Olfactory detection of viruses shapes brain immunity and behavior in zebrafish

  1. Aurora Kraus
  2. Benjamin Garcia
  3. Jie Ma
  4. Kristian J. Herrera
  5. Hanna Zwaka
  6. Roy Harpaz
  7. Ryan Y. Wong
  8. Florian Engert
  9. Irene Salinas

  • Curated by eLife

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    This study presents a useful description of transcriptional responses in adult zebrafish olfactory bulb microglia and neurons following exposure to infectious hematopoietic necrosis virus. This solid work advances our understanding of central nervous system responses to viral infection and provides an inventory of gene expression changes in particular cell types that can be used as hypothesis generators for future studies. Experiments to assess behavioral and neural responses to the virus in adults and larvae are inadequate and would benefit from a clearer conceptual framework that connects these avenues of investigation both to published literature and to the authors' single cell RNA sequencing results.

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Abstract

Olfactory sensory neurons (OSNs) are constantly exposed to pathogens, including viruses. However, serious brain infection via the olfactory route rarely occurs. When OSNs detect a virus, they coordinate local antiviral immune responses to stop virus progression to the brain. Despite effective immune control in the olfactory periphery, pathogen-triggered neuronal signals reach the CNS via the olfactory bulb (OB). We hypothesized that neuronal detection of a virus by OSNs initiates neuroimmune responses in the OB that prevent pathogen invasion. Using zebrafish ( Danio rerio ) as a model, we demonstrate viral-specific neuronal activation of OSNs projecting into the OB, indicating that OSNs are electrically activated by viruses. Further, behavioral changes are seen in both adult and larval zebrafish after viral exposure. By profiling the transcription of single cells in the OB after OSNs are exposed to virus, we found that both microglia and neurons enter a protective state. Microglia and macrophage populations in the OB respond within minutes of nasal viral delivery followed decreased expression of neuronal differentiation factors and enrichment of genes in the neuropeptide signaling pathway in neuronal clusters. Pituitary adenylate-cyclase-activating polypeptide ( pacap ), a known antimicrobial, was especially enriched in a neuronal cluster. We confirm that PACAP is antiviral in vitro and that PACAP expression increases in the OB 1 day post-viral treatment. Our work reveals how encounters with viruses in the olfactory periphery shape the vertebrate brain by inducing antimicrobial programs in neurons and by altering host behavior.

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  1. Version published to 10.7554/elife.88271.1 on eLife
  2. Version published to 10.7554/elife.88271 on eLife
  3. eLife

    eLife assessment

    This study presents a useful description of transcriptional responses in adult zebrafish olfactory bulb microglia and neurons following exposure to infectious hematopoietic necrosis virus. This solid work advances our understanding of central nervous system responses to viral infection and provides an inventory of gene expression changes in particular cell types that can be used as hypothesis generators for future studies. Experiments to assess behavioral and neural responses to the virus in adults and larvae are inadequate and would benefit from a clearer conceptual framework that connects these avenues of investigation both to published literature and to the authors' single cell RNA sequencing results.

  4. eLife

    Reviewer #1 (Public Review):

    Kraus et al. investigated transcriptional responses to transient exposure to infectious hematopoietic necrosis virus in the brain of adult zebrafish using single cell RNA-Seq methods. The authors discovered valuable evidence for immune responses in microglial clusters within minutes of viral exposure, and longer term changes in neuronal populations one day after viral treatment. The strength of the study is the RNA-Seq data which will act as a valuable resource for the zebrafish community. Their discoveries from the RNA-Seq studies are convincing, where they find a neuropeptide called PACAP enriched in neuronal populations a day after viral exposure, which exhibit antiviral activity.

    The authors select the 1 day time point post-infection based on initial behavioral experiments, the evidence for which is modest at best. While the experiments with larval animals are more substantiated, they use adults for their RNA-Seq experiments. The behavioral phenotype in adults is a marginal decrease in velocity 1 day after infection. The authors could have performed other tests associated with sickness behaviors, or even characterized the locomotion in the open field experiment with more in-depth analysis (for example, the larval experiments had more information regarding turning angles).

