PONE-D-21-17173

Canine Bocavirus-2 and its possible association with encephalopathy in domestic dogs

PLOS ONE

Dear Dr. Piewbang,

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.

Many thanks for submitting your manuscript to PLOS One

It was reviewed by two experts in the field, and they have recommended some modifications be made prior to acceptance

I therefore invite you to make these changes and to write a response to reviewers which will expedite revision upon resubmission

I wish you the best of luck with your modifications

Hope you are keeping safe and well in these difficult times

Thanks

Simon

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Kind regards,

Simon Clegg, PhD

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: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: No

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: This study indicates the possible association of the CboV-2 with encephalopathy in dogs. This research is of certain significance. But the paper needs to be thoroughly revised because of a number of spelling and grammar errors.

Reviewer #2: You have a nice and interesting manuscript here which has some very interesting findings in it which will be of interest to the wider scientific community. I have added some comments in below which hopefully are useful. They are mainly just typographical comments which I have tried to correct for you. But overall, a nice paper

Line 24- (CBoVs) have been recognised ….

Line 26- across is spelt incorrectly

Line 36- also detected in the intestine, lymphoid organs and the heart ….. (reword)

Line 40- CBoV-2

Line 47- belongs to the Parvoviridae family (reword)

Line 57- 2012 in dogs, has recently been considered to be pathogenic ….. (reword)

Line 86- except for one exception (reword)

Line 89- where ethe CBoV sites of replication (reword)

Line 114- buffered formalin manufacturer needed

Line 114- For the histopathology study … (reword)

Line 117- homogenised in what?

Line 120- nucleic acid

Line 120- quantified rather than quantitated may sound better?

Line 123- please define FFPE

Line 132- were taken from our previous publication (reword)

Line 133- please include the manufacturer and contents of the GoTaq Green Master mix

Line 136- the positively amplified fragments (reword)

Line 144- any reason why rabies wasn’t tested for?

Line 149- including the PCR reagents and conditions here would be useful

Line 155- coding sequences

Line 158- reference for MEGA 7

Line 166- reference or link for RDP may be useful

Line 170- manufacturer needed for Simplot

Line 173- how were the models chosen for the trees to be produced?

Line 176- used as a template … (reword)

Line 179- canine brains from this study…(reword)

Line 181- jModelTest reference needed

Line 194- reword this as unclear

Line 195- maybe selected rather than selective?

Line 199- 440bp of the NS1 gene .. (reword)

Line 221- was performed can be removed

Line 216- reword as unclear

Line 216-217- the selected two CBoV positive brain sections …(reword)

Line 217- virus particles in brain … (reword)

Line 235- just be careful with screening for genomes, you didn’t screen for a full genome, just a part of it. Maybe better to say PCR screening?

Line 240- why not included the sequence results here?

Line 241- Among the positive samples ….(reword)

Line 243- comma after (15/15)

Line 257- is this based on genome or genes- this needs to be made clear

Line 264- isn’t this complete genomes?

Line 271- comma after study

Line 271- is it really 2000 BC? Over 4000 years ago?

Line 272- you mention there being 6 before- why only 5 now?

Line 282- red circles represented CBoV strains collected in this study (reword)

Line 292- neutrophils (typo)

Line 302-303- what about the other samples?

Line 307- in the cerebellum and the brain stem (reword)

Line 308- using a feline parvovirus …. (reword)

Line 317- used as a counterstain (C) and (D). (reword and correct full stops)

Line 328- demonstration of an infected brain … (reword)

Line 330- delete ‘that’

Line 335- including the intestines (reword)

Line 334-336- this may be nice shown in a table

Line 337- is this genomes or genes?

Line 340- is this epithelia rather than epitheliums?

Line 354- bocaviruses (reword)

Line 361- definitively is spelled wrong

Line 381- genotypes (reword)

Line 383- epithelia rather than epitheliums

And line 385

Line 390- Our results also showed a tissue tropism of CBoV-2 in the myocardium (reword)

Line 392- is associated with myocarditis (reword)

Line 394- did you detect virus in the blood?

Line 397- also details viral gene/ genome detection (reword)

Line 398- other parts of the body (reword)

Line 399- may be caused by differing sensitivities (reword)

Line 403- has been investigated in several …. (reword)

Line 414- this would be useful to have a reference for

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Reviewer #1: No

Reviewer #2: No

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Canine Bocavirus-2 infection and its possible association with encephalopathy in domestic dogs

PONE-D-21-17173R1

Dear Dr. Piewbang,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Simon Clegg, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

Many thanks for resubmitting your manuscript to PLOS One

As you have addressed all the comments and the manuscript reads well, I have recommended it for publication

You should hear from the Editorial Office shortly.

It was a pleasure working with you and I wish you the best of luck for your future research

Hope you are keeping safe and well in these difficult times

Thanks

Simon

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: All comments have been addressed

**********

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: 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: This study indicates the possible association of the CboV-2 with encephalopathy in dogs. This research is of certain significance. The author has answered my previous questions and I think this article can be accepted.

Reviewer #2: I wish to thank the authors for their diligence in addressing my previous comments. I believe that the manuscript reads much better now.

There are three very minor comments which could be addressed during final editing as I dont feel that they warrant delaying the publication of this interesting manuscript.

With best wishes

Line 167- delete one of the s in settings

Line 225- from a previous study (reword)

Line 358- typo in definitively.

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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