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Detection of novel coronaviruses in bats in Myanmar

Identifieur interne : 000296 ( Pmc/Checkpoint ); précédent : 000295; suivant : 000297

Detection of novel coronaviruses in bats in Myanmar

Auteurs : Marc T. Valitutto [États-Unis] ; Ohnmar Aung [États-Unis] ; Kyaw Yan Naing Tun [États-Unis] ; Megan E. Vodzak [États-Unis] ; Dawn Zimmerman [États-Unis] ; Jennifer H. Yu [États-Unis] ; Ye Tun Win [Birmanie] ; Min Thein Maw [Birmanie] ; Wai Zin Thein [Birmanie] ; Htay Htay Win [Birmanie] ; Jasjeet Dhanota [États-Unis] ; Victoria Ontiveros [États-Unis] ; Brett Smith [États-Unis] ; Alexandre Tremeau-Brevard [États-Unis] ; Tracey Goldstein [États-Unis] ; Christine K. Johnson [États-Unis] ; Suzan Murray [États-Unis] ; Jonna Mazet [États-Unis]

Source :

RBID : PMC:7144984

Abstract

The recent emergence of bat-borne zoonotic viruses warrants vigilant surveillance in their natural hosts. Of particular concern is the family of coronaviruses, which includes the causative agents of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and most recently, Coronavirus Disease 2019 (COVID-19), an epidemic of acute respiratory illness originating from Wuhan, China in December 2019. Viral detection, discovery, and surveillance activities were undertaken in Myanmar to identify viruses in animals at high risk contact interfaces with people. Free-ranging bats were captured, and rectal and oral swabs and guano samples collected for coronaviral screening using broadly reactive consensus conventional polymerase chain reaction. Sequences from positives were compared to known coronaviruses. Three novel alphacoronaviruses, three novel betacoronaviruses, and one known alphacoronavirus previously identified in other southeast Asian countries were detected for the first time in bats in Myanmar. Ongoing land use change remains a prominent driver of zoonotic disease emergence in Myanmar, bringing humans into ever closer contact with wildlife, and justifying continued surveillance and vigilance at broad scales.


Url:
DOI: 10.1371/journal.pone.0230802
PubMed: 32271768
PubMed Central: 7144984


Affiliations:


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PMC:7144984

Le document en format XML

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<wicri:regionArea>One Health Institute, School of Veterinary Medicine, University of California, Davis, California</wicri:regionArea>
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<author>
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<addr-line>One Health Institute, School of Veterinary Medicine, University of California, Davis, California, United States of America</addr-line>
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<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>One Health Institute, School of Veterinary Medicine, University of California, Davis, California</wicri:regionArea>
<placeName>
<region type="state">Californie</region>
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<author>
<name sortKey="Murray, Suzan" sort="Murray, Suzan" uniqKey="Murray S" first="Suzan" last="Murray">Suzan Murray</name>
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<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Global Health Program, Smithsonian’s National Zoological Park and Conservation Biology Institute, Washington, District of Columbia</wicri:regionArea>
<wicri:noRegion>District of Columbia</wicri:noRegion>
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</author>
<author>
<name sortKey="Mazet, Jonna" sort="Mazet, Jonna" uniqKey="Mazet J" first="Jonna" last="Mazet">Jonna Mazet</name>
<affiliation wicri:level="2">
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</nlm:aff>
<country xml:lang="fr">États-Unis</country>
<wicri:regionArea>One Health Institute, School of Veterinary Medicine, University of California, Davis, California</wicri:regionArea>
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<region type="state">Californie</region>
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<series>
<title level="j">PLoS ONE</title>
<idno type="eISSN">1932-6203</idno>
<imprint>
<date when="2020">2020</date>
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<front>
<div type="abstract" xml:lang="en">
<p>The recent emergence of bat-borne zoonotic viruses warrants vigilant surveillance in their natural hosts. Of particular concern is the family of coronaviruses, which includes the causative agents of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and most recently, Coronavirus Disease 2019 (COVID-19), an epidemic of acute respiratory illness originating from Wuhan, China in December 2019. Viral detection, discovery, and surveillance activities were undertaken in Myanmar to identify viruses in animals at high risk contact interfaces with people. Free-ranging bats were captured, and rectal and oral swabs and guano samples collected for coronaviral screening using broadly reactive consensus conventional polymerase chain reaction. Sequences from positives were compared to known coronaviruses. Three novel alphacoronaviruses, three novel betacoronaviruses, and one known alphacoronavirus previously identified in other southeast Asian countries were detected for the first time in bats in Myanmar. Ongoing land use change remains a prominent driver of zoonotic disease emergence in Myanmar, bringing humans into ever closer contact with wildlife, and justifying continued surveillance and vigilance at broad scales.</p>
