Consecutive deletions in a unique Uruguayan SARS-CoV-2 lineage evidence the genetic variability potential of accessory genes

Yanina Panzera; Lucía Calleros; Natalia Goñi; Ana Marandino; Claudia Techera; Sofía Grecco; Natalia Ramos; Sandra Frabasile; Gonzalo Tomás; Emma Condon; María Noel Cortinas; Viviana Ramas; Leticia Coppola; Cecilia Sorhouet; Cristina Mogdasy; Héctor Chiparelli; Juan Arbiza; Adriana Delfraro; Ruben Pérez

doi:10.1371/journal.pone.0263563

Peer Review History

Original SubmissionOctober 8, 2021
7 Jan 2022 Decision Letter - Herman Tse, Editor PONE-D-21-32425Consecutive deletions in a unique Uruguayan SARS-CoV-2 lineage evidence the genetic variability potential of accessory genesPLOS ONE Dear Dr. Perez, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. As pointed out by the reviewer, while the technical analysis and writing are done very well, there is an overabundance of speculative discussion in the manuscript. It would take much more data and analysis to untangle the combined effect of viral fitness, population genetics, and public health measures on the spread and prevalence of particular SARS-CoV-2 lineages in this pandemic. 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If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: 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: In this manuscript, the authors present evidence of circulation of strains with 2 deletions within the ORF7a gene in 2020. They speculate that the functional consequence of these deletions is the expression of a putative ORF7ab gene product. ORF7 deletions in SARS-CoV-2 (even to the extent of complete absence) have been reported from various parts of the world. The significance of such deletions on SARS-CoV-2 transmissibility or clinical manifestations is not known. The methodology of this study is clear and the results are convincing. The epidemiological and clinical significance of these ORF7-del variants is uncertain. But, I think there is considerable speculation in parts of the manuscript that needs to be revised. Specific comments below: - Please cite other papers describing ORF7b deletions: Tse H et al, Emergence of a Severe Acute Respiratory Syndrome Coronavirus 2 virus variant with novel genomic architecture in Hong Kong, Clin Infect Dis; Mazur-Panasiuk N et al, Expansion of a SARS-CoV-2 Delta variant with an 872 nt deletion encompassing ORF7a, ORF7b and ORF8, Poland, July to August 2021, Euro Surveillance. - How many samples were analyzed in total to obtain the few with 12-nt deletion in table 1? Unable to gauge how common this variant is. - Any epidemiological link between patients listed in table 1? - Were any of the patients listed in table 1 immunocompromised? - "This potential functionality could explain why the sizeable Δ12+68 change could be transmitted at least between four individuals." this seems to imply that these four patients transmitted this variant between each other, which I don't think is the case? Please clarify. - The delta-68 deletion could also have occurred within 4 hosts independently (intra-host evolution) without being transmitted? So, if there is no epidemiological link between these 4 cases, the authors should not speculate on the transmissibility of this variant. - "The topology of the phylogenetic tree implies that the Δ12 variant emerged early in the N.7 lineage during its initial differentiation in Uruguay (the first case was reported in July 2020); the Δ12+68 variant emerged subsequently by the acquisition of a second deletion (Δ68) from a Δ12 individual." I agree this is probably most likely, but any possibility of under-sequenced communities or provinces where an earlier emergence of the Δ68 might have been missed? - I find the discussion on structure and function of the putative ORF7ab gene product to be very speculative (line 280 onwards). Should be edited and condensed heavily to avoid speculations (such as "the Δ12 mutation in ORF7a could be slightly deleterious and temporarily tolerated in a low-competitive population; after acquiring the additional deletion (Δ68)"). Instead, just state the type of experiments that could be performed to determine effects of deletions observed. - Should add a section on limitations of the study. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [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.0263563.r001
Revision 1
20 Jan 2022 Author Response The following comments were uploaded as "Response to Reviewers" file. Reviewer #1: In this manuscript, the authors present evidence of circulation of strains with 2 deletions within the ORF7a gene in 2020. They speculate that the functional consequence of these deletions is the expression of a putative ORF7ab gene product. ORF7 deletions in SARS-CoV-2 (even to the extent of complete absence) have been reported from various parts of the world. The significance of such deletions on SARS-CoV-2 transmissibility or clinical manifestations is not known. The methodology of this study is clear and the results are convincing. The epidemiological and clinical significance of these ORF7-del variants is uncertain. But, I think there is considerable speculation in parts of the manuscript that needs to be revised. Specific comments below: Please cite other papers describing ORF7b deletions: Tse H et al, Emergence of a Severe Acute Respiratory Syndrome Coronavirus 2 virus variant with novel genomic architecture in Hong Kong, Clin Infect Dis; Mazur-Panasiuk N et al, Expansion of a SARS-CoV-2 Delta variant with an 872 nt deletion encompassing ORF7a, ORF7b and ORF8, Poland, July to August 2021, Euro Surveillance. Answer: We thank the reviewer for pointing out these two important papers; both references were added to the manuscript discussion. - How many samples were analyzed in total to obtain the few with 12-nt deletion in table 1? Unable to gauge how common this variant is. Answer: We screened 51 samples that were available in our Uruguayan biobank of positive samples (this information was added to the manuscript). Therefore, the frequency of the double deletion is 13/51 (approximately 25%). However, we recognize that this number could be biased because we do not have access to all the samples from Uruguay. Therefore, we added a sentence about frequency in the manuscript and clarified this possible bias in the "limitation of the study" statement. - Any epidemiological link between patients listed in table 1? Answer: In the study period, the epidemic in Uruguay was characterized by different outbreaks, with an average of about 20 new COVID-19 cases per day. Because of the reduced number, sixty-seven percent of patients had a contact record with a confirmed case in the national territory, and thirteen percent had known contact with a traveler (https://www.gub.uy/ministerio-salud-publica/comunicacion/noticias/informe-epidemiologico-covid-19-del-30-noviembre-2020). The genetic homogeneity of the samples also supports a few transmission clusters. We obtained and added new data to Table 1, evidencing the epidemiological link among some patients. Regretfully, this epidemiological data were not available for all patients. Therefore, we added a sentence clarifying this point and included the bias in the limitation of the study section. - Were any of the patients listed in table 1 immunocompromised? Answer: We could obtain only detailed clinical information of the two patients that required admission to the intensive care unit. Both have preexisting conditions, one had a cerebrovascular stroke, and the other had cancer. Unfortunately, these patients died in the health center. These data were included in Table 1 and the manuscript's text. The data of the other patients did not indicate evidence of immunosuppression. - "This potential functionality could explain why the sizeable Δ12+68 change could be transmitted at least between four individuals." this seems to imply that these four patients transmitted this variant between each other, which I don't think is the case? Please clarify. Answer: Two of the four Δ12+68 individuals are from the same family (new data added to Table 1). The four individuals were likely part of one transmission cluster, but the referee is right that we cannot establish the exact connection among them. Therefore, we removed this sentence from the discussion. - The delta-68 deletion could also have occurred within 4 hosts independently (intra-host evolution) without being transmitted? So, if there is no epidemiological link between these 4 cases, the authors should not speculate on the transmissibility of this variant. Answer: We obtained the information that two cases with the Δ12+68 variant were epidemiologically closely related (new data in Table 1). These patients were part of one of the few transmission clusters in Uruguay during that period; we added this information in the results. - "The topology of the phylogenetic tree implies that the Δ12 variant emerged early in the N.7 lineage during its initial differentiation in Uruguay (the first case was reported in July 2020); the Δ12+68 variant emerged subsequently by the acquisition of a second deletion (Δ68) from a Δ12 individual." I agree this is probably most likely, but any possibility of under-sequenced communities or provinces where an earlier emergence of the Δ68 might have been missed? Answer: The emergence of Δ12 before Δ68 is supported by: i) the few cases occurring in the period or analysis, ii) the fact that Δ12 appeared two months earlier, and iii) the double-deleted samples were genetically homogenous and epidemiologically related (at least two of the four cases). Furthermore, considering that all N7 we analyzed have the Δ12 deletion, the later emergence of Δ68 seems more parsimonious. We agree with the reviewer that there is a chance of a different scenario, but our data does not support it. - I find the discussion on structure and function of the putative ORF7ab gene product to be very speculative (line 280 onwards). Should be edited and condensed heavily to avoid speculations (such as "the Δ12 mutation in ORF7a could be slightly deleterious and temporarily tolerated in a low-competitive population; after acquiring the additional deletion (Δ68)"). Instead, just state the type of experiments that could be performed to determine effects of deletions observed. Answer: We reduced and moderated the tone of the discussion from line 280 onwards. We highlighted in yellow the paragraphs that were modified in the discussion section. Thanks for the suggestion; we believe the discussion is now clearer and concise. - Should add a section on limitations of the study. Answer: We added a statement in the discussion commenting on the limited number of samples due to Uruguay's low prevalence that underpowers statistical analysis (i.e., frequency of cases). Our analysis, therefore, represents a snapshot in time before the emergence of variants of concern that replace the N.7 lineage. Another limitation was the method of sampling. Individuals submitted for the report may not fully reflect the prevalence of infections in the overall population. Nevertheless, the data obtained were from different geographic origins, and the sample number can be considered relatively representative. Finally, another potential limitation of the study may be the lack of functional studies to conclