SARS-CoV-2 Infects Peripheral and Central Neurons Before Viremia, Facilitated by Neuropilin-1

  1. Jonathan D. Joyce
  2. Greyson A. Moore
  3. Poorna Goswami
  4. Telvin L. Harrell
  5. Tina M. Taylor
  6. Seth A. Hawks
  7. Jillian C. Green
  8. Mo Jia
  9. Neeharika Yallayi
  10. Emma H. Leslie
  11. Nisha K. Duggal
  12. Christopher K. Thompson
  13. Andrea S. Bertke

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Abstract

Neurological symptoms associated with COVID-19, acute and long-term, suggest SARS-CoV-2 affects both central and peripheral nervous systems. Although studies have shown olfactory and hematogenous entry into the brain and neuroinflammation, little attention has been paid to the susceptibility of the peripheral nervous system to infection or to alternative routes of CNS invasion. We show that neurons in the central and peripheral nervous system are susceptible to productive infection with SARS-CoV-2. Infection of K18-hACE2 mice, wild-type mice, golden Syrian hamsters, and primary neuronal cultures demonstrate viral RNA, protein, and infectious virus in peripheral nervous system neurons and satellite glial cells, spinal cord, and specific brain regions. Moreover, neuropilin-1 facilitates SARS-CoV-2 neuronal infection. Our data show that SARS-CoV-2 rapidly invades and establishes a productive infection in the peripheral and central nervous system via direct invasion of neurons prior to viremia, which may underlie some cognitive and sensory symptoms associated with COVID-19.

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  1. Version published to 10.1101/2022.05.20.492834v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2022.05.20.492834: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Ethics statement: This study was approved by the Virginia Polytechnic Institute & State University Institutional Animal Care and Use Committee (Protocol # 20-228, approved 02/01/2021).
    Field Sample Permit: Tissue types collected, methods of collection, and downstream assays were the same as described for the mice.
    Sex as a biological variableMouse infections: Eight to ten-week-old male and female B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (Stock # 034860; Jackson Laboratory; n=12, 2 groups of 6 mice), and their wild-type C57BL/6J counterparts (n=12, 2 groups of 6 mice) were inoculated intranasally with SARS-CoV-2 isolate USA-WA1/2020 (Extended Data Fig 1a).
    RandomizationAnimals were randomly assigned to either inoculum group or control group ensuring the groups were age-matched.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    SARS-CoV-2 N protein was visualized using an Alexa Fluor® 488 conjugated rabbit monoclonal anti-SARS-CoV-2 nucleocapsid antibody at a 1:1000 concentration (NBP2-90988AF488; Novus Biologicals).
    anti-SARS-CoV-2
    suggested: None
    hACE2 was visualized using an Alexa Fluor® 594 conjugated mouse monoclonal anti-ACE2 antibody at a 1:1000 concentration (sc-390851 AF594; Santa Cruz Biotechnology).
    anti-ACE2
    suggested: (Santa Cruz Biotechnology Cat# sc-7385 AF594, RRID:AB_2847914)
    NeuN was visualized using an Alexa Fluor® 647 conjugated rabbit monoclonal anti-NeuN antibody at a 1:1000 concentration (ab190565; Abcam).
    anti-NeuN
    suggested: (Abcam Cat# ab190565, RRID:AB_2732785)
    Tyrosine hydroxylase was visualized using an Alexa Fluor® 594 conjugated mouse monoclonal anti-TH antibody at a 1:500 concentration (818004; Biolegend).
    anti-TH
    suggested: (LSBio (LifeSpan Cat# LS-C40052-500, RRID:AB_972074)
    Glutamine synthetase was visualized using a mouse monoclonal anti-GS antibody at a 1:100 concentration (MA5-27750; Invitrogen) followed by an Alexa Fluor® 594 conjugated goat anti-mouse monoclonal antibody at a 1:1000 concentration (A11005; Invitrogen).
    anti-GS
    suggested: None
    anti-mouse
    suggested: (Molecular Probes Cat# A-11005, RRID:AB_141372)
    Neuropilin-1 was visualized using a goat polyclonal anti-NRP1 antibody at a 15 μg/mL concentration (AF566; R&D Systems) followed by an Alexa Fluor® 647 conjugated donkey anti-goat monoclonal antibody at a 1:1000 concentration (ab150135; Abcam).
    anti-NRP1
    suggested: None
    anti-goat
    suggested: (Abcam Cat# ab150135, RRID:AB_2687955)
    Experimental Models: Cell Lines
    SentencesResources
    The undiluted tissue homogenate as well as a ten-fold dilution of homogenate was inoculated in duplicate onto confluent monolayers of Vero E6 cells in 24-well plates.
    Vero E6
    suggested: None
    Neurons were pretreated with 100 μM of the NRP-1 antagonist EG00229 (6986; Tocris) dissolved in DMSO prior to infection as described for Caco-2 cells24.
    Caco-2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse infections: Eight to ten-week-old male and female B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (Stock # 034860; Jackson Laboratory; n=12, 2 groups of 6 mice), and their wild-type C57BL/6J counterparts (n=12, 2 groups of 6 mice) were inoculated intranasally with SARS-CoV-2 isolate USA-WA1/2020 (Extended Data Fig 1a).
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Uninfected K18-hACE2 control mice (n=2) and C57BL/6J wild-type control mice (n=2) were housed in a separate on campus ABSL-1 facility.
    C57BL/6J
    suggested: RRID:IMSR_JAX:000664)
    Software and Algorithms
    SentencesResources
    Images were imported into ImageJ and contrast and brightness was adjusted identically across all images within tissue types.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    All statistical analyses were performed in JMP Pro 16 (SAS Institute) and confirmed in GraphPad Prism version 8 during figure creation.
    SAS Institute
    suggested: (Statistical Analysis System, RRID:SCR_008567)
    GraphPad
    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 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 10.1101/2022.05.20.492834v1 on bioRxiv