Short report: First whole-genome evidence of dengue virus in field-caught mosquitoes from southern Brazil

PLOS Neglected Tropical Diseases

Dear Dr. Giovanetti,

Thank you for submitting your manuscript to PLOS Neglected Tropical Diseases. After careful consideration, we feel that it has merit but does not fully meet PLOS Neglected Tropical Diseases's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within by Jan 13 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosntds@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pntd/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Kinley Wangdi, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Michael Holbrook

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

Additional Editor Comments:

Abstract

The aim should be made clear.

Introduction

The aim is not clear; it should be made clear. Is the aim mosquito-trapping or quantify the circulating dengue virus serotypes.

The study

1. The process of sampling is very detailed, summarize the process.

2. Limitation of study is needed.

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Maria Eduarda Calazans Rodrigues, and Isaque João da Silva de Faria. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plosntds/s/authorship#loc-author-contributions

2) We do not publish any copyright or trademark symbols that usually accompany proprietary names, eg ©,  ®, or TM  (e.g. next to drug or reagent names). Therefore please remove all instances of trademark/copyright symbols throughout the text, including:

- ® on page: 4.

3) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines:

https://journals.plos.org/plosntds/s/figures

4) We notice that your supplementary figures are uploaded with the file type 'Table'. Please amend the file type to 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list.

5) Some material included in your submission may be copyrighted. According to PLOSu2019s copyright policy, authors who use figures or other material (e.g., graphics, clipart, maps) from another author or copyright holder must demonstrate or obtain permission to publish this material under the Creative Commons Attribution 4.0 International (CC BY 4.0) License used by PLOS journals. Please closely review the details of PLOSu2019s copyright requirements here: PLOS Licenses and Copyright. If you need to request permissions from a copyright holder, you may use PLOS's Copyright Content Permission form.

Please respond directly to this email and provide any known details concerning your material's license terms and permissions required for reuse, even if you have not yet obtained copyright permissions or are unsure of your material's copyright compatibility. Once you have responded and addressed all other outstanding technical requirements, you may resubmit your manuscript within Editorial Manager.

Potential Copyright Issues:

- Figure 1. Please (a) provide a direct link to the base layer of the map (i.e., the country or region border shape) and ensure this is also included in the figure legend; and (b) provide a link to the terms of use / license information for the base layer image or shapefile. We cannot publish proprietary or copyrighted maps (e.g. Google Maps, Mapquest) and the terms of use for your map base layer must be compatible with our CC BY 4.0 license.

Note: if you created the map in a software program like R or ArcGIS, please locate and indicate the source of the basemap shapefile onto which data has been plotted.

If your map was obtained from a copyrighted source please amend the figure so that the base map used is from an openly available source. Alternatively, please provide explicit written permission from the copyright holder granting you the right to publish the material under our CC BY 4.0 license.

If you are unsure whether you can use a map or not, please do reach out and we will be able to help you. The following websites are good examples of where you can source open access or public domain maps:

* U.S. Geological Survey (USGS) - All maps are in the public domain. (http://www.usgs.gov)

* PlaniGlobe - All maps are published under a Creative Commons license so please cite u201cPlaniGlobe, http://www.planiglobe.com, CC BY 2.0u201d in the image credit after the caption. (http://www.planiglobe.com/?lang=enl)

* Natural Earth - All maps are public domain. (http://www.naturalearthdata.com/about/terms-of-use/).

6) Please amend your detailed Financial Disclosure statement. This is published with the article. It must therefore be completed in full sentences and contain the exact wording you wish to be published.

- State the initials, alongside each funding source, of each author to receive each grant. For example: "This work was supported by the National Institutes of Health (####### to AM; ###### to CJ) and the National Science Foundation (###### to AM)."

- State what role the funders took in the study. If the funders had no role in your study, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.".

If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

Reviewers' Comments:

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 manuscript demonstrated the whole-genome evidence of dengue virus in field-caught mosquitoes from southern Brazil. The method is precise; however, some issues need to be clarified.

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: (No Response)

Reviewer #2: The results presented is not clear and insufficient.

The figure 1, is not informative and very busy to comprehend. The authors should reconstruct the phylogenetic tree with the concise needed information (fewer reference sequences from the Genbank) and outgroup. The authors should also describe each clade clearly for the readers.

Line 135 -140: Not clearly demonstrated in the Figure 1B-C, very difficult to follow.

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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: * The manuscript currently does not include a clearly defined Discussion section. Adding this section would help contextualize the findings and relate them to existing literature.

* The manuscript would also benefit from the inclusion of a brief statement addressing the limitations of the study. Highlighting these limitations will provide a more balanced interpretation of the results and strengthen the overall presentation.

**********

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: * In the Background section, it would be helpful to include additional general information about the virus, such as the approximate size of its genome.

* The abbreviation “NS” should be formally defined at first mention.

* The manuscript presents genomes sequenced from specimens collected in 2023 and shown on a phylogenetic tree spanning isolates from 1991 to 2023 (Figure 1B). However, the compressed time scale makes it appear that the 2023 genomes are closer to the August 2021 time point, which may cause confusion. It would be useful to clearly mark or label the 2023 point on the timeline.

* The formatting of the Supplemental Tables needs correction. Several tables contain truncated text, overlapping content across pages, and columns that are too narrow—causing numerical values to appear as “###.” Please adjust the layout to ensure all text and data are clearly visible.

* The manuscript aims to address an important knowledge gap regarding DENV strains circulating in underrepresented regions of Brazil. While the methods used for genomic sequencing appear generally adequate, the methodological details are interspersed with the results and discussion. This formatting choice makes it difficult to clearly and critically evaluate the methods. The authors are encouraged to present the Methods section separately to improve clarity and reproducibility.

