PONE-D-24-41674Neuroinvasive and neurovirulent potential of SARS-CoV-2 in the acute and post-acute phase of intranasally inoculated ferretsPLOS ONE

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This work was funded in part by a fellowship from the Netherlands Organization for Scientific Research (VIDI contract 91718308).  Ferret experiments were previously published and supported by funding from the National Institutes of Health (AI146980, AI121349, NS091263, AI114736, HHSN272201400008C).  

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This work was funded in part by a fellowship from the Netherlands Organization for Scientific Research (VIDI contract 91718308). Ferret experiments were previously published and supported by funding from the National Institutes of Health (AI146980, AI121349, NS091263, AI114736, HHSN272201400008C).

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This work was funded in part by a fellowship from the Netherlands Organization for Scientific Research (VIDI contract 91718308).  Ferret experiments were previously published and supported by funding from the National Institutes of Health (AI146980, AI121349, NS091263, AI114736, HHSN272201400008C). 

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript titled “Neuroinvasive and neurovirulent potential of SARS-CoV-2 in the acute and post-acute phase of intranasally inoculated ferrets” by Benavides et al. examines the potential of SARS-CoV-2 to invade the brain and cause neurological changes in a ferret model. The study offers valuable insights into how even mild respiratory infections might impact the brain, highlighting subtle changes in microglial and astrocytic activity. However, several methodological limitations need to be addressed to enhance the robustness of the findings.

Major Concerns:

1. While the study detects viral RNA in the brain through qPCR, this is not confirmed by in situ hybridization (ISH) or immunohistochemistry (IHC). This weakens the claim of neuroinvasion. Employing electron microscopy, at earlier time points (e.g., days 2–4) could provide stronger evidence.

2. Although changes in microglial and astrocytic activity are reported, the study does not explore functional consequences such as cognitive or behavioral changes. Including behavioral data or electrophysiological studies in future work would significantly enhance the interpretation of these findings. If these experiments are not feasible at this stage, the manuscript should include a discussion of potential follow-up studies to explore functional outcomes and current limitations.

3. The small sample size limits the statistical power and generalizability of the results. The authors should acknowledge this limitation and propose including larger cohorts in future research to address inter-individual variability.

4. Sampling at 7 and 21 dpi may overlook critical early events of neuroinvasion or inflammatory response. Including earlier time points (e.g., 1–4 dpi) in future studies is recommended. The authors should discuss this limitation in the current manuscript.

5. Incorporating systemic cytokine profiling would provide valuable information about potential indirect mechanisms driving neurovirulence, adding depth to the findings.

Minor Concerns:

1. While the figures and tables are informative, adding more detailed statistical annotations would improve clarity and accessibility for readers.

2. The extent of GFAP changes in areas beyond the olfactory bulb is unclear. This should be clarified.

Conclusion:

This study offers important insights into the neurological effects of SARS-CoV-2 in a mild respiratory disease model and underscores the relevance of the ferret model in this context. However, the absence of direct evidence for neuroinvasion, limited exploration of functional consequences, and other methodological constraints reduce its impact. Addressing these concerns, either experimentally or through a detailed discussion, would strengthen the manuscript and make its contributions clearer to the readership.

Recommendation: Revisions required.

Reviewer #2: The authors describe a study on the neuroinvasive and neurovirulent potential of SARS-CoV-2 conducted on the ferret animal model, specifically those that had subclinical or mild infection for which it is currently unknown. It was noted that tissues analysed in the present work were collected from a previous study (ref 29) on intranasally inoculated SARS-CoV-2 ferrets. Findings on the limited neuroinvasive potential of the virus warrants further work on the neurological effects of subclinical/mild disease.

Methods and results were generally well described however, the writing would benefit from editing/language checks with the following observed upon review:

Line 64: “In experimentally inoculation animals…” suggest to check the term used.

Line 91: "...infection is currently unknown, Therefore we investigated the neuroinvasive..."

Other comments:

Line 71: Authors described the mechanisms of neurovirulence, but in what way are microglia/astrocytes activated with SARS-CoV-2 infection? E.g. neuroinflammatory response, activation of mast cells?

Line 298: A number of references were listed i.e. (4,5,14,16,17,29) - it would be worth providing some description of other similar studies e.g. in ferrets, relating to the results of this study and how they differ/align.

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Reviewer #1: No

Reviewer #2: No

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Neuroinvasive and neurovirulent potential of SARS-CoV-2 in the acute and post-acute phase of intranasally inoculated ferrets

PONE-D-24-41674R1

Dear Dr. van Riel,

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Kind regards,

Victor C Huber

Academic Editor

PLOS ONE

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