PONE-D-21-02395

Investigation of SARS-CoV-2 Infection in Dogs and Cats of Humans Diagnosed with COVID-19 in Rio de Janeiro, Brazil

PLOS ONE

Dear Dr. Calvet,

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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We look forward to receiving your revised manuscript.

Kind regards,

Maged Gomaa Hemida, ph.D

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear Authors,

The reviewers agree that your manuscript have some merits and I really encourage you to address their comments point by point in your revised manuscript if you decided to do so.

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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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: No

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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

Reviewer #3: 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 manuscript “Investigation of SARS-CoV-2 Infection in Dogs and Cats of Humans Diagnosed with COVID-19 in Rio de Janeiro, Brazil”. Overall, the manuscript is pertinent and deserves publication after miner revisions (listed below) are done. The manuscript has stated objectives:

1) To assess the exposure, infection, and persistence of SARS-CoV-2 in nasopharyngeal and oropharyngeal secretions (nasal and oral swabs) and feces (rectal swab) of dogs and cats living in households with a COVID-19 human case.

2) To investigate clinical and laboratory alterations (complete blood count and serum biochemistry) in pets associated with the SARS-CoV-2 infection.

3) To evaluate risk factors associated with SARS-CoV-2 infection in pets.

However, the objective 2 does not appear to be pertinent to the context of the manuscript and its results were not really discussed. I recommend the second objective and related parts of the manuscript be removed.

144, 210, 212, 414 etc.: “… human’s samples” should be “… human samples”

145-147: “All VTM samples had viral RNA extracted manually by the QIAamp Viral RNA

Mini kit (Qiagen), or automatedly by Perkin-Elmer Chemagic machine/chemistry…” should be re-phrased because QIAamp Viral RNA kit actually extracts both DNA and RNA, that is total nucleic acids (no DNase treatment). If Perkin-Elmer Chemagic does not include DNase than it would be appropriate to say:

“Nucleic acids were extracted from VTM samples by QIAamp Viral RNA Mini kit (Qiagen), or by Chemagic instrument (Perkin-Elmer)…”

Also please specify the instrument model – was it Chemagic Prime, Chemagic 360 or Chemagic Prepito?

150: “Amplifications were conducted …” should be changed to “Reverse transcription and amplification were conducted…”

213-215: “…tested by a RT-PCR designed to amplify N1 and N2 genes of SARS-CoV-2, as previously described [22]. Pet samples were only considered positive for SARS-CoV-2 when at least two genes were amplified.” This should be re-phrased because: (i) it does not match the reference which describes detection of RdRp, E and N genes by real-time PCR assays; (ii) SARS-CoV-2 does not have N1 and N2 genes.

244-145 “The PRNT is a highly specific serologic test and can be carried out on samples to

confirm the presence of neutralizing antibodies to SARS-CoV-2 [24].” Should be re-phrased because stating “to confirm the presence” implies a screening test was done before, therefore it is more appropriate to state” The PRNT is a highly specific serologic test for detection of neutralizing antibodies to SARS-CoV-2 [24].”

252: research facility of biosafety level 3 platform of IOC/Fiocruz. – define “IOC”

Reviewer #2: This manuscript strengthen previous studies reporting transmission of SARS-CoV-2 from human to dogs and cats, living in the same household . Twenty eight percent of dogs and 40% of cat from 47.6% of households, included in the study, were positive to SARS-CoV-2. The positivity lasted from 11 to 51 days after the human index COVID-19 case onset of symptoms. Risk factors associated with positivity were discussed.

. The longitudinal approach adopted in this study, with the wide sampling window, added more insights on the dynamic of SARS-CoV-2 transmission from human patients to their pets animals. Such finding are of great practical value in protecting pets animals and containing the current of SARS-CoV-2 pandemic, which is in line with one health approach.

Comments to the Authors:

- The material and methods section was extensively written, Please reduce it

- Have you considered sampling dogs and cat from nearby households with negative SARS-CoV-2 owners , as a mean to add a control element to the study.

- Line 58: replace (infected/exposed) with (positive)

- In the entire text replace the word “exposed” and use the world “ infected or positive” because the demarcation between the two words is not sufficiently clear in the current prospective.

-Line 342: the use of plasma in serological testing is not recommended because of the possibility of nonspecific result. Please discard, if any, serological results based on the use of plasma.

Line 514 this sentence should be removed “It was also experimentally demonstrated that cats can become infected and 515 transmit SARS-CoV-2 to other cats” because of its poor relation to the rest of the paragraph.

Reviewer #3: Dear editor,

Many thanks for giving me the opportunity to review the manuscript titled (Investigation of SARS-CoV-2 Infection in Dogs and Cats of Humans Diagnosed with COVID-19 in Rio de Janeiro, Brazil). The manuscript is presenting interesting da about the dynamics of SARS-CoV-2 infection in some companion animals in the close proximity of positive SARS-CoV-2 infected patients. These types of research is of high importance these days to get more information about the roles of these animals in the transmission of SARS-CoV-2 to human as well as the back-zoonotic impacts of human to infect animals. Although the manuscript is presenting large sets of data about the dynamics of SARS-CoV-2 infection in some pets in household of positive cases, some points should be considered before acceptance is granted.

1- One of the major concerns is the lack of description and comparison of the obtained sequences from animals to that of the circulating strains in humans at the time of their longitudinal study. It is highly recommended to sequence the full-length genome or at least the full-length S gene sequences of some isolates from animals and compare it to that of human. Such information will help great impacts in improving the quality of this manuscript. Meanwhile, presenting theses sequences and making them publically available is of high demands.

2- The authors must map in more precise details the sampling schedules from both human and animals.

3- A detailed description of the outcomes of the viral infection in animals in correlation to human must be presented.

4- The statistical analysis in the M&M is long. Please consider shortening this section and use key references instead.

5- The discussion section is very long. Some paragraphs are too vague. Please consider shortening theses sections and mainly focusing this section on discussing your findings in comparison to other international groups that have conduced similar studies especially from USA, Asia, and Europe.

6- Please consider deleting Objectiv-2 since it is not adding too much to the main theme of this manuscript.

7- Please try to highlight the roles of the One Health concept as an important approach for studying this type of research and its potential roles in the mitigation and control of the emerging and re-emerging diseases.

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Reviewer #1: No

Reviewer #2: Yes: Abdulmohsen A. Alnaeem

Reviewer #3: No

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Investigation of SARS-CoV-2 Infection in Dogs and Cats of Humans Diagnosed with COVID-19 in Rio de Janeiro, Brazil

PONE-D-21-02395R1

Dear Dr. Calvet,

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,

Maged Gomaa Hemida, ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear authors, I am pleased to let you know that your manuscript is greatly improved after a rigor round of revision and now is accepted for publication in PLos One

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 #2: All comments have been addressed

Reviewer #3: 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 #2: Partly

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: N/A

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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 #2: Yes

Reviewer #3: Yes

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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 #2: Yes

Reviewer #3: 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 #2: the anthers adequately addressed my comments raised in a previous round. Generally. this manuscript met the criteria of well designed and conducted study,

Reviewer #3: Although the authors did not show any virus sequence from human came in close contact with positive SARS-CoV2

animals, they listed this point as one of the major limitation oh the current study. The revised manuscript is greatly improved

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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 #2: No

Reviewer #3: Yes: Maged Gomaa Hemdia