Isolation and Characterization of SARS-CoV-2 strains circulating in Eastern India

  1. Bharati Singh
  2. Kiran Avula
  3. Sanchari Chatterjee
  4. Ankita Datey
  5. Arup Ghosh
  6. Saikat De
  7. Supriya Suman Keshry
  8. Soumyajit Ghosh
  9. Amol Suryawanshi
  10. Rupesh Dash
  11. Shantibhusan Senapati
  12. Tushar K. Beuria
  13. Punit Prasad
  14. Sunil Raghav
  15. Rajeeb Swain
  16. Ajay Parida
  17. Gulam Hussain Syed
  18. Soma Chattopadhyay

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Abstract

Emergence of SARS-CoV-2 as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts and rapid emergence of new variants urge for establishment of research infrastructure to facilitate rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, five SARS-CoV2 strains (ILS01, ILS02, ILS03, ILS15 and ILS24) of four different clades (19A, 19B, 20A and 20B) were isolated from patient swab samples collected during the 1 st COVID-19 wave in Odisha, India. The viral isolates were adapted to in-vitro cultures and further characterized to identify strain specific variations in viral growth characteristics. All the five isolates showed substantial amount of virus induced CPE however ILS03 belonging to 20A clade displayed highest level of CPE. Time kinetics experiment revealed spike protein expression was evident after 16th hours post infection in all five isolates. ILS03 induced around 90% of cytotoxicity. Further, the susceptibility of various cell lines (human hepatoma cell line (Huh-7), CaCo2 cell line, HEK-293T cells, Vero, Vero-E6, BHK-21, THP-1 cell line and RAW 264.7 cells) were assessed. Surprisingly, it was found that the human monocyte cells THP-1 and murine macrophage cell line RAW 264.7 were permissive to all the SARS-CoV-2 isolates. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government run vaccine drive in India. The micro-neutralization assay suggested that both Covaxin and Covishield vaccines were equally effective (100% neutralization) against all of the isolates. The whole genome sequencing of culture adapted viral isolates and viral genome from patient oropharyngeal swab sample suggested that repetitive passaging of SARS-CoV2 virus in Vero-E6 cells did not lead to emergence of many mutations during the adaptation in cell culture. Phylogenetic analyses revealed that the five isolates clustered to respective clades. The major goal was to isolate and adapt SARS-CoV-2 viruses in in-vitro cell culture with minimal modification to facilitate research activities involved in understanding the molecular virology, host-virus interactions, application of these strains for drug discovery and animal challenge models development which eventually will contribute towards the development of effective and reliable therapeutics.

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  1. ScreenIT

    SciScore for 10.1101/2021.12.13.472526: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: The current studies involving swab samples from the human participants were reviewed and approved by the Institutional Human Ethics Committee, Institute of Life Sciences.
    Consent: The written consent form duly signed by the participants/ legal guardian was taken into consideration for the concerned study Virus Isolation: Oropharyngeal swab samples of confirmed COVID-19 patients were used for isolation of the virus.
    IACUC: This study has been approved by the Institutional biosafety committee (IBSC) (IBSC file no. V-122-MISC/2007-08/01).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were then incubated with SARS-CoV-2 rabbit anti-nucleocapsid (Abgenex, cat. No. 11-2003) antibody for 1-2 hour followed by 3x wash with PBS and 1 hr incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG.
    anti-nucleocapsid
    suggested: None
    anti-rabbit IgG
    suggested: None
    The Non-Linear fit log (inhibitor) vs. response - Variable slope was used to determine the percentage inhibition of virus infection due to vaccine-induced antibody-mediated neutralization.
    antibody-mediated neutralization.
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and Viruses: Vero E6, Vero, BHK-21, HEK293T and Huh7 cells were maintained in high glucose DMEM supplemented with 10% fetal bovine serum and 1X Pencillin/Streptomycin.
    Vero E6
    suggested: RRID:CVCL_XD71)
    BHK-21
    suggested: None
    HEK293T
    suggested: None
    Huh7
    suggested: None
    CaCo2 cells were maintained in DMEM supplemented with 20% fetal bovine serum and 1X Pencillin/Streptomycin.
    CaCo2
    suggested: None
    THP-1 and RAW 264.7 were maintained in RPMI supplemented with 10% fetal bovine serum, 1X Pencillin/Streptomycin, 10 mM sodium pyruvate, 1M HEPES, and glucose.
    THP-1
    suggested: None
    RAW 264.7
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical Analysis: Statistical analysis was performed using the GraphPad Prism software version 5.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: 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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

    Results from scite Reference Check: We found no unreliable references.

    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 10.1101/2021.12.13.472526v1 on bioRxiv