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<article-id pub-id-type="pmid">32271768</article-id>
<article-id pub-id-type="pmc">7144984</article-id>
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<contrib-group>
<contrib contrib-type="author">
<contrib-id authenticated="true" contrib-id-type="orcid">http://orcid.org/0000-0001-7567-2635</contrib-id>
<name>
<surname>Valitutto</surname>
<given-names>Marc T.</given-names>
</name>
<role content-type="http://credit.casrai.org/">Formal analysis</role>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Project administration</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<role content-type="http://credit.casrai.org/">Visualization</role>
<role content-type="http://credit.casrai.org/">Writing – original draft</role>
<role content-type="http://credit.casrai.org/">Writing – review & editing</role>
<xref ref-type="aff" rid="aff001">
<sup>1</sup>
</xref>
<xref ref-type="corresp" rid="cor001">*</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Aung</surname>
<given-names>Ohnmar</given-names>
</name>
<role content-type="http://credit.casrai.org/">Data curation</role>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Project administration</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<role content-type="http://credit.casrai.org/">Writing – review & editing</role>
<xref ref-type="aff" rid="aff001">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Tun</surname>
<given-names>Kyaw Yan Naing</given-names>
</name>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Project administration</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<xref ref-type="aff" rid="aff001">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Vodzak</surname>
<given-names>Megan E.</given-names>
</name>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Project administration</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<xref ref-type="aff" rid="aff001">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="currentaff001">
<sup>¤</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zimmerman</surname>
<given-names>Dawn</given-names>
</name>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Project administration</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<xref ref-type="aff" rid="aff001">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<contrib-id authenticated="true" contrib-id-type="orcid">http://orcid.org/0000-0002-4176-6825</contrib-id>
<name>
<surname>Yu</surname>
<given-names>Jennifer H.</given-names>
</name>
<role content-type="http://credit.casrai.org/">Formal analysis</role>
<role content-type="http://credit.casrai.org/">Writing – original draft</role>
<role content-type="http://credit.casrai.org/">Writing – review & editing</role>
<xref ref-type="aff" rid="aff001">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Win</surname>
<given-names>Ye Tun</given-names>
</name>
<role content-type="http://credit.casrai.org/">Project administration</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<role content-type="http://credit.casrai.org/">Writing – review & editing</role>
<xref ref-type="aff" rid="aff002">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Maw</surname>
<given-names>Min Thein</given-names>
</name>
<role content-type="http://credit.casrai.org/">Formal analysis</role>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Project administration</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<role content-type="http://credit.casrai.org/">Validation</role>
<role content-type="http://credit.casrai.org/">Writing – review & editing</role>
<xref ref-type="aff" rid="aff002">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Thein</surname>
<given-names>Wai Zin</given-names>
</name>
<role content-type="http://credit.casrai.org/">Formal analysis</role>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<role content-type="http://credit.casrai.org/">Validation</role>
<xref ref-type="aff" rid="aff002">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Win</surname>
<given-names>Htay Htay</given-names>
</name>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Validation</role>
<xref ref-type="aff" rid="aff002">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Dhanota</surname>
<given-names>Jasjeet</given-names>
</name>
<role content-type="http://credit.casrai.org/">Formal analysis</role>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Validation</role>
<xref ref-type="aff" rid="aff003">
<sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Ontiveros</surname>
<given-names>Victoria</given-names>
</name>
<role content-type="http://credit.casrai.org/">Formal analysis</role>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Validation</role>
<xref ref-type="aff" rid="aff003">
<sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Smith</surname>
<given-names>Brett</given-names>
</name>
<role content-type="http://credit.casrai.org/">Formal analysis</role>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Validation</role>
<xref ref-type="aff" rid="aff003">
<sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Tremeau-Brevard</surname>
<given-names>Alexandre</given-names>
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<role content-type="http://credit.casrai.org/">Formal analysis</role>
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<xref ref-type="aff" rid="aff003">
<sup>3</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Goldstein</surname>
<given-names>Tracey</given-names>
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<role content-type="http://credit.casrai.org/">Data curation</role>
<role content-type="http://credit.casrai.org/">Formal analysis</role>
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<xref ref-type="aff" rid="aff003">
<sup>3</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Johnson</surname>
<given-names>Christine K.</given-names>
</name>
<role content-type="http://credit.casrai.org/">Conceptualization</role>
<role content-type="http://credit.casrai.org/">Data curation</role>
<role content-type="http://credit.casrai.org/">Formal analysis</role>
<role content-type="http://credit.casrai.org/">Funding acquisition</role>
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<xref ref-type="aff" rid="aff003">
<sup>3</sup>
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<contrib contrib-type="author">
<name>
<surname>Murray</surname>
<given-names>Suzan</given-names>
</name>
<role content-type="http://credit.casrai.org/">Funding acquisition</role>
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<xref ref-type="aff" rid="aff001">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Mazet</surname>
<given-names>Jonna</given-names>
</name>
<role content-type="http://credit.casrai.org/">Conceptualization</role>
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<sup>3</sup>
</xref>
</contrib>
</contrib-group>
<aff id="aff001">
<label>1</label>
<addr-line>Global Health Program, Smithsonian’s National Zoological Park and Conservation Biology Institute, Washington, District of Columbia, United States of America</addr-line>
</aff>
<aff id="aff002">
<label>2</label>
<addr-line>Livestock Breeding and Veterinary Department, Ministry of Agriculture, Livestock and Irrigation, Naypyitaw, Myanmar</addr-line>
</aff>
<aff id="aff003">
<label>3</label>
<addr-line>One Health Institute, School of Veterinary Medicine, University of California, Davis, California, United States of America</addr-line>
</aff>
<contrib-group>
<contrib contrib-type="editor">
<name>
<surname>Chan</surname>
<given-names>Renee W.Y.</given-names>
</name>
<role>Editor</role>
<xref ref-type="aff" rid="edit1"></xref>
</contrib>
</contrib-group>
<aff id="edit1">
<addr-line>Chinese University of Hong Kong, HONG KONG</addr-line>
</aff>
<author-notes>
<fn fn-type="COI-statement" id="coi001">
<p>
<bold>Competing Interests: </bold>
The authors have declared that no competing interests exist.</p>
</fn>
<fn fn-type="current-aff" id="currentaff001">
<label>¤</label>
<p>Current address: Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America</p>
</fn>
<corresp id="cor001">* E-mail:
<email>ValituttoM@si.edu</email>
</corresp>
</author-notes>
<pub-date pub-type="epub">
<day>9</day>
<month>4</month>
<year>2020</year>
</pub-date>
<pub-date pub-type="collection">
<year>2020</year>
</pub-date>
<volume>15</volume>
<issue>4</issue>
<elocation-id>e0230802</elocation-id>
<history>
<date date-type="received">
<day>17</day>
<month>12</month>
<year>2019</year>
</date>
<date date-type="accepted">
<day>9</day>
<month>3</month>
<year>2020</year>
</date>
</history>
<permissions>
<license xlink:href="https://creativecommons.org/publicdomain/zero/1.0/">
<license-p>This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the
<ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/publicdomain/zero/1.0/">Creative Commons CC0</ext-link>
public domain dedication.</license-p>
</license>
</permissions>
<self-uri content-type="pdf" xlink:href="pone.0230802.pdf"></self-uri>
<abstract>
<p>The recent emergence of bat-borne zoonotic viruses warrants vigilant surveillance in their natural hosts. Of particular concern is the family of coronaviruses, which includes the causative agents of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and most recently, Coronavirus Disease 2019 (COVID-19), an epidemic of acute respiratory illness originating from Wuhan, China in December 2019. Viral detection, discovery, and surveillance activities were undertaken in Myanmar to identify viruses in animals at high risk contact interfaces with people. Free-ranging bats were captured, and rectal and oral swabs and guano samples collected for coronaviral screening using broadly reactive consensus conventional polymerase chain reaction. Sequences from positives were compared to known coronaviruses. Three novel alphacoronaviruses, three novel betacoronaviruses, and one known alphacoronavirus previously identified in other southeast Asian countries were detected for the first time in bats in Myanmar. Ongoing land use change remains a prominent driver of zoonotic disease emergence in Myanmar, bringing humans into ever closer contact with wildlife, and justifying continued surveillance and vigilance at broad scales.</p>
</abstract>
<funding-group>
<award-group id="award001">
<funding-source>
<institution-wrap>
<institution-id institution-id-type="funder-id">http://dx.doi.org/10.13039/100000200</institution-id>
<institution>United States Agency for International Development</institution>
</institution-wrap>
</funding-source>
<award-id>GHN-A-OO-09-00010-00</award-id>
<principal-award-recipient>
<name>
<surname>Mazet</surname>
<given-names>Jonna</given-names>
</name>
</principal-award-recipient>
</award-group>
<funding-statement>This study was made possible by the generous support of the American people through the United States Agency for International Development (USAID) Emerging Pandemic Threats PREDICT project (cooperative agreement number AID-OAA-A-14-00102 and GHN-A-OO-09-00010-00 to JM).
