Divergent SARS-CoV-2 variant emerges in white-tailed deer with deer-to-human transmission

  1. Bradley Pickering
  2. Oliver Lung
  3. Finlay Maguire
  4. Peter Kruczkiewicz
  5. Jonathon D. Kotwa
  6. Tore Buchanan
  7. Marianne Gagnier
  8. Jennifer L. Guthrie
  9. Claire M. Jardine
  10. Alex Marchand-Austin
  11. Ariane Massé
  12. Heather McClinchey
  13. Kuganya Nirmalarajah
  14. Patryk Aftanas
  15. Juliette Blais-Savoie
  16. Hsien-Yao Chee
  17. Emily Chien
  18. Winfield Yim
  19. Andra Banete
  20. Bryan D. Griffin
  21. Lily Yip
  22. Melissa Goolia
  23. Matthew Suderman
  24. Mathieu Pinette
  25. Greg Smith
  26. Daniel Sullivan
  27. Josip Rudar
  28. Oksana Vernygora
  29. Elizabeth Adey
  30. Michelle Nebroski
  31. Guillaume Goyette
  32. Andrés Finzi
  33. Geneviève Laroche
  34. Ardeshir Ariana
  35. Brett Vahkal
  36. Marceline Côté
  37. Allison J. McGeer
  38. Larissa Nituch
  39. Samira Mubareka
  40. Jeff Bowman

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Abstract

Wildlife reservoirs of broad-host-range viruses have the potential to enable evolution of viral variants that can emerge to infect humans. In North America, there is phylogenomic evidence of continual transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer ( Odocoileus virginianus ) through unknown means, but no evidence of transmission from deer to humans. We carried out an observational surveillance study in Ontario, Canada during November and December 2021 ( n  = 300 deer) and identified a highly divergent lineage of SARS-CoV-2 in white-tailed deer (B.1.641). This lineage is one of the most divergent SARS-CoV-2 lineages identified so far, with 76 mutations (including 37 previously associated with non-human mammalian hosts). From a set of five complete and two partial deer-derived viral genomes we applied phylogenomic, recombination, selection and mutation spectrum analyses, which provided evidence for evolution and transmission in deer and a shared ancestry with mink-derived virus. Our analysis also revealed an epidemiologically linked human infection. Taken together, our findings provide evidence for sustained evolution of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.

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  1. Version published to 10.1038/s41564-022-01268-9
  2. Version published to 10.1101/2022.02.22.481551v3 on bioRxiv
  3. Version published to 10.1101/2022.02.22.481551v2 on bioRxiv
  4. ScreenIT

