Sequence analysis of SARS-CoV-2 in nasopharyngeal samples from patients with COVID-19 illustrates population variation and diverse phenotypes, placing the in vitro growth properties of B.1.1.7 and B.1.351 lineage viruses in context

  1. Tessa Prince
  2. Xiaofeng Dong
  3. Rebekah Penrice-Randal
  4. Nadine Randle
  5. Catherine Hartley
  6. Hannah Goldswain
  7. Benjamin Jones
  8. Malcolm G. Semple
  9. J. Kenneth Baillie
  10. Peter J. M. Openshaw
  11. Lance Turtle
  12. ISARIC4C Investigators
  13. Grant L. Hughes
  14. Enyia R. Anderson
  15. Edward I. Patterson
  16. Julian Druce
  17. Gavin Screaton
  18. Miles W. Carroll
  19. James P. Stewart
  20. Julian A. Hiscox

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Abstract

New variants of SARS-CoV-2 are continuing to emerge and dominate the regional and global sequence landscapes. Several variants have been labelled as Variants of Concern (VOCs) because of perceptions or evidence that these may have a transmission advantage, increased risk of morbidly and/or mortality or immune evasion in the context of prior infection or vaccination. Placing the VOCs in context and also the underlying variability of SARS-CoV-2 is essential in understanding virus evolution and selection pressures. Sequences of SARS-CoV-2 in nasopharyngeal swabs from hospitalised patients in the UK were determined and virus isolated. The data indicated the virus existed as a population with a consensus level and non-synonymous changes at a minor variant. For example, viruses containing the nsp12 P323L variation from the Wuhan reference sequence, contained minor variants at the position including P and F and other amino acids. These populations were generally preserved when isolates were amplified in cell culture. In order to place VOCs B.1.1.7 (the UK ‘Kent’ variant) and B.1.351 (the ‘South African’ variant) in context their growth was compared to a spread of other clinical isolates. The data indicated that the growth in cell culture of the B.1.1.7 VOC was no different from other variants, suggesting that its apparent transmission advantage was not down to replicating more quickly. Growth of B.1.351 was towards the higher end of the variants. Overall, the study suggested that studying the biology of SARS-CoV-2 is complicated by population dynamics and that these need to be considered with new variants.

Importance

SARS-CoV-2 is the causative agent of COVID-19. The virus has spread across the planet causing a global pandemic. In common with other coronaviruses, SARS-CoV-2 genetic material (genomes) can become quite diverse as a consequence of replicating inside cells. This has given rise to multiple variants from the original virus that infected humans. These variants may have different properties and in the context of a widespread vaccination program may render vaccines less ineffective. Our research confirms the degree of genetic diversity of SARS-CoV-2 in patients. By isolating viruses from these patients, we show that there is a 100-fold range in growth of even normal variants. Interestingly, by comparing this to the pattern seen with two Variants of Concern (UK and South African variants), we show that at least in cells the ability of the B.1.1.7 variant to grow is not substantially different to many of the previous variants.

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    SciScore for 10.1101/2021.03.30.437704: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Ethics and clinical information: The patients from which the virus samples were obtained gave informed consent and were recruited under the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC) WHO Clinical Characterisation Protocol CCP.
    IRB: Ethical approval for data collection and analysis by ISARIC4C was given by the South Central-Oxford C Research Ethics Committee in England (reference 13/SC/0149), and by the Scotland A Research Ethics Committee (reference 20/SS/0028).
    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
    Human ACE2-A549 (hACE2-A549), a lung epithelial cell line which overexpresses the ACE-2 receptor, were the kind gift of Oliver Schwartz (23) and were cultured in DMEM with 10% FBS and 0.05mg/ml gentamicin with the addition of 10μg/ml Blasticidin (Invitrogen).
    ACE2-A549
    suggested: None
    Briefly, confluent 24-well plates of Vero E6 cells were inoculated with serial ten-fold dilutions of the stocks in duplicate for one hour at 37°C/5% CO2.
    Vero E6
    suggested: None
    Virus growth kinetics: Vero E6, Vero/hSLAM and hACE2-A549 cells were grown in 96 well plates for viral growth kinetic experiments.
    hACE2-A549
    suggested: None
    Software and Algorithms
    SentencesResources
    RNA from viral stocks and from 72-hour post infection cultures were sequenced by Oxford Nanopore long read length sequencing on flow cells run on MinION or GridION.
    MinION
    suggested: (MinION, RRID:SCR_017985)
    These bam files were further analysed using DiversiTools (http://josephhughes.github.io/btctools/) with the “-orfs” function to generate the ratio of amino acid change in the reads and coverage at each site of protein in comparison to the reference SARS-CoV-2 genome (NC_045512.2).
    DiversiTools
    suggested: None

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    ISRCTN66726260NANA

    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

    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 10.1101/2021.03.30.437704v1 on bioRxiv