Dear Mr. De Kesel,

Thank you very much for submitting your manuscript "Antibodies against medically relevant arthropod-borne viruses in the ubiquitous African rodent Mastomys natalensis" 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.

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

[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.

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.

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,

Ran Wang, M.D.

Academic Editor

PLOS Neglected Tropical Diseases

Michael Holbrook

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

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

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

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

-Were correct statistical analysis used to support conclusions? Yes

-Are there concerns about ethical or regulatory requirements being met? No

Reviewer #2: (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: The analysis presented match the analysis plan and results are clearly presented using good quality figures.

Reviewer #2: 1. Page 18, line 348 - Please correct grammar.

2. Page 23, line 453-458 - The authors showed that the DBS samples were cross-reactive with different viral proteins like NS1 of DENV and E2 of CHIKV, and they discussed the cross-reactivity to between different two proteins. So to well understand that point, please indicate the amino acid homology rate between NS1 and E2 proteins.

3. Common experimental mouse has different sensitivity to viral infections between sex. Do authors think the sensitivity affect the seropositivity in your results?

4. Authors showed the seroprevalence of Arbovirus you listed. Are there reports that those viruses experimentally infect to mouse, and induced antibodies to NS1 or E2 proteins?

5. Please correct Fig 2, especially Zika virus. Authors Zika virus belongs to Togaviridae. This is not correct.x

6. How is the sensitivity of this high-throughput multiplex immune assay?

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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: The authors conclude that M.natalensis play an important role in sustaining arbovirus transmission and this is supported by the data presented in the results (an overall seroprevalence of 24%). The paper clearly stated the limitations in their analysis such as using negative control from a breeding colony that could potentially over/under-estimate the sero-prevalence.

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

Is public health relevance addressed? Yes

Reviewer #2: (No Response)

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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: Though the paper is well-written i think some background on the multiplex immunoassay will help to understand why the authors chose this method.

The method section is well-written however it would read better if the authors could consider adding subtitles to subsections e.g. Screening for IgG antibodies to arboviruses, recombinant arbovirus proteins etc

More details on beads coupling (if it was done)

Reviewer #2: (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: De Kesel and co-authors present an interesting study that address a key gap in the existing knowledge of arboviral diseases transmission dynamics. The authors hypothesize that rodents specifically M.natalensis may serve as amplifying hosts of arboviruses. To this end, they screened blood samples from M.natalensis individuals for antibodies against 15 medically important arboviruses and reported an overall arbovirus seroprevalence of 24% suggesting that M.natalensis plays a role in the transmission of multiple arboviruses. The main strength of this paper is that the authors chose a perfect rodent species to study arboviruses transmission dynamics. Besides being a reservoir host of numerous pathogens and also the most abundant rodent species in SSA, M.natalensis lives in close proximity to humans increasing the risk of pathogen spillover to humans. As such, the paper identifies rodents as potential sources of arbovirus infections that pose a threat to human health. One of the weaknesses or rather a limitation is that the authors used negative controls obtained from a breeding colony instead of natural positive controls which could have affected the cut-off values. I enjoyed reading this manuscript and i think the paper is well written. However, some background information on the immunoassay used is missing and the analysis, set up and protocol in the methods section could be strctured into subsections with subtitles.

Reviewer #2: The authors developed a high-throughput multiplex immune assay using Luminex technology. The system employed the viral proteins such as NS1 protein for Flaviviridae and E2 protein for Togaviridae to evaluate the seroprevalence for 15 species of arboviruses in multimammate mouse and Mastomysnatalensis in Tanzania. They showed the these african small mammals had highly seropositive rate to several arboviruses, such as Dengue virus type 2 (8.44%), Usutu virus (4.61%), Chikungunya virus (6.17%), and etc. Moreover, they presented the difference in seropositivity between sex or age. These results suggest that these small mammals play a role of transmission of arboviruses in African continent. This work is necessary to understand the viral circulations. This accumulates our knowledge to maintain public health.

If the authors continue this study, I'd like to try to isolate the arboviruses from African small animals.

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Reviewer #1: No

Reviewer #2: 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 Mr. De Kesel,

We are pleased to inform you that your manuscript 'Antibodies against medically relevant arthropod-borne viruses in the ubiquitous African rodent Mastomys natalensis' 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,

Ran Wang, M.D.

Academic Editor

PLOS Neglected Tropical Diseases

Michael Holbrook

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 #1: yes

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? Yes.

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

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

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

-Were correct statistical analysis used to support conclusions? Yes.

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

**********

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: yes

Reviewer #2: -Does the analysis presented match the analysis plan? Yes.

-Are the results clearly and completely presented? Yes.

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

**********

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: yes

Reviewer #2: -Are the conclusions supported by the data presented? Yes.

-Are the limitations of analysis clearly described? Yes.

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

-Is public health relevance addressed? Yes.

**********

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: Accept

Reviewer #2: The authors have responded appropriately to the reviewers' comments, and their replies have enabled us to fully understand this paper. I recommend that this paper be accepted.

**********

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: All comments are addressed in a satisfactory manner.

Reviewer #2: I don't have no more comments. This paper is well written.

**********

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: No

Reviewer #2: No