Dear Dr. Ning,

Thank you very much for submitting your manuscript "Senecavirus A-induced glycolysis facilitates virus replication by promoting lactate production that attenuates the interaction between MAVS and RIG-I" 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, who found your study interesting. 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' and editor's comments.

- We encourage you to corroborate the main cell culture findings (Fig 5D-F) with TCDID50 or plaque assay. From the RNA and protein levels the reader can clearly see the trends but not the effect size. To which extent was the replication inhibited?

- Does glycolysis facilitate senecavirus reproduction solely via RIG-I signaling or other pathways involved as well? To this end, an experiment with infection in RIG-I knockdown (e.g., shRNA or CRISPRi) plus/minus glycolysis inhibitors plus/minus lactate would be very informative.

- More information on tissue pathology in mice is required, as suggested by Reviewer #2.

- Lines 251-254: Please correct name superscripts of virus proteins, e.g. 2B, 2C etc, (see PMID: 28884666  for guidance and links).

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,

Peter V Lidsky

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 manuscript reports investigation of effect of glucose metabolism on the SVA infection. The authors performed in vitro and in vivo characterization of glycolysis related pathway in SVA infection. They showed that SVA infection can induce glycolysis and production of lactate, which can inhibit RLR signaling. Overexpression of glycolytic enzymes promoted SVA replication. The findings are very interesting.

Reviewer #2: In the manuscript "Senecavirus A-induced glycolysis facilitates virus replication by promoting lactate production that attenuates the interaction between MAVS and RIG-I", the authors evaluated the new mechanism used by SVA to enhance its replication from the perspective on the relationship between metabolism and innate immunity of the host. The results also suggest glycolysis or lactate may be new targets against this swine emerging virus. The work is well designed and the manuscript is clearly written and structured.

Reviewer #3: Senecavirus causes porcine idiopathic vesicular disease, which is harmful to the pig industry, and forms persistent infection and weaken the immune system of the pig. This manuscript focused on the effect of Senecavirus on innate immunity, i.e., IFN production. The novel idea was started from the aspect that viral infection affected cellular sugar metabolism, which then affected IFN production, and a very positive result was obtained. Therefore, the manuscript provided a new idea for controlling the disease from the aspect of metabolic control.

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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: Its clinical value to control SVA in pigs remains further confirmed.

Are the findings of this study limited to the single strain used or to all SVA strains? Is there any unique changes in the CH-GDFS-2018 strain? This needs to be discussed.

Reviewer #2: 1. Cell lines commonly used for SVA include PK-15 and HEK293T cells. Whether SVA infection-mediated glycolysis exists in non-porcine origin cells, HEK293T? It is better to clearly state.

2. In Figure 8, schematic overview, this figure has clearly defined the whole research context. It is recommended to remove the dotted line outside the cell membrane.

3. In animal experiments, the results showed the difference of virus load in tissues collected with various treatment in different groups. Readers would like to know whether this change of viral load caused by different treatment lead to pathological or histopathological change? This is related to the effect of different factors on the pathogenicity of the virus.

Reviewer #3: 1. It is mentioned that glycolysis promotes viral replication, but for viral replication, the authors measures the expression of SVA VP2 protein. It suggests that viral titers should be preferably determined. Otherwise, explain the feasibility of VP2 protein test.

2. In Figure 1, differences in mRNA levels of some proteins related to glycolysis such as HK2, PFKM, PKM, PGK1 and HIF-1α were detected after SVA virus infection, but only PGK1 and PKM were detected during protein level detection. Please explain why the latter two were selected for protein level detection

**********

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: Page 2, abbreviations of many molecules and pathways needed spell out.

The manuscript needs to be improved since several places lack of scientific writing. Lines 241-243, what is the meaning of “this first outbreak”?

Reviewer #2: 1. Abstract Page 2, line 35 : Please specify what experiments while not “Further”;

2. Page 7, lines: 141-145. Please note that contents in brackets for 2DG, oxamate and DCA was information belongs to the methods;

3. Page 25, lines: 605. “proteins” should be “detected proteins”.

Reviewer #3: FIG. 1C, the ordinate icon "Relative viral RNA" should be modified to "Relative expression levels of mRNA".

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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.

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Reviewer #1: No

Reviewer #2: Yes: Yingjun Lv

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

Dear Dr. ning,

Thank you very much for submitting your manuscript "Senecavirus A-induced glycolysis facilitates virus replication by promoting lactate production that attenuates the interaction between MAVS and RIG-I" 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 recommendations below and Reviewer #3 request:

Please fix the protein names that are misrepresented: Lines 251/254: “2B proteinase (2Bpro), 2C proteinase (2Cpro), 3C proteinase (3Cpro), 3D proteinase (3Dpro)”.

Proteinase = protease, it means a specific enzymatic activity, ability of the protein to cleave other proteins. For all picornaviruses, this activity was ascribed only to 3C protein, that can have "pro" superscript: 3Cpro. Likewise, 2C is a helicase and 3D is a polymerase, they should be spelled as 2Chel and 3Dpol, respectively. 2B does not have enzymatic activity and should be spelled without a superscript. Please see this paper for guidance doi:10.1099/jgv.0.000911 .

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,

Peter V Lidsky

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

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

Dear Dr. Ning,

We are up to accept the manuscript in its present form. No additional experiments are required.

Please fix the protein names that are misrepresented: Lines 251/254: “2B proteinase (2Bpro), 2C proteinase (2Cpro), 3C proteinase (3Cpro), 3D proteinase (3Dpro)”.

Proteinase = protease, it means a specific enzymatic activity, ability of the protein to cleave other proteins. For all picornaviruses, this activity was ascribed only to 3C protein, that can have "pro" superscript: 3Cpro. Likewise, 2C is a helicase and 3D is a polymerase, they should be spelled as 2Chel and 3Dpol, respectively. 2B does not have enzymatic activity and should be spelled without a superscript. Please see this paper for guidance doi:10.1099/jgv.0.000911 .

With regards,

Peter Lidsky

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: my comments were addressed

Reviewer #2: The authors answered all my questions, I have no questions at present.

Reviewer #3: The article has been modified according to the questions raised by the reviewers. The data is completed and the article has been well improved.

**********

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: I think no other experiments are needed.

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: (No Response)

Reviewer #2: No

Reviewer #3: In Figure 3 E and F. For consistency, it is suggested to place HG column on the left and LG column on the right.

**********

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. ning,

We are pleased to inform you that your manuscript 'Senecavirus A-induced glycolysis facilitates virus replication by promoting lactate production that attenuates the interaction between MAVS and RIG-I' 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,

Peter V Lidsky

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 Comments (if any, and for reference):