PONE-D-24-09462Epidemiological, Serological, and Virological Analysis of an Outbreak of Elephant Hemorrhagic Disease in SwitzerlandPLOS ONE

Dear Dr. Ackermann,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Vishwanatha R. A. P. Reddy

Academic Editor

PLOS ONE

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Reviewers' comments:

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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: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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: Thank you for sharing our data - this is a good addition to the current knowledge availabe.

Please find the comments on your paper in the attached.

Please consider a major revision, especially of the results section (abridge), and move excessive explanations to suppl materials.

Reviewer #2: PONE-D-24-09462

EEHV outbreak[s] in Switzerland

Elephants are hosts to several closely related herpesviruses known as Elephant endotheliotropic herpesviruses (EEHVs). These viruses can cause Elephant hemorrhagic disease (EHD), which is frequently fatal, and poses a risk to the precious and carefully managed herds of elephants that live in zoos around the world.

As a group, EEHVs have been classified as members of the betaherpesvirus subfamily, but they have genetic properties that make them distinct from other herpesviruses. Based on their DNA sequences, isolates of EEHV have been informally classified into several types, e.g., EEHV1A. Within types, strain-specific sequences have been detected. At present, the available information has been insufficient to motivate formal action related to classification of EEHV strains into distinct virus species.

This manuscript describes a very sad story about an outbreak of EHD that killed three young elephants in a Swiss zoo.

The paper is important because it is a carefully detailed description of the outbreak and the actions taken to understand and respond to what happened. It is important that this thoughtfully compiled information be peer-reviewed and made available in a public archive, such as PLOS One, as a resource for whoever needs to deal with such a problem in the years to come.

This work is likely to have long-term positive impact, but probably not of the sort measured by citation frequency.

Suggested modifications:

L30: “This virus probably re-emerged” (it is not a “new” virus”)

L73: “knowledge of …is also important”

L80. Define “ECPK”

L85: delete “at least”

L103: “to admit”

L183-186. A real-time PCR assay was used to obtain relative quantitation of virus and host genome copy numbers. The virus target is present at one copy per virus genome, and the host target is present at two copies per diploid genome. The text should be modified to make this clear, as well as its influence on data interpretation.

L315. Is “at least” needed here?

L403. I do not know the meaning of “27’488 and 4679 NNNs”.

L405. A denominator is needed for the 56 nt mismatch.

L417. Not sure what is meant by “non-core” likely tegument protein.

L420. Roseoloviruses also encode OBPs.

The L417 and L420 items end up being mentioned in lines 431-434, but those areas might be able to be more tightly integrated.

L448. Is the hypervariability within isolates or strains, or between isolates or strains?

The complex string spanning lines 509-513 contains what seem to be inconsistencies in usage of commas, semicolons, periods, and parentheses.

L528. p. 22. Not sure what is meant by “triplex patterns”, which are mentioned here and on p. 22 line 528.

L642. “routing” to “rooting”

L670. “sequence of Kimba contained”

L672. “address their biological function in the future”

L675. delete “considered”

L676-677. “enzyme, thereby terminating its activity, or”

L682. I don’t understand the sentence that begins with “Interestingly”

L695. delete “single one”

Having defined its abbreviation in line 702, use “gB” thereafter.

L713. “of gB subtype 1B”

L728. “revealed much” to “detected”

L762. Is “apparently” needed?

L778. “homology” to “similarity”

L786. “new” to “previously undescribed”

L792. “convey information about”

L793. “If all” to “Since”

L802. “Despite the long”

Lines 805, 807, and 809 say the same thing three different ways, which is confusing. “U38 DNA polymerase”, “U38 protein”, and U38 DNA pol. Pick a winner.

Other comments:

1. It seems like Umesh’s sequence should be included in Table 2.

2. In Fig. 1, the orange used for Thai, is too similar to Indi’s yellow for the parentage of Umesh to be unambiguous. A similar situation exists for Ruwani. It would be helpful to indicate which animals died from EHD. Are the two prior cases those of Xian and Aishu? If so, please say so.

3. Parts of the case descriptions are difficult to follow because of the several nomenclatures used. Lines 382-390 provide several illustrations of this:

• In line 382, “EP07” is used as a name for an animal. In line 389, “EP55” is described as the name of an isolate. In Table 2, “EP55” is described as a “Case”. From context, I wondered whether similar designations connected to Genbank entries. Too much work is required to decipher the information. It would be helpful to adopt a consistent scheme for such things.

• It would also be helpful to include a comprehensive expanded version of Table 2 that includes all of the animals mentioned in the paper, their birth and death years, virus and strain information, sequence accessions, lengths, and genes (for short sequences), etc.

4. A single, large “EEHV1 Loads” could be added parallel to the y-axes on the left side of Fig. 3

5. Fig. 4 would be easier to understand if it included the names of the animals, in place of, or at least in addition to codes such as “E361A_EP07”.

6. Fig. 5 would be improved by addition of animal names and where they are from (American, Zurich, or European).

7. Fig. 6 would be improved by a lighter color scheme in the upper right half and a larger font in the lower left half. The color scheme for the aligned genome fraction provides little information due to the high degree of similarity across the collection. A color gradient that ranges from 80% or 90% to 100% might be more informative.

8. The red text in Figure 7 is illegible. A larger font will increase the vertical dimension of the figure, but that is OK.

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Reviewer #1: No

Reviewer #2: Yes:  Philip E Pellett

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Attachments

Attachment

Submitted filename: PONE-D-24-09462 - review 1.pdf

PONE-D-24-09462R1Epidemiological, Serological, and Viral Genomic Analysis of an Outbreak of Elephant Hemorrhagic Disease in SwitzerlandPLOS ONE

Dear Dr. Ackermann,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jan 24 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Vishwanatha R. A. P. Reddy

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear authors,

Thanks for resubmission of the reviewed manuscript, and this is now reviewed by two reviewers. After careful consideration of the reviewers comments, unfortunately, in the present form it is not acceptable in the present format, and I would like to invite you to address the comments. Please adapt the below comments.

1) The introduction needs to succinct and not a full literature review on the EEHV genome.

2) Interpretation of the data should be restricted to the discussion

3) The treatment details should be added to this paper, as these are referred to in the discussion

4) The abstract needs to clearly reflect the main findings

Best wishes,

Vishwanatha Reddy

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. 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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

6. 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: These are some very interesting findings, but the manuscript in its present state is somewhat challenging to read. I hope my suggestions are helpful to reach a wider audience:

Introduction is 6 pages A4, please abridge to max 3 pages but referring to appropriate references. The introduction is too comprehensive, and should only cover

1) The global issue of EEHV

2) The difficulties with the genome, in broad lines

3) The presenting case, which leads into:

4) What this study is looking at

Try to remove all animal names from the text. Use Calf A, Calf B, Calf C. Bull 1, Bull 2, etc. You can have a tablet or figure in which these neutral phrases are connected to the animal’s name. By doing so, it will be easier for the reader to critically assess the course of the disease and results of what you’ve found.

Results – keep these factual by only reporting them, not interpretating them until the Discussion section, excluding the comparisons part of your study. Do not use emotive words, or interpretative words in this section.

Treatment of the calves is touched on in the discussion but details are not included. Either include the treatment in your case description, or if it is novel and warrants a separate publication, publish both papers in tandem so that you can refer too it.

Reviewer #2: Ackermann and colleagues have provided a detailed account of things learned while dealing with a slow-moving outbreak of elephant hemorrhagic disease in captive and free-living elephants. The work includes extensive use of virus genome sequencing to track what turned out to be several strains of elephant endotheliotropic herpesviruses (EEHV). This is a complex outbreak report, not a hypothesis-test. It is important that this work be archived in an internationally accessible venue such as PLOS ONE, as guidance for whoever might encounter another such situation, be it with EEHV in elephants or some other unusual combination of agent and host.

The authors are to be commended for the meticulous work done in dealing with the unfortunate clinical situations and in the depth and manner in which they have organized the compiled associated dataset and narrative, including submission of the numerous DNA sequences to Genbank.

The original reviewers made numerous suggestions for improving the manuscript. As detailed in their Rebuttal letter, the authors have done a very commendable job of responding to the suggestions.

I have nothing to add.

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7. 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: Yes:  Phil Pellett

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: PONE-D-24-09462_R1 - review 2.pdf

Epidemiological, Serological, and Viral Genomic Analysis of an Outbreak of Elephant Hemorrhagic Disease in Switzerland

PONE-D-24-09462R2

Dear Dr. Ackermann,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Vishwanatha R. A. P. Reddy

Academic Editor

PLOS ONE