Dear Dr. Yu,

Thank you very much for submitting your manuscript "Aggressive organ penetration and high vector transmissibility of epidemic dengue virus-2 Cosmopolitan genotype in a transmission mouse model" for consideration at PLOS Pathogens. 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.

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,

Mehul Suthar

Associate Editor

PLOS Pathogens

Ana Fernandez-Sesma

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Here, the authors have produced a paper investigating DENV-2 strain-dependent pathogenicity and mosquito infection. The authors used a combination of experiments that relied on both mosquito inoculation and natural mosquito feeding to characterize a new strain of DENV-2 detected during an outbreak in Taiwan in 2015. Using chimeric viruses they were able to determine that both enhanced mosquito infection and enhanced mouse virulence mapped principally to prM and E. While these results are interesting, I believe there are several areas that need to be addressed to facilitate understanding by the reader and a few scientific issues that need to be addressed. Critically, there are no line numbers, which makes it difficult to call out specific areas that need improvement. Please include line numbers. Specific points follow.

Reviewer #2: In this manuscript, the authors examine the virulence determinants of dengue virus which increase host and vector transmissibility. The authors characterized a novel clinical isolate of Dengue-2 (TW2015) and found this virus is pathogenic in mice lacking type I and II interferon and STAT1 signaling. This analysis included increased mortality and higher virus virus replication in peripheral tissues as compared to the NGC strain. Next, the authors performed transmission studies with mosquitoes and mice and found that the virus can infect mice from mosquitoes and that the virus can be transmitted from mice to mosquitoes. Lastly, the authors identified that the PrM-E region is the main virulence determinant in mice. Overall, this is a nice study that includes analysis from clinical isolate characterization, infection of mice, infection of mosquitoes, and identification of virulence determinants. However, there are concerns about the rigor of several experiments (many of which it is unclear whether they were performed once or multiple times). Further, there were also some concerns about the growth kinetics of the chimeric viruses.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: Comments:

1. Figure 2- Was infectious virus recovered from the spleen and other organs with TW2015?

2. Figure 3 A/B- These data are intersting, but are rather underpowered.

3. Figure 5- The authors mapped the virulence determinants using an infectious clone strategy. However, for several of the analyses, these data are rather preliminary and underpowered. Further, it is unclear whether these experiments were repeated or performed once.

4. Figure 5- Does the PrM/E chimeric virus have similar virus growth kinetics to the parental strains in Vero cells and other primary mouse/human cells? Does the PrM/E chimeric viruses alter cell infection (binding and entry)?

5. Figure 7B- Again, some of these findings are underpowered.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Abstract: it is unclear what the author’s are referring to when stating the TW2015 16681 virus. Is this a chimera between DENV-2 strain TW2015 and strain 16681?

Summary: “dengue severe hemorrhagic fever” is not a clinical term.

Introduction: the authors are remiss in not discussing the hypotheses and mechanisms that explain severe and fatal dengue. This would properly frame the rationale of the study.

Methods: What is the passage history of all virus isolates used in this study and were they verified by sequencing? Similarly what was the passage history and sequence of the virus used to make the 16681 clone. Both NGC and 16681 are typically considered high passage viruses.

Methods: please specify what “normal” housing conditions are for mosquito experiments, including temperature, relative humidity, and light cycle.

Fig 1A: This does not show titers, this is a representative image of a plaque assay. Also, it is not exactly clear what the authors are trying to demonstrate here and in the first line of the results. Is this simply the titer after stock generation?

Fig1D: This panel is illegible. Is there a more informative, preferably quantitative way to show that the mice had DENV NS1 present.

Fig1E: why was only DENV-NGC used as a control for these experiments and not 16681?

Fig 2: why was a different inoculum dose used for these experiments?

Fig 4F and G: For the transmission back to uninfected mice, how many mosquitoes were allowed to feed on the naive mice?

For the reverse genetics experiments, why was a clone based on the DENV strain 16681 strain used for chimera generation? It is fine if this was the most straightforward path but some justification for use of this strain, which up until this point was not included in any other experiment is warranted.

Fig 5B: Plaque size should be measured and compared using a statistical test.

Discussion: the discussion could be improved to place the results in the appropriate context. Perhaps discussing previous DENV circulation in Taiwan and speculating about mechanisms that led to displacement of previous strains with this new strain.

Reviewer #2: (No Response)

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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.

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

Dear Dr. Yu,

We are pleased to inform you that your manuscript 'Aggressive organ penetration and high vector transmissibility of epidemic dengue virus-2 Cosmopolitan genotype in a transmission mouse model' has been provisionally accepted for publication in PLOS Pathogens.

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 Pathogens.

Best regards,

Mehul Suthar

Associate Editor

PLOS Pathogens

Ana Fernandez-Sesma

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: No general comments, reviewers responded thoughtfully to reviewers concerns.

The authors made a majority of the recommended changes requested during initial peer-review of this manuscript and if the changes were not made, a sufficient explanation was provided. The changes significantly enhanced the credibility and scientific nature of the manuscript. The readers of the article can now fully understand the scientific methods used throughout this study and accurately interpret the scientific findings without bias or incomplete information. I recommend that this article should be accepted for publication without additional major changes to the manuscript.

Reviewer #2: The authors have addressed the previous reviewers concerns. No additional concerns were noted.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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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