Torquetenovirus in saliva: A potential biomarker for SARS-CoV-2 infection?

Maria C. Mendes-Correa; Tania Regina Tozetto-Mendoza; Wilton S. Freire; Heuder G. O. Paiao; Andrea B. C. Ferraz; Ana C. Mamana; Noely E. Ferreira; Anderson V. de Paula; Alvina C. Felix; Camila M. Romano; Paulo H. Braz-Silva; Fabio E. Leal; Regina M. Z. Grespan; Ester C. Sabino; Silvia F. Costa; Steven S. Witkin

doi:10.1371/journal.pone.0256357

Peer Review History

Original SubmissionFebruary 20, 2021
13 Apr 2021 Decision Letter - Baochuan Lin, Editor PONE-D-21-05788 Torquetenovirus in saliva: a biomarker for progression or resolution of SARS-CoV-2 infection? PLOS ONE Dear Dr. Tozetto Mendoza, 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. I have received the reviews of your manuscript. While your paper addresses an interesting question, the reviewers stated several concerns about your study and did not recommend publication in its present form. One reviewer has concern regarding the sample collection and control population, another reviewer also has concern about the matched control group. Moreover, other reviewers have identified numerous issues where additional experimentation and documentation is needed. Please see the reviewers’ insightful comments for details and these comments need to be addressed carefully. In addition, I would like the authors to address whether human subject research approval is needed for collecting the saliva samples? Also, the figure legend needs more detail in describing the symbols. Additionally the quality of the language needs to be improved, there are quite a few awkward sentences throughout the manuscript. Please have a fluent, preferably native, English-language speaker thoroughly copyedit your manuscript for language usage, spelling, and grammar. Please submit your revised manuscript by May 28 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, Baochuan Lin, 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. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information. 3. Thank you for stating the following financial disclosure: "NO, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." At this time, please address the following queries: Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” If any authors received a salary from any of your funders, please state which authors and which funders. If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [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: Partly Reviewer #2: Partly Reviewer #3: Yes Reviewer #4: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A Reviewer #3: Yes Reviewer #4: 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: No Reviewer #3: Yes Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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: Thanks for the opportunity to review the manuscript. The research idea was novel and attractive. However, the title is not completely compatible with the data and the methods that was provided in the manuscript. I have some comments and questions: 1- About the SARS-CoV-2 sampling, as I found the patients were instructed and did the sampling by themselves, using nasopharyngeal swabs. This method (nasopharyngeal swabs) is a sensitive sampling method but is painful and disgusting. How could you be sure that samples were collected correctly? I mean, how could you be sure that the negative samples were not false negative results? 2- We are in the pandemic of COVID-19, could we assume a symptomatic individual as a control just with a single SARS-CoV-2 negative PCR? 3- It was a good idea to select the controls among symptomatic individuals who had negative SARS-CoV-2 PCR, but we need more information about the cause of disease in controls because an allergic rhinitis would be different from common cold. Moreover, some of the controls could have other strains of corona virus. I afraid the controls are not homogenous. 4- In line 97, you wrote “Several individuals positive for SARS-CoV-2 in their saliva were requested to collect additional saliva samples over a 30-day period.”, what about individuals with negative PCR? 5- About the clinical sign and symptoms, did the patient measured his/her body temperature? If yes, is this a sensitive method to divide patients to febrile and afebrile? 6- Could we have more information about the drug history and background disease of both cases and controls? Because it might affect the TTV viral load. 7- In table 3, you showed that the SARS-CoV-2-positive patients had the highest median TTV viral load on day 8-9 while the viral load was lower in day 6-7, how could you interpret this finding considering the normal immunologic response to SARS-CoV-2 infection? 8- Could you describe, why you divided the study course to days 3-5, 6-7, 8-9, 10-16,…? Why the intervals are not equal? 9- You only provided data about the kinetics of SARS-CoV-2 virus, what about the clinical course of disease in different time points, data on hospital admission, or mortality? 10- About the figure 1, this is a spaghetti plot showing the kinetics of TTV, if each shape is representative for a single patient, I afraid you had a lot of missing data. Could you provide the figure legend with more details about the plot and shapes? 11- You might need to provide the immunologic markers, as well as more information about the clinical course of disease, if you aim to conclude that TTV is a marker of local immune status and as predictors of prognosis. Reviewer #2: In this manuscript, Mendes-Correa et al. focuse on the attempt to demonstrate that TTV level in saliva from COVID-19 patients might be used as biomarker for progression or resolution of SARS CoV-2 infection. Although the topic may be of interest, the way it is treated is disappointing and a number of issues makes weak the manuscript. Overall, in the current form, the manuscript does not reach the quality standard needed for publication Reviewer #3: Interesting study from a single center cohort about the association of TTV titer in saliva with SARS-CoV-2 status. 1) the major limitation is already discussed in the manuscript: the SARS-CoV-2 negative group - all with "mild respiratory symptoms" was not further characterized. Especially testing with a multiplex-PCR for other respiratory viruses would help to interpret the data in the manuscript, especially since TTV levels seem to stay unchanged in the SARS-CoV-negative group day 10-16 and >16 in contrast to the SARS-CoV-2 positive group. If saliva samples are still available, I would recommend performing these analyses in both groups. The manuscript could be further improved if a matched control group without respiratory symptoms would also be tested for TTV in saliva. 2) line 49: it is true that TTV has been described as an indicator of immune function mainly after organ transplantation, but there is also literature on TTV as a marker of immune function in chronic diseases such as liver cirrhosis or HIV. This could be briefly elaborated by the authors to show that looking at TTV levels could also have a value outside of organ transplantation. 3) line 53/54: should be probably ref. 6 (TTV in HSCT recip.); "predicting lymphocyte status" is somewhat obscure to me, would suggest immune function, immunocompetence or immunreconstitution. 4) line 191: it would be helpful to provide the number of subjects with more than one saliva sample, as this is rather small and therefore a cautious interpretation is required 5) line 203: would remove "anti-SARS-CoV-2" as no rationale is provided that TTV level reflects specific anti-SARS-CoV-2 immune activity - considering the literature, it is more likely a marker of overall immunocompetence Reviewer #4: The authors describe the evolution of torque tenovirus viral load in saliva related to Sars-CoV 2 viral load in nasopharyngeal swabs, in an effort to find markers for immunosuppression. They also compare TTV levels at different times post initiation of symptoms for the covid 19 population (mild covid) and a control population with a mild respiratory disease. Second, the authors show in a subpopulation of the covid 19 group that TTV viral load decreases with the disparition of symptoms. As a consequence, they consider that TTV viral load in saliva may be suitable to describe rhinopharyngeal local immune response. And they refer to studies comparing blood TTV viral load and evolution of immunity /rejection in transplant patients. The idea is interesting, because simple tests to evaluate local immunity are very important to identify potential patients that could evolve toward high viral loads and covid worsening. The data concerning the evolution of TTV viral load in relation to SARS-CoV 2 viral load is convincing. This is, to my knowledge, the first paper describing TTV load in saliva for covid patients . Although, the paper suffers from some weaknesses that could be overcome: First : the authors did not justify the use of saliva instead of the Nasopharyngeal samples for TTV viral load.Although they refer to its use in previous comparisons with plasma levels in transplant patients to predict global immunity which is very different. They use the same extraction method . We understand of course that saliva is easier and potentially more reproducible for auto collection, but the TTV load values, in saliva, are highly variable in both populations and as NOP sampling was performed, they could have compared TTV load in both samples, to comfort their hypothesis. Second : The large panel of viralloads at each time, coming from the fact that there was only one sample per patient and thus at each time we compare different patients make the group comparison less significant, the authors should comment on this and provide a graph in addition to the table to show the repartition of viral loads within time in both groups. Third : The lowering of TTV viral load from sequential samples in the covid patients cannot be compared with that of the non-covid group. In addition we do onot have the diagnosis (VRS