SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture

  1. Jessica E. Agius
  2. Jessica C. Johnson-Mackinnon
  3. Winkie Fong
  4. Mailie Gall
  5. Connie Lam
  6. Kerri Basile
  7. Jen Kok
  8. Alicia Arnott
  9. Vitali Sintchenko
  10. Rebecca J. Rockett

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Abstract

Low frequency intrahost single nucleotide variants (iSNVs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been increasingly recognised as predictive indicators of positive selection. Particularly as growing numbers of SARS-CoV-2 variants of interest (VOI) and concern (VOC) emerge. However, the dynamics of subgenomic RNA (sgRNA) expression and its impact on genomic diversity and infection outcome remain poorly understood. This study aims to investigate and quantify iSNVs and sgRNA expression in single and longitudinally sampled cohorts over the course of mild and severe SARS-CoV-2 infection, benchmarked against an in vitro infection model.

Methods

Two clinical cohorts of SARS-CoV-2 positive cases in New South Wales, Australia collected between March 2020 and August 2021 were sequenced. Longitudinal samples from cases hospitalised due to SARS-CoV-2 infection (severe) ( n = 16) were analysed and compared with cases that presented with SARS-CoV-2 symptoms but were not hospitalised (mild) ( n = 23). SARS-CoV-2 genomic diversity profiles were also examined from daily sampling of culture experiments for three SARS-CoV-2 variants (Lineage A, B.1.351, and B.1.617.2) cultured in VeroE6 C1008 cells ( n = 33).

Results

Intrahost single nucleotide variants were detected in 83% (19/23) of the mild cohort cases and 100% (16/16) of the severe cohort cases. SNP profiles remained relatively fixed over time, with an average of 1.66 SNPs gained or lost, and an average of 4.2 and 5.9 low frequency variants per patient were detected in severe and mild infection, respectively. sgRNA was detected in 100% (25/25) of the mild genomes and 92% (24/26) of the severe genomes. Total sgRNA expressed across all genes in the mild cohort was significantly higher than that of the severe cohort. Significantly higher expression levels were detected in the spike and the nucleocapsid genes. There was significantly less sgRNA detected in the culture dilutions than the clinical cohorts.

Discussion and Conclusion

The positions and frequencies of iSNVs in the severe and mild infection cohorts were dynamic overtime, highlighting the importance of continual monitoring, particularly during community outbreaks where multiple SARS-CoV-2 variants may co-circulate. sgRNA levels can vary across patients and the overall level of sgRNA reads compared to genomic RNA can be less than 1%. The relative contribution of sgRNA to the severity of illness warrants further investigation given the level of variation between genomes. Further monitoring of sgRNAs will improve the understanding of SARS-CoV-2 evolution and the effectiveness of therapeutic and public health containment measures during the pandemic.

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  1. Version published to 10.3389/fmicb.2022.824217
  2. ScreenIT

    SciScore for 10.1101/2021.11.23.21266789: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Mycoplasma testing was routinely conducted to exclude contamination of the culture media and cell line.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    CostarÒ 24-well clear tissue culture-treated multiple well plates (CorningÒ, Corning, NY, USA) were seeded at 40% confluence with Vero C1008 cells [Vero 76, clone E6, Vero E6] (ATCC® CRL-1586(tm)) in Dulbecco’s minimal essential medium (DMEM, Lonza Bioscience, Alpharetta, GA, USA), and supplemented with 9% foetal bovine serum (FBS, HyClone, Cytiva, Sydney,
    Vero C1008
    suggested: BCRC Cat# 60476, RRID:CVCL_0574)
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Cells were incubated at 37°C in 5% CO2 for 4 days (days 0 to 3), and were sealed with AeraSealä Film (Excel Scientific, Inc., Victorville, CA, USA) to minimise evaporation, spillage, and well-to-well cross-contamination.
    Excel Scientific
    suggested: None
    The reads were demultiplexed and quality trimmed using Trimmomatic version 0.36 (minimum read quality score of 20, leading/trailing quality of 5).
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    Reference mapping and consensus calling was performed using iVar version 1.2.1.
    iVar
    suggested: None
    Variant filtering and analysis: The frequency and positions of variants (SNPs and iSNVs) in all samples were determined using Varscan version 2.3.9.
    Varscan
    suggested: (VARSCAN, RRID:SCR_006849)

    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.

    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.

  3. Version published to 10.1101/2021.11.23.21266789v1 on medRxiv