool embedded in COOT82and figures were generated with PyMOL (www.pymol.org).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    Results from OddPub: Thank you for sharing your code and 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 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.

  3. Version published to 10.1101/2021.03.08.21253075v1 on medRxiv