Short communication: Evaluation of charged membrane filters and buffers for concentration and recovery of infectious salmon anaemia virus in seawater

Simon Chioma Weli; Haitham Tartor; Bjørn Spilsberg; Ole Bendik Dale; Atle Lillehaug

doi:10.1371/journal.pone.0253297

Peer Review History

Original SubmissionJanuary 13, 2021
6 Apr 2021 Decision Letter - Uwe Fischer, Editor PONE-D-21-01328 Evaluation of Charged Membrane Filters and Buffers for Concentration and Recovery of Infectious Salmon Anaemia Virus in Seawater PLOS ONE Dear Dr. Weli, 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. As a comment to the standard text I would like to add that the paper needs minor revision in terms of the scientific content as suggested by reviewer 2. However, as reviewer 1 has commented, a similar paper has already been published with another virus (SAV) by your group. Please consider to shorten the current manuscript accordingly, referring on the SAV paper, stating the modifications of the methods described for SAV recovery from sea water. Actually, I ask myself why both manuscripts weren't combined in one paper. I am sorry for the delayed reviewing process, but seven other potential reviewers rejected to deal with the manuscript or where uninvited because of missing responses. Please submit your revised manuscript by May 21 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. 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. 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, Uwe Fischer Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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. 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. [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: 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: 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: Evaluation of Charged Membrane Filters and Buffers for Concentration and Recovery of Infectious Salmon Anaemia Virus in Seawater. The manuscript describes the method for detection and concentration of Infectious salmon anaemia virus in natural and artificial seawater using charged membrane filters. The methods and use of filters including the experimental design and set up is very similar to published work by authors group for salmonid alphavirus as: Weli, Simon Chioma, et al. "Development and evaluation of a method for concentration and detection of salmonid alphavirus from seawater." Journal of Virological Methods 287 (2021): 113990. The novel finding in the current manuscript is applicability of previously described method for concentration and recovery of Infectious Salmon Anaemia Virus. The experimental set up is already available in public domain. In my opinion, this work could not be accepted for publication in current form and should be revised highlighting the findings related to ISAV as short communication with reference to published work for methodology. Reviewer #2: The present study evaluated different membrane filters and buffer for recovery of ISAV from natural and artificial seawater. The authors postulate that routine surveillance of seawater for the presence of ISAV can supplement traditional surveillance programs based on the detection of clinical ISA disease, and provide early warning. The study demonstrated that the negative charged filters combined with NucliSENS® lysis buffer produced the highest ISAV recoveries from natural seawater – and further studies were therefore directed toward the MF-/buffer 1 combination. The authors went on to show that virus recover using either qPCR or ddPCR was generally better at lower viral concentrations. And finally a LOQ was defined for this assay. In general, this is a well-conceived and excellent written manuscript. Its focus is largely technically based, and addresses an important aspect of eDNA surveillance programs for viruses. I have raised only minor comments/edits that should be considered. Line 134-137 – The did the researchers include a positive control either directly in the RNA extraction or spiked at the lysis stage? This would help control for loss of RNA during extraction process. Line 141 – Only 1 ml of lysate was used for the RNA extraction. However, the volume of lysis buffer added to the filters was 4ml therefore only ¼ of the sample was extracted. Was the yield of ISAV adjusted for this? From the equation on line 139 it would appear not. Line 155 – the authors need to explain what they mean by inhibition and how it was quantified and what was the threshold. Additional info also needs to be provided for the ratios 1:1 and 1:4, was this RNA:seawater in the 2ul template (i.e. 1ul RNA + 1 ul seawater = 1:1). Furthermore, and related to the comment above, if RNA was diluted was the yield calculations adjusted accordingly? Line 165 – three not tree Line 213 – what do the authors mean by ‘reliably detected’. Was there a specific threshold used? Discussion – I would prefer to have seen some discussion putting the results of this study into the context of what we know about ISAV viral shedding. For example, Gregory et al. 2009 (DOI: 10.1111/j.1365-2761.2009.00999.x) has described the shedding rate and minimal infectious dose of ISAV in experimental challenges and reported their results a TCID 50. In the present study the LOQ was defined, but it remains unclear to this reviewer how this compares to the minimum infectious dose and shedding rate. If the LOQ is higher than the minimum infectious dose then the utility of this assay would be limited. The manuscript would be greatly improved if additional discussion concerning these points could be included. ****** 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: Yes:** Kalpana Agnihotri 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.0253297.r001
Revision 1
18 May 2021 Author Response PONE-D-21-01328 Dear Editor,     Thank you for the positive feedback, and all the constructive comments from you and the reviewers to the manuscript: Evaluation of Charged Membrane Filters and Buffers for Concentration and Recovery of Infectious Salmon Anemia Virus in Seawater Authors: Simon Chioma Weli, Haitham Tartor, Bjørn Spilsberg, Ole Bendik Dale, Atle Lillehaug We have revised the manuscript as recommend by you and the two reviewers by addressing all the comments raised. We have attached the following files: 1. A revision showing edits made in this revision as tracked changes (in Word doc format), file name: “Revised Manuscript with Track Changes”. 2. A revision with all changes accepted ('clean' file, in Word doc format), which is an unmarked version of the revised manuscript without tracked changes. File labeled “manuscript”. 3. A response letter, file name: “Response to Reviewers”, which is a rebuttal letter that responds to each point raised by the academic editor and reviewers. Editor’s comments: As a comment to the standard text I would like to add that the paper needs minor revision in terms of the scientific content as suggested by reviewer 2. However, as reviewer 1 has commented, a similar paper has already been published with another virus (SAV) by your group. Please consider to shorten the current manuscript accordingly, referring on the SAV paper, stating the modifications of the methods described for SAV recovery from sea water. Actually, I ask myself why both manuscripts weren't combined in one paper. Author’s response: As suggested by reviewer 1, we have shortened the manuscript and referred to the method as described for SAV recovery from seawater. With regard to why both manuscript (SAV and ISAV) were not combined in one paper. The reason was that, although the entire funding for the research was for developing a method for concentration and recovery of fish viruses (ISAV and SAV) from seawater, the SAV aspects of the research work was specifically organized as part of a PhD thesis. Combining both manuscript (SAV and ISAV) in one paper would be problematic for the student. It will mean that the PhD student would have to defend a subject that is not part of her PhD topic, as published articles derived from PhD thesis work are normally attached and defended during dissertation. Journal Requirements: We have revised the manuscript to meet PLOS ONE format and style as requested by the academic editor. We have reviewed the reference. Change to the reference list: Reference #21 was updated to reflect correct article publication date Reviewer #1 comment: In my opinion, this work could not be accepted for publication in current form and should be revised highlighting the findings related to ISAV as short communication with reference to published work for methodology. Author’s response: As suggested by reviewer 1, we have shortened the manuscript (as short communication) without omitting details that will allow readers to have full understanding of the work. We have also referenced the method as described for SAV recovery from seawater. The reduction where mainly focused on the materials and method section. Reviewer #2: Reviewer comment: Line 134-137 Did the researchers include a positive control either directly in the RNA extraction or spiked at the lysis stage? This would help control for loss of RNA during extraction process. Author’s response: No, we did not include positive control directly during RNA extraction or spiked at the lysis stage in this particular (in vitro) experiment. However, in our field study, we did add Mengovirus to evaluate extraction efficiency of SAV recovery from seawater. We agree that addition of spike positive control, would have probably helped to check for loss of RNA during extraction process. Reviewer comment: Line 141 – Only 1 ml of lysate was used for the RNA extraction. However, the volume of lysis buffer added to the filters was 4ml therefore only ¼ of the sample was extracted. Was the yield of ISAV adjusted for this? From the equation on line 139 it would appear not. Author’s response: Lines 126 – 130: Yes, the recovery was adjusted. Retrospective (back) calculations was used to calculate ISAV copy number in 4 or 2.4 ml concentrates by taken into consideration, that ISAV was spiked in 1L of seawater, concentrated and eluted in either 4 or 2.4 ml (lysate) and that from the 4 or 2.4ml, RNA was extracted from 1ml of the lysate. The extracted RNA was eluted in 50 µl and 2 µl of RNA was used for PCR. Reviewer comment: Line 155: The authors need to explain what they mean by inhibition and how it was quantified and what the threshold was. Additional info also needs to be provided for the ratios 1:1 and 1:4, was this RNA: seawater in the 2ul template (i.e. 1ul RNA + 1 ul seawater = 1:1). Furthermore, and related to the comment above, if RNA was diluted was the yield calculations adjusted accordingly? Author’s response: Line 145 – 152: We explained inhibition and how it was quantified in the manuscript. Additional, we have also provided detailed information about the dilutions in the manuscript. Environmental samples, such seawater are particularly challenging and usually contain complex mixtures of a variety of inhibitory substances that could impact the RT-qPCR and target quantification. For this reason, RNA was therefore, analyzed undiluted (1:1) and diluted (1:4) in duplicates, by RT-qPCR. Potential inhibition was detected when the Cq difference between the 1:1 and 1:4 samples was found to be less than 2 Cq. For the samples that falls within this threshold, the 1:4 dilution was used to estimate virus quantities. The ratios 1:1 indicate stock RNA (2 µL template) while the 1:4 ratios was stock RNA: RNA elution buffer (i.e. 1ul RNA + 3 µL RNA elution buffer = 1:4). Yes, when 1:4 dilution was used to estimate virus quantities, virus recovery calculations was adjusted accordingly. Reviewer comment: Line 165: Three not tree Author’s response: Line 165: Corrected from tree to three Reviewer comment: Line 213: What do the authors mean by ‘reliably detected? Was there a specific threshold used? Author’s response: Line 213: The statement ‘reliably detected’ has been rephrased to read “detected”, as there was no threshold used Reviewer comment: Discussion – I would prefer to have seen some discussion putting the results of this study into the context of what we know about ISAV viral shedding. For example, Gregory et al. 2009 (DOI: 10.1111/j.1365-2761.2009.00999.x) has described the shedding rate and minimal infectious dose of ISAV in experimental challenges and reported their results a TCID 50. In the present study the LOQ was defined, but it remains unclear to this reviewer how this compares to the minimum infectious dose and shedding rate. If the LOQ is higher than the minimum infectious dose then the utility of this assay would be limited. The manuscript would be greatly improved if additional discussion concerning these points could be included. Author’s response: We agree with the reviewer suggestion; however, the authors deliberately decided to avoid ISAV virus shedding as we have a separate manuscript on ISAV in experimental challenges study where we focused on ISAV viral shedding by comparing virus recovery from seawater and tissues (gills and heart) over a time period. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0253297.r002
21 May 2021 Decision Letter - Uwe Fischer, Editor PONE-D-21-01328R1 Evaluation of Charged Membrane Filters and Buffers for Concentration and Recovery of Infectious Salmon Anaemia Virus in Seawater PLOS ONE Dear Dr. Weli, 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 see my comments below. Please submit your revised manuscript by Jul 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. 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. 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, Uwe Fischer 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): I think you have properly addressed the reviewer's comments. To convert the manuscript into a follow-up short communication is a good compromize. Please find attached some minor points: Line 111 and 114: “were used” Line 144: “copy numbers” “by taking” Line 166: “such as” Line 169: “therefore” can be omitted. Line 171: “that fall” Line 173: “ratios were” Line 442: “Graph shows” Please double-check the manuscript for similar mistakes (e.g. connected with singular/plural mix-ups). [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.0253297.r003
Revision 2
27 May 2021 Author Response PONE-D-21-01328 Dear Editor,     Thank you again for the positive feedback, and all the constructive comments to the manuscript: Evaluation of charged membrane filters and buffers for concentration and recovery of infectious salmon anemia virus in seawater Authors: Simon Chioma Weli, Haitham Tartor, Bjørn Spilsberg, Ole Bendik Dale, Atle Lillehaug We have revised the manuscript as recommend by addressing all the comments you raised. We have attached the following files: 1. A revision showing edits made in this revision as tracked changes (in Word doc format), file name: “Revised Manuscript with Track Changes”. 2. A revision with all changes accepted ('clean' file, in Word doc format), which is an unmarked version of the revised manuscript without tracked changes. File labeled “manuscript”. 3. A response letter, file name: “Response to Reviewers”, which is a rebuttal letter that responds to each point raised by the academic editor and reviewers. Editor’s comments: Please double-check the manuscript for similar mistakes (e.g. connected with singular/plural mix-ups). Please double-check the manuscript for similar mistakes (e.g. connected with singular/plural mix-ups). Author’s response: As suggested by the editor, we have gone through the manuscript for mistakes. In lines 111 and 114: Was has been changed to “were used” In line 144: We changed copy number to “copy numbers” and taken has been changed to “by taking” In line 166: We have added such “as” In Line 169: We have removed “therefore”. In line 171: We have changed “falls” to fall” In line 173: We have “ratios were” In line 442: We change Graph show t0 “Graph shows” We have also reviewed the reference list to ensure that they are complete and correct. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0253297.r004
2 Jun 2021 Decision Letter - Uwe Fischer, Editor Short Communication: Evaluation of charged membrane filters and buffers for concentration and recovery of infectious salmon anaemia virus in seawater PONE-D-21-01328R2 Dear Dr. Weli, 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, Uwe Fischer Academic Editor PLOS ONE Additional Editor Comments (optional): Dear authors, thank you for implementing changes as suggested. Indicated lines for changes do not match with the present document which is probably due to shifts in text length resulting from the respective changes. I think the manuscript has now reached a quality so that it can be published in PLOS ONE. With best regards, Uwe Fischer. Reviewers' comments: https://doi.org/10.1371/journal.pone.0253297.r005
Formally Accepted
8 Jun 2021 Acceptance Letter - Uwe Fischer, Editor PONE-D-21-01328R2 Short Communication: Evaluation of charged membrane filters and buffers for concentration and recovery of infectious salmon anaemia virus in seawater Dear Dr. Weli: 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. Uwe Fischer Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0253297.r006

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