<ext-link ext-link-type="uri" xlink:href="https://www.usaid.gov/news-information/fact-sheets/emerging-pandemic-threats-program">https://www.usaid.gov/news-information/fact-sheets/emerging-pandemic-threats-program</ext-link>
The contents are the responsibility of the authors and do not necessarily reflect the views of USAID or the United States Government. The sponsor did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Support for the preparation of this manuscript was provided by the Morris Animal Foundation and Dennis and Connie Keller through a training partnership, as well as Judy and John W. McCarter, Jr., and James and Jamie Coss. This content has not been reviewed or endorsed by the Morris Animal Foundation, and the views expressed herein do not necessarily reflect the views of the Foundation, its officers, directors, affiliates, or agents.</funding-statement>
</funding-group>
<counts>
<fig-count count="1"></fig-count>
<table-count count="1"></table-count>
<page-count count="11"></page-count>
</counts>
<custom-meta-group>
<custom-meta id="data-availability">
<meta-name>Data Availability</meta-name>
<meta-value>All sequences are available from the GenBank database. The accession numbers have been included in a supporting document.</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
<notes>
<title>Data Availability</title>
<p>All sequences are available from the GenBank database. The accession numbers have been included in a supporting document.</p>
</notes>
</front>
<sub-article id="pone.0230802.r001" article-type="aggregated-review-documents">
<front-stub>
<article-id pub-id-type="doi">10.1371/journal.pone.0230802.r001</article-id>
<title-group>
<article-title>Decision Letter 0</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Chan</surname>
<given-names>Renee W.Y.</given-names>
</name>
<role>Academic Editor</role>
</contrib>
</contrib-group>
<permissions>
<copyright-statement>© 2020 Renee W.Y. Chan</copyright-statement>
<copyright-year>2020</copyright-year>
<copyright-holder>Renee W.Y. Chan</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<license-p>This is an open access article distributed under the terms of the
<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p>
</license>
</permissions>
<related-article id="rel-obj001" ext-link-type="doi" xlink:href="10.1371/journal.pone.0230802" related-article-type="reviewed-article"></related-article>
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<meta-name>Submission Version</meta-name>
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</front-stub>
<body>
<p>
<named-content content-type="letter-date">7 Jan 2020</named-content>
</p>
<p>PONE-D-19-34833</p>
<p>Detection of Novel Coronaviruses in Bats in Myanmar</p>
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<p>[Note: HTML markup is below. Please do not edit.]</p>
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<bold>Comments to the Author</bold>
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<p>Reviewer #1: Yes</p>
<p>Reviewer #2: Yes</p>
<p>**********</p>
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<p>Reviewer #1: Yes</p>
<p>Reviewer #2: N/A</p>
<p>**********</p>
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<p>Reviewer #1: Yes</p>
<p>Reviewer #2: Yes</p>
<p>**********</p>
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<p>**********</p>
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<p>Reviewer #1: I have uploaded my comments on the manuscript for the author to review and take into consideration when re-writing the manuscript. importantly the phylogenetic tree to show the relationship between the corona viruses.</p>
<p>Reviewer #2: Studies of “Bat borne” coronaviruses are vital for the effective mitigation and prevention of zoonotic coronavirus outbreaks. It is likely that the currently circulating alphacoronaviruses and betacoronaviruses in mammals have their evolutionary ancestral viruses originated from different bat species. Meanwhile most recent coronaviruses that cause human infections like the MERS-CoV or future viruses could possibly have their ancestral relatives in bats. With the ever-expanding human activity and habitat that continue to overlap habitats of bats there is bound to be future coronavirus spillover and subsequent outbreaks at some point. Therefore, we need to urgently invest to conduct long-term coronavirus surveillance studies in bats as well as in other wildlife and livestock to maintain our vigilance. In this context, the authors here report about their viral surveillance attempts in Myanmar to identify viruses that would pose a risk for potential spill over into human population. Despite the modest samples size the authors identify three new alphacoronaviruses and three new betacoronaviruses in bats in Myanmar that warrants further explorations on this. They found many of these positives in guano samples indicating an important transmission route. Overall, I believe this is an important work that needs to be published. However, this report could to be further improved and my comments are as below.</p>
