Olfactory detection of viruses shapes brain immunity and behavior in zebrafish

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).

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.

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.