Dear Dr. Kepple,

Thank you very much for submitting your manuscript "Comparative transcriptomics reveal differential gene expression among Plasmodium vivax geographical isolates and implications on erythrocyte invasion mechanisms" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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[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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Thank you again for your submission to our journal. 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,

Ananias A. Escalante, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Walderez Dutra

Section Editor

PLOS Neglected Tropical Diseases

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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: Objectives and methods are properly described. Statistical analysis seems appropriate. No ehtical concerns were identified.

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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: Overall, the manuscript is well written and results are prearly presented and discussed.

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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: Comparing Plasmodium vivax transcriptomes obtained with different study protocols is necessarily challenging. This authors might perhaps discuss the potential limitations of the comparative approach in greater detail.

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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: None.

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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: Kepple and colleagues have generated 10 new transcriptomes of Plasmodium vivax isolates from Ethiopia and compare their results with previously published data from Cambodia (4 transcriptomes) and Brazil (2 transcriptomes).

In two of the studies (Ethiopia and Brazil), cryopreserved, patient-derived isolates were thawed and maintained in short-term culture ex-vivo for approximately 40-44 hours, under slightly different conditions, for schizont maturation; in one study (Cambodia), fresh parasites were used for ex-vivo maturation. The key question is whether differences in transcriptomic profiles among parasites were mostly due to the geographic origin of parasites (Horn of Africa vs. Southeast Asia vs. Amazon Basin in South America) or to differences in sample preparation protocols prior to transcriptomic analysis. This question deserves deeper discussion throughout the manuscript.

Interestingly, CIBERSORTx deconvolution analysis shows the presence of some ring-stage transcripts in Brazilian isolates (Figure 1), which is unexpected after the ex-vivo maturation process. However, the difference in the proportion of late trophozoites and schizonts among samples of different geographic origin did not reach statistical significance.

Why are transcriptomes of Cambodian and African parasites more similar to each other than they are to those from South American samples is not entirely clear. Population genomic analyses (e.g., doi: 10.1038/ng.3588) typically show greater similarity between African and American lineages of P. vivax , compared with Southeast Asian lineages. Again, can this be due to differences in study protocols used for samples processing and transcriptomic analyses rather than geographic differences in transcription patterns?

Two minor comments:

1. Because the genes coding for the PIR family in P. vivax had originally been described as the vir multigene family in P. vivax, the nomenclature may be a bit confusing.

2. The authors coreectly state that "malnourishment has a protective effect to P. vivax infections in people from the western Brazilian Amazon (70)". Yes, there is a single cross-sectional report of such an association in children, with suboptimal controlling for confounders, which cannot be taken as evidence for a causal association between malnutrition and protection from malaria. I suggest these data are not overinterpreted in this context (differences between the transcriptomes of two P. vivax lineages from adults in Brazil and those from other geographic regions) as population-based nutritional status data from the study sites are not considered here.

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Reviewer #1: No

Figure Files:

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr. Kepple,

We are pleased to inform you that your manuscript 'Comparative transcriptomics reveal differential gene expression among Plasmodium vivax geographical isolates and implications on erythrocyte invasion mechanisms' 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,

Walderez O. Dutra, PhD.

Section Editor

PLOS Neglected Tropical Diseases

Walderez Dutra

Section Editor

PLOS Neglected Tropical Diseases

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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: (No Response)

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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: (No Response)

**********

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)

**********

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: (No Response)

**********

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: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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: Yes: Marcelo U. Ferreira