Dear Dr. Gutierrez,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’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.

There were some minor comments and clarifications requested by reviewer 1 which would improve this manuscript.

Please submit your revised manuscript by 07 January 2024 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 plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• 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 applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Kelli L. Barr, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating in your Funding Statement: 

“This work was supported by the Montpellier University of Excellence programme, 2018 (ArboSud project; authors receiving the grant: P.V.T., R.D., T.B., S.G.). D.A.P.K. was the recipient of a funding by European Union’s Horizon 2020 research and innovation programme (Infravec2 project, grant agreement 731060). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. 

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

“This work is part of the ArboSud project funded by the 2018 call of the Montpellier University of Excellence (MUSE) program. D. Kabore acknowledges funding from the European Union’s Horizon 2020 research and innovation program (Infravec2 project, grant agreement 731060).”

We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

“This work was supported by the Montpellier University of Excellence programme, 2018 (ArboSud project; authors receiving the grant: P.V.T., R.D., T.B., S.G.). D.A.P.K. was the recipient of a funding by European Union’s Horizon 2020 research and innovation programme (Infravec2 project, grant agreement 731060). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. Thank you for stating the following in your Competing Interests section: 

“The authors declare no competing interests”

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now 

This information should be included in your cover letter; we will change the online submission form on your behalf.

5. Thank you for uploading your study's underlying data set. Unfortunately, the repository you have noted in your Data Availability statement does not qualify as an acceptable data repository according to PLOS's standards.

At this time, please upload the minimal data set necessary to replicate your study's findings to a stable, public repository (such as figshare or Dryad) and provide us with the relevant URLs, DOIs, or accession numbers that may be used to access these data. For a list of recommended repositories and additional information on PLOS standards for data deposition, please see https://journals.plos.org/plosone/s/recommended-repositories.

6. In the online submission form, you indicated that your data will be submitted to a repository upon acceptance. We strongly recommend all authors deposit their data before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire minimal dataset will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption.

7. We note that Figure 7 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 7 to publish the content specifically under the CC BY 4.0 license. 

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

8. 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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: General comments

This work looks like the first of its kind in Western Africa. In general, and with the first detection of SINV in Burkina Faso this manuscript is a valuable contribution to increasing knowledge on the prevalence of viruses in West Africa. As this knowledge is still very limited in many African regions, every report including genetic information by sequencing can be useful to close this gap.

In anticipation of the result and discussion section, the authors should not underestimate the value of this report. The authors discuss the use of metagenomic screening surveys, such as the one presented in the study, as an alternative to targeted diagnostics for arbovirus diversity in mosquitoes. However, it would be more appropriate to frame this work as a supplementary or pilot study to assess viral prevalence in this region. With this and after knowing that the virus is prevalent in the vector, follow-up studies (in vectors or humans) with a targeted detection approach can serve as a more specific and less expensive detection approach.

The paper is well-structured. The English language level is good but some words and sentences need editing.

Specific comments

In the author's summary of the work, in lines 51-52, it was stated that this is the first time SINV has been found in Culex quinquefasciatus. Is this so in Burkina Faso or worldwide?

Introduction

1. In lines 81-83, the authors should check this statement and consider these questions: Are arboviruses specific to vectors in terms of species? For the disease to be endemic, it means such vectors should be found in that area, are the vectors globally distributed? Or geographically oriented? How then did they result in outbreaks? Or is this statement a general observation for the spread of any virus not just arboviruses?

2. In line 88, there is a grammatical error. The use of no specific ...... and unavailable. If no treatments are available, it is either "specific treatments... are unavailable" or "no specific treatments... are available." In that same line, the statement is a bit confusing. What do the authors mean by unnecessary diagnostic testing? Are arboviral infections disguisable by signs and symptoms? Or do you mean about other causative organisms apart from arboviruses?

3. The second statement in line 104, can be backed with literature? It would give some knowledge of human behavior's effect on arboviral diversity.

Methods

Regarding the general methodology, the study design and implementation were conducted appropriately. However, there is some need for clarification in the methods and results section.

1. The first statement for sampling sites is great and simple but it will be a bit inconvenient to go and read that paper on mosquito diversity before getting the rationale for the choice of sampling sites. I think the authors can give a brief reason then the citation can be attached if further reading is needed.

2. Three trapping methods were used in this work, were all methods used at each of the different sites? How does this affect the sampling in terms of the number of catches? There is no description of the number of mosquitoes each caught.

3. One major point is the pooling strategy of mosquitoes and the resulting number of libraries that were prepared. According to Table 1, sometimes 3 libraries were prepared from 3 pools, other times 3 libraries were prepared from 5 to 7 pools. The strategy behind this is not clear from the methodology or result section.

Results/Discussion

1. In addition to that, in the result section (line 258) it was stated that the same number of libraries per mosquito species was prepared, but very different numbers of mosquitos were pooled. This results in a different sequencing depth per single mosquito. To achieve a more comparable screening between the pools the same sequencing depth should be targeted across all samples.

2. In line 261, the number of reads per library is given (min/max = 0.8/7.65 million reads per library). The sequencing depth per library varies significantly, is this due to the different numbers of pools per library? A clearer explanation or comment on that would be needed.