<p>In the Methods section,</p>
<p>it is not clear how much of the initial sample volume used for RNA extraction from each of the oral, rectal and guano samples. Although one would expect, this is specially important since the oral swabs yielded the lowest.</p>
<p>Additionally, if deposited in GenBank or elsewhere the sequence data accession numbers need to be provided and mentioned here.</p>
<p>In the Results and discussion,</p>
<p>Authors need to indicate if at all any virus culture been attempted and if any of these viruses has been isolated and characterized.</p>
<p>The absence of at least RdRp partial sequence phylogenetic analysis is a shortcoming for this report that needs to be discussed or addressed. On a similar note, a phylogenetic analysis derived from spike(S) and receptor-binding domain (RBD) would be informative to include here.</p>
<p>Moreover, this report could be further improved if they consider providing information on the protein sequence alignment of coronavirus RdRps or S proteins with the conserved motifs and other unique signatures indicated as necessary.</p>
<p>Although their limited sequencing results may not allow, if the novel viruses found in this study would constitute recent recombination events from existing coronaviruses needs to be at least discussed since such recombination is rather common and are thought to contribute to the emergence of novel coronaviruses.</p>
<p>**********</p>
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<p>Reviewer #2: No</p>
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<supplementary-material content-type="local-data" id="pone.0230802.s002">
<label>Attachment</label>
<caption>
<p>Submitted filename:
<named-content content-type="submitted-filename">Reviewer Comments.docx</named-content>
</p>
</caption>
<media xlink:href="pone.0230802.s002.docx">
<caption>
<p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
</body>
</sub-article>
<sub-article id="pone.0230802.r002" article-type="author-comment">
<front-stub>
<article-id pub-id-type="doi">10.1371/journal.pone.0230802.r002</article-id>
<title-group>
<article-title>Author response to Decision Letter 0</article-title>
</title-group>
<related-article id="rel-obj002" ext-link-type="doi" xlink:href="10.1371/journal.pone.0230802" related-article-type="editor-report"></related-article>
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<meta-name>Submission Version</meta-name>
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</front-stub>
<body>
<p>
<named-content content-type="author-response-date">27 Feb 2020</named-content>
</p>
<p>RESPONSES TO REVIEWERS (Comments to the Author)</p>
<p>Reviewer #1: </p>
<p>I have uploaded my comments on the manuscript for the author to review and take into consideration when re-writing the manuscript. Importantly the phylogenetic tree to show the relationship between the corona viruses.</p>
<p>• Line 65 needs literature quotation</p>
<p>• Lines 68-70 need literature quotation</p>
<p>• Lines 90-92 need literature quotation</p>
<p>Thank you for finding these errors. The manuscript has been revised to include literature citations for these lines listed. </p>
<p>• Line 110 Animal capture and sampling: General comment is to clarify to the reader if there was any form of anesthesia application on the big chiropterans that couldn’t be handled manually during sampling. </p>
<p>Thank you for identifying this omission. All animals were handled manually; no anesthetic or immobilization agents were used to assist with capture or restraint, and the manuscript has been corrected to reflect this.</p>
<p>• Also Line 131-132: after how long were the bats released after capture and sampling. Time estimate here would be good for the readers.</p>
<p>Thank you for the suggestion. We have noted in the manuscript the following text: “Bats were released within 1 km of the capture site as soon as possible upon completion of each sampling event, with net capture and pillowcase restraint between 5 to 30 minutes and handling times less than 5 minutes for each individual.” </p>
<p>• Line 162 Results general comment is mainly on seasonality. Can the season/months be indicated in the table of results as it is stated at length in the results section. </p>
<p>Thank you for this very reasonable suggestion. However, the authors note that seasonality was intentionally discussed at greater length in the results of the main body of the paper, as the authors did not find a way to naturally insert the data into Table 1, given that the species captured and sites spanned both dry- and wet-seasons. Results not easily depicted in the table were discussed at greater length in the main body. </p>
<p>• Still in the results section; - a phylogenetic tree is needed to indicate to the reader the relationship between the beta and alpha corona viruses with the known and unknown corona viruses.</p>
