Dear Dr. Cassar,

Thank you very much for submitting your manuscript "Southern African Origin for HTLV-1 in Romania" 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.

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).

Important additional instructions are given below your reviewer comments.

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,

Adly M.M. Abd-Alla, Prof asso.

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

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)

Reviewer #2: The main method of this study consists in the molecular analysis of these new HTLV sequences. Therefore, it is essential to include in methods how the sequences were obtained, and all phylogenetic analysis done. This information is currently spread in results session. Authors need to include it in methods.

First statement of results belongs to methods. And authors need to expand briefly on the method used (reference 12).

“DNA samples were obtained from peripheral blood buffy coats. The LTR sequence was obtained for the 11 samples, as previously described (12)”

Figure 1 and 2 legends also contain important information that needs to be in methods.

The following statement in results also should be in methods.

“The DNA samples were subjected to four series of PCR (F1-F4) using both a high-fidelity hot-start DNA polymerase and primers designed from highly conserved regions for the major HTLV-1 subtypes. In brief, we obtained four different HTLV-1 proviral genomic regions, each about 2.5 kb in length: F1, LTR-Gag; F2, pro-pol; F3, pol-env and F4, tax-LTR. PCR products were then directly sequenced on both strands using a set of 16 specific primers (12). After alignment of sense and antisense sequences using the Clustal W algorithm (Mac Vector 18.6.1 software, Oxford molecular), comparison of each generated segment was implemented to derive a consensus sequence.”

Minor points:

- How many patients were tested for HBV?

- How many mothers were tested?

- Include in methods information about transfusion (the first time this is mentioned is in discussion). Was it done in Romania?

- If available, it would be interesting to know how long they have been out of Romania.

Reviewer #3: There appear no problems in the objectives, methods and interpretation of the results.

Ethical or regulatory requirements are properly met.

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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)

Reviewer #2: Results

Authors says that all sequences except ROU7 belong to a-TC-Mozambique – Southern Africa A subgroup. What about the outlier (ROU7) ?

Authors did whole genome sequence of 10/11 sequences, please inform the reader which sample was missing.

Authors say that stop mutations are quite frequent in ATL. This needs to go to discussion, and a reference is needed.

Authors say that 9/10 Romanian strain form a specific “Romania” clade. Please add in the text which sample is the exception.

The last paragraph of result can be moved to discussion.

Suggest replacing “They” at the beginning of the second paragraph of results for something more specific Newly characterised sequences…

Reviewer #3: Figure 1 and 2: The figures are blurred and the texts in the figures unreadable.

The analysis itself is appropriate and the results are described clearly.

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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: Authors need to include a conclusion session.

Reviewer #3: 1. Page 14: I understand that all the viruses identified in Romania seem to originate from Mozambique, but there is little discussion on the background and reasons for this. Is this because of immigrants? Also, given that multiple individuals have been infected, shouldn't there be a discussion of how and when the virus entered the country and how it spread within the country?

2. Page 14 and figure 2: The possible origin of the remaining one specimen should be discussed

3. Page 14, lines 11-13: Since many cases of ATL from horizontal infection have been confirmed in Japan, should the wording be weakened?

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: As mentioned above, the figures in the PDF are very poor to understand what are described. This may be due to the problems in making PDF, however, the clear figures need to be provided to the reviewers.

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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: (No Response)

Reviewer #2: This manuscript brings novel and interesting data that sheds light on HTLV-1 spread to Romania, a high endemic country in Europe. The molecular analysis is done at high standard and shows convincing evidence of a recent African origin.

However, some methods are currently described in result session or Figure legends. Discussion can also be expanded.

Authors could include discussion about current situation of HTLV response in Romania. Are they testing Blood donors? Are they testing organ donors? They can also discuss strategies that can limit the spread of this virus in Romania (as they suggested this should be done).

Another interesting point to discuss is how to limit spread to other countries. Recent blood donor data indicates high prevalence of HTLV in Moldova and brings concern about other countries within the region where no HTLV prevalence data is available. Authors have extensive experience on HTLV epidemiology and could expand on this topic. Another question that this study brings is if the origin of HTLV in Moldova is the same as they have revealed for Romania.

Reviewer #3: It is significant that the origin of HTLV-1 in Romania has been clarified.

However, there appears some room for improvement in interpretation and discussion of the data、in addition to the poor quality of the figures.

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Reviewer #1: No

Reviewer #2: Yes: Carolina Rosadas

Reviewer #3: Yes: Toshiki Watanabe

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

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Attachments

Attachment

Submitted filename: Reviewer_comments.docx

Dear Dr. Cassar,

We are pleased to inform you that your manuscript 'Southern African Origin of HTLV-1 in Romania' 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,

Adly M.M. Abd-Alla, Prof asso.

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

***********************************************************

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 #2: NA

Reviewer #3: The description of the method has been improved significantly with revisions suggested by the reviewers. Thus, readers can understand them clearly.

**********

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 #2: NA

Reviewer #3: Responding to the reviewers’ comments, revised descriptions have been improved so that the readers can clearly understand the information.

**********

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 #2: NA

Reviewer #3: The manuscript does not have an independent section of conclusions; however, interpretation and the significance of the results is presented in the discussion section, which appear to be reasonable and understandable to the readers.

**********

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 #2: (No Response)

Reviewer #3: They are improved in the revised manuscript.

**********

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 #2: I appreciate the changes made by authors, I believe they answer all questions appropriately and I am happy with the revised version of the manuscript.

Reviewer #3: The abstract section provides the summary and comments, which are reasonable based on the results obtained

**********

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 #2: Yes: Carolina Rosadas

Reviewer #3: No