3. In line 284 authors state that they only tested Cx. quinquefasciatus mosquitoes in RT-qPCR. For a more comprehensive result, it would have been interesting to also test the other pools of Aedes mosquitoes for SINV. Even reports of negative results would support the thesis that this mosquito species is not the typical vector.

4. Regarding the phylogenetic reconstruction, the general methodology was appropriate. However, the phylogenetic tree in this work is based on concatenated NS and S ORFs. The six SINV genotypes (G1-G6) are usually determined based on E2 gene phylogenies only even when they were first described (Lundström JO, Pfeffer M. 2010. Phylogeographic structure and evolutionary history of Sindbis virus. Vector Borne Zoonotic Dis 10:889–907. doi: 10.1089/vbz.2009.0069). Would a calculation based on the E2 gene change the outcome of phylogenetic reconstruction and cluster the strains differently? This should be tested before considering all the following comments on the phylogeny.

5. In lines 294 – 301 and the corresponding phylogenetic tree, only the cluster of the Burkina Faso strains with other African strains as well as another cluster including the strain from Algeria are described in detail and highlighted. The relation to other strains including the European sequences is not described and also not discussed later. Only in line 341, do the authors hypothesize different genotypes involved in the spread in North and Northwest Africa as well as different scenarios regarding the spread to Europe. However, according to the authors, this phylogeny involves only SINV Genotype 1 sequences. This does not match with the previous result presentation, where SINV sequences were identified as genotype 1. If the authors assume the presence of another SINV genotype (2 – 6), then representative strains should be included in the phylogenetic reconstruction. In case this only refers to the difference in the sequences of Burkina Faso and Algeria strain genomes then this should be explained more precisely because the phylogeny only includes Genotype 1 sequences and show the clusters within this genotype 1.

6. Did the authors also check for major differences in the amino acid sequences of the different ORFs, for example, in a multiple alignment with all the strains that are included in the phylogeny? This can sometimes show patterns of amino acid changes or deletions for a set of strains. It would be interesting to see if there is a notable difference between the sequences from Europe (causing sporadic outbreaks) to those from Burkina Faso and/or African strains in general.

Reviewer #2: I joined the review of the article after the first round of revision, and perhaps that is why the manuscript left the most favorable impression. The authors clearly presented the research methods and results, carefully and self-critically analyzed the data, outlined the limitations of their study, and discussed the significance of their results. It was a stroke of luck to find Sindbis virus in mosquitoes collected in West Africa without any information about an ongoing outbreak. The prevalence estimate in the paper highlights how difficult it is to make such a finding. An important advantage of the work is that the authors not only conducted a metagenomic search, PCR screening, and whole-genome sequencing but also isolated the virus in cell culture. I have no doubts about the importance of the find, and I am grateful to the authors for such a careful and thorough description of the work done.

Then I went through the comments of the first-round reviewers and the authors' responses to them, step by step. And only after this, it became clear that the authors had done a lot of work revising the manuscript. In my opinion, after this conducted revision, the manuscript can be accepted for publication.

There are only 2 small comments, the answers to which do not require a separate round of review:

Lines 154-155. "August/September" means "from August to September" or "only in August and only in September"? Same for "June/October". Please rephrase this text so there are no misunderstandings.

General comment: Based on the previous work (Mosquito (Diptera: Culicidae) populations in contrasting areas of the western regions of Burkina Faso: species diversity, abundance and their implications for pathogen transmission), the authors collected numerous mosquitoes of different species, but PCR screening for Sindbis virus was undertaken only for Cx. quinquefasciatus. It might make sense to extend PCR screening to other mosquito species/genera from Rural 1 zone 2020. It is possible that other species of collected mosquito may also be involved in the circulation of SINV.

**********

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: Yes:  Marat Makenov

**********

[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.]

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. Registration is free. 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 PLOS at figures@plos.org

A metagenomics survey of viral diversity in mosquito vectors allows the first detection of Sindbis virus in Burkina Faso

PONE-D-24-41081R1

Dear Dr. Gutierrez,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Please during final proofing make the changes suggested by reviewer 2, which are small enough to be introduced during copyediting.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Rhys Harold Parry

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #2: "August/September" and "June/October" are still confusing, but that's a minor quibble. At the discretion of the authors

Reviewer #3: I commend the authors for their valuable contribution to arbovirus surveillance in mosquito vectors. Arboviruses in vectors remain significantly understudied, particularly in Africa, where their circulation and potential impact on public health are not well documented. The experiments have been conducted rigorously, and the study provides valuable insights.

That said, I noticed that a comment from Reviewer 2 regarding Lines 157–158 has not been fully addressed. The phrasing “August/September 2019,” “June/October 2020,” and “May/June 2021” remains ambiguous. To enhance clarity, I recommend explicitly stating “from August to September”,“from June to October” and “from May to June “ to prevent any misinterpretation.

Additionally, I recommend including the sampling years in the abstract. Specifying the study period upfront would improve clarity and accessibility, ensuring that readers do not have to search for this information in the methodology section. This would make the abstract more informative and enhance readability.

Otherwise, the manuscript is well-structured and presents significant findings that contribute to the field

**********

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: No

Reviewer #3: No

**********