* Line 117: The authors state that “outliers were removed to improve dating accuracy.” Please provide additional detail on the parameters or criteria used to identify and remove these outliers, as this information is essential for evaluating the validity of the temporal analysis.d

**********

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 manuscript demonstrated the whole-genome evidence of dengue virus in field-caught mosquitoes from southern Brazil. The method is precise; however, some issues need to be clarified and more discussed as follows.

The authors should provide citations to their statements below:

Line 58-59 “Environmental and anthropogenic factors……….”

Line 75-76 “ In 2023, 38,176 cases………”

The authors mentioned they pooled both males and females into tubes. What is the essence of including males in the study?

How long did the authors preserved the samples without significant RNA degradation and what is the medium of preservation (solution?)?

Line 103: typo “for” “for” delete one.

Regarding the RNA extraction, it can be found that the genomic DNA, if not digested, can be present in the eluted RNA, resulting possible positive amplification of the gene of interest in tested specimens. Did the authors decontaminate the DNA before amplification?

The authors should provide the serotype-specific primers used in the study?

The pooling method described in the study is inappropriately described. Most samples in the tubes analyzed were individual (1) samples, yet the authors mentioned they are pools. Pool samples indicates combination of more than 1 sample together in a tube.

Also, the one (1) sample in a tubes, is it male or female?

From the Table_S1, the study has unequal sample pools from 2-15 pools per tube, and individual (1) sample tubes.

What is the total number of males and females’ flies included in the study?

The 2 pools the author mentioned that were positive for both DENV I and 2, how many samples in each pool? All males or females?

The authors claimed that they had 33 positive samples out of 41 pools for DENV: 29 for DENV-1 and 4 for DENV-2. How accurate is this high infection rate? Does the author understand the epidemiological implication of this claim. The chance of possible transmission is very high in the study areas. These results are discussable.

How did the authors avoid contaminations amongst sample analyzed?

Line 127: Were the CT values analyzed by the comparative threshold cycle method, and normalized to the endogenous/reference control?

The figure 1, is not informative and very busy to comprehend. The authors should reconstruct the phylogenetic tree with the concise needed information (fewer reference sequences from the Genbank) and outgroup. The authors should also describe each clade clearly for the readers.

Line 135 -140: Not clearly demonstrated in the Figure 1B-C, very difficult to follow.

Overall, the observed results needs more discussion.

Reviewer #3: * There could be substantial additional value to the findings reported in this manuscript if the authors extended their analysis. While they allude to the importance of understanding the impact of the variants identified in the sequenced DENV genomes, they do not perform the bioinformatic analyses necessary to address this knowledge gap. With the availability of advanced structural modeling tools (e.g., AlphaFold3), it would be feasible to assess the potential structural or functional implications of the identified variants using a reference DENV strain as a model template. Including such analyses would considerably strengthen the study’s impact and interpretive depth.

**********

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: Jian-Wei Shao

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

Figure resubmission:

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies 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. 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: Comments for PNTD-D-25-01609.docx

Response to ReviewersRevised Manuscript with Track ChangesManuscript

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

Additional Editor Comments:Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Maria Eduarda Calazans Rodrigues. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plosntds/s/authorship#loc-author-contributions

2) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines:

https://journals.plos.org/plosntds/s/figures

3) Tables should not be uploaded as individual files. Please remove these files and include the Tables in your manuscript file as editable, cell-based objects. For more information about how to format tables, see our guidelines:

https://journals.plos.org/plosntds/s/tables

4) Please amend your detailed Financial Disclosure statement. This is published with the article. It must therefore be completed in full sentences and contain the exact wording you wish to be published.

- State the initials, alongside each funding source, of each author to receive each grant. For example: "This work was supported by the National Institutes of Health (####### to AM; ###### to CJ) and the National Science Foundation (###### to AM)."

- State what role the funders took in the study. If the funders had no role in your study, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.".

If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

Reviewers' comments:

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 objectives of the study clearly articulated with a clear testable hypothesis stated

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: (No Response)

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: (No Response)

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: Having reviewed the authors' responses to my previous concerns, I find that all issues have been satisfactorily addressed. I now recommend that the manuscript be accepted for publication.

Reviewer #2: (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 #2: (No Response)

Reviewer #3: The manuscript has improved considerably with the current revision. The authors have done a commendable job expanding the discussion and acknowledging the study’s shortcomings through the addition of a limitations section. However, a few minor issues remain that should be addressed prior to acceptance.

On lines 155–157, the authors state that the distributions of DENV-1 and DENV-2 occur in different geographic locations, as shown in Figure 1A. However, all data points appear in the same color, preventing the reader from distinguishing between DENV-1 and DENV-2 by region as suggested in the text.

Additionally, many values in Supplemental Table 1 are still displayed as “####” in the CT values, total reads, and Deep columns. The authors indicated that this issue was corrected during revision, but it does not appear to have been fully resolved.

Finally, there are multiple instances throughout the manuscript of extra spaces and an occasional additional period. The manuscript would benefit from another careful proofreading to address these minor typographical issues and to ensure consistent use of abbreviations throughout.

**********

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: Jian-Wei Shao

Reviewer #2: No

Reviewer #3: No

Figure resubmission:

Reproducibility:--> -->-->To enhance the reproducibility of your results, we recommend that authors of applicable studies 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. 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-->?>

Dear Dr. Giovanetti,

We are pleased to inform you that your manuscript 'First Whole-Genome Detection of Dengue Virus in Urban Aedes aegypti from Southern Brazil' 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,

Kinley Wangdi, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Michael Holbrook

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; line-height: 16.0px; font: 14.0px Arial; color: #323333; -webkit-text-stroke: #323333}span.s1 {font-kerning: none