Insights into the genetic diversity, recombination, and systemic infections with evidence of intracellular maturation of hepadnavirus in cats

Chutchai Piewbang; Sabrina Wahyu Wardhani; Surangkanang Chaiyasak; Jakarwan Yostawonkul; Poowadon Chai-in; Suwimon Boonrungsiman; Tanit Kasantikul; Somporn Techangamsuwan

doi:10.1371/journal.pone.0241212

Peer Review History

Original SubmissionJuly 23, 2020
24 Aug 2020 Decision Letter - Haitao Guo, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-20-22914 Insights into the genetic diversity, recombination, and systemic infections with evidence of intracellular maturation of hepadnavirus in cats 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. Please submit your revised manuscript by Oct 08 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. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2.Thank you for stating the following in the Acknowledgments Section of your manuscript: [C.P. was supported by the Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University. S.W. is financially afforded by scholarship program for ASEN countries, Chulalongkorn University. This research was funded by The Thailand Research Fund (RSA6180034), Grant for Joint Funding of External Research Project, Ratchadaphisek Somphot Endowment Fund and Veterinary Science Research Fund (RES_61_364_31_037), Chulalongkorn University, and Veterinary Pathogen Bank, Faculty of Veterinary Science, Chulalongkorn University.] We note that you have provided funding information that is not currently declared in your Funding Statement. 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Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A 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 ****** 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 ****** 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 described the detection of the recently discovered domestic cat hepadnavirus (DCH) in cat sera and cats died from hepatic diseases. The authors obtained and characterized the full-length viral genomes and revealed that nine DCH sequences phylogenetically formed three distinct monophyletic clades. They found a putative DCH recombinant strain. They also performed IHC staining of DCH and showed a wide-spread positivity of DCH in multiple tissues. The writing is clear and the results interesting. 1. Table 1, the viral copy number in various organs would be better presented as copies per milligram or gram of tissue. 2. A polyclonal anti-human HBc was used in the IHC staining. The authors should verify the cross-reactivity and specificity of this antibody to DCH core protein using Western blot. Reviewer #2: The authors explored the genetic diversity of the recently discovered mammalian hepadnavirus, tentatively named domestic cat hepadnavirus (DCH), among a collection of cat sera and cat tissues using PCR. Their full-length genome characterization of revealed the nine Thai DCH sequences obtained formed three distinct monophyletic clades. They found a putative DCH recombinant strain, suggesting a possible role of recombination in DCH evolution. They also used qPCR to determine the viral genome copy numbers in various organs of the cats with liver diseases and were able to localize the putative viral capsid protein in tissues using a polyclonal antibody against the HBV capsid protein via immunohistochemistry (IHC). In addition to the liver, positive-DCH immunoreactivity was found in various other organs, including kidneys, lung, heart, intestine, brain, and lymph nodes, suggesting possible systemic infection. This report adds some interesting new information regarding a relatively new mammalian hepadnavirus. I have a couple of questions for the authors. 1. Did the authors ever verify the specificity of the polyclonal antibody against the capsid by western blot analysis? Although the IHC control showed some evidence of specificity, western blot analysis will be more definitive. Inclusion of an alignment of the DCH and HBV capsid protein sequence would also help to evaluate the specificity of the polyclonal antibody. 2. The EM images are rather poor and not definitive. The putative capsids claimed by the authors might be surface antigen spheres, given the reported diameter of 23-25 nm, which is closer to the HBsAg spheres (ca. 22 nm) than the HBV capsids (ca. 30 nm). If feasible, immuno-EM using the cross-reactive capsid antibody would help to identify the capsid particles, which at the same time will also help to verify the specificity of the antibody. ****** 6. 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. https://doi.org/10.1371/journal.pone.0241212.r001
Revision 1
23 Sep 2020 Author Response Rebuttal response on a revision of PONE-D-20-22914 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 Response: Thank you for your positive reviews to our manuscript. 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes Response: Thank you for your positive reviews to our manuscript. 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 Response: Thank you for your positive reviews to our manuscript. 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 Response: Thank you for your positive reviews to our manuscript. 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 described the detection of the recently discovered domestic cat hepadnavirus (DCH) in cat sera and cats died from hepatic diseases. The authors obtained and characterized the full-length viral genomes and revealed that nine DCH sequences phylogenetically formed three distinct monophyletic clades. They found a putative DCH recombinant strain. They also performed IHC staining of DCH and showed a wide-spread positivity of DCH in multiple tissues. The writing is clear and the results interesting. 