Sequence analysis of Indian SARS-CoV-2 isolates shows a stronger interaction of mutated receptor binding domain with ACE2 receptor

  1. Pujarini Dash
  2. Jyotirmayee Turuk
  3. Santosh Ku. Behera
  4. Subrata Ku. Palo
  5. Sunil K. Raghav
  6. Arup Ghosh
  7. Jyotsnamayee Sabat
  8. Sonalika Rath
  9. Subhra Subhadra
  10. Debadutta Bhattacharya
  11. Srikant Kanungo
  12. Jayasingh Kshatri
  13. Bijaya kumar Mishra
  14. Saroj Dash
  15. Namita Mahapatra
  16. Ajay Parida
  17. Sanghamitra Pati

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Abstract

SARS-CoV-2 is a RNA Coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). It has affected the whole world including Odisha, a state in eastern India. Many people migrated in the state from different countries as well as states during this SARS-CoV-2 pandemic. As per the protocol laid by ICMR and Health & Family welfare of India, all the suspected cases were tested for SARS-CoV-2 infection. The aim of this study was to analyze the RNA binding domain (RBD) sequence of spike protein from the isolates collected from the throat swab samples of COVID-19 positive cases and further to assess the RBD affinity with ACE2 of different species including human.

Whole genome sequencing for 35 clinical SARS-CoV-2 isolates from COVID-19 positive patients was performed using ARTIC amplicon based sequencing. Sequence analysis and phylogenetic analysis was carried out for the Spike and RBD region of all isolates. The interaction between the RBD and ACE2 receptor of five different species was also analysed.

Except three isolates, spike region of 32 isolates showed one/multiple alterations in nucleotide bases in comparison to the Wuhan reference strain. One of the identified mutation at 1204 (Ref A, RMRC 22 C) in the RBD of spike protein was identified which depicted a stronger binding affinity with human ACE2 receptor compared to the wild type RBD. Furthermore, RBDs of all the Indian isolates are capable of binding to ACE2 of human, bat, hamster and pangolin.

As mutated RBD showed stronger interaction with human ACE2, it could potentially result in higher infectivity. The study shows that RBDs of all the studied isolates have binding affinity for all the five species, which suggests that the virus can infect a wide variety of animals which could also act as natural reservoir for SARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.28.271601: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    The libraries were prepared for WGS using ACTIC amplicon based sequencing kits from Qiagen as per manufacturer recommended protocol.
    WGS
    suggested: None
    The detailed information and method have been described by Raghav et al., 20209 Sequence alignment of RMRC spike genes with reference sequence: For sequence alignment, full length genome sequence of SARS-CoV-2 isolate of Wuhan-Hu-1 (Accession no.NC_045512) was downloaded from NCBI database and used as reference sequence for all further analysis.
    NCBI
    suggested: (NCBI, RRID:SCR_006472)
    Alignment of all 35 RMRC spike nucleotide sequences with the reference genome was carried out using BLASTN (align two/more
    BLASTN
    suggested: (BLASTN, RRID:SCR_001598)
    Multiple sequence alignment of all 35 RBDs (amino acid sequences) was carried out using Clustal X tool.
    Clustal X tool
    suggested: None
    A phylogenetic tree was generated using the MEGA software version 6 with 1000 bootstrap replications as instructed in MEGA software.
    MEGA
    suggested: (Mega BLAST, RRID:SCR_011920)
    , 805 amino acids), Indian bat (Cynopterus sphinx) (Uniprot QZF77831, 807 amino acids) (probable natural hosts for SARS-CoV-2) and human (Homo Sapiens) ACE2 (UniprotKB Q9BYF1
    UniprotKB
    suggested: (UniProtKB, RRID:SCR_004426)
    Protein Data Bank (PDB) did not provide any experimental structure of ACE2 receptors of pangolin, hamster, Chinese and Indian bat, which has prompted us to predict their three dimensional (3D) structure through homology modelling using Modeller v 9.19 tool followed by structure validation.
    Modeller
    suggested: (MODELLER, RRID:SCR_008395)
    Suitable templates were identified for 3D model building of ACE2 using BLASTp11 search against PDB (Protein Data Bank) database.
    BLASTp11
    suggested: None
    Side chain optimization was performed using WHATIF13 and GalaxyRefine14 tool.
    GalaxyRefine14
    suggested: None
    In silico translation of RBD sequences and their interaction with ACE2 receptor of different natural reservoirs: Four nucleotide sequences of RMRC RBDs were selected and translated to protein sequence using EMBOSS Transeq tool of European Bioinformatics Institute (EBI).
    EMBOSS
    suggested: (EMBOSS, RRID:SCR_008493)

    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 pages 17, 18 and 20. 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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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  2. Version published to 10.1101/2020.08.28.271601 on bioRxiv