Estimated quantity of swine virus genomes based on quantitative PCR analysis in spray-dried porcine plasma samples collected from multiple manufacturing plants

Elena Blázquez; Joan Pujols; Joaquim Segalés; Carmen Rodríguez; Joy Campbell; Louis Russell; Javier Polo

doi:10.1371/journal.pone.0259613

Peer Review History

Original SubmissionOctober 21, 2021
2 Feb 2022 Decision Letter - Caryn L Heldt, Editor PONE-D-21-33668Estimated quantity of swine virus genomes based on quantitative PCR analysis in spray-dried porcine plasma samples collected from multiple manufacturing plants.PLOS ONE Dear Dr. Polo Pozo, 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 address all comments made by the reviewers. In particular, the use of low, medium, and high is too subjective. Please change all reference to this qualitative result to quantitative results. Also, please differentiate if Table 1 is log TCID50 or log reduction values (LRVs) in TCID50 ============================== Please submit your revised manuscript by Mar 19 2022 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'. 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Kind regards, Caryn L Heldt, Ph.D. 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. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 4. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 3 in your text; if accepted, production will need this reference to link the reader to the Table. 5. 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. 6. 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: 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: 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: This manuscript provides valuable information on the presence of genomic information for porcine viruses of concern in spray dried porcine plasma. The estimation of TCID50 based on standard curves is also of interest, as is the % positive sample information for SDPP from different geographical regions. I have annotated the manuscript with suggestions for improving English language usage and reader understanding. Please use these suggestions if you agree. I have also asked some questions that can be addressed in your revision. Reviewer #2: This is a well-written manuscript that contributes greatly to the literature and warrants publication. However, there is heavy discussion and conclusions that the estimated level of contamination was very low or low, whereas the data suggest otherwise. There was no prior establishment of what constitutes 'high' 'moderate' or 'low' levels of contamination, so these conclusions are entirely that of the authors, which have a conflict of interest in the data interpretation to be low. In reality, up to 100% contamination and relatively low Ct (below that of the established infectious dose) suggest there is risk in this ingredient, but the abstract, discussion, and conclusions try to convince the reader otherwise. It would be more appropriate to recognize there are a high number of positive samples with Ct below the known infectious dose, but that it is likely these are nonviable gene fragments that were inactivated during the manufacturing process. ****** 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:** Raymond W. Nims 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-21-33668_reviewer.pdf https://doi.org/10.1371/journal.pone.0259613.r001
Revision 1
23 Feb 2022 Author Response 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Sorry we did not know how to include the funding information in the system. We add this information in the revised version of the manuscript 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. We apologized for the misunderstanding. In fact, we provide the data of all the information included in the manuscript. We clarified this subject in the revised version. 4. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 3 in your text; if accepted, production will need this reference to link the reader to the Table. The Table 3 was already included in the text of the initial submission. Lines 234-235 as Tables 2 and 3. In the new revised version appears in line 236-237 as Table 2 and Table3. 5. 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. Captions have been included for the Supporting information files. We have identified the supplementary tables as: S1 Table -SDPP. Ct values and estimated virus genome presence in SDPP per months during the years 2018-2019. S2 Table – Raw Plasma. Estimated virus genome presence in raw plasma per months during the years 2018-2019. Reviewer’s Comments to the Author Reviewer #1: This manuscript provides valuable information on the presence of genomic information for porcine viruses of concern in spray dried porcine plasma. The estimation of TCID50 based on standard curves is also of interest, as is the % positive sample information for SDPP from different geographical regions. The authors appreciated the comments from Reviewer #1 because confirms our believe that the information provided in the manuscript can be of relevance for the swine sector, not only for understanding the presence of swine viral genome in raw or SDPP but also as a picture of the presence of different viruses in the different geographical areas studied. This information can be of importance for surveillance studies because provide quantitative information about the incidence of the different virus of concern for the swine industry. I have annotated the manuscript with suggestions for improving English language usage and reader understanding. Please use these suggestions if you agree. I have also asked some questions that can be addressed in your revision. The authors appreciated all the grammar suggestions provided by the Reviewer #1. We incorporated all of them in the revised manuscript. Regarding the questions from reviewers #1. Find below the authors response to these questions. Line # 55. “…multiple hurdles that…”. The reviewer is suggesting to change the word “hurdles” by “steps” throughout the manuscript. To the authors, the term “multiple hurdles” is a common term to describe food manufacturing systems as can be found in the publication of Leistner (2000). Int. J. Food Microb. 55:181-186. https://edisciplinas.usp.br/pluginfile.php/128773/mod_resource/content/1/Leistner.2000.pdf Therefore, since this is a usual wording for the field being here studied, the authors prefer to keep the term “multiple hurdles” throughout the article. Line #56. “…SD, 80oC throughout substance…”. How much time? Throughout substance means that the particle achieves 80ºC during the drying process but the time that the particle is at that temperature is unknown and is variable depending on the driers design and capacity. The safety heat treatment is to achieve this 80ºC. It is the same safety concept that in case of meat for human consumption, the heat treatment at a minimum temperature of 80°C, which must be reached throughout the meat but without specifying the time at that temperature (European Council Directive 2002/99/EC. Annex III). Line #56. “…ultraviolet light (UV) treatment (3000 J/L)…”. J/m2 No, the values are correctly expressed as J/L and not J/m2 as suggested by the reviewer #1. We refer to the paper of Blázquez et al., 2019. Reference [17] in the manuscript. In the case of treating liquid plasma, the UV system is a continuous flow reactor designed for exposing a liquid to UV light. Therefore, J/L is the appropriate unit of measure. Line #139. “…After 1.5 hours at 37ºC, inoculum was removed, and 30 mL of medium were added. Titration was done in triplicate obtaining a final titer of 105.48 TCID50/mL.”. Two significant figures is enough. The titration assay is not accurate to three significant figures, I think. The authors did not fully understand the comment from the reviewer #1. If he/she is referring that expressing the virus titer with maximum two decimals and not three as appear in some cases, we corrected this mistake in the revised version of the manuscript. We assume that some were expressed with three significant figures due to the effect of the mathematical mean. We apologize for the mistake and thank you for correcting it. Line #186. “…A viral suspension was obtained and titrated in triplicate, obtaining a final viral solution of 106.64 TCID50 /mL. “ Provide incubation duration, is CPE the endpoint? Sorry, we forgot to provide this information. Thank you for your observation. Once the flasks were inoculated, they were incubated at 37ºC for four days, at which time a clear CPE is observed and the infection was stopped. We add this information on line 188 of the revised manuscript: “After that time, the contents of the tube were transferred to a 175 cm2 flask, in which 40 mL of MEM-E supplemented with 1% pyruvate were added. Inoculated flasks were incubated for four days at 37ºC until CPE was observed. A viral suspension was obtained and titrated in triplicate, obtaining a final viral solution of 106.64 TCID50 /mL. “ Line #197. “… Each kit contained a genome quantified standard for the different vi 195 ruses tested: PRRSV (PRRSV-I dtec-RT-qPCR, PRRSV-II dtec-RT-qPCR), PEDV (PEDV dtec-RT-qPCR), PPV (PPV-1 dtec-RT-qPCR) and SIV (SIV dtec-RT-qPCR).” Add information for PCV? We do not provide information about PCV-2 in this section since in the case of PCV-2 it was not necessary to perform a quantitative PCR again due that the first PCR performed (PCV-2 (LSI VetMAXTM Porcine Circovirus Type 2 Quantification, Thermo Fisher Scientific, MA, USA)) was already quantitative. Line #247. “…effective identification of non-symptomatic animals probably contributed to the absence of SVA genome in the tested SDPP samples.”. How are non-symptomatic animals identified? SVA has been associated with lameness and cutaneous vesicles, as well as increased mortality in the first weeks of age. Therefore, the absence of these clinical signs, at least the ones affecting finishers would be one of the arguments by which SVA might not be detected. On the other hand, animals at abattoir are inspected ante-mortem, and presumably only animals non-displaying clinical signs are sacrificed and used for human consumption. The reviewer is right that non-symptomatic animals is difficult to identified. We changed this paragraph in the revised version of the manuscript as following “Despite SVA infected animals have been sporadically detected on-farm and at abattoirs during ante-mortem inspection [33], effective identification of farm outbreaks and surveillance system in place probably contributed to the absence of SVA genome in the tested SDPP samples” Line #291. “…In all regions, the estimated level of PCV2- was low (<2 log10 TCID50/g SDPP that corresponds to less than 5 virus particles per g of raw plasma), while PPV presence was slightly higher (<2.0 log10 TCID50/g liquid plasma).” This value is the same as that for PCV-2. Am I missing something? The value for PCV-2 was indicated per g of SDPP while the value for PPV was refer to liquid plasma. In the revised version we put both values as g of SDPP. This sentence has been changed in the revised version of the manuscript as following: “In all regions, the estimated level of PCV-2 was <2 log10 TCID50/g SDPP, while PPV presence was <3.0 log10 TCID50/g SDPP.” Line #308. “…It is speculated that differences in stunning method, design of collection trough or slower line speed of abattoirs in Europe and Brazil compared to that in US and Canada may contribute to different levels of SIV contamination.” How? Slower line speed of abattoirs in Europe and Brazil may affect to have higher residence time during the collection period and therefore more time for potential contamination of blood with SIV at the slaughter line. We changed this paragraph in the revised version of the manuscript as following: “…It is speculated that slower line speed of abattoirs in Europe and Brazil compared to that in US and Canada resulting in longer time for blood collection that may contribute to increased levels of SIV contamination.” Line #338. “…The authors have read the journal's policy and the authors of this manuscript have the following competing interests: EB, CR, and JPolo are employed by APC Europe, S.L.U.” JPujols? Dr. Joan Pujols is a researcher of IRTA-CReSA and his never has been employed of APC Europe, S.L.U. or APC companies. Line #584. Table 3. The reviewer #1 is asking why the range of the values of PEDV, PPV and PPRS-US are negative. We appreciate the comment from reviewer #1. As indicated in the title of the Table 3, these values are expressed in log10 and therefore a negative log10 values do not mean that the virus particle is negative, only that is less than 1 particle/g of liquid plasma. For example, log10 -1.76 TCID50/ g liquid plasma in fact, means 0.017 TCID50/g liquid plasma Reviewer #2: This is a well-written manuscript that contributes greatly to the literature and warrants publication. However, there is heavy discussion and conclusions that the estimated level of contamination was very low or low, whereas the data suggest otherwise. There was no prior establishment of what constitutes 'high' 'moderate' or 'low' levels of contamination, so these conclusions are entirely that of the authors, which have a conflict of interest in the data interpretation to be low. In reality, up to 100% contamination and relatively low Ct (below that of the established infectious dose) suggest there is risk in this ingredient, but the abstract, discussion, and conclusions try to convince the reader otherwise. It would be more appropriate to recognize there are a high number of positive samples with Ct below the known infectious dose, but that it is likely these are nonviable gene fragments that were inactivated during the manufacturing process. We appreciated the comment received from the reviewer #2. It is true that the term high, moderate, or low levels of contamination is not defined and is subjective for each expert in the field. However, from the authors, having average values of log10 0.1 for PEDV, 1.56 for PCV-2, -1.4 for PRRSV US strain or 0.17 for PRRSV EU strain are relatively low, especially when compare with values of these viruses as the peak of viremia. Importantly, those values are considered low from the point of view of their potential infectiousness in an animal model. For example, 5.6 × 101 TCID50/g (1.75 log10 TCID50/g) was the minimum PEDV dose in feed to be infective for pigs (Schumacher et al., 2016, Am J Vet Res 77(10):1108-13). For PRRSV USA, a 2-mL inoculum containing 101 fluorescent foci units (equivalent to TCID50) of virus per milliliter was found sufficient to achieve infection by either route, but it was the very minimal dose to get it (Yoon et al., 1999 Vet Res 30(6):629-38). Therefore, all values obtained in plasma at slaughter should be considered within the low range of viral loads, most of them below the infectiousness levels as demonstrated when using one of the most susceptible routes of inoculation, the intraperitoneal one (Blázquez et al., 2019 Vet Microbiol 239:108450). Moreover, the likelihood of infection through the oral route is usually much lower compared to other routes such as drinking water or intranasal/oropharyngeal exposure as demonstrated with ASFV for example (Niederwerder et al., 2021 AnimaIs 11, 792). Therefore, all together would point out to low levels of contamination, also assuming this can be considered a subjective appraisal. In any case, throughout the manuscript we have tried to eliminate this ambiguity of low or high levels of contamination. You can find these changes in the revised version of the manuscript. Finally, although it is true that some co-authors have conflict of interest, all the data from the study is readily available in tables and supplementary material for the readers and it cannot be underestimated that other co-authors signing this manuscript are worldwide very well-recognized virology researchers in the field. Therefore, the authors consider that there is no bias with the information provided in this manuscript. Attachments Attachment Submitted filename: Response to reviewers-R1bis.docx https://doi.org/10.1371/journal.pone.0259613.r002
14 Mar 2022 Decision Letter - Caryn L Heldt, Editor PONE-D-21-33668R1Estimated quantity of swine virus genomes based on quantitative PCR analysis in spray-dried porcine plasma samples collected from multiple manufacturing plants.PLOS ONE Dear Dr. Polo Pozo, 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 Apr 28 2022 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: https://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, Caryn L Heldt, 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): Thank you for addressing the reviewers comments. I ask that you fix the significant figures and add errors for your stock titers. For all of the titers given in the methods, the significant figures are too high and errors are needed. Example, it says for PRRS (3268 EU strain) that the final titer was 10^5.48 TCID50/ml and done in triplicate. An error needs to be associated with the final titer and then the significant digits are so that there is only one significant digit in the error. So, if the error is 10^0.5, then the titer is 10^5.5 ± 10^0.5. The error should also be fixed in Table 2 and Table 3. The error should have one significant digit and this will tell you how many digits you can use in the number reported. See https://www.ruf.rice.edu/~bioslabs/tools/data_analysis/errors_sigfigs.html. Error and significant figures need also be fixed in the supplemental data. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: [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.0259613.r003
Revision 2
23 Mar 2022 Author Response Additional Editor Comments (if provided): Thank you for addressing the reviewers comments. I ask that you fix the significant figures and add errors for your stock titers. For all of the titers given in the methods, the significant figures are too high and errors are needed. Example, it says for PRRS (3268 EU strain) that the final titer was 10^5.48 TCID50/ml and done in triplicate. An error needs to be associated with the final titer and then the significant digits are so that there is only one significant digit in the error. So, if the error is 10^0.5, then the titer is 10^5.5 ± 10^0.5. The authors appreciated the suggestion from the Academic Editor. We have included in the new version of the manuscript the error for the titers in the inoculum. As example the new reported information for the PRRS EU strain (3268EU) inoculum is as follow: ”… final titer of 105.5±0.2 TCID50/mL.” The error should also be fixed in Table 2 and Table 3. The error should have one significant digit and this will tell you how many digits you can use in the number reported. See https://www.ruf.rice.edu/~bioslabs/tools/data_analysis/errors_sigfigs.html. Error and significant figures need also be fixed in the supplemental data. The authors really appreciated the help from the Academic Editor to clarify the request and provide the significant digit for all data included in Table2 and Table 3 and Supplementary Table 1 and Suplementary Table 2. We are deeply in debt for her willing to review all the data in advance to the new submission. The new revised version of the manuscript includes the correct format for all the data in the mentioned tables. Attachments Attachment Submitted filename: Response to reviewers-2.docx https://doi.org/10.1371/journal.pone.0259613.r004
6 Apr 2022 Decision Letter - Caryn L Heldt, Editor Estimated quantity of swine virus genomes based on quantitative PCR analysis in spray-dried porcine plasma samples collected from multiple manufacturing plants. PONE-D-21-33668R2 Dear Dr. Polo Pozo, 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. Thank you for all of your work on the error analysis. 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, Caryn L Heldt, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0259613.r005
Formally Accepted
11 Apr 2022 Acceptance Letter - Caryn L Heldt, Editor PONE-D-21-33668R2 Estimated quantity of swine virus genomes based on quantitative PCR analysis in spray-dried porcine plasma samples collected from multiple manufacturing plants. Dear Dr. Polo Pozo: 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. Caryn L Heldt Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0259613.r006

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