Dear Dr Lambert,

Thank you very much for submitting your manuscript "Investigating the dose-dependency of the midgut escape barrier using a mechanistic model of within-mosquito dengue virus population dynamics" 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,

Louis Lambrechts

Guest Editor

PLOS Pathogens

Alexander Gorbalenya

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'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: This paper looks at dose dependence of within-mosquito DENV kinetics through a combination of experimental and mathematical work. The manuscript addresses a gap in knowledge regarding this topic, and both the experimental and computational experiments support their overall conclusions. However, there are some minor concerns regarding the organization of the output.

Reviewer #2: Please see attached word document

Reviewer #3: The paper presents a novel mechanistic within-vector model of dengue infection in Aedes mosquitoes. The model is fitted to data on dengue infection, including single and double blood feeds. The topic is very relevant and not extensively examined for viral infections. Overall, the paper is well written, with clear justification of modeling choices, and acknowledgement of limitations. While the model is simplistic, it is an excellent first step with a nice road map of ways to improve the model (e.g. stochasticity). In particular, the careful attention to detail on the model formulation, providing all the necessary components for replication, is rare and is much appreciated.

**********

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: None

Reviewer #2: Please see attached word document

Reviewer #3: No major issues.

**********

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: Introduction/Discussion

See Christofferson et al. “Characterizing the likelihood of dengue emergence and detection in naïve populations” for an example of DENV dose-dependence and transmission.

See Veronesi et al. “Estimating the Impact of Consecutive Blood Meals on Vector Competence of Aedes albopictus for Chikungunya Virus” for additional observations of a second blood meal not affecting CHIKV.

M&M:

“… this provides semi-continuous data…” Three data points are longitudinal, but was a curve fit to these data to achieve semi-continuous?

What was the titer of the initial bloodmeal so that the dilutions have a titer associated with them? This is not clear. If this does address this point, consider moving this above.

Page 7: Mayton et al. “A method for repeated, longitudinal…” might also be relevant.

Results:

“At 3 DPI, mosquitoes feeding on the 1:1 viral dilution show higher midgut DENV titers but this early lead is

transient, and the viral titer in the midgut layer is largely independent of the initial blood meal dose by 8 DPI.” – provide a statistical test for this statement.

In text, the reference to dashed lines for Figure 5 is a bit confusing, consider saying “within the vertical dashed lines”

Line 435-437 – this paper shows a single timepoint (not the latest) at which dissemination was higher in Ae. albopictus, so “substantially” may not be warranted

Lines 430-431 – this section makes a lot of supposition about mutations. Other external forces affect EIP and vector competence, including temperature which might affect mosquito metabolism rather than viral kinetics directly… Here you are modeling a phenotype, there is no evidence to suggest the A226V mutation affects any of the parameters being toggled here. That’s not to say that this computational experiment is not interesting or informative, but I worry putting it into an assumed mechanism from a mutation will be overinterpreted and mis-cited in future. Consider softening the association with mutations and just put it in the context of alternative phenotypes.

Reviewer #2: Please see attached word document

Reviewer #3: - It is unusual to call the model “time-dependent” as done in the methods and discussion. The equations are autonomous, with no explicit dependence on time. It would be more accurate to call the model a system of autonomous ODEs and remove “time-dependent”

- Why is a Hill coefficient of 2 chosen for the rate that the virus invades the midgut? This typically indicates cooperation. This is one of the few modeling choices not discussed.

- In general, all figures could do with larger font.

- In Figures 2 and 3, the colors can be hard to distinguish (especially for the points). Consider using different marker symbols.

- In Figure 5 B and C, the font is small to the point of being difficult to read.

- In Figures 6, S1, S4, S5, S6, S7, write out what DPI means.

- In Figure 7, label the axis with words in addition to symbols. The symbols are not mentioned in the caption either.

- In Figure S2, what does *** indicate?

- The final paragraph seems out of place. While this reviewer agrees with the sentiment, it’s not clear it’s appropriate at this point in the manuscript.

**********

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

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 on PLOS Biology: 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: ReviewComments.docx

Dear Dr Lambert,

Thank you very much for submitting your manuscript "Investigating the dose-dependency of the midgut escape barrier using a mechanistic model of within-mosquito dengue virus population dynamics" 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. 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.

Most of the previous comments have been satisfactorily addressed. The stylistic choice to keep the manuscript as is, rather than change to a purely results section without interpretation, is fine. However, Reviewer #2 has pointed out a couple of minor corrections that remain to be made.

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,

Louis Lambrechts

Guest Editor

PLOS Pathogens

Alexander Gorbalenya

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

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

Most of the previous comments have been satisfactorily addressed. The stylistic choice to keep the manuscript as is, rather than change to a purely results section without interpretation, is fine. However, Reviewer #2 has pointed out a couple of minor corrections that remain to be made.

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: The revision clarified some of ambiguity in parts of the manuscript.

Reviewer #2: No further comments

Reviewer #3: The paper presents a novel mechanistic within-vector model of dengue infection in Aedes mosquitoes. The model is fitted to data on dengue infection, including single and double blood feeds. The topic is very relevant and not extensively examined for viral infections, as evidenced by another recently published paper addressing a similar topic. Overall, the paper is well written, with clear justification of modeling choices, and acknowledgement of limitations.

**********

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: None

Reviewer #2: There are no major issues

Reviewer #3: None.

**********

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: All comments were addressed.

Reviewer #2: I still think the manuscript could be made somewhat more concise by restricting the results section just to the results and therefore easier to digest, but accept that this is just a difference in opinion concerning style.

From line 48 statement is still not correct:

While some studies measure dissemination as the proportion of all blood-fed mosquitoes with disseminated infection [21, 22], this makes it impossible to separate out the impact of changes to the bloodmeal titer on infection and subsequent dissemination. Thus we measured dissemination as the proportion of mosquitoes with infected midguts which have disseminated infection.

Both studies measure and present results on the proportion of infected mosquitoes that have a disseminated infection, which makes it possible to separate out the impact of changes.

Fig 1. D. ‘other tissues’ should be h(t) not m(t)

Reviewer #3: The authors have addressed all concerns of this reviewer during revision.

**********

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

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 Dr Lambert,

We are pleased to inform you that your manuscript 'Investigating the dose-dependency of the midgut escape barrier using a mechanistic model of within-mosquito dengue virus population dynamics' 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,

Louis Lambrechts

Guest Editor

PLOS Pathogens

Alexander Gorbalenya

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Editor Comments:

All final comments have been addressed, congratulations to the authors on a nice study !