Dear Dr. Weger-Lucarelli:

Thank you very much for submitting your manuscript "Host Nutritional Status Affects Alphavirus Virulence, Transmission, and Evolution" (PPATHOGENS-D-19-01146) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

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If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

Richard J. Kuhn, PhD

Associate Editor

PLOS Pathogens

Mark Heise

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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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: This is an important piece of work that is aimed at understanding the effects of nutrition on alphavirus infections. The effects of host nutrition on virus infections in general is an understudied and under appreciated area. Therefore this paper has important implications for human health in an increasingly obese environment. However, there are some concerns with the paper that need to addressed.

Reviewer #2: In their manuscript, Weger-Lucarelli et. al. describe their study of nutritional influences on alphavirus infection mouse models. The authors find some interesting correlations between obesity/undernutrition and alphavirus infection. The paper is well written and clear and, overall, very good work. The topic is extremely novel and timely, especially with the global syndemic of malnutrition and endemic and emerging infectious diseases. However, some questions/issues exist and should be addressed in the context of these studies.

Reviewer #3: This study attempts to identify an important and previously unexplored link between host nutritional status and alphavirus infection. The study is straightforward and clearly outlined. The results are well presented. Statistics are acceptable. The discussion is well done with the exception of lines 400-409 which is essentially a repeat of the results. See below for specific comments:

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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: 1. Supplementary Table 1 was not included with the manuscript. This table described the nutrient content of the diets, so this is critical missing piece. I am assuming this was just an oversight by the authors

2. In the introduction, the authors state that they fed mice two high fat diets-45% and 61%, but the data presented only includes 45% high fat diets. Did they use the 61% diet? And if so, did the results mirror the 45% diet? This would be important information to know.

3. Authors should present actual weights (figures 1 and 2) of mice rather than just percent loss or percent gain of weight. It can be added as supplementary data, but it needs to be there. The readers have no idea if the starting weights were identical, and how much weight in grams were lost or gained. How many grams were the obese mice?

4. The authors mention diabetes in the discussion, but no metabolic markers were provided. What were the glucose levels of the mice? Were they, in fact, diabetic?

5. The authors measure leptin, but it’s unclear what the point is. It’s well known that leptin levels increase with adiposity. They did not see a leptin difference between low protein and control diet-what is the significance of this finding? Does it matter? This data could be removed, unless the authors provide more context. It seems to be just “thrown in” as a piece of data.

6. The legend for the stats on figure 1A is not clear. You have to read the text to understand the purpose of the one star that is shown on the data. The stats information should be clearly stated in the figure legend.

7. With the CHIKV challenge, why were weight actually increasing after infection for the non-obese groups?

8. For the pathology slides in Supp Fig 2, it would be helpful for the readers to use arrows to point out the inflammation, and to provide what histopathological score the photos represent.

9. The authors wanted to see if their results would be similar using a strain of mouse different from the B6 mice. This is a logical step, however, they also had to treat the mice with antibody to block type-I IFN-again, a major departure from the B6 mice. So this is not a good comparison between strains.

10. In the discussion section, they claim that the model of RRV led to 60% of the undernourished mice and 40% of the obese died from the infection, yet all the lean mice lived. However, in the results section, they state that 2 mice from the obese group and 3 mice from the LP group died-out of a total of 7 mice. This is not 60% mortality. And, they also state that the results were not statistically significant, so this does not rise to a level that needs to be discussed.

Reviewer #2: 1) Type of fat included in the diet can be very influential on the outcomes of experimental infection. How was this particular diet chosen as a proxy for biological relevance? Also, it may be good include a genetically obese model in the experimentation to show that obesity itself and not diet, can result in similar changes as observed in these studies. This would also be a very interesting question to help cover the influence of leptin on outcomes of these experiments as the authors have chosen to use this measure as a proxy of nutritional status.

2) Since adipose tissue is a highly vascularized organ, it is possible that the virus is being trapped in the extra adipose and that is why the authors do not observe differences (or even lowered) viremia? Was any attempt made to measure viral particles in other tissues (especially adipose) aside from the footpad and serum? Similar studies would be interesting for the persistence of viral RNA. Please consider doing this experimentation.

Reviewer #3: None

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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: Some paragraphs in the discussion need to be broken up for better readability.

Reviewer #2: 1) In the introduction, the authors refer to four different diets (Line 90), but only discuss three diets in the feeding methodology described on lines 140-141. Please consider revising for consistency. The remainder of the paper only refers to the 45% diet. If data is available for the 61% diet, please include in the paper.

2) Please give references in the methodology text for successful employment of these diets in previous studies.

3) Was any kind of clinical score (semi-quantitiative) taken during the mouse infections to monitor health of the animals? Hunching, coat quality, etc all could be monitored to increase knowledge of morbidity in these animals.

4) Lines 256: “weight gain” should just be “weight”?

5) Please show all data from C57 versus Balb/c experimentation, including leptin measurements. This data is especially important as these animals are generally less susceptible to dietary treatment that the more widely used C57Bl/6 mice.

6) The data from the serological viral titer determination and the luminescence studies seems to be contradictory. Was viremia/RNAemia measured at day 6 between obese and healthy models?

7) Is 2 days generally accepted as the period of time with the most changes in the viral quasispecies? This would be especially important to ask if a virus from a LP or obese mouse cause differential morbidity or mortality in a lean host aside from changes in fitness?

8) How do the genetic changes in the viruses correlate to pathogenicity of the viral species themselves aside from fitness?

9) While the LP diet is interesting (as it represents one of the main types of malnutrition, kwashiorkor), other types of malnutrition exist in areas endemic for these virsues including complete marasmus, caloric restriction, and micronutrient deficiencies. Can the authors further comment on how different types of undernutrition could further change the results of this study?

Reviewer #3: How much Protein is in the control diet? Currently, only obese and lean are described.

Figure 2: There is an interesting bi- or tri-phasic behavior in the foot pad swelling. Is there an explanation for this? It seems consistent in MAYV and CHIKV both. Also, line 255 says CHIKV and RRV, but only CHIKV is discussed. Was the data and discussion for RRV missed? It would be important to mention since E and F have a different mouse with RRV. Readers may be interested in the RRV C57bL/6 data for comparison.

Figure 4: B,C and D, labels difficult to see. What A represents is unclear. What ‘flux’ are we measuring?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Dear Dr. Weger-Lucarelli,

We are pleased to inform that your manuscript, "Host Nutritional Status Affects Alphavirus Virulence, Transmission, and Evolution", has been editorially accepted for publication at PLOS Pathogens. 

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Best regards,

Richard J. Kuhn, PhD

Associate Editor

PLOS Pathogens

Mark Heise

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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

The authors have responded to each of the three reviewers' questions and concerns. Significant improvements and clarification were made as a result. The response was appropriate and the manuscript has been improved as a consequence and is now acceptable for publication.

Reviewer Comments (if any, and for reference):