Genetic Characteristics and Phylogeny of 969-bp S Gene Sequence of SARS-CoV-2 from Hawaii Reveals the Worldwide Emerging P681H Mutation

  1. David P. Maison
  2. Lauren L. Ching
  3. Cecilia M. Shikuma
  4. Vivek R. Nerurkar

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Abstract

COVID-19 pandemic has ravaged the world, caused over 1.8 million deaths in the first year, and severely affected the global economy. Hawaii is not spared from the transmission of SARS-CoV-2 in the local population, including high infection rates in racial and ethnic minorities. Early in the pandemic, we described in this journal various technologies used for the detection of SARS-CoV-2. Herein we characterize a 969-bp SARS-CoV-2 segment of the S gene downstream of the receptor-binding domain. At the John A. Burns School of Medicine Biocontainment Facility, RNA was extracted from an oropharyngeal swab and a nasal swab from two patients from Hawaii who were infected with the SARS-CoV-2 in August 2020. Following PCR, the two viral strains were sequenced using Sanger sequencing, and phylogenetic trees were generated using MEGAX. Phylogenetic tree results indicate that the virus has been introduced to Hawaii from multiple sources. Further, we decoded 13 single nucleotide polymorphisms across 13 unique SARS-CoV-2 genomes within this region of the S gene, with one non-synonymous mutation (P681H) found in the two Hawaii strains. The P681H mutation has unique and emerging characteristics with a significant exponential increase in worldwide frequency when compared to the plateauing of the now universal D614G mutation. The P681H mutation is also characteristic of the new SARS-CoV-2 variants from the United Kingdom and Nigeria. Additionally, several mutations resulting in cysteine residues were detected, potentially resulting in disruption of the disulfide bridges in and around the receptor-binding domain. Targeted sequence characterization is warranted to determine the origin of multiple introductions of SARS-CoV-2 circulating in Hawaii.

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    SciScore for 10.1101/2021.01.06.425497: (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 variablePatient PID 00498 and patient PID 00708 are both males, one is caucasian and the other is Japanese/Okinawan/Filipino, and their mean age is 29.5 years.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Patient PID 00498 and patient PID 00708 are both males, one is caucasian and the other is Japanese/Okinawan/Filipino, and their mean age is 29.5 years.
    Patient PID 00498
    suggested: None
    Software and Algorithms
    SentencesResources
    The resulting sequences were input into and verified using both MEGAX19,20 and SnapGene software (Insightful Science, snapgene.com) and aligned using MUSCLE program21 to define the contiguous sequence.
    MEGAX19,20
    suggested: None
    Coronavirus sequences were aligned with the 969-bp S gene region with SnapGene using MUSCLE and the corresponding region was used for future analysis.
    SnapGene
    suggested: (SnapGene, RRID:SCR_015052)
    Upon finding the P681H mutation among the two Hawaii strains in this study, the GISAID database22,23 was used to filter worldwide SARS-CoV-2 sequences by the P681H mutation to create a ratio of sequences containing the P681H mutation to all sequences reported in the GISAID database for a given month.
    GISAID
    suggested: (GISAID, RRID:SCR_018279)
    D614G frequency were conducted and verified using GraphPad Prism version 9.0.0 for Mac (GraphPad Software, San Diego, California USA, www.graphpad.com), JASP version 0.14,24 and RStudio version 1.3.1093.25 Phylogenetic Tree: After the SNP analysis, incomplete sequences were removed prior to the construction of the phylogenetic tree.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    JASP
    suggested: (JASP, RRID:SCR_015823)
    RStudio
    suggested: (RStudio, RRID:SCR_000432)
    The phylogenetic tree was constructed using MEGAX.
    MEGAX
    suggested: None
    19 The alignment was first done using the program MUSCLE.
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)
    The output tree from MEGAX was rooted using FigTree version 1.4.4 based on alpha coronavirus human 299E (KF514433).
    FigTree
    suggested: (FigTree, RRID:SCR_008515)

    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 10.1101/2021.01.06.425497v1 on bioRxiv