PONE-D-19-16699

Detection of CWD in Cervids by RT-QuIC Assay of Third Eyelids

PLOS ONE

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National Institute of Health

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R01-NS06190 EAH, P01-AI-077774 EAH, R01-NS076894 EAH, and R01AI112956 CKM.

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Additional Editor Comments:

Thank you for submitting your manuscript to PLoS One. Your manuscript has been reviewed by two experts in the field, each of whom saw value in your study. At the same time the reviewers have raised a number of questions and suggestions for improving your manuscript. With respect to Reviewer 2’s comment on Figure 5 and associated text, I have followed up to find out that the problem is with the Reviewer’s difficulty in reconciling all of the numbers in the text with the data in the figure. Accordingly, I encourage you to submit a revised manuscript that addresses the points raised by the reviewers to the best of your ability. If you choose not to follow the suggestions, please explain.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: No

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5. 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: This manuscript applies the third eyelid assay for prions (previously described for scrapie-infected sheep) to the detection of CWD in deer, adding RT-QuIC as a means to enhance detection. The test shows relatively good sensitivity in clinical deer with the 96GG Prnp genotype. The test is less effective in preclinical animals, particularly those with polymorphisms at amino acid 96.

1) A table comparing all the samples would be very helpful (providing times post-infection or symptomatic/pre-symptomatic) to clarify all the various incubation periods/genotypes etc.

2) Were any live animals tested? Will this assay work as an ante-mortem test?

3) Any thoughts as to why the seeding activity is much higher in GS and SS than GG third eyelids late in the disease course but not detectable in the early samples? Does this affect the utility of the test on field samples?

Minor points:

Line 141-142: The sentence "However, RT-QuIC analysis..." needs rewriting, it is awkwardly phrased.

Line 252: Clarification needed--was the test performed by the USDA or just required to be done by the USDA?

Reviewer #2: This study is proposed to diagnose CWD in deer by detecting accumulation of disease associated PrPCWD in the 3rd eyelid lymphoid follicles by RT-QuIC to ease the sample collection protocol over the current protocol which requires detailed anatomical knowledge and dissection skills to collect precise retrophyngeal lymph node tissues. This study showed that PrPCWD detection as early as one month after inoculation of CWD inoculated deer in the 3rd eyelid by RT-QuIC. Interestingly, CWD infected deer with 96GG started accumulating PrPCWD earlier than CWD infected deer with 96GS in the 3rd eyelid. This finding might be expanded in future to address whether the 3rd eyelid is appropriate test samples for all types of CWD infected deer or deer with specific genotypes.

Even though this is an interesting study, insufficient labels and explanations of the figures and table, it is hard to understand the data. For instance, in abstract, line 36 -37 ”IHC detected…5 of 10 deer (50%) whereas third eyelids of all these animals…”, which data shows 5/10 by IHC and 10/10 by RT-QuIC in the third eyelids in clinical deer?

In Introduction, add a rational(s) behind comparing 96GG, GS, and SS genotypes in CWD disease development in deer.

Line 48-50, “ Disease begins when …. (PrPC) into a disease-associated, protease-resistant form (PrPCWD) which….” This is a general definition of PrPRES, and references sited here (12-14) are also covered a general term. So change PrPCWD to PrPRES and add another sentence to define PrPCWD, which is PrPRES found in CWD infected corvid.

Line 64 “…the third eyelid, a nictitating membrane found in….” after defined what is the third eyelid, use one for the rest of the manuscript. For example, Line 65-69 used “the third eyelid” and line 70 used “nictitating membrane” in the same paragraph. Line 120 is another example. Please check the entire manuscript to be consistent in terminology.

Results, request to improve figure and table labels and legends in order to easily understand data.

Figure1A-E

- Explain what was compared using two-tailed Mann-Whitney test?

- Figure 1 B-E, which samples are 96GG or GS? If all GG, explain.

Figure 1B

- 812 looked like a negative control in Fig 1E, are there any reasons behind? Explain.

Line 91 define an abbreviation first time you use, “Brain per os,” oral administration

Line 100-102, hard to follow. Please make points clear.

Line 102 “…in the RPLN, obex, and the third eyelid.” Be consistent in order of the Figure 2, “ the obex, RPLN, and third eyelid…” or change the order of the IHC images.

Line117-119, adding the genotype GG, GS, SS labels to Table 1 will help to understand “4 of 8 96GG deer” data in the table.

Figure 3, report significant values for one star, three stars and four stars.

Line285-289, “A two-tailed Mann-Whitney test was… to corresponding negative controls.” and p-values should be in figure legend.

Explain case 1305 in Figure 4 for lower seeding activity unlike others

Figure 5 and its results section needs to be improved.

The first paragraph in Discussion should be in introduction.

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Reviewer #1: No

Reviewer #2: No

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Detection of CWD in Cervids by RT-QuIC Assay of Third Eyelids

PONE-D-19-16699R1

Dear Dr. Hoover,

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Byron Caughey

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: