PONE-D-24-20796Effect of a loss of the mda5/ifih1 gene on the antiviral resistance in a Chinook salmon Oncorhynchus tshawytscha cell linePLOS ONE

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The manuscript is of interest for the PLOS ONE readers, however, the authors should correct and resolve all the questions raised by the reviewers, in order to be accepted for publication.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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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

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The present manuscript explores the role of Melanoma Differentiation-Associated protein 5

(MDA5) in antiviral defense in Chinook salmon cells, using CRISPR/Cas9 to create a cell line lacking the

ifih1 gene. The study found that while the induction of type I interferon (IFN1) was impaired in these

modified cells upon viral infection, there was no increase in cytopathic effects. These results suggest

that the cells have redundant antiviral defense mechanisms that compensate for the loss of MDA5.

Undoubtedly, a lot of work has gone into this paper and it is characterized by novelty. However,

some questions arise. Could the authors explain an alternative mechanism path?

Given that RIG-I and MDA5 target different dsRNA lengths, could this variability be affecting the discussion?

In lines 70-81, the text is overly verbose. Since other species also possess the specific gene, I strongly recommend moving this section to the supplementary material. The authors could also include other fish species (e.g.see genus: Gadus, Perka) for a comparative sequencing analysis.

In the manuscript, the authors state "data not shown" three times. They should either provide

this data in the supplementary material or omit these repeatead references to refine the text. Are there

any available results from at least the wild type analysis? Also, the position 20 is not significant on the

structure for protein functionality.

Although the maximum likelihood method is accurate for this type of phylogenetic analysis, the

authors should discuss the results more thoroughly and could consider using another model for the matrix.

Based on these facts, I suggest the conditional acceptance of this manuscript for publication in PlosOne.

Reviewer #2: The present report explores knockdown of a key receptor in PAMP recognition, contributing to antiviral immunity. Knockdown MDA5 cells were created using CRISPR/Cas9 and cells were infected with several important fish pathogens to look at differences in responses. Feedback is given in point form below.

- In all figures the knockdown cells are not compared to wildtype cells in figures. It was comment upon in the results that there were no differences between MDA5C2 and WT cells (225). This is misleading to then continue describing only the knockdown clones. This data must be shown in all figures.

- Manuscript needs to be thoroughly edited for grammar, many awkward sentences and atypical word choices (examples include the first sentence of the abstract, the abstract sentence "invalidated for the ifih1 gene", figure 3A y-axis label includes "et" instead of and)

- If cells were plated and then allowed to grow to confluence, how was MOI determined? Were cells re-counted prior to infection?

- Was beta-actin check for stability as a housekeeping gene?

- For SHRV and SAV 15ul of RNA was used, how was it confirmed that this RNA came from the same number of cells?

- NNV details are missing, how was RNA isolated?

- Clarify in line 186 how FBS was decomplemented

- Describe all abbreviations in figure legends

- Discussion should be re-written for flow, several "hanging" paragraphs are present that aren't integrated into the discussion, as an example line 289-291.

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Reviewer #1: No

Reviewer #2: No

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Effect of a loss of the mda5/ifih1 gene on the antiviral resistance in a Chinook salmon Oncorhynchus tshawytscha cell line

PONE-D-24-20796R1

Dear Dr. Collet,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Maria del Mar Ortega-Villaizan

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have satisfactorily revised the manuscript and it is now acceptable for publication.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have satisfactorily addressed all previous concerns.The present form of the manuscript (PONE-D-24-20796R1) is suitable for publication in PLOS ONE .

Reviewer #2: (No Response)

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Sarah Poynter

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