Dear Dr. Yoshinaga,

Thank you very much for submitting your manuscript "Plasma lipidome profiling of newborns with antenatal exposure to Zika virus" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

The reviewers found this to be a well written and executed study, but did have some suggestions that could strengthen the manuscript. One reviewer asked for an additional experiment that seems reasonable and should be highly considered by the authors.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Doug E Brackney, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Rebecca Rico-Hesse

Deputy Editor

PLOS Neglected Tropical Diseases

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The reviewers found this to be a well written and executed study, but did have some suggestions that could strengthen the manuscript. One reviewer asked for an additional experiment that seems reasonable and should be highly considered by the authors.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The authors investigate lipidomic profile of ZIKA congenitally infected nowborns by using high performance liquid chromatography. The method suffers from two limitations (unability to diciminate HODE isoforms and single point sampling) but they are discussed and do not critically change the manuscript message.

Reviewer #2: (No Response)

Reviewer #3: This study compares the plasma lipidome profiles of Zika virus exposed microcephalic and normocephalic newborns compared to uninfected controls. A key oxidized lipid, hydroxyoctadeconoic acid (HODE) derived from linoleic acid was observed to be higher in microcephalic newborns compared to the norpmocephalic group and controls. HODE was also positively associated with increased circulating free fatty acids providing a preliminary basis for a plasma lipidome signature. An additional interesting finding was the observation that lysophosphatidylcholine was higher in ZIKV exposed normocephalic newborns linking possibilities that polyunsaturated fatty acid transport may be hindered. This study is very well written and results clearly explained. The lipidomics and the statistical analyses are clearly carried out and written well in the materials and methods. Concerns regarding sample pool size is addressed in the discussion including other limitations. No concerns about ethical or regulatory requirements.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The data sound and are presented clearly. Figures and tables are well designed and allow nice understanding of the results.

Reviewer #2: (No Response)

Reviewer #3: The results are clearly explained and limitations addressed.

It would be very valuable to the readership if the authors could also provide a table of information of all the data from the untargeted analyses with included observed mass, retention time, putative IDs, accurate mass, ppm error etc so that a complete picture of the observed plasma lipidome of these newborns could be assessed with the understanding that accurate identification of the masses require further analyses.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The conclusions based on this limited dataset are valid and are well discussed compared to the current literature.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: There is a reference displayed as "Kikut et al., 2020", line 281, unlike the others, displayed as numbers.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Viral infection during pregnacy may associate with alteration of lipid metabolism, thereby threatening homeostasis and development. Here the authors show that the lipidomic profile of newborns congenitally infected by ZIKV differs from that of control subjects and show increased HODE levels. The findings provide new insight on ZIKV infection pathophysiology. They disclose new clues to understand neurological sequelae since elevated HODE levels have been already associated with HCMV infection, even though no causality can be formally established.

Reviewer #2: (No Response)

Reviewer #3: While this is a limited data set and limited number of samples that are challenging to acquire, the information resulting from this study are clearly presented and can drive future analyses of how lipids might control newborn brain development as well as how ZIKV might alter this process.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

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Attachments

Attachment

Submitted filename: comments.docx

Dear Dr. Yoshinaga,

I have reviewed the revisions and added material and don't believe it requires being disseminated to reviewers for a second round of reviews. Therefore, we are pleased to inform you that your manuscript 'Plasma lipidome profiling of newborns with antenatal exposure to Zika virus' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Doug E Brackney, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Rebecca Rico-Hesse

Deputy Editor

PLOS Neglected Tropical Diseases

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