Zika virus isolation, propagation, and quantification using multiple methods

Worawat Dangsagul; Kriengsak Ruchusatsawat; Apiwat Tawatsin; Don Changsom; Pirom Noisumdaeng; Sukontip Putchakarn; Chayawat Phatihattakorn; Prasert Auewarakul; Pilaipan Puthavathana

doi:10.1371/journal.pone.0255314

Peer Review History

Original SubmissionApril 8, 2021
6 May 2021 Decision Letter - Juan Carlos de la Torre, Editor PONE-D-21-11564 Zika virus isolation, propagation, and quantification using multiple methods PLOS ONE Dear Prof. Puthavathana 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. It would be critically important addressing the issue raised by the reviewer about the genotype of the ZIKV isolates exhibiting different plaque phenotypes. In addition, the reviewer cited a number of minor issues that you need to adequately address in the revised version of the paper. Please submit your revised manuscript by Jun 20 2021 11:59PM. 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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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 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 ****** 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: No ****** 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: In this manuscript “Zika virus isolation, propagation, and quantification using multiple methods” by Dangsagul et al., the authors attempted to isolate Zika virus (ZIKV) from a total of 270 specimens (92 sera, 171 urines, and 7 autopsy tissue samples) by intrathoracic inoculation of samples into Toxorhynchitis splendens mosquitoes, followed by three passages in C6/36 mosquito cells. From these 270 samples, the authors were able to isolate ZIKV from 11 samples. Isolated ZIKV were titrated by plaque (plaque forming units, PFU) and immunofluorescence (focus forming units) assays in Vero cells and viral genome copies were determined by real time RT-PCR and digital RT-PCR. The manuscripts demonstrate the feasibility of isolate ZIKV using inoculation of Toxorhynchitis splendens mosquitoes followed by passage in C6/36 mosquito cells, and that plaque and immunofluorescence assays results in similar viral titers while quantification by real-time or digital droplet RT-PCR were higher than those obtained using plaque or immunofluorescence assays. Overall, this is a descriptive manuscript that could probably fit better in a methodology journal since provide with technical approaches for isolation and titration of ZIKV. Notably, although the authors have been able to isolate and observe differences in the plaque phenotype of the isolated ZIKV, genome sequence of the viral genomes have not been provided. The manuscript will improve if the authors provide with the sequence analysis of the different isolated ZIKV that could explain the differences in the plaque phenotype. This is particularly important because the virus with the biggest plaque phenotype (MUMT-1/2016) was isolated from a brain sample, contrary to the other ZIKV that were isolated from either urine or serum samples. There are also some minor concerns: 1) The authors could provide with plaque assays for the 11 isolated ZIKV (Table 1) rather than a selected set of 3 of them (Figure 1). 2) The authors indicate that some of the ZIKV isolates presented with viruses with different plaque sizes, suggesting the presence of mix population of viruses. However, plaque assays shown in Figure 1 suggest a similar plaque phenotype in the three isolated ZIKV MUM-1/2016 (A), MU-DMSC-1/2017 (B) and MU-DMSC-6/2016. 3) The authors should revise the manuscript to provide with some missing information (e.g. source of Vero cell lines) and to keep consistency with the nomenclature (e.g. ZIKV vs. Zika virus). 4) Line 219 indicate Table 2 but I think the authors refer to Table 1. ****** 6. 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Revision 1
23 Jun 2021 Author Response Response to Reviewer PONE-D-21-11564 Article title: Zika virus isolation, propagation, and quantification using multiple methods 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: Partly ________________________________________ 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ________________________________________ 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 ________________________________________ 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: No Response: We have Dr. Arthur Brown, a native English speaker, editing our language in the revised manuscript as shown in acknowledgement. ________________________________________ 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: In this manuscript “Zika virus isolation, propagation, and quantification using multiple methods” by Dangsagul et al., the authors attempted to isolate Zika virus (ZIKV) from a total of 270 specimens (92 sera, 171 urines, and 7 autopsy tissue samples) by intrathoracic inoculation of samples into Toxorhynchitis splendens mosquitoes, followed by three passages in C6/36 mosquito cells. From these 270 samples, the authors were able to isolate ZIKV from 11 samples. Isolated ZIKV were titrated by plaque (plaque forming units, PFU) and immunofluorescence (focus forming units) assays in Vero cells and viral genome copies were determined by real time RT-PCR and digital RT-PCR. The manuscripts demonstrate the feasibility of isolate ZIKV using inoculation of Toxorhynchitis splendens mosquitoes followed by passage in C6/36 mosquito cells, and that plaque and immunofluorescence assays results in similar viral titers while quantification by real-time or digital droplet RT-PCR were higher than those obtained using plaque or immunofluorescence assays. Overall, this is a descriptive manuscript that could probably fit better in a methodology journal since provide with technical approaches for isolation and titration of ZIKV. Notably, although the authors have been able to isolate and observe differences in the plaque phenotype of the isolated ZIKV, genome sequence of the viral genomes have not been provided. The manuscript will improve if the authors provide with the sequence analysis of the different isolated ZIKV that could explain the differences in the plaque phenotype. This is particularly important because the virus with the biggest plaque phenotype (MUMT-1/2016) was isolated from a brain sample, contrary to the other ZIKV that were isolated from either urine or serum samples. Response: We conducted complete genome sequencing of these 11 ZIKV isolates by the Sanger method and deposited the sequencing data in the GenBank database. The sequencing data will be released in June 2021 using the accession numbers as shown in Table 1. The information on molecular characterization of these 11 viruses together with the other ZIKV isolates in Thailand is shown in the other manuscript under reviewing process by the other publisher. In response to this important comment from the reviewer, we add more information about the genetics of these 11 ZIKV isolates in the Materials and Methods, and the Discussion of the revised manuscript as follows. Line Nos. 201-204: Genomic characteristics of the ZIKV isolates Complete genome sequencing by the Sanger method showed that all 11 ZIKV isolates belonged to the Asian lineage. These sequences can be retrieved from the GenBank database using the accession numbers shown in Table 1. Line Nos. 276-281: Nevertheless, using the Sanger method for nucleotide sequencing, we did not see any double peaks in the chromatograms, a marker of a virus quasi-species. Moreover, difference in plaque sizes across the viral isolates, particularly the larger plaque size of an isolate from the brain stem, may be related to the viral virulence. We are analyzing the virulence determinants and phylogenetic relationship of our genomic sequences against the others. There are also some minor concerns: 1) The authors could provide with plaque assays for the 11 isolated ZIKV (Table 1) rather than a selected set of 3 of them (Figure 1). Response: We have included the plaque characteristics of 8 more ZIKV isolates in the S1 Fig. We have included this information in Line No. 223. 2) The authors indicate that some of the ZIKV isolates presented with viruses with different plaque sizes, suggesting the presence of mix population of viruses. However, plaque assays shown in Figure 1 suggest a similar plaque phenotype in the three isolated ZIKV MUM-1/2016 (A), MU-DMSC-1/2017 (B) and MU-DMSC-6/2016. Response: The three ZIKV isolates mentioned displayed different plaque sizes, but the limitation of photograph resolution may have made the small plaque sizes not clearly visible. We put the arrows to make the pictures clearer. 3) The authors should revise the manuscript to provide with some missing information (e.g. source of Vero cell lines) and to keep consistency with the nomenclature (e.g. ZIKV vs. Zika virus). Response: The C6/36 cell line (ATCC CRL-1660) and Vero cell line (ATCC CCL-81) were used. Please see Line no.112, and 149, respectively. We replace the nomenclature of Zika virus with ZIKV. 4) Line 219 indicate Table 2 but I think the authors refer to Table 1. Response: Thank you. We change “Table 2” in line 221 to “Table 1”. ________________________________________ Attachments Attachment Submitted filename: Response to Reviewers_10-06-2021.docx https://doi.org/10.1371/journal.pone.0255314.r002
28 Jun 2021 Decision Letter - Juan Carlos de la Torre, Editor PONE-D-21-11564R1 Zika virus isolation, propagation, and quantification using multiple methods PLOS ONE Dear Dr. Puthavathana, Thank you for submitting your manuscript to PLOS ONE. After careful consideration of your revised version of the paper, we concluded that still needs a minor revision to thoroughly address the issue raised by the reviewer about the different plaque size phenotypes (see comments for Author). Therefore, we invite you to submit a revised version of the manuscript that addresses this minor issue. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Juan Carlos de la Torre, Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments (if provided): This revised version of a paper by Dangsagul and colleagues has addressed the main concerns raised by the reviewer of the original submission, with exception of the question raised by the reviewers about the plaque size phenotype. The response provided by the authors is not satisfactory. The authors need to provide additional information about the characterization of the plaque size phenotype by confirming a stable plaque size phenotype of plaque purified viral populations exhibiting different plaque size shown in figure 1 (red and green arrows). [Note: HTML markup is below. Please do not edit.] [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.0255314.r003
Revision 2
10 Jul 2021 Author Response PONE-D-21-11564R1 - [EMID:d7f4c12d9bb6967e] Article title: Zika virus isolation, propagation, and quantification using multiple methods Additional Editor Comments (if provided): This revised version of a paper by Dangsagul and colleagues has addressed the main concerns raised by the reviewer of the original submission, with exception of the question raised by the reviewers about the plaque size phenotype. The response provided by the authors is not satisfactory. The authors need to provide additional information about the characterization of the plaque size phenotype by confirming a stable plaque size phenotype of plaque purified viral populations exhibiting different plaque size shown in figure 1 (red and green arrows). Response to additional comment Plaque variants or the mixed plaque size phenotype are common in several virus isolates of early passages. This phenotype may be due to the difference in fitness of the individual infectious virus to grow in new hosts. Usually, the virus with a larger plaque size replicates faster and will overgrow and replace the smaller plaque size after sub-passaging. Consequently, the virus preparation will contain a homogeneous population of larger plaque size. Virologists performed plaque purification and characterization for specific purposes, such as to study the viral virulence determinants or when the purified virus population is essential for vaccine development or molecular cloning. Plaque purification and characterization by themselves are sufficient to serve as the specific aims in an article, as exampled below. 1. Kato F, Tajima S, Nakayama E, Kawai Y, Taniguchi S, Shibasaki K, et al. Characterization of large and small-plaque variants in the Zika virus clinical isolate ZIKV/Hu/S36/Chiba/2016. Sci Rep. 2017;7(1):16160. https://doi.org/10.1038/s41598-017-16475-2. 2. Mandary MB, Masomian M, Ong SK, Poh CL. Characterization of plaque variants and the involvement of quasi-species in a population of EV-A71. Viruses. 2020;12(6):651. https://doi.org/10.3390/v12060651. 3. Jia Y, Moudy RM, Dupuis AP, Ngo KA, Maffei JG, Jerzak GV, et al. Characterization of a small plaque variant of West Nile virus isolated in New York in 2000. Virology. 2007;367(2):339-47. https://doi.org/10.1016/j.virol.2007.06.008. Nevertheless, the objectives of our manuscript are to isolate Zika viruses from clinical specimens and determine the virus titers using multiple techniques: plaque assay, focus forming unit assay, droplet digital RT-PCR, real time RT-PCR. The plaque assay demonstrated that our Zika virus isolates were consisted of the population with mixed plaque sizes. Individual plaque, regardless of size or virulence was count as one infectious unit. Therefore, the objective of our manuscript on virus titration was fulfilled. Plaque purification and purification can take a year to finish, and the result gained does not add up much information for this manuscript. The technique should be carried out to serve a specific purpose that is more worthwhile than the virus quantification. According to the explanation described above, we do not change the revised version of our manuscript. Please reconsider. Attachments Attachment Submitted filename: Additional Editor Comments_11-07-2021.docx https://doi.org/10.1371/journal.pone.0255314.r004
14 Jul 2021 Decision Letter - Juan Carlos de la Torre, Editor Zika virus isolation, propagation, and quantification using multiple methods PONE-D-21-11564R2 Dear Dr. Puthavathana, 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, Juan Carlos de la Torre, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): In this second revision of their paper, the authors have addressed the comment raised by the reviewer about the plaque size phenotype. Although the response is far from satisfactory, the plaque size phenotype represents only a minor component of the paper and therefore it has a limited impact on the overall content of the paper. Reviewers' comments: https://doi.org/10.1371/journal.pone.0255314.r005
Formally Accepted
21 Jul 2021 Acceptance Letter - Juan Carlos de la Torre, Editor PONE-D-21-11564R2 Zika virus isolation, propagation, and quantification using multiple methods Dear Dr. Puthavathana: 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. Juan Carlos de la Torre Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0255314.r006

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