PONE-D-20-27563

Phylogenetic and evolutionary analysis of Dengue Virus serotypes circulating at the Colombian–Venezuelan border during 2015–2019

PLOS ONE

Dear Dr. Martinez-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.

Despite the good intentions of your group in obtaining a certification of readability, I regret to inform you that the job of editing was not done satisfactorily.  I find that the errors are so numerous that I cannot in good conscience invite reviewers to wade through your manuscript.  Indeed, even I have not read the whole thing. So far, I have noticed the following

l. 99 spelling of "April"

l. 97 since 'eastern" is an adjective, a noun needs to follow.

l. 98  Villa Rosario is at (not "has")

l 101 personnel, not "personal"

l 104 them --> they

l. 162 was and exponential ???

These examples were enough to notify me that this manuscript is not acceptable as it now stands. I did get the impression that, if extensively rewritten, there may be information worthy of publication. Certainly, any resubmission will need thorough peer review.

Please submit your revised manuscript by Nov 05 2020 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.

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Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

Despite the good intentions of your group in obtaining a certification of readability, I regret to inform you that the job of editing was not done satisfactorily. I find that the errors are so numerous that I cannot in good conscience invite reviewers to wade through your manuscript. Indeed, even I have not read the whole thing. So far, I have notice the following

l. 99 spelling of "April"

l. 97 since 'eastern" is an adjective, a noun needs to follow.

l. 98 Villa Rosario is at (not "has")

l 101 personnel, not "personal"

l 104 them --> they

l. 162 was and exponential ???

These examples were enough to notfy me that this mansucript is not acceptable as it now stands. I did get the impression that, if extensively rewritten, there may be information worthy of publication. Certainly, any resubmission will need thorough peer review.

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

Reviewers' comments:

[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. Please note that Supporting Information files do not need this step.

PONE-D-20-27563R1

Phylogenetic and evolutionary analysis of Dengue Virus serotypes circulating at the Colombian–Venezuelan border during 2015–2019

PLOS ONE

Dear Dr. Martinez-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.

==============================

1. In this study, the authors collected the clinical samples during 2015-2016 and 2018-2019. The use of 2015–2019 in the Title was inappropriate.
2. The sample size of 229 suspected DENV samples was relatively small. In particular, of 41 samples positive for DENV, only 10 E gene sequences were obtained. Why so few sequences to be obtained? The proportion of successfully sequencing was substantially lower than a previous report (J Travel Med. 2020, 27(7): taaa195). Could the authors use RT-nested PCR to amplify the E gene (Emerg Infect Dis. 2012, 18(11):1850-7) ?  Too few sequences do not result in a solid result or conclusion. This point should be mentioned as a limitation.
3. The molecular epidemiology of four DENV serotypes in America (especially in Venezuela and Colombia) should be mentioned in Introduction. If four DENV serotypes are co-circulating in a continent/region, cross-border transmission of all four serotypes were often observed among neighboring countries. The compelling evidences are from Asia, where all four DENV serotypes were introduced into China from neighboring countries along land border ports (e.g. China-Myanmar border), and/or surrounding countries via air travel (e.g. between Shanghai, China and some Southeastern Asian countries) (Emerg Infect Dis 2018; 24:1756–8，J Travel Med. 2020, 27: taaa195). If all four serotypes co-circulating in America, why no DENV-3 and DENV-4 to be detected? And also no cross-border transmission of DENV-3 and DENV-4 in these regions? This point should be mentioned and discussed (maybe as a limitation) in the paper. 
4. the cross-border transmission of all four serotypes were observed between
5. The structure of the Results should be reorganized. Too many sections are included in Results. Phylogenetic analysis, Phylogenetic analysis of DENV-1, and Phylogenetic analysis of DENV-2 should be merged into one section (e.g. Phylogenetic analysis). Similarly, Evolutionary analysis, DENV-1 E gene phylogeography and DENV-2 E gene phylogeography should be merged into one section of “Phylogeographic analyses”.
6. In the Phylogenetic analyses of DENV-1 and -2, it makes no sense to further divide genotypes into clades and even sub-clades. I suggest to remove these classification.
7. In the phylogeographic analysis of DENV-1, first introduction should not be traced back to 1973 (it should be later than 1973, maybe about 1980). In addition, the identification of second, third and fourth introductions was incorrect. The so-called three introduction events were more likely to be a single introduction event with a tMRCA in around 1994 (at the next node of the 1993 node).
8. In the phylogeographic analysis of DENV-2, there might be two independent introduction events of DENV-2 from Venezuela to Colombia in around 1998 and 2001. Since then, frequent cross-border transmissions of DENV-2 between Venezuela and Colombia were observed. So, the description of the so-called fifth and sixth introduction events were inappropriate.  
9. In the phylogeographic analyses, the sequences from other continents (e.g. Asia, Africa) should be included.

==============================

Please submit your revised manuscript by May 06 2021 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.

Please include the following items when submitting your revised manuscript:

• 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 you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Chiyu Zhang, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

1. In this study, the authors collected the clinical samples during 2015-2016 and 2018-2019. The use of 2015–2019 in the Title was inappropriate.

2. The sample size of 229 suspected DENV samples was relatively small. In particular, of 41 samples positive for DENV, only 10 E gene sequences were obtained. Why so few sequences to be obtained? The proportion of successfully sequencing was substantially lower than a previous report (J Travel Med. 2020, 27(7): taaa195). Could the authors use RT-nested PCR to amplify the E gene (Emerg Infect Dis. 2012, 18(11):1850-7) ? Too few sequences do not result in a solid result or conclusion. This point should be mentioned as a limitation.

3. The molecular epidemiology of four DENV serotypes in America (especially in Venezuela and Colombia) should be mentioned in Introduction. If four DENV serotypes are co-circulating in a continent/region, cross-border transmission of all four serotypes were often observed among neighboring countries. The compelling evidences are from Asia, where all four DENV serotypes were introduced into China from neighboring countries along land border ports (e.g. China-Myanmar border), and/or surrounding countries via air travel (e.g. between Shanghai, China and some Southeastern Asian countries) (Emerg Infect Dis 2018; 24:1756–8，J Travel Med. 2020, 27: taaa195). If all four serotypes co-circulating in America, why no DENV-3 and DENV-4 to be detected? And also no cross-border transmission of DENV-3 and DENV-4 in these regions? This point should be mentioned and discussed (maybe as a limitation) in the paper.

4. the cross-border transmission of all four serotypes were observed between

5. The structure of the Results should be reorganized. Too many sections are included in Results. Phylogenetic analysis, Phylogenetic analysis of DENV-1, and Phylogenetic analysis of DENV-2 should be merged into one section (e.g. Phylogenetic analysis). Similarly, Evolutionary analysis, DENV-1 E gene phylogeography and DENV-2 E gene phylogeography should be merged into one section of “Phylogeographic analyses”.

6. In the Phylogenetic analyses of DENV-1 and -2, it makes no sense to further divide genotypes into clades and even sub-clades. I suggest to remove these classification.

7. In the phylogeographic analysis of DENV-1, first introduction should not be traced back to 1973 (it should be later than 1973, maybe about 1980). In addition, the identification of second, third and fourth introductions was incorrect. The so-called three introduction events were more likely to be a single introduction event with a tMRCA in around 1994 (at the next node of the 1993 node).

8. In the phylogeographic analysis of DENV-2, there might be two independent introduction events of DENV-2 from Venezuela to Colombia in around 1998 and 2001. Since then, frequent cross-border transmissions of DENV-2 between Venezuela and Colombia were observed. So, the description of the so-called fifth and sixth introduction events were inappropriate.

9. In the phylogeographic analyses, the sequences from other continents (e.g. Asia, Africa) should be included.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this paper authors track the migration of the dengue strain DENV in Central America, especially in the Columbia – Venezuela border. The paper is well-written. I am not expert in the phylogenetics approaches, but with whatever knowledge I have, I think methods are well done. Results are presented well, but I did not understand, why there is suddenly figure legend in the result text, I hope, this legend will go below the figure in the final version.

Also, I have suggestion to give. In the discussion, I was lost in reading how the dengue strain travelled through Columbia, Venezuela and other parts of Central America. I was thinking if this information can be depicted through a map, it will be beneficial to readers. Specially for people who are not familiar to the Central America geography.

Reviewer #2: The authors conduct a phylogenetic analysis of DENV strains circulating in the border of

Colombia and Venezuela. The results of this study provide some support for DENV cross-border transmission and suggests that border surveillance and characterization of imported and exported strains are very important. However, this study has many limitations.

1. The number of sequences included in the study was too small to illuminate the genetic characteristics of the border-prevalent strains. So the conclusions, geographical proximity between Colombia and Venezuela is favourable for the export and import of different cannot be strongly supported.

2. Is there more Asian strains included for the evolutionary rate calculating? Too few sequences will lead to bias in the calculation results. There are a large number of dengue sequences from Asia in the database.

3. It will be better to annotate the meanings of the different colors in Figure 2 and Figure 3 in the figure.

**********

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-27563R2

Phylogenetic and evolutionary analysis of dengue virus serotypes circulating at the Colombian–Venezuelan border during 2015-2016 and 2018-2019

PLOS ONE

Dear Dr. Martinez-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.

==============================

Please carefully check the Figures 4 and 5, and see my new comments. 

==============================

Please submit your revised manuscript by Jun 23 2021 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.

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• 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 you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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: http://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,

Chiyu Zhang, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comments (if provided):

1. Please carefully check the Figures 4 and 5. Do the authors sure that the sampling year of the latest samples (the terminal braches in the MCC trees) were about 2025? Therefore, all tMRCA of the key nodes were wrong. In addition, please carefully determine which nodes can reflect the tMRCA of the strains from Colombia.

2. Please clearly label each introduction event (at corresponding node).

3. Line141：previously reported primers: please provide refs.

4. Line 315: sequences from Santander reported during 2010–2016) and Venezuela (2005–2015): please check this sentence.

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

[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.]

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Phylogenetic and evolutionary analysis of dengue virus serotypes circulating at the Colombian–Venezuelan border during 2015-2016 and 2018-2019

PONE-D-20-27563R3

Dear Dr. Martinez-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.

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.

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Kind regards,

Chiyu Zhang, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

line351:"(95% HPD: 1995–1995)" should be " (95% HPD: 1992–1995)".

Reviewers' comments: