Genotypes and phylogenetic analysis of adenovirus in children with respiratory infection in Buenos Aires, Argentina (2000–2018)

Débora N. Marcone; Andrés C. A. Culasso; Noelia Reyes; Adriana Kajon; Diana Viale; Rodolfo H. Campos; Guadalupe Carballal; Marcela Echavarria

doi:10.1371/journal.pone.0248191

Peer Review History

Original SubmissionNovember 17, 2020
22 Dec 2020 Decision Letter - Dong-Yan Jin, Editor PONE-D-20-36189 Genotypes And Phylogenetic Analysis Of Adenovirus In Children With Respiratory Infection In Buenos Aires, Argentina (2000 - 2018) PLOS ONE Dear Dr. Echavarria, 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. Particularly, the questions and comments raised by Reviewer 2 should be addressed. Please submit your revised manuscript by Feb 05 2021 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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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 ******** 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: No 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 article entitled “Genotypes And Phylogenetic Analysis Of Adenovirus In Children With Respiratory Infection In Buenos Aires, Argentina (2000 - 2018)” by Débora N. Marcone et al. is a cross-sectional study using HAdV positive respiratory samples from children with ARI, in Buenos Aires. The topic of the article is interesting and medically important. The manuscript is well written. My comments to the authors are as follows: Abstract: OK. Introduction: Line 44: Describe the common adenoviral and occasional adenoviral disease separately. Methods: It will be well understood for the reader if the whole method is shown with the help of a flowchart. Line 111, 112: Explain the rationale of direct PCR-sequencing of L1 (HVR1-6) of the hexon. Line: 135-37: REA enzyme profile can be added as a supplementary file if available. Result: OK Discussion and conclusion: OK Reviewer #2: This manuscript reported genotypes and phylogenetic analysis of adenovirus in children with respiratory infection in Buenos Aires, Argentina (2000-2018). Authors performed phylogenetic analysis based on sequences from the hexon gene region obtained from 141 patients and confirmed the circulation of the four species (B, C, D and E) represented by 11 genotypes, with predominance in species B and C and shifts in their proportion in the studied period (2000 to 2018). The following are some comments and suggestions for the authors: 1. Did authors checked their sequences for possible recombination before phylogenetic analysis? 2.How long is the hexon gene fragment been used in the phylogenetic analysis? Please state this clearly. 3. It is advised to use second method (e.g. Bayesian inference) to construct another phylogenetic tree. Comparing the phylogenies constructed by different methods is essential to avoid systemic errors in evolutionary study. 4. The phylogenetic trees should be simplified. There are a lot of very similar sequences from the same region and/or years making the trees very large and very difficult to inspect. Too many very similar sequences did not benefit to the phylogenetic analysis but rather interfere the inspection of tree topology. I would suggest to omit some identical sequences if any. 5. Why did the authors not include 7b reference sequences since 7b is one of the major genotypes either? 6. How did the authors distinguish B7 and B66? As I see, they cannot be distinguished in the phylogenetic tree based on the hexon gene fragment. ****** 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. Attachments Attachment Submitted filename: PONE-D-20-36189_review_14-12-2020.docx https://doi.org/10.1371/journal.pone.0248191.r001
Revision 1
4 Feb 2021 Author Response Dear reviewers In this document you will find the response to all your comments. To easy the reading, they were written in blue arranged in line with your questions (in black). Additionally, we would like to reinforce that all data associated to this study are supplied as supporting material, published at Mendeley database and GenBank: • Marcone, Debora; Culasso, Andres; Campos, Rodolfo; Echavarria, Marcela (2019), “ADENOVIRUS GENOTYPES TO BE USED IN PHYLOGENETIC ANALYSIS”, Mendeley Data, v1 http://dx.doi.org/10.17632/prf5vffgd2.1 • Culasso, Andrés; Marcone, Debora (2020), “Bibliographic Source for HAdV sequences”, Mendeley Data, V1, doi: 10.17632/jnh663g7w9.1 http://dx.doi.org/10.17632/jnh663g7w9.1 • Sequence data are available in GenBank under accessions numbers: MG000707 to MG000784 and MK913783 to MK913843. Responses • Reviewer #1: The article entitled “Genotypes And Phylogenetic Analysis Of Adenovirus In Children With Respiratory Infection In Buenos Aires, Argentina (2000 - 2018)” by Débora N. Marcone et al. is a cross-sectional study using HAdV positive respiratory samples from children with ARI, in Buenos Aires. The topic of the article is interesting and medically important. The manuscript is well written. My comments to the authors are as follows: Abstract: OK. Introduction: Line 44: Describe the common adenoviral and occasional adenoviral disease separately. The common and occasional adenoviral diseases were described separately Methods: It will be well understood for the reader if the whole method is shown with the help of a flowchart. An additional supporting figure (Supporting Fig 1), containing a flowchart of data acquisition and analyses was added. Line 111, 112: Explain the rationale of direct PCR-sequencing of L1 (HVR1-6) of the hexon. Direct PCR-Sequencing of HVR 1-6 was used as a molecular alternative to neutralization assay as it was suggested by Lu and Erdman (2006). It represents a quick approach and requires few resources (laboratory and trained personnel). According to these authors, the analyzed region is thought to contain most of the antigenic determinants of the neutralization assays. Line: 135-37: REA enzyme profile can be added as a supplementary file if available. REA enzyme profiles as captured by photography are not available. Result: OK Discussion and conclusion: OK • Reviewer #2: This manuscript reported genotypes and phylogenetic analysis of adenovirus in children with respiratory infection in Buenos Aires, Argentina (2000-2018). Authors performed phylogenetic analysis based on sequences from the hexon gene region obtained from 141 patients and confirmed the circulation of the four species (B, C, D and E) represented