PONE-D-20-31124

Isolation and characterization of lumpy skin disease virus from cattle in India

PLOS ONE

Dear Dr. Kumar,

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 take in account the comments of the two reviewers and take care to the spelling errors in some place. As noted by the two reviewers, please provide a few more information/ explanation in the comparison of VERO and MDBK cells cultures used in this study. In addition, please check the repository status. please provide a full explanation refering to page and line for modification in the manuscript corresponding to the answers to the reviewers in your rebutal letter.

Please submit your revised manuscript by Dec 24 2020 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Pierre Roques, Ph.D.

Academic Editor

PLOS ONE

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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: N/A

Reviewer #2: No

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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: Yes

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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: The authors describe the first successful isolation of LSDV from a natural outbreak in India. LSDV as causative agent was confirmed by clinical observation, PCR, partial sequencing and serum neutralization assay. In addition, three different primary cell lines were examined regarding their susceptibility towards LSDV, and utilization of an alternative permanent cell line (Vero cells) for propagation of LSDV was analyzed. Moreover, authors compared viral genome load and virus titer in cell culture systems at different time points post infection to gain insight in the life cycle of LSDV.

General

• l. 184: phosphate buffered saline

• l. 211: subcutis

• l. 236: LSDV/SPV/GPV

• l. 255: PGT

• l. 256: Why the MDBK cells were not used for virus isolation in comparison?

• l. 257: brackets should be deleted

• l. 295: maybe better “Serum samples from all the clinically affected animals that showed presence of…” instead of brackets

• l. 298: titer of 1:64 to 1:1024

• l. 308: “from India in 2019”

• l. 311: “(until this paper is being written)” is not necessary

• l. 352: TCID50

• l. 379: phase

• be consistent with spelling “titer” (AE) or titre (BE) during the manuscript

Figures and Tables

• Figure 2: brackets not necessary

• Figure 3: could be given as supplemental information

• Figure 5: cold be given as supplemental information

• Table 2

o footnotes (*, #) should be described

o information about how many scabs were taken from an individual and how many scabs were taken at the same farm would be helpful

o please specify scab 22 “1 month later”, is this related to an individual animal/farm (which one?) or in general?

o In place of Viremia, better: LSDV genome (blood)

o Antibody titer

o Antibody titer should be indicated as 1:xx or transferred into ND50/ml

• Figure legend Figure 2: explain red triangles

Results

• Structure outbreak and later cell culture work together – in detail:

o “reactivity of LSDV to the sera from LSDV-infected animals” should be between “Identification of the agent” and “phylogenetic analysis” as it belongs to the examination of the outbreak and results are additionally presented in Table 2

Reviewer #2: What are the main claims of the paper and how significant are they for the discipline?

• The paper describes outbreaks of LSDV in India, and contains interesting details such as the existence of serologically positive animals which display no clinical signs, morbidity data, and phylogenetic analyses of the virus isolated. This information will be important to researchers in the field and policy makers who are trying to control the current LSD epidemic in Asia.

Are the claims properly placed in the context of the previous literature? Have the authors treated the literature fairly?

• The literature is cited well, and most of the claims are properly placed in the context. However the utility and widespread use of MDBK cells to isolate and analyse capripoxviruses is not highlighted sufficiently.

Do the data and analyses fully support the claims? If not, what other evidence is required?

• The majority of the claims are well supported. The only unsupported claim is the use of Vero cells for the propagation of LSDV (line 367-370 and elsewhere). The authors clearly show that Vero cells cannot support propagation of field strains of LSDV (line 264-269). The virus had to be passaged 15 times in order for CPE and viral plaques to be detected on Vero cells. The literature, however, shows that MDBK cells do not require such virus adaptation and CPE can be detected on first passage. The authors should therefore have concluded that Vero cells are not permissive for LSDV isolation and are inferior to MDBK cells in this respect. Their conclusion that Vero cells are suitable for the propagation of LSDV is incorrect and not supported by their results.

• Figure 5C is very indistinct, can the authors provide a better quality one? The plaques are not distinguishable on the current figure

• Please provide statistical analyses for the comparisons between Vero cells and MDBK cells in Figure 6. Are the differences reported statistically significant?

• Please re-examine the legend for Figure 6. It is not correct. A and B are growth curves. C and D (not currently mentioned in the legend) are PCRs.

• Make it clear in the legend and text for Figure 6 that the data presented is for the cell culture adapted strain of LSDV, not a field strain. This is very important for diagnosticians who may read the manuscript and not realise straight away that Vero cells do not support LSDV isolation.

• Do not use PFU/ml as a synonym for TCID50, as described in the legend of Figure 6. They are very different things. If you counted plaques (or foci) then use PFU. If you examined cells for CPE, use TCID50.

PLOS ONE encourages authors to publish detailed protocols and algorithms as supporting information online. Do any particular methods used in the manuscript warrant such treatment? If a protocol is already provided, for example for a randomized controlled trial, are there any important deviations from it? If so, have the authors explained adequately why the deviations occurred?

• Not applicable

If the paper is considered unsuitable for publication in its present form, does the study itself show sufficient potential that the authors should be encouraged to resubmit a revised version?

• Not application

Are original data deposited in appropriate repositories and accession/version numbers provided for genes, proteins, mutants, diseases, etc.?

• The LSDV strain has been deposited in a repository (lines 261-262) although this could not be confirmed on the website of the depository

Are details of the methodology sufficient to allow the experiments to be reproduced?

• Yes

Is the manuscript well organized and written clearly enough to be accessible to non-specialists?

• Yes, it is very nicely written.

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Reviewer #1: No

Reviewer #2: No

[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.

PONE-D-20-31124R1

Isolation and characterization of lumpy skin disease virus from cattle in India

PLOS ONE

Dear Dr. Kumar,

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.

As stated by the reviewer 2, the usage of VERO cells to isolation and propagation of wild type LSDV is still a mater of debate. Thus you have to clearly indicate as requested that adaptation to this cell line is needed to allow usage of VERO cells until you were able to shown multiple isolation of LSDV from field sampling using this cell line and not the classical MDBK cells. The fact that amplification of a reference adaptated LSDV strain in VERO can be done is of interest but the limitation of such a strain remains to be highlighted to people that are more interested in the epidemiological aspect of the LSDV emergence in India to avoid any missinterpretation.

Please submit your revised manuscript by Feb 05 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Pierre Roques, Ph.D.

Academic Editor

PLOS ONE

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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: All comments have been addressed

Reviewer #2: (No Response)

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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: Partly

**********

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: Yes

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: Yes

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: The performed changes in the revised manuscript are fine for me. This revised version is acceptable for publication.

Reviewer #2: The authors have addressed the majority of the comments by the reviewers. However they need to make very clear throughout the manuscript that they are analysing their laboratory adapted strain of LSDV in figure 3, and not a wildtype strain. The inability of Vero cells to support propagation of LSDV needs to be clearly stated. The authors also need to make clear that MDBK cells are a suitable and widely used cell line for LSDV isolation and propagation. This information is very important for diagnosticians who may read the manuscript and not realise straight away that Vero cells do not support LSDV isolation. Thus please take in account the comments from the reviewer 2.

Please correct “LSDV” to “Vero-cell adapted strain of LSDV” on line 35, 39, 292, 295, 298, 302, 394, 398 (twice) and 536. Also in the legend to Figure 3.

Add MDBK cells to the list of cells used for LSDV isolation on line 349-350.

Delete lines 379-380 and 396 which state that Vero cells are suitable for the propagation of LSDV. They are clearly not.

Line 95-96 Delete the statement “However, a plaque assay to precisely quantify infectious LSDV is still lacking.” This plaque assay has been described in Fay et al 2020.

Figure S2 (b) the images are out of focus, which precludes examination of the CPE at p15. Do the authors have a better image?

Figure S2 (b) LSDV is spelt incorrectly in the figure heading.

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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: No

[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.

Isolation and characterization of lumpy skin disease virus from cattle in India

PONE-D-20-31124R2

Dear Dr. Kumar,

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,

Pierre Roques, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

thank you for your comments and have a good new year and a happy Christmas

Reviewers' comments: