The post-acute phase of SARS-CoV-2 infection in two macaques species is associated with signs of ongoing virus replication and pathology in pulmonary and extrapulmonary tissues

  1. Kinga P. Böszörményi
  2. Marieke A. Stammes
  3. Zahra C. Fagrouch
  4. Gwendoline Kiemenyi-Kayere
  5. Henk Niphuis
  6. Daniella Mortier
  7. Nikki van Driel
  8. Ivonne Nieuwenhuis
  9. Richard A. W. Vervenne
  10. Tom Haaksma
  11. Boudewijn Ouwerling
  12. Deborah Adema
  13. Roja Fidel Acar
  14. Ella Zuiderwijk-Sick
  15. Lisette Meijer
  16. Petra Mooij
  17. Ed J. Remarque
  18. Herman Oostermeijer
  19. Gerrit Koopman
  20. Alexis C. R. Hoste
  21. Patricia Sastre
  22. Bart L. Haagmans
  23. Ronald E. Bontrop
  24. Jan A.M. Langermans
  25. Willy M. Bogers
  26. Ivanela Kondova
  27. Ernst J. Verschoor
  28. Babs E. Verstrepen

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Abstract

The post-acute phase of SARS-CoV-2 infection was investigated in rhesus macaques ( Macaca mulatta ) and cynomolgus macaques ( Macaca fascicularis ). During the acute phase of infection, SARS-CoV-2 was shed via nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, as suggested by the absence of viral RNA in nasal and tracheal swabs, computed tomography (CT) and positron emission tomography (PET)-CT were able to reveal pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, like conjunctiva, cervical and mesenteric lymph nodes.

However, 5-6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans.

Author summary

More than a year after the start of the pandemic, the long-term consequences of SARS-CoV-2 infection start to surface. The variety of clinical manifestations associated with post-acute COVID-19 suggests the involvement of multiple biological mechanisms. In this study, we show that rhesus and cynomolgus macaques shed virus from their respiratory tract, generate virus-specific humoral immune responses, and show signs of SARS-CoV-2-induced lung pathology. PET-CT revealed that both species showed ongoing mild to moderate pulmonary disease, even after the virus was no longer detectable in nasal and tracheal swabs. Five to six weeks after infection, necropsy confirmed minimal to mild histopathological manifestations in various tissues, like the lungs, heart, lymph nodes, and conjunctiva. We detected Viral RNA in the heart, respiratory tract, and tracheobronchial lymph nodes, and subgenomic messenger RNA in the lungs and surrounding lymph nodes, indicative of ongoing virus replication. We show widespread tissue dissemination of SARS-CoV-2 in infected macaques and the presence of replicating virus in lungs and surrounding lymph nodes after alleged convalescence of infection. This finding is intriguing in the light of long-COVID disease symptoms seen in humans as it has been hypothesized that persistent infection may contribute to this phenomenon.

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  1. Version published to 10.1101/2020.11.05.369413v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.11.05.369413: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Upon positive advice by the independent ethics committee (DEC-BPRC) the competent authorities (CCD, Central Committee for Animal Experiments) issued a project license (license AVD5020020209404).
    IACUC: Upon positive advice by the independent ethics committee (DEC-BPRC) the competent authorities (CCD, Central Committee for Animal Experiments) issued a project license (license AVD5020020209404).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    To detect macaque-IgM, the same protocol was followed, but the secondary antibody used was an anti-Monkey IgM (μ-chain specific)-Peroxidase antibody produced in goat (Sigma-Aldrich, Merck KGaA, MI, USA) at 1:5000 dilution.
    anti-Monkey IgM
    suggested: None
    μ-chain specific)-Peroxidase
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    This strain was isolated from a patient who traveled from China to Germany, and an aliquot of a Vero E6 cell culture was made available through the European Virus Archive-Global (EVAg).
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Ethics and Biosafety Statement: All housing and animal care procedures took place at the Biomedical Primate Research Centre (BPRC) in Rijswijk, the Netherlands.
    BPRC
    suggested: None

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    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 10.1101/2020.11.05.369413v1 on bioRxiv