SARS-CoV-2 genomic and quasispecies analyses in cancer patients reveal relaxed intrahost virus evolution

  1. Juliana D. Siqueira
  2. Livia R. Goes
  3. Brunna M. Alves
  4. Pedro S. de Carvalho
  5. Claudia Cicala
  6. James Arthos
  7. João P.B. Viola
  8. Andréia C. de Melo
  9. Marcelo A. Soares
  10. on behalf of the INCA COVID-19 Task Force

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Abstract

Numerous factors have been identified to influence susceptibility to SARS-CoV-2 infection and disease severity. Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. We have determined the full-length SARS-CoV-2 genomic sequences of cancer patients and healthcare workers (HCW; non-cancer controls) by deep sequencing and investigated the within-host viral quasispecies of each infection, quantifying intrahost genetic diversity. Naso- and oropharyngeal SARS-CoV-2 + swabs from 57 cancer patients and 14 healthcare workers (HCW) from the Brazilian Cancer Institute were collected in April–May 2020. Complete genome amplification using ARTIC network V3 multiplex primers was performed followed by next-generation sequencing. Assemblies were conducted in Geneious R11, where consensus sequences were extracted and intrahost single nucleotide variants (iSNVs) were identified. Maximum likelihood phylogenetic analysis was performed using PhyMLv.3.0 and lineages were classified using Pangolin and CoV-GLUE. Phylogenetic analysis showed that all but one strain belonged to clade B1.1. Four genetically linked mutations known as the globally dominant SARS-CoV-2 haplotype (C241T, C3037T, C14408T and A23403G) were found in the majority of consensus sequences. SNV signatures of previously characterized Brazilian genomes were also observed in most samples. Another 85 SNVs were found at a lower frequency (1.4-19.7%). Cancer patients displayed a significantly higher intrahost viral genetic diversity compared to HCW (p = 0.009). Intrahost genetic diversity in cancer patients was independent of SARS-CoV-2 Ct values, and was not associated with disease severity, use of corticosteroids, or use of antivirals, characteristics that could influence viral diversity. Such a feature may explain, at least in part, the more adverse outcomes to which cancer/COVID-19 patients experience.

Author Summary

Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. In this study, phylogenetic and variation analysis of SARS-CoV-2 genomes from cancer patients and non-cancer healthcare workers at the Brazilian National Cancer Institute were characterized by deep sequencing. Viral genomes showed signatures characteristic of Brazilian viruses, consistent with the hypothesis of local, community transmission rather than virus importation from abroad. Despite most genomes in patients and healthcare workers belonging to the same lineage, intrahost variability was higher in cancer patients when compared to non-cancer counterparts. The intrahost genomic diversity analysis presented in our study highlights the relaxed evolution of SARS-CoV-2 in a vulnerable population of cancer patients. The high number of minor variations can result in the selection of immune escape variants, resistance to potential drugs, and/or increased pathogenicity. The impact of this higher intrahost variability over time warrants further investigation.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.26.267831: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Ethics Statement: All participants agreed to be enrolled in the study and signed an informed consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    SARS-CoV-2 near full-length consensus sequence and nucleotide variations: All analyses were conducted using Geneious R11 software (Biomatters, Auckland, New Zealand), where the reads were trimmed to achieve an error rate below 0.1% and assembled to the Wuhan-Hu-1 reference sequence genome (GenBank number MN908947).
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    An alignment including the consensus sequences generated and genomes from Brazilian sequences available on the GISAID Database classified as B1, B1.1 and the Brazilian clusters B1.1-BR/ B1.1-EU/BR (S1 Table) were submitted to a maximum likelihood phylogenic reconstruction using PhyML v.
    PhyML
    suggested: (PhyML, RRID:SCR_014629)
    All graphical representations and statistical analyses were performed using Geneious R11 (Biomatters) and GraphPad Prism v.8.0.1 (
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad Software Inc.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your code.

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

    About SciScore

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  2. Version published to 10.1101/2020.08.26.267831 on bioRxiv