    SciScore for 10.1101/2022.02.22.481551: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Deer sample collection and study area: Between November 1 and December 31, 2021, adult and yearling free-ranging WTD were sampled as part of the Ontario Ministry of Northern Development, Mines, Natural Resources and Forestry’s (NDMNRF) annual Chronic Wasting Disease (CWD) surveillance program.
    Sex as a biological variablenot detected.
    RandomizationPhylogenetic analyses were performed using CFIA-NCFAD/scovtree Nextflow workflow (v1.6.0) (https://github.com/CFIA-NCFAD/scovtree/) with the consensus sequences contextualised with closely related sequences identified by UShER and randomly sampled representative sequences from major WHO SARS-CoV-2 clades from GISAID (Shu & McCauley, 2017) (downloaded 2022-02-10).
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    HEK293T cells (ATCC) were cultured in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma), 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.3 mg/mL L-glutamine (Invitrogen) and maintained at 37°C, 5% CO2 and 100% relative humidity.
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    All constructs were cloned in pCAGGS and verified by Sanger sequencing.
    pCAGGS
    suggested: RRID:Addgene_127347)
    HEK293T seeded in 10-cm dishes were co-transfected with lentiviral packaging plasmid psPAX2 (gift from Didier Trono, addgene #12260)
    psPAX2
    suggested: RRID:Addgene_12260)
    lentiviral vector pLentipuro3/TO/V5-GW/EGFP-Firefly Luciferase (gift from Ethan Abela, addgene#119816), and plasmid encoding the indicated S construct at a 5:5:1 ratio using jetPRIME transfection reagent according to the manufacturer protocol.
    pLentipuro3/TO/V5-GW/EGFP-Firefly
    suggested: RRID:Addgene_119816)
    Software and Algorithms
    SentencesResources
    Reverse-transcription polymerase chain reaction (RT-PCR) was performed using the Luna Universal Probe One-Step RT-qPCR kit (New England BioLabs; https://www.neb.ca).
    New England BioLabs
    suggested: (New England Biolabs, RRID:SCR_013517)
    Quantstudio 3 software (Thermo Fisher Scientific; https://www.thermofisher.com) was used to determine the cycle threshold (Ct).
    Thermo Fisher Scientific
    suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
    Paired-end (2×150 bp) sequencing was performed on a MiniSeq with a 300–cycle reagent kit (Illumina, USA) with a negative control library with no input SARS-CoV-2 RNA extract.
    MiniSeq
    suggested: None
    WGS performed at CFIA used extracted nucleic acid quantified using the Qubit™ RNA High Sensitivity (HS) Assay Kit on a Qubit™ Flex Fluorometer (Thermo Fisher Scientific).
    WGS
    suggested: None
    (Di Tommaso et al., 2017; Ewels et al., 2020; Patel et al., 2022) which ran: FASTQC (v0.11.9) (Andrews, 2010) read-level quality control, fastp (v0.20.1) (Chen et al., 2018) quality filtering and adapter trimming, Bowtie2 (v2.4.2) (Langmead & Salzberg, 2012) read mapping to Wuhan-Hu-1 (MN908947.3) (Wu et al., 2020) SARS-CoV-2 reference, Mosdepth (v0.3.1)(Pedersen & Quinlan, 2018)/Samtools (v.1.12) (Li et al., 2009) read mapping statistics calculation, iVar (v1.3.1) (Grubaugh et al., 2019) ARTIC V4 primer trimming, variant calling, and consensus generation; SnpEff (v5.0) (Cingolani, Platts, et al., 2012)/SnpSift (v4.3t) (Cingolani, Patel, et al., 2012) for variant effect prediction and annotation; and Pangolin (v3.1.20) (O’Toole et al., 2021) with PangoLEARN (2022-01-05), Scorpio (v0.3.16) (Colquhoun & Jackson, 2021), and Constellations (v.0.1.1) was used for PANGO lineage (Rambaut et al., 2020) assignment. iVar primer trimmed soft-clipped read alignments were converted to hard-clipped alignments with fgbio ClipBam (http://fulcrumgenomics.github.io/fgbio/).
    FASTQC
    suggested: (FastQC, RRID:SCR_014583)
    Bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    SnpEff
    suggested: (SnpEff, RRID:SCR_005191)
    This workflow generated a multiple sequence alignment using Nextalign CLI (v1.10.1) (Aksamentov et al., 2021) and inferred a maximum-likelihood (ML) phylogeny using IQ-TREE (v2.2.0_beta) (Minh et al., 2020) using the GTR model for visualisation with Phylocanvas (Abudahab et al., 2021) via shiptv (v0.4.1) (https://github.com/CFIA-NCFAD/shiptv) and ggtree (Yu et al., 2017).
    IQ-TREE
    suggested: (IQ-TREE, RRID:SCR_017254)
    Multiple sequence alignment of this subset of sequences was performed with MAFFT (v7.490)
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Additional figures were generated and annotated using BioRender (BioRender, 2022) and Inkscape (Inkscape Project, 2020)
    BioRender
    suggested: (Biorender, RRID:SCR_018361)
    Neutralization half-maximal inhibitory dilution (ID50) was calculated using Graphpad Prism and represents the plasma dilution that inhibits 50% of pseudotype transduction in 293T-ACE2.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    , EHDV 6 isolate OV208 (NCBI MG886400 - MG886409), and Elk circovirus Banff/2019 (NCBI MN585201) (Fisher et al., 2020) were imported into Geneious (v.9.1.8) (Kearse et al., 2012).
    Geneious
    suggested: (Geneious, RRID:SCR_010519)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    It should be noted there were some limitations in genome quality and coverage that may have resulted in failure to detect additional mutations that were present. All Ontario WTD clade samples (including the associated human case) had missing terminal domains and contained internal regions with no or low coverage when sequenced using the ARTIC v4 amplicon scheme. This is a widespread issue that may explain the rarity of the 3’ proximal ORF10:L37F in GISAID. Significantly in our samples this meant there was no or <10x coverage in all 5 WTD sequences from ∼27000-27177 (dropout of ARTICv4 amplicons 90-91) which includes regions of the M gene. However, by combining the ARTIC v4 sequencing with additional sequencing using probe-based enrichment we were able to compensate for this dropout and generate high coverage and completeness (<100 positions with no coverage in all WTD and <100 positions with <10X coverage in 3/5 WTD genomes; see Table S3). The neutral non-synonymous to synonymous mutation ratio (dN/dS ∼ 1) and evenly distributed mutations in the WTD lineage contrasts with the signatures of strong selection in the equivalently divergent Omicron VOC (dN/dS ∼6 and accumulation of spike mutations). In combination with the most recent common ancestor being phylogenetically distant and dating from 2020, we infer that the WTD lineage likely diverged in 2020 and has been maintained in wildlife under relatively neutral selection since that time. It is possible that the absence of pre-...

    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.

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  5. Version published to 10.1101/2022.02.22.481551v1 on bioRxiv