<p>Thank you for the suggestion. However, the authors note that only short fragments of the RdRp gene (328bp and 434 bp, depending on the assay used) were amplified. Given the short sequence fragment of a conserved gene, phylogenetic analyses would likely be uninformative and limited in value. The authors agree that this is a shortcoming of the paper. However, the purpose of the study was not to fully characterize specific viruses, but to broadly screen for viruses in wildlife (especially bats) living in close proximity to human communities, and thus to better understand potential sources of zoonotic viral transmission in the context of these human-wildlife interfaces. The discussion has been expanded to further discuss this.</p>
<p>• Line 178 Since you used cyt B for species identification: why are the four host species of the pooled guano sampled aren’t identified? The reader might wonder.</p>
<p>Thank you very much for identifying this error! It has come to the attention of the authors that the initial table and figure were created with an older version of the data, without the final host species barcoding incorporated. This has been corrected in the body of the manuscript, in Table 1, and in Figure 1. As such, there are no positive samples where the host species was not identified by barcoding. The four pooled guano samples referred to in Line 178 were attributed to Chaerephon plicatus. </p>
<p>Reviewer #2: </p>
<p>• In the Methods section, it is not clear how much of the initial sample volume used for RNA extraction from each of the oral, rectal and guano samples. Although one would expect, this is specially important since the oral swabs yielded the lowest. </p>
<p>The authors agree with this assessment. The manuscript has been revised to reflect our methodology: samples were collected in 500 μl Trizol, and 250 μl was used from each sample for extraction. One aliquot was used for extraction per the RNA extraction kit, and an additional aliquot was stored to ensure that sufficient volume was available for a second extraction, or other downstream analyses if needed. 8 μl RNA (i.e. the maximum volume for the Superscript III kit) was used for cDNA transcription. </p>
<p>• Additionally, if deposited in GenBank or elsewhere the sequence data accession numbers need to be provided and mentioned here. </p>
<p>The authors are in agreement with the reviewer. The sequence data have been uploaded to NCBI GenBank since submission of the first draft of the manuscript, and the accession numbers have been added in a supplemental table. At the time of resubmission, the sequences have not yet been published.</p>
<p>• In the Results and discussion, Authors need to indicate if at all any virus culture been attempted and if any of these viruses has been isolated and characterized.</p>
<p>Thank you for identifying this omission. The authors have corrected the manuscript to reflect that viral culture and isolation were not pursued in this study. </p>
<p>• The absence of at least RdRp partial sequence phylogenetic analysis is a shortcoming for this report that needs to be discussed or addressed. On a similar note, a phylogenetic analysis derived from spike(S) and receptor-binding domain (RBD) would be informative to include here. Moreover, this report could be further improved if they consider providing information on the protein sequence alignment of coronavirus RdRps or S proteins with the conserved motifs and other unique signatures indicated as necessary. </p>
<p>Thank you for the suggestions. The authors note that only short fragments of the RdRp gene (328bp and 434 bp, depending on the assay used) were amplified. Given the short sequence fragment of a conserved gene, protein sequence analysis would not be informative. This is the same reason why a phylogenetic analysis was not pursued. The authors agree that this is a shortcoming of the paper. However, the purpose of the study was not to fully characterize specific viruses, but to broadly screen for viruses in wildlife (especially bats) living in close proximity to human communities, and thus to better understand potential sources of zoonotic viral transmission in the context of these human-wildlife interfaces. The discussion has been expanded to further discuss this.</p>
<p>• Although their limited sequencing results may not allow, if the novel viruses found in this study would constitute recent recombination events from existing coronaviruses need to be at least discussed since such recombination is rather common and are thought to contribute to the emergence of novel coronaviruses. </p>
<p>Thank you very much for the suggestion. However, the authors note that it is not possible to assess recombination from a short fragment of the RdRp gene as was performed in this study; thus, those analyses were not performed and therefore not included in the discussion. We agree that should complete genomes have been amplified, or the complete spike gene sequence amplified, those analyses to identify recombination and associated discussion may have been appropriate. The discussion has been expanded to further discuss this.</p>
