Dear Ms Liu,

Thank you very much for submitting your manuscript "Orthohantavirus infections in humans and rodents in the Yichun region, China, from 2016 to 2021" 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.

Both reviewers have requested substantial additional details for methodology, which would be necessary for a proper evaluation of the results after revision. Adding more detail in the discussion of the biology of the hosts and transmission in the introduction and discussion would also help to strengthen the paper. Reviewer 2 has provided more detailed comments directly in the manuscript file, so please ensure that you review those carefully. The authors do not need to strictly adhere to every suggestion about which sentences belong in which sections, but please consider the revisions outlined. Please also consider asking a native english speaker to revise the manuscript for grammar, as suggested by reviewers.

Reviewer 1's additional comments are below:

1. The MS should be revised by native english speaker.

2. line 58, add rodent-associated before viruses.

3. lines 74 and 77, The Yichun region? or The Yichun city?

4. line 106, add collection after Samples

5. line 143 and others, the primers sequences should be listed in a table.

6. Fig 2C and 2D, virus infection in rodents and humans detected in this study should be presented in another figure.

7. line 253, P should be itanlicized.

8. line 156, add S before segment

9. line 263, were the real-time RT-PCR products sequenced?

10. line 265, were Dabieshan orthohantavirus (DBSV) identified by primers for HTNV or SEOV? please clarify.

11. lines 276-277, "The cytochrome b gene sequences of rodents were deposited into GenBank under accession no. OQ187773-OQ187792." should be shown in line 240. In line 240, the rodent species idenfied by cytb gene should be adeed.

12. The description for genetic analysis and phylogenetic trees should be revised.

13. rodent name: full genus name at first mention, later with abbreviated genus name throughout the text (exception: at the beginning of a sentence)

14. Partial S should be amplified by PCR. Particularly, the genetic characteristic of SEOV infected in humans should be clarified.

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,

Bruce A. Rosa

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

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

Both reviewers have requested substantial additional details for methodology, which would be necessary for a proper evaluation of the results after revision. Adding more detail in the discussion of the biology of the hosts and transmission in the introduction and discussion would also help to strengthen the paper. Reviewer 2 has provided more detailed comments directly in the manuscript file, so please ensure that you review those carefully. The authors do not need to strictly adhere to every suggestion about which sentences belong in which sections, but please consider the revisions outlined. Please also consider asking a native english speaker to revise the manuscript for grammar, as suggested by reviewers.

Reviewer 1's additional comments are below:

1. The MS should be revised by native english speaker.

2. line 58, add rodent-associated before viruses.

3. lines 74 and 77, The Yichun region? or The Yichun city?

4. line 106, add collection after Samples

5. line 143 and others, the primers sequences should be listed in a table.

6. Fig 2C and 2D, virus infection in rodents and humans detected in this study should be presented in another figure.

7. line 253, P should be itanlicized.

8. line 156, add S before segment

9. line 263, were the real-time RT-PCR products sequenced?

10. line 265, were Dabieshan orthohantavirus (DBSV) identified by primers for HTNV or SEOV? please clarify.

11. lines 276-277, "The cytochrome b gene sequences of rodents were deposited into GenBank under accession no. OQ187773-OQ187792." should be shown in line 240. In line 240, the rodent species idenfied by cytb gene should be adeed.

12. The description for genetic analysis and phylogenetic trees should be revised.

13. rodent name: full genus name at first mention, later with abbreviated genus name throughout the text (exception: at the beginning of a sentence)

14. Partial S should be amplified by PCR. Particularly, the genetic characteristic of SEOV infected in humans should be clarified.

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: Partial S should be amplified by PCR. Particularly, the genetic characteristic of SEOV infected in humans should be clarified.

Reviewer #2: -The article lack a clear objective.

-The methods are incomplete.

-Some methodology are write in the Introduction and in Results.

-The statistical analysis are not included in methods.

--------------------

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: -the epigraphs of tables and figures are not right writes.

-Some results are not presented in methods.

--------------------

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: The public health relevance is clear but the biology of the hosts, the importance of the different rodent species in the transmission, how are the transmission to human and other important topics are lacked in the discussion that help to understand the dynamic of the HFRS and how could prevent it.

The discussion are incompleted.

--------------------

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)

--------------------

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: The topic of this manuscript is relevant to the public health, but the form to expose the analyses, data and results require a lot of changes because the sentences are write in not correct sections.

Additionally, there are not a clear objetive, the introduction is so short (lack of information detailed in the attach revised file) and Methods and Result have to improve.

The discussion have to improve too, adding more biological informations about the hosts.

All specific comments are added in the attach file.

I consider that the authors have to rewrite the manuscript for the readers can extract the valuable of their data (that are truly valuable).

--------------------

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

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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Attachments

Attachment

Submitted filename: PNTD-D-23-00074_revised1.pdf

Dear Ms Liu,

Thank you very much for submitting your manuscript "Orthohantavirus infections in humans and rodents in the Yichun region, China, from 2016 to 2021" 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.

The reviewers have had a chance to review the revised manuscript. They are mostly positive about it now, but there are just a few revisions suggested from reviewer 3 that should be considered, including trying to look at amino acid sequence changes, and more clearly stating the public relevance of the findings.

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,

Bruce A. Rosa

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

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

The reviewers have had a chance to review the revised manuscript. They are mostly positive about it now, but there are just a few revisions suggested from reviewer 3 that should be considered, including trying to look at amino acid sequence changes, and more clearly stating the public relevance of the findings.

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 #3: The objectives of the study articulates the testible hypothesis. Study design is appropriate.

It remains unclear what was the reason for using neighbor-joining method for phylogenetic analysis?

Sample size is sufficient to address the hypothesis.

--------------------

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 #3: 1. Analysis of amino acid sequences would be more informative compared to nucleotide sequences. That is because some nucleotide substitutions may not change amino acid resulting in limited changes in protein function. Also, without comparison of amino acid sequences the statement of finding new lineages of Hantaan orthohantavirus has limited support.

2. Strain SGHu02/2020 has different location on S and M segment phylogenetic tree. How authors could explain this.

--------------------

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 #3: Public relevance should be clearly stated.

--------------------

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 #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: (No Response)

Reviewer #3: (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: 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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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 Ms Liu,

We are pleased to inform you that your manuscript 'Orthohantavirus infections in humans and rodents in the Yichun region, China, from 2016 to 2021' 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,

Bruce A. Rosa

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

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

The authors have sufficiently addressed reviewer concerns.