PONE-D-20-17798

Coronavirus testing indicates transmission risk increases along wildlife supply chains for human consumption in Viet Nam, 2013-2014

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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

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: No

Reviewer #2: Yes

Reviewer #3: 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

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: Huong et al. present a coronavirus detection study in the rats and bats in Vietnam. They used PCR method for detection and amplicon sequencing for verification and characterization of the evolutionary origin of the detected coronaviruses. They found a significant prevalence of coronavirus in these animals. Interestingly, the positive rates in rat increase from farm, to markets and to restaurants. This result supports that the virus spillover risk increases along the wildlife consumption chain by human. The study is nice, and the manuscript was well written. The only things I hope could be improved are below:

In the phylogenetic tree (Figure 6) I did not see the benefit, but indeed confusing, of putting the clade name in front of each of the sequences produced by this study. E.g. Coronavirus PREDICT CoV-35/VN23F0226 and Scotophilus bat coronavirus 512 2005/PREDICT-VN13F0333. “Coronavirus PREDICT CoV-35” and “Scotophilus bat coronavirus 512 2005” are the virus that is representative of the clades where the virus strain VN23F0226 and VN13F0333 are falling into (This is what I guessed). I could not find “Coronavirus PREDICT CoV-35” nor “Scotophilus bat coronavirus 512 2005” representative strains in the tree, and I must say these are not widely-used clade names but some clades defined by one or two previous paper. I am not trying to say these clade designation are invalid, but their representative strains should be included in the tree and provided with GenBank accession number so that other researchers could follow. Using “CoV-512 Clade” etc in the vertical bar designation of the lineage are great, but then you don’t have to repeat “Scotophilus bat coronavirus 512 2005” for every strain such as VN13F0333, VN13F0161, etc. Please use some more widely used standard format for virus naming E.g. Rat/VN13F0333/Vietnam/2014. This more understandable. Putting “Scotophilus bat coronavirus 512 2005” in front of “VN13F0333” just confuse reader that it was sampled from bats, but in fact this was sampled from rats.

In the median-joining network (Figure 7), some branches did not show with the number of the mutations as specified in the legend. It would be useful if the branch are drawn in scale with the number of mutations.

Reviewer #2: This is an interesting study examining the prevalence of coronaviruses at various points in the animal/human interface in Vietnam. Although timely in the context of COVID-19, I have some issues with the sampling techniques and data presentations as below:

Major comments

• A number of terms are used to describe rodent sampling sites. ‘Trader’, ‘rodent farms’, ‘live rodent trade supply chain’, ‘Large Market’, ‘Restaurant’, ‘Live rodent trade sites’, ‘rodent wildlife farm sites’, ‘wildlife farms’, ‘rodents in the trade’ and ‘field rat trade’. On the other hand, the analysis in figure 5 clearly classifies the sites into only three types: traders, large markets and restaurants. The sampling site terminology should be clarified with straightforward definitions and the authors should stick with these definitions throughout the manuscript. I understand that the authors are trying to analyse the data according to interface or sub-interface, but it is very confusing as currently presented.

• Figure 6 shows a tree based on a 387 bp RdRp fragment. Several of the bat-derived 512 2005 and rodent-derived 512 2005 appear to have an identical sequence (see CoV-512 clade). This is rather astonishing if they are really infecting coronavirus strains from different animals. I would interpret this as a signal of cross-contamination due to amplicon carry-over in the laboratory. If the cross-contamination occurred in the field, I would expect some nucleotide differences in this fragment as I gather that the sites of rodent sampling and bat sampling are geographically distinct?? I don't think it is reasonable to conclude that bat/ avian coronaviruses can infect rodents based on the evidence presented.

• Another concern I have is cross-contamination during sampling. Faeces collected from the ground of cages (even of individually housed animals) should ideally be called environmental samples as there is no way to tell if they really originate from the animal in the cage at the time. I believe this is confirmed by the presence of IBV and bat coronaviruses in rodent farms.

• Also, if a butcher/ vendor/ chef is using the same knife to handle several animals, there would be extensive cross-contamination if one of them is positive. Would this explain the high PCR positive rates?

• The sampling is comprehensive, but also very heterogeneous in terms of type of sample collected. Why wasn’t a standard sample type applied across all sites? I am concerned whether the type of sample collected at different settings might have contributed to the apparent variation in coronavirus detection rate between settings.

Minor comments:

• The introduction could be a bit more concise.

• Line 151: Are these 28 rodent farm sites classified as ‘traders’?

• Line 174 – 175 mentions 41 + 30 = 71 sites sampled. However, line 262 and 270 mention 70 sites. Why is this?

• Figure 4: Bars representing Sciuridae and R. argentiventer are not presented in this figure. Why are they in the legend?

• Figure 5: Could add between-group comparisons by chi-square to this figure.

Reviewer #3: In this manuscript, the authors have performed a surveillance study on the presence of the coronaviruses in the wildlife and the wildlife-human interfaces in Viet Nam. They detected the coronaviruses by consensus PCR and discovered the infected cases were increased along the supply chain. Although this study is performed in 2013-2014, its findings have a very good insight into the current COVID-19 outbreak, especially showing the potential risk of spreading the coronaviruses along the wild animal trade supply chain. Their findings can help to dissect how coronaviruses can spread from wild animals to humans by social activities. In view of this, I support to publish this manuscript in PLoS-One.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Coronavirus testing indicates transmission risk increases along wildlife supply chains for human consumption in Viet Nam, 2013-2014

PONE-D-20-17798R1

Dear Dr. Olson,

I have put your manuscript in the fast track in both rounds of review. Your revised paper has now been reviewed by one original reviewer and he promptly recommended acceptance of your work for publication, We’re therefore 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. Congratulations!

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Kind regards,

Dong-Yan Jin

Academic Editor

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

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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: Yes

**********

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

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 #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 #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 #2: Thanks for addressing the comments. The manuscript has been improved considerably with the clarifications of the terminology.

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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: Yes: Siddharth Sridhar