<supplementary-material content-type="local-data" id="pone.0230802.s003">
<label>Attachment</label>
<caption>
<p>Submitted filename:
<named-content content-type="submitted-filename">Response to Reviewers_Valitutto et al..docx</named-content>
</p>
</caption>
<media xlink:href="pone.0230802.s003.docx">
<caption>
<p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
</body>
</sub-article>
<sub-article id="pone.0230802.r003" article-type="aggregated-review-documents">
<front-stub>
<article-id pub-id-type="doi">10.1371/journal.pone.0230802.r003</article-id>
<title-group>
<article-title>Decision Letter 1</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Chan</surname>
<given-names>Renee W.Y.</given-names>
</name>
<role>Academic Editor</role>
</contrib>
</contrib-group>
<permissions>
<copyright-statement>© 2020 Renee W.Y. Chan</copyright-statement>
<copyright-year>2020</copyright-year>
<copyright-holder>Renee W.Y. Chan</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<license-p>This is an open access article distributed under the terms of the
<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p>
</license>
</permissions>
<related-article id="rel-obj003" ext-link-type="doi" xlink:href="10.1371/journal.pone.0230802" related-article-type="reviewed-article"></related-article>
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<custom-meta>
<meta-name>Submission Version</meta-name>
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</custom-meta-group>
</front-stub>
<body>
<p>
<named-content content-type="letter-date">10 Mar 2020</named-content>
</p>
<p>Detection of Novel Coronaviruses in Bats in Myanmar</p>
<p>PONE-D-19-34833R1</p>
<p>Dear Dr. Valitutto,</p>
<p>We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. In addition,
<bold>please amend the '2019-nCoV' and the related nomenclature into SARS-CoV-2 and COVID within the text, whenever appropriate in your finalized version.</bold>
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<p>With kind regards,</p>
<p>Renee W.Y. Chan, Ph.D.</p>
<p>Academic Editor</p>
<p>PLOS ONE</p>
<p>Reviewers' comments:</p>
<p>Reviewer's Responses to Questions</p>
<p>
<bold>Comments to the Author</bold>
</p>
<p>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.</p>
<p>Reviewer #2: All comments have been addressed</p>
<p>**********</p>
<p>2. Is the manuscript technically sound, and do the data support the conclusions?</p>
<p>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. </p>
<p>Reviewer #2: Yes</p>
<p>**********</p>
<p>3. Has the statistical analysis been performed appropriately and rigorously? </p>
<p>Reviewer #2: Yes</p>
<p>**********</p>
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<p>Reviewer #2: Yes</p>
<p>**********</p>
<p>5. Is the manuscript presented in an intelligible fashion and written in standard English?</p>
<p>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.</p>
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<p>**********</p>
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<p>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)</p>
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<p>Reviewer #2: No</p>
</body>
</sub-article>
<sub-article id="pone.0230802.r004" article-type="editor-report">
<front-stub>
<article-id pub-id-type="doi">10.1371/journal.pone.0230802.r004</article-id>
<title-group>
<article-title>Acceptance letter</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Chan</surname>
<given-names>Renee W.Y.</given-names>
</name>
<role>Academic Editor</role>
</contrib>
</contrib-group>
<permissions>
<copyright-statement>© 2020 Renee W.Y. Chan</copyright-statement>
<copyright-year>2020</copyright-year>
<copyright-holder>Renee W.Y. Chan</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<license-p>This is an open access article distributed under the terms of the
<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p>
</license>
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<related-article id="rel-obj004" ext-link-type="doi" xlink:href="10.1371/journal.pone.0230802" related-article-type="reviewed-article"></related-article>
</front-stub>
<body>
<p>
<named-content content-type="letter-date">26 Mar 2020</named-content>
</p>
<p>PONE-D-19-34833R1 </p>
<p>Detection of novel coronaviruses in bats in Myanmar </p>
<p>Dear Dr. Valitutto:</p>
<p>I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. </p>
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<p>Thank you for submitting your work to PLOS ONE.</p>
<p>With kind regards,</p>
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<p>on behalf of</p>
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<p>Academic Editor</p>
<p>PLOS ONE</p>
</body>
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