  5. eLife

    Reviewer #2 (Public Review):

    Kraus, Aurora et al. investigated the potential immune response of the olfactory bulb after exposure of the infectious hematopoietic necrosis virus (IHNV), via the olfactory epithelia. Specifically, they show that a) viral-specific neuronal activation of "OSNs" (Crypt cells), b) changes in behaviour of both adult and larval zebrafish after viral exposure, c) Pituitary adenylate-cyclase-activating polypeptide (PACAP), was enriched when assayed by single cell transcriptomic profiling of cells in the OB after OSNs are exposed to IHNV

    Although the paper does have strengths in principle, the weaknesses of the manuscript are that these strengths are not directly demonstrated and the referencing of the manuscript omits many references important for the understanding of the questions and the results of the study. Furthermore, the data presented are not sufficient to fully support the key claims in the manuscript. In particular:

    a) Viral-specific neuronal activation of OSNs:
    What type of neurons? The authors are a bit elusive and do not clearly state that the neurons are crypt cells (Sepahi et al.: rainbow trout) which have a very specific axonal projection to the brain and whose response characteristics are not well characterized (see work of Korsching lab). Crypt cells are not present in the olfactory epithelia of mammals. Furthermore, in their previous work the crypt cells die; so how do they think the (inflammatory) virus response is transmitted to the olfactory bulbs in order to protect the brain?
    The authors state from previous work that they never detected virus in the brain, but why would they? Does INHV move trans-synaptically?
    The neuronal activity was monitored using a pan-neuronal marker thus these data are of limited use when trying to understand the role of neuronal activity (crypt cells) in the IHNV-triggered activity: the authors may be looking at a generalized inflammation response, and the image presented is not particularly informative it is difficult to decipher the results. The authors assume IHNV is an odorant without carefully ruling out the possibility of a generalized inflammation response.
    b) Changes in behaviour of both adult and larval zebrafish after viral exposure:
    What is the motivating question for looking at behaviour of the virus infected animals? Do we know the effects of crypt cell loss on the behaviour in any fish species? Authors need to build a better conceptual framework for the behaviour experiments.

    c) Pituitary adenylate-cyclase-activating polypeptide (PACAP) was enriched when assayed by single cell transcriptomic profiling of cells in the OB after OSNs are exposed to IHNV. Authors draw many generous conclusions from limited data. Authors seem to have forgotten to cite papers previously published showing that PACAP-38 has anti-viral activities in fish (VHSV: trout) such as: Velasquez et al 2020, First in vivo evidence of pituitary adenylate cyclase-activating polypeptide antiviral activity in teleost.
    The histology for PACAP presented in the manuscript is not convincing. The antibody is against the human form of PACAP thus any labelling should be treated with caution (and called PACAP-38-like).

    Summary: The authors need to better develop their model (perhaps a diagram would be helpful) explaining exactly which neurons are transmitting the information. Because of the elusive nature of some referencing and the skirting of important issues such as clearly stating which neurons are affected (crypt cells), what the point of the behaviour is (relate to neuronal type infected by virus), and, the lack of an antibody specific to the zebrafish protein, the model appears to be built on an unstable base.

  6. eLife

    Reviewer #3 (Public Review):

    Using the zebrafish model, this paper by Kraus A. et al., described the anti-virus response in the Olfactory bulb (OB) neurons and microglia. This paper used the behavioral test, neuron calcium imaging, and single-cell transcriptomic analysis. Importantly, this paper discovered that following IHNV infection, the OB neuron increased Pacap expression, which likely protects the neuron cells and mediates the anti-viral defense response. Overall, the findings presented in this paper are quite interesting.

    Major strength:
    (1) The author demonstrated for the first time that zebrafish OSN neurons sense the IHNV viruses and transmit the viral signal to OB neurons. The zebrafish can be used as a new system to investigate the viral-neuron interaction and understand the mechanisms of how the neurons in the CNS to viral infection through the peripheral chemosensory system.

    (2) This paper generated the first zebrafish OB sc-RNA sequencing data. The sc-RNA sequencing data generated in this paper will also help other zebrafish researchers who study the OB neurons.

    Major weakness:
    The experiment results presented in this paper are not well-integrated. For example, it is unclear how the behavioral phenotype is connected to the neuronal calcium phenotype. It is also unclear how the behavioral or neuronal calcium imaging results is connected to the scRNA sequencing result.

  7. Version published to 10.1101/2023.03.17.533129v2 on bioRxiv
  8. Version published to 10.1101/2023.03.17.533129v1 on bioRxiv