1. Table 1, the viral copy number in various organs would be better presented as copies per milligram or gram of tissue. Response: Thank you for your positive reviews to our manuscript. We have revised the unit of the viral load to per gram of extracted tissue and re-calculated the viral copy number as a revision found in the Table 1. (Page 16, lines 364-365) 2. A polyclonal anti-human HBc was used in the IHC staining. The authors should verify the cross-reactivity and specificity of this antibody to DCH core protein using Western blot. Response: Thank you for your concern and suggestion. We have tested the cross-reactivity of the anti-HBcAg antibody with the DCH-positive samples, compared to the result of HBV-infected tissue using western blot analysis. Furthermore, we have provided the specificity of the antibody by performing the western blotting with the extracted protein derived from DCH-negative liver samples (from cats with/without hepatitis). The details and results of the western blotting were given in the Methods (page 10, lines 221-241) and Results section (pages 19-20, lines 454-469), respectively. Reviewer #2: The authors explored the genetic diversity of the recently discovered mammalian hepadnavirus, tentatively named domestic cat hepadnavirus (DCH), among a collection of cat sera and cat tissues using PCR. Their full-length genome characterization of revealed the nine Thai DCH sequences obtained formed three distinct monophyletic clades. They found a putative DCH recombinant strain, suggesting a possible role of recombination in DCH evolution. They also used qPCR to determine the viral genome copy numbers in various organs of the cats with liver diseases and were able to localize the putative viral capsid protein in tissues using a polyclonal antibody against the HBV capsid protein via immunohistochemistry (IHC). In addition to the liver, positive-DCH immunoreactivity was found in various other organs, including kidneys, lung, heart, intestine, brain, and lymph nodes, suggesting possible systemic infection. This report adds some interesting new information regarding a relatively new mammalian hepadnavirus. I have a couple of questions for the authors. 1. Did the authors ever verify the specificity of the polyclonal antibody against the capsid by western blot analysis? Although the IHC control showed some evidence of specificity, western blot analysis will be more definitive. Inclusion of an alignment of the DCH and HBV capsid protein sequence would also help to evaluate the specificity of the polyclonal antibody. Response: Thank you for your positive review on our manuscript. As your suggestion, we have further elucidated the cross-reactivity throughout the specificity of the antibody used for IHC analysis by using the western blotting. Details and results of the western blotting were described in the Methods (page 10, lines 221-241) and Results section (pages 19-20, lines 454-469), respectively. 2. The EM images are rather poor and not definitive. The putative capsids claimed by the authors might be surface antigen spheres, given the reported diameter of 23-25 nm, which is closer to the HBsAg spheres (ca. 22 nm) than the HBV capsids (ca. 30 nm). If feasible, immuno-EM using the cross-reactive capsid antibody would help to identify the capsid particles, which at the same time will also help to verify the specificity of the antibody. Response: We sincerely respected with this comment since the EM images are quite not definitive. With the respects of TEM performing on the dead animals and fixative agents are not definitively appropriated for TEM analysis, resulting in poor-defined images. However, we performed the TEM from the FFPE section by locating the areas that were positive to the IHC analysis using Pop-off technique, supporting the particles found in those areas would be the targeting virus. With the respect of reviewer’s concern, we have revised that the detected particles would be the surface antigen spheres as suggestion by reviewer and also added further image of the EM. We honestly said that we are not able to do the immune-EM in this recent and we have performed the western blotting to test the specificity of the antibody used for IHC as described in Methods and Results sections. 6. 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 Response: We respected with the reviewer’s decision. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0241212.r002
12 Oct 2020 Decision Letter - Haitao Guo, Editor Insights into the genetic diversity, recombination, and systemic infections with evidence of intracellular maturation of hepadnavirus in cats PONE-D-20-22914R1 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, Haitao Guo Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0241212.r003
Formally Accepted
14 Oct 2020 Acceptance Letter - Haitao Guo, Editor PONE-D-20-22914R1 Insights into the genetic diversity, recombination, and systemic infections with evidence of intracellular maturation of hepadnavirus in cats Dear Dr. Piewbang: I'm 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. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Haitao Guo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0241212.r004

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