by 11 genotypes, with predominance in species B and C and shifts in their proportion in the studied period (2000 to 2018). The following are some comments and suggestions for the authors: 1. Did authors checked their sequences for possible recombination before phylogenetic analysis? As usual before phylogenetic analyses, the presence of recombination was assessed. The use of traditional methods like RDP (recombination detect programs) is difficult due to the large number of possible parental genomes (+90) and the fact that some of the parentals (reference sequences) are recognized have a recombinant origin (for example HAdV B 16 and E 4). Additionally, the analyzed region is relatively small (~700 to ~800 depending on HAdV species and genotype) and it is not known to include recombination hotspot (that are located near the genomes ends). Our approach was to carefully examine the results of the FASTA36 local alignments made to classify the sequence to the species. In the case of a recombinant sequence, the program may inform secondary hits with different reference sequences. In our analyses, the only “multiple” hits results were in cases of well know shared hexon regions, like HAdV B 3 and 68, B 7 and 66, and C 2 and 89. Finally, in the phylogenetic analyses, no sequence was suspiciously divergent from the references, and genetic distances were too low to reliably test for recombination events. 2.How long is the hexon gene fragment been used in the phylogenetic analysis? Please state this clearly. According to Lu and Erdman 2006, depending on the actual species and genotype the product of the nested PCR protocol should have between 688 and 821 bp. Information regarding the hexon gene fragment amplified and used in the phylogenetic analysis are now described in Methods (line 122). 3. It is advised to use second method (e.g. Bayesian inference) to construct another phylogenetic tree. Comparing the phylogenies constructed by different methods is essential to avoid systemic errors in evolutionary study. Supplementary phylogenetic trees using Bayesian inference were constructed, to be compared with those obtained using maximum likelihood, and presented as supporting fig 2 (genotype assignment) and supporting fig 4 (genetic diversity assessment). Although the topologies obtained with both methods are almost identical, differences in branch lengths were observed. Mainly, very short (but not insignificant branch lengths) were obtained in Bayesian methods while near-zero lengths were rounded up to zero in ML trees. Additionally, it should be note that due to the sequence similarity, the number of sequences and the star-like topology, the result of the Bayesian method should be taken with extreme care due to the Star Tree Paradox issue caused by the default priors on star-like topologies. 4. The phylogenetic trees should be simplified. There are a lot of very similar sequences from the same region and/or years making the trees very large and very difficult to inspect. Too many very similar sequences did not benefit to the phylogenetic analysis but rather interfere the inspection of tree topology. I would suggest to omit some identical sequences if any. The fig 2 was now simplified. The full version of these trees is depicted in Supporting Figure 3 and 4, for ML and Bayesian method respectively. In the new version all the identical sequences from GenBank were represented by a single sequence and added the suffix “[+XXX]” to indicate the number of additional sequences that lied in the same position of the tree. However, all sequences from this study were included in this new version. 5. Why did the authors not include 7b reference sequences since 7b is one of the major genotypes either? The reference sequence for 7b is now included in our reference dataset (http://dx.doi.org/10.17632/prf5vffgd2.1) and in the phylogenetic tree of figure 1. Originally, the reference sequence present in Harrach’s list was only those for 7a reference (a vaccine strain), we further included 7p (prototype: Gomen’s strain) and 7d2 (alternate vaccine strain). Since our REA results showed no 7b sequences we initially don’t include this reference sequence. At the analyzed region the 7b reference sequence is identical to 7a, 7d2 and 66. 6. How did the authors distinguish B7 and B66? As I see, they cannot be distinguished in the phylogenetic tree based on the hexon gene fragment. Indeed, as stated in the previous response the sequences for B7 and B66 are identical at the hexon region. The fiver region of B66 is identical to B3 and thus, the antigenic / genetic differences are concentrated in the penton region. A similar situation is found between the hexon region of B3 and B68. Because of these, we now include a note in the line 169 of the revised manuscript explaining that the genotype assignment includes both, phylogenetic and REA results. Attachments Attachment Submitted filename: Response letter.docx https://doi.org/10.1371/journal.pone.0248191.r002
22 Feb 2021 Decision Letter - Dong-Yan Jin, Editor Genotypes And Phylogenetic Analysis Of Adenovirus In Children With Respiratory Infection In Buenos Aires, Argentina (2000 - 2018) PONE-D-20-36189R1 Dear Dr. Echavarria, 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. 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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: (No Response) ****** 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: In their revised manuscript (PONE-D-20-36189_R1) entitled “Genotypes And Phylogenetic Analysis Of Adenovirus In Children With Respiratory Infection In Buenos Aires, Argentina (2000 - 2018)”, Débora N. Marcone et al., addressed the reviewers query meaningfully. The incorporation of reviewers' suggestions improved the manuscript a lot. I have no further suggestion. Reviewer #2: (No Response) ****** 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: Yes:** Arun Kumar Adhikary Reviewer #2: No https://doi.org/10.1371/journal.pone.0248191.r003
Formally Accepted
26 Feb 2021 Acceptance Letter - Dong-Yan Jin, Editor PONE-D-20-36189R1 Genotypes And Phylogenetic Analysis Of Adenovirus In Children With Respiratory Infection In Buenos Aires, Argentina (2000 - 2018) Dear Dr. Echavarria: 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 Professor Dong-Yan Jin Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248191.r004

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