Dear Prof. Takada,

Thank you very much for submitting your manuscript "Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Zambia" 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 revised version that takes into account the reviewers' comments.

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[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,

Jonas Klingström

Associate Editor

PLOS Neglected Tropical Diseases

Jeremy Camp

Deputy 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: The design of the study was simple and appropriate to address the author’s objective. The methods used in the study were also appropriate. One request from the reviewer is to provide the primer sets (and the target position) for the L gene. Although authors described that they could not detect L gene, it should be helpful to provide that information.

Reviewer #2: The authors describe the first evidence of CCHFV in Zambia based on the detection of IgG antibodies to nucleocapsid protein in 88 cattle sera collected 2012-2015 and the presence of viral RNA in 11 Hyalomma ticks collected 2015-2016. The objective and the public health relevance are clearly described and the manuscript is well written.

Can the authors please provide a statement in Methods confirming that the experimental protocol/protocols for sampling cattle were approved by a licensing committee (name the authorizing body and ethical permit number). If the authors feel that this is not relevant to their study, depending on the regulations in Zambia, can the authors please provide a statement in Methods explaining why there is no need for such a permit.

The authors refer to reference 8 for the cattle samples. The paper referred to describes the collection of 942 samples in 2014. Can the authors please add information on the remaining samples or provide an additional reference describing their origin?

The authors use an in-house IF method for detection of IgG antibodies to recombinant N protein expressed in HeLa cells. As the authors present the first evidence of CCHFV in Zambia, providing more information on the specificity and sensitivity of the assay using sera from cattle, a publication describing the use of this assay with cattle sera, or confirming the results by detection of anti-glycoprotein antibodies would further strengthening the manuscript.

Reviewer #3: (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 results presented in this study was shown clearly.

Reviewer #2: Lines 176-179, 269-276, and 273-274: The authors present and discuss an estimation of the seroprevalence in different districts, can the authors please provide the 95% CI for these estimations.

Reviewer #3: (No Response)

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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: Conclusion of the study was clear and well supported from the presented data.

Reviewer #2: The authors have drawn somewhat strong conclusions from the phylogenetic analyses which are based on a limited number of sequences. Can the authors please address these limitations in the Discussion.

Reviewer #3: Authors conclude that CCHFV has moderate circulation throughout Zambia and discuss how their data add to the field's understanding of CCHFV circulation in Africa.

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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: The reassortant between African and Asian CCHFVs is one of the exciting finding in this manuscript. Reviewer requested to author to discuss a little bit more about the reassortant. Is there any CCHFV reassortment reported of African M segment in Asia? Why only M segment is reassorted in the African CCHFV? Is it possible that L segment is also originated from Asian CCHFV in African CCHFV?

Reviewer #2: Line 96: Please clarify if the 1047 sera were collected from individual animals or not.

Lines 180-181: Please clarify if the 526 sera tested by nested RT-PCR was part of the 1047 sera tested for antibodies. In Methods, provide information regarding the treatment and RNA extraction of the sera prior to PCR analysis.

Lines 202-203: Provide the statistical test used and the p-value for determining possible correlation between prevalence and tick species. Also, in Methods add information on which software that was used to perform the analysis.

Please add information in Methods on the BSL facility and the procedures and conditions used for the attempts to isolate CCHF from tick homogenates (described in Results, line 203).

Lines 218-219: Although it was not possible to obtain S segment full-length sequences from the other 10 RT-PCR positive ticks, did the authors try to recover partial sequences of the M segment from these ticks to confirm the result?

Line 224-237: Parts of the data presented here are/confirm previous findings from reference 16, please move these parts to the Discussion.

Please provide GenBank accession numbers for the sequences used in the phylogenetic analysis. These accession numbers can either be added to the trees or be presented in a table in supplementary linking the names used in the trees with their respective accession number.

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: Authors reported the circulation of CCHFV in Zambia for the first time. Although authors could not isolate the live virus from their samples, the data shown in here with the serologic data and the genetical data, was enough to support their conclusion. Only few requests were provided from the reviewer above to support their discussion.

Reviewer #2: (No Response)

Reviewer #3: The manuscript by Kajihara et al investigates the seroprevalence of CCHF in cattle and presence of CCHFV RNA in ticks within Zambia, a country that has not been reported as CCHFV endemic. The manuscript is well written and data well presented. Data adds to our understanding of the circulation of CCHFV within Africa, an understudied region for CCHFV.

Minor comments:

Line 203: Authors state that no infectious virus was recovered from any tick sample, what method was used to attempt to recover infectious virus?

Table 2: Were ticks collected from individual cattle or were multiple ticks collected from the same cow? Could positive ticks have come from the same cow?

Line 134 - 148: Were no template controls included?

What is the level of movement of cattle within Zambia? Cattle movement between Zambia and Namibia or South Africa where the Asian M segment viruses have been reported?

Figure 1: Authors should consider adding a symbol such as a star to indicate sampling sites with positive CCHFV tick or cattle samples to make it easier to understand geographically where CCHFV was detected.

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Reviewer #1: Yes: Shuzo Urata

Reviewer #2: No

Reviewer #3: No

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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Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

Dear Prof. Takada,

We are pleased to inform you that your manuscript 'Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Zambia' 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,

Jonas Klingström

Associate Editor

PLOS Neglected Tropical Diseases

Jeremy Camp

Deputy 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: Methods were clear.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

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: Results were shown clearly.

Reviewer #2: (No Response)

Reviewer #3: (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: Conclusions were well supported by the results.

Reviewer #2: (No Response)

Reviewer #3: (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: N/A

Reviewer #2: The authors have carefully considered the reviewers comments/questions and where appropriate, made changes to the manuscript to enhance clarity.

Reviewer #3: (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: Well described.

Reviewer #2: (No Response)

Reviewer #3: Authors have satisfactorily addressed all my comments.

**********

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

Reviewer #3: No