From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer

  1. Mathias Martins
  2. Paola M. Boggiatto
  3. Alexandra Buckley
  4. Eric D. Cassmann
  5. Shollie Falkenberg
  6. Leonardo C. Caserta
  7. Maureen H. V. Fernandes
  8. Carly Kanipe
  9. Kelly Lager
  10. Mitchell V. Palmer
  11. Diego G. Diel

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in humans, has a broad host range, and is able to infect domestic and wild animal species. Notably, white-tailed deer (WTD, Odocoileus virginianus ), the most widely distributed cervid species in the Americas, were shown to be highly susceptible to SARS-CoV-2 in challenge studies and reported natural infection/exposure rates approaching 30–40% in free-ranging WTD in the U.S. Thus, understanding the infection and transmission dynamics of SARS-CoV-2 in WTD is critical to prevent future zoonotic transmission to humans, at the human-WTD interface during hunting or venison farming, and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2 B.1 lineage, WTD fawns (~8-month-old) shed infectious virus up to day 5 post-inoculation (pi), with high viral loads shed in nasal and oral secretions. This resulted in efficient deer-to-deer transmission on day 3 pi. Consistent a with lack of infectious SARS-CoV-2 shedding after day 5 pi, no transmission was observed to contact animals added on days 6 and 9 pi. We have also investigated the tropism and sites of SARS-CoV-2 replication in adult WTD (3–4 years of age). Infectious virus was detected up to day 6 pi in nasal secretions, and from various respiratory-, lymphoid-, and central nervous system tissues, indicating broad tissue tropism and multiple sites of virus replication. The study provides important insights on the infection and transmission dynamics of SARS-CoV-2 in WTD, a wild animal species that is highly susceptible to infection and with the potential to become a reservoir for the virus in the field.

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  1. Version published to 10.1371/journal.ppat.1010197
  2. ScreenIT

    SciScore for 10.1101/2021.12.14.472547: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Animal studies: All animal work and procedures performed in this study were approved by the National Animal Disease Center (NADC) Institutional Animal Care and Use Committee for both the fawn study (protocol ARS-2020-902) and the adult deer study (protocol ARS-2020-861)
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    BlindingCell cultures with no CPE were frozen, thawed, and subjected to two additional blind passages/inoculations in Vero E6/TMPRSS2 cell cultures.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For SARS-CoV-2 detection, tissue sections were incubated with anti-mouse biotinylated secondary antibody followed by incubation with the Vectastain Elite ABC HRP reagent.
    anti-mouse biotinylated secondary
    suggested: None
    Then, were incubated for 45 min at rt using a rabbit polyclonal antibody (pAb) anti-ACE2 (Abcan ref # ab15348) and a mouse monoclonal antibody (mAb) anti-TMPRSS2 (Santa Cruz Biotechnology, Inc. ref # sc-515727).
    anti-ACE2
    suggested: (Abcam Cat# ab15348, RRID:AB_301861)
    anti-TMPRSS2
    suggested: (Santa Cruz Biotechnology Cat# sc-515727, RRID:AB_2892118)
    Followed by 30 min incubation at rt with a goat anti-rabbit IgG (goat anti-rabbit IgG, Alexa Fluor® 594) and a goat anti-mouse IgG antibody (goat anti-mouse IgG, Alexa Fluor® 488).
    anti-rabbit IgG
    suggested: None
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and Virus: Vero E6 (ATCC® CRL-1586™), and Vero E6/TMPRSS2 (JCRB Cell Bank, JCRB1819) were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine (2mM), penicillin (100 U.ml−1), streptomycin (100 μg.ml−1) and gentamycin (50 μg.ml−1) for both cell lines.
    Vero E6
    suggested: None
    Positive samples on viral isolation were subjected to end point titrations by limiting dilution using the Vero E6/TMPRSS2 cells.
    Vero E6/TMPRSS2
    suggested: None
    Following incubation of serum and virus, 50 µl of a cell suspension of Vero cells was added to each well of a 96-well plate and incubated for 48 h at 37 °C with 5% CO2.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    SARS-CoV-2 MinION whole genome sequencing (WGS) and genetic analysis: The genetic make-up of SARS-CoV-2 following replication in WTD over viral transmission was investigated by whole genome sequencing.
    WGS
    suggested: None
    For this, nasal secretions collected on days 2 to 9 pi from inoculated and oronasal secretions on days 2 and 3 pc in contact animals were subjected to MinION-based targeted SARS-CoV-2 WGS.
    SARS-CoV-2
    suggested: (BioLegend Cat# 946101, RRID:AB_2892515)
    Low frequency variants were initially called using LoFreq [42] and subsequently filtered using Variabel [43].
    LoFreq
    suggested: (LoFreq, RRID:SCR_013054)
    Statistical analysis and data plotting were performed using the GraphPad Prism software (Version 9.0.1).
    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:
    • 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.

    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 10.1101/2021.12.14.472547 on bioRxiv