may be different from rhinovirus for example in terms of local immunity, samely pharyngitis or thracheitis may be different from coryza?) Theu could add the diagnosis if available. Fourth : The authors have choosen a mild covid group for a better comparison with mild respiratory disease. Could they correlate the initial TTV load or the maximum TTV load , with the duration of symptoms , or the delay to SARS-CoV 2 undetectability? This could be very interesting to predict persistent infections. Minor comments : some text errors or form errors are present in the text and bibliography, and a cautious review is necessary before submission. ****** 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:** Omid Rezahosseini Reviewer #2: No Reviewer #3: No Reviewer #4: 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.0256357.r001
Revision 1
7 Jun 2021 Author Response Baochuan Lin, Ph.D. Academic Editor, PLOS ONE - PONE-D-21-05788R1 Dear Dr. Lin, We thank you and the reviewers for the positive and constructive comments on our manuscript. The individual comments and our responses are listed below. Reviewer #1: Thanks for the opportunity to review the manuscript. The research idea was novel and attractive. However, the title is not completely compatible with the data and the methods that was provided in the manuscript. I have some comments and questions: Answer: The title has been revised and now reads: Torquetenovirus in saliva: a potential biomarker for SARS-CoV-2 infection? 1- About the SARS-CoV-2 sampling, as I found the patients were instructed and did the sampling by themselves, using nasopharyngeal swabs. This method (nasopharyngeal swabs) is a sensitive sampling method but is painful and disgusting. How could you be sure that samples were collected correctly? I mean, how could you be sure that the negative samples were not false negative results? Answer: As stated in the text, subjects were given a detailed video that clearly demonstrated the correct way to collect samples. Follow-up of subjects confirmed that all negative assays were truly negative. We included the term “nasal-oropharyngeal swab sample” in order to clarify the appropriated procedure which we used in this study. 2- We are in the pandemic of COVID-19, could we assume a symptomatic individual as a control just with a single SARS-CoV-2 negative PCR? Answer: As mentioned above we have had follow-up contact with all negative controls to verify that they were not infected. We also included a sentence in the lines 111 to 113 to clarify such aspect. 3- It was a good idea to select the controls among symptomatic individuals who had negative SARS-CoV-2 PCR, but we need more information about the cause of disease in controls because an allergic rhinitis would be different from common cold. Moreover, some of the controls could have other strains of corona virus. I afraid the controls are not homogenous. Answer: We thank the reviewer for this comment that requires clarification. The totality of symptoms in each of the controls were characteristic of the common cold. Those with allergic rhinitis were excluded. This is now stated in Methods (in the lines 85 to 86). We did not test for the presence of other coronavirus strains in the controls. Their presence was unlikely since the prevalence of these viruses is rare in our population. 4- In line 97, you wrote “Several individuals positive for SARS-CoV-2 in their saliva were requested to collect additional saliva samples over a 30-day period.”, what about individuals with negative PCR? Answer: We only collected additional samples from SARS-CoV-2-positive patients. It probably would have been optimal to also collect additional samples from the controls. However, it was apparent that there was no clinical indication of symptom worsening or the presence of Covid in this group. 5- About the clinical sign and symptoms, did the patient measured his/her body temperature? If yes, is this a sensitive method to divide patients to febrile and afebrile? Answer: Body temperature was self-collected and, we agree, may not always be accurate. However, this was not the only parameter evaluated. 6- Could we have more information about the drug history and background disease of both cases and controls? Because it might affect the TTV viral load. Answer: We have now added to the text in Methods that none of the subjects used antibiotics or other medications in the two weeks prior to sample collection (in the lines 87 and 88). In addition, they were all healthy with no underlying pathology. 7- In table 3, you showed that the SARS-CoV-2-positive patients had the highest median TTV viral load on day 8-9 while the viral load was lower in day 6-7, how could you interpret this finding considering the normal immunologic response to SARS-CoV-2 infection? Answer: The viral loads on days 6-7 and 8-9 are very similar and statistically indistinguishable. 8- Could you describe, why you divided the study course to days 3-5, 6-7, 8-9, 10-16,…? Why the intervals are not equal? Answer: The interval was chosen to have approximately the same number of subjects at each time point. We believed that this provided the most reliable indication of changes. 9- You only provided data about the kinetics of SARS-CoV-2 virus, what about the clinical course of disease in different time points, data on hospital admission, or mortality? Answer: As stated in the text, this is a study of patients with mild symptoms that resolved during the study period. None required hospitalization admission. 10- About the figure 1, this is a spaghetti plot showing the kinetics of TTV, if each shape is representative for a single patient, I afraid you had a lot of missing data. Could you provide the figure legend with more details about the plot and shapes? Answer: The figure legend has now been implemented to state more explicitly that the graph measures the TTV titer in saliva at each time point for each subject where more than one sample was obtained (in the lines 213 to 216). 11- You might need to provide the immunologic markers, as well as more information about the clinical course of disease, if you aim to conclude that TTV is a marker of local immune status and as predictors of prognosis. Answer: Immune markers were not measured in these subjects and so we only speculate that the reduction in TTV as a predictor of prognosis occurred at least partially by a mechanism of immune activation. This was based on other studies relating TTV titer to immune system status. We have now clarified in Discussion that an immune-mediated mechanism was not directly tested and that our findings are consistent with an increased overall immune competence (in the lines 252 and 253). Reviewer #2: In this manuscript, Mendes-Correa et al. focuse on the attempt to demonstrate that TTV level in saliva from COVID-19 patients might be used as biomarker for progression or resolution of SARS CoV-2 infection. Although the topic may be of interest, the way it is treated is disappointing and a number of issues makes weak the manuscript. Overall, in the current form, the manuscript does not reach the quality standard needed for publication Answer: We appreciate the reviewer’s concerns and trust that our responses to the specific comments pf the other reviewers will alleviate the reviewer’s concerns. Reviewer #3: Interesting study from a single center cohort about the association of TTV titer in saliva with SARS-CoV-2 status. 1) the major limitation is already discussed in the manuscript: the SARS-CoV-2 negative group - all with "mild respiratory symptoms" was not further characterized. Especially testing with a multiplex-PCR for other respiratory viruses would help to interpret the data in the manuscript, especially since TTV levels seem to stay unchanged in the SARS-CoV-negative group day 10-16 and >16 in contrast to the SARS-CoV-2 positive group. If saliva samples are still available, I would recommend performing these analyses in both groups. The manuscript could be further improved if a matched control group without respiratory symptoms would also be tested for TTV in saliva. Answer: Unfortunately, the subjects in this study were not tested for other respiratory viruses and the samples are no longer available for this purpose. As the reviewer states, and as we acknowledged in Discussion, this as a study limitation. While it would certainly be of interest to determine the prevalence and titer of TTV in healthy individuals, and this is planned for the near future, that information is not essential for data analysis in the present investigation. 2) line 49: it is true that TTV has been described as an indicator of immune function mainly after organ transplantation, but there is also literature on TTV as a marker of immune function in chronic diseases such as liver cirrhosis or HIV. This could be briefly elaborated by the authors to show that looking at TTV levels could also have a value outside of organ transplantation. Answer: We thank the reviewer for this suggestion and have now added the additional information to Introduction with references (in the lines 55 to 57). 3) line 53/54: should be probably ref. 6 (TTV in HSCT recip.); "predicting lymphocyte status" is somewhat obscure to me, would suggest immune function, immunocompetence or immunreconstitution. Answer: The text has been changed as suggested to now read “immunocompetence” and reference 8 has been added (in the line 61). 4) line 191: it would be helpful to provide the number of subjects with more than one saliva sample, as this is rather small and therefore a cautious interpretation is required Answer: The number of subjects with more than one saliva sample is evident from Figure 1. We have now added this, 10 subjects, to the text as suggested (in the lines 213 to 216). 5) line 203: would remove "anti-SARS-CoV-2" as no rationale is provided that TTV level reflects specific anti-SARS-CoV-2 immune activity - considering the literature, it is more likely a marker of overall immunocompetence Answer: We agree, and has been stated in comment 11 for Reviewer #1, a mechanism revolving around altered immune status was speculative and not based on collection of immune-related parameters. The term “anti-SARS-CoV-2” has been deleted. Reviewer #4: The authors describe the evolution of torque tenovirus viral load in saliva related to , 10Sars-CoV 2 viral load in nasopharyngeal swabs, in an effort to find markers for immunosuppression. They also compare TTV levels at different times post initiation of symptoms for the covid 19 population (mild covid) and a control population with a mild respiratory disease. Second, the authors show in a subpopulation of the covid 19 group that TTV viral load decreases with the disparition of symptoms. As a consequence, they consider that TTV viral load in saliva may be suitable to describe rhinopharyngeal local immune response. And they refer to studies comparing blood TTV viral load and evolution of immunity /rejection in transplant patients. The idea is interesting, because simple tests to evaluate local immunity are very important to identify potential patients that could evolve toward high viral loads and covid worsening. The data concerning the evolution of TTV viral load in relation to SARS-CoV 2 viral load is convincing. This is, to my knowledge, the first paper describing TTV load in saliva for covid patients . Answer: We thank the Reviewer for acknowledging our unique hypothesis regarding TTV analysis in Covid patients and its eventual clinical potential. Although, the paper suffers from some weaknesses that could be overcome: First : the authors did not justify the use of saliva instead of the Nasopharyngeal samples for TTV viral load. Although they refer to its use in previous comparisons with plasma levels in transplant patients to predict global immunity which is very different. They use the same extraction method . We understand of course that saliva is easier and potentially more reproducible for auto collection, but the TTV load values, in saliva, are highly variable in both populations and as NOP sampling was performed, they could have compared TTV load in both samples, to comfort their hypothesis. Answer: We thank the reviewer for this comment. Unfortunately, the NOP samples were not available for TTV testing when the study was designed. Second : The large panel of viral loads at each time, coming from the fact that there was only one sample per patient and thus at each time we compare different patients make the group comparison less significant, the authors should comment on this and provide a graph in addition to the table to show the repartition of viral loads within time in both groups. Answer: We agree with the Reviewer that the use of saliva from different subjects at different time points is not optimal, and this is acknowledged in the Discussion as a study limitation as requested. We disagree with the Reviewer that in addition to the Table we should also provide a graph. We think this is unnecessarily repetitious and does not provide any additional information. Third : The lowering of TTV viral load from sequential samples in the covid patients cannot be compared with that of the non-covid group. In addition we do onot have the diagnosis (VRS may be different from rhinovirus for example in terms of local immunity, samely pharyngitis or thracheitis may be different from coryza?) Theu could add the diagnosis if available. Answer: We responded to similar comments by Reviewer 1, comment #3 and Reviewer #3, comment 1 and agree that the control group might possibly be heterogeneous. We did not test the control subjects for the possible presence of different rhinoviruses as acknowledged as a study limitation in Discussion. Based solely on their symptoms and recovery time without treatment it appeared most likely that they had the common cold, and not an allergic rhinitis or a bacterial pharyngitis or tracheitis. We have now added a comment in Methods to further clarify this limitation. Fourth : The authors have choosen a mild covid group for a better comparison with mild respiratory disease. Could they correlate the initial TTV load or the maximum TTV load , with the duration of symptoms , or the delay to SARS-CoV 2 undetectability? This could be very interesting to predict persistent infections. Answer: We thank the Reviewer for this very constructive suggestion. Unfortunately, in our study these additional comparisons were not possible. Our sample size was too small to perform this analysis with a high degree of validity. Minor comments : some text errors or form errors are present in the text and bibliography, and a cautious review is necessary before submission. Answer: We apologize for these errors and have now corrected them in the revision. We are available to send you any further information you may require. Yours faithfully, Tania Regina Tozetto-Mendoza and co-authors. Attachments Attachment Submitted filename: PLoS One response to reviewers_03_05_21.docx https://doi.org/10.1371/journal.pone.0256357.r002
8 Jul 2021 Decision Letter - Baochuan Lin, Editor PONE-D-21-05788R1 Torquetenovirus in saliva: a potential biomarker for SARS-CoV-2 infecton? PLOS ONE Dear Dr. Tozetto Mendoza, 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. The revised manuscript has addressed most of the reviewers' comments, however, the reviewers still have concerns that need to be addressed. Please see reviewers' comments below. Please submit your revised manuscript by Aug 22 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, Baochuan Lin, 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. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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 #3: 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: No Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: Yes ****** 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: No Reviewer #3: 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 #3: 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: Thank you for having addressed my comments. Although some of the responses could not satisfy me. For example about the response to comment 3, you responded, "We did not test for the presence of other coronavirus strains in the controls. Their presence was unlikely since the prevalence of these viruses is rare in our population.". As I can see in literature, prevalence of human coronaviruses (HCoVs) is about 11.5% (https://doi.org/10.1590/S1517-83822013000100049). Although the kinetics of TTV could be a possible marker of local immunity, but considering your results, it is difficult to predict prognosis using TTV. Reviewer #3: While the lack of testing for other respiratory viruses and healthy controls somewhat limit the scientific value, the data should be still of interest for the scientific community. ****** 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:** Omid Rezahosseini Reviewer #3: 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.0256357.r003
Revision 2
17 Jul 2021 Author Response RESPONSE TO REVIEWERS Baochuan Lin, Ph.D. Academic Editor PLOS ONE Dear Dr. Lin: Thank you and the reviewers for the responses to our revised manuscript, PONE-D-21-05788R1, “Torquetenovirus in saliva: a potential biomarker for SARS-CoV-2 infection?” The remaining reviewer comments and our responses are listed below. Reviewer #1 "About the response to comment 3, you responded, "We did not test for the presence of other coronavirus strains in the controls. Their presence was unlikely since the prevalence of these viruses is rare in our population". As I can see in literature, prevalence of human coronaviruses (HCoVs) is about 11.5% (https://doi.org/10.1590/S1517-83822013000100049)." Answer: We appreciate the reviewer pointing out to us this relevant publication that we unfortunately had not previously seen. It is now cited in the revised text and included in the Reference section (reference 25, lines 341-343). The Discussion section has been extensively revised to more accurately reflect our observations and study limitations, as well as to bolster our argument that other viruses that may have been present in the nasopharynx likely did not have an appreciable influence on our findings. A portion of the revised Discussion is presented here: “Other viruses (reference 24), including different coronavirus species (reference 25), may also have been present in the nasopharynx of our study population. Thus, the possible influence of viral co-infections on SARS-C0V-2-related salivary TTV levels remains undetermined. We would stress, however, that the uniform responses in the SARS-CoV-2 positive and negative patients suggest that any influence of other co-infections is likely to be relatively minor. A parallel diminution in TTV titer over time after resolution of respiratory symptoms was not observed in the SARS-CoV-2-negative individuals. This strongly suggests that immune alterations associated with a mild respiratory tract infection are insufficient to have a noticeable influence on TTV levels in saliva”. Reviewer #1: Although the kinetics of TTV could be a possible marker of local immunity, but considering your results, it is difficult to predict prognosis using TTV. Answer: We agree with the reviewer that quantitation of TTV in saliva may not ultimately prove to be a sensitive indicator of SARS-CoV-2 prognosis. Our inclusion of a question mark at the end of the title of our manuscript signifies this possibility. It is our strong belief, however, that this possibility is worthy of further investigation and that publication of our observations will bring this possibility to the attention of the research community. We also believe that our revised Discussion further supports our argument that the kinetics of TTV is a valid maker of local immunity (in the manuscrip is in the lines 228 to 334 and lines 250 to 260). Reviewer #3 While the lack of testing for other respiratory viruses and healthy controls somewhat limit the scientific value, the data should be still of interest for the scientific community. Answer: We thank the reviewer for acknowledging the interest of our findings for the scientific community. Please see the comments to Reviewer #1 for our revisions that we believe further increase their scientific value. We are available to send you any further information you may require. Yours faithfully, Tania Regina Tozetto-Mendoza and co-authors Attachments Attachment Submitted filename: RESPONSE TO REVIEWERS_D-21-05788R1_PLoS One comments.docx https://doi.org/10.1371/journal.pone.0256357.r004
22 Jul 2021 Decision Letter - Baochuan Lin, Editor PONE-D-21-05788R2 Torquetenovirus in saliva: a potential biomarker for SARS-CoV-2 infecton? PLOS ONE Dear Dr. Tozetto Mendoza, 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. The revised manuscript is scientifically sound, however, the quality of the language needs to be improved since PLoS ONE does not perform copyediting of manuscripts at any later stage in the publication process.<o:p></o:p> We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.<o:p></o:p> Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free. Please submit your revised manuscript by Sep 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, Baochuan Lin, 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. [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.0256357.r005
Revision 3
27 Jul 2021 Author Response Response to the editor comment Baochuan Lin, Ph.D. Academic Editor PLOS ONE Dear Dr. Lin: Thank you for suggestion to the manuscript, PONE-D-21-05788R2, “Torquetenovirus in saliva: a potential biomarker for SARS-CoV-2 infection?” Our version of the manuscript PONE-D-21-05788R2 was reviewed by an experienced scientist whose native language is English, professor emeritus Steven S. Witkins. Yours faithfully, Tania Regina Tozetto-Mendoza and co-authors Attachments Attachment Submitted filename: RESPONSE TO REVIEWERS_D-21-05788R1_PLoS One comments.docx https://doi.org/10.1371/journal.pone.0256357.r006
28 Jul 2021 Decision Letter - Baochuan Lin, Editor PONE-D-21-05788R3 Torquetenovirus in saliva: a potential biomarker for SARS-CoV-2 infecton? PLOS ONE Dear Dr. Tozetto Mendoza, 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. The revised manuscript has shown significant improvement, however, there quite a few things that still need edition/clarification. Specific comments: 1. Line 37 & 38, suggest changing "more frequent" to "more prevalent". 2. Line 42, suggest changing "...TTV was initially present the TTV..." to "TTV was initially present, the TTV..." 3. Line 68, suggest adding "," after resolved. 4. Line 80, please use the same font. 5. Line 83 - 84, suggest deleting "Ethical approval was given to the project." Redundant statement. 6. Line 83, suggest changing "...informed written consent." to "...written informed consent." 7. Line 84, suggest changing "...a convenience sample..." to "...samples..." 8. Line 88 - 89, this sentence is awkward, please rephrase for clarity. 9. Line 90, suggest changing "They were all healthy with no underlying pathology." to "All subjects have no underlying condition." 10. Line 236, correct "All Individuals..." to "All individuals..." 11. Line 250, correct "An Alternate..." to "An alternate..." Please submit your revised manuscript by Sep 11 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, Baochuan Lin, 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): [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.0256357.r007
Revision 4
30 Jul 2021 Author Response RESPONSE TO REVIEWERS - PONE-D-21-05788R4 Baochuan Lin, Ph.D. Academic Editor PLOS ONE Dear Editor, Thank you for the specific comments to our revised manuscript, PONE-D-21-05788R3, “Torquetenovirus in saliva: a potential biomarker for SARS-CoV-2 infection?” All changes were marked in blue color in the revised manuscript to each point raised by the academic editor. We apologize for these errors and have now corrected them in the revision. Yours faithfully, Tania Regina Tozetto-Mendoza, PhD Laboratory of Virology, Institute of Tropical Medicine School of Medicine, University of São Paulo, Brazil E-mail: tozetto@usp.br July, 30, 2021 Attachments Attachment Submitted filename: RESPONSE TO REVIEWERS - PONE-D-21-05788R4.docx https://doi.org/10.1371/journal.pone.0256357.r008
5 Aug 2021 Decision Letter - Baochuan Lin, Editor Torquetenovirus in saliva: a potential biomarker for SARS-CoV-2 infecton? PONE-D-21-05788R4 Dear Dr. Tozetto Mendoza, 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, Baochuan Lin, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0256357.r009
Formally Accepted
16 Aug 2021 Acceptance Letter - Baochuan Lin, Editor PONE-D-21-05788R4 Torquetenovirus in saliva: a potential biomarker for SARS-CoV-2 infection? Dear Dr. Tozetto-Mendoza: 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. Baochuan Lin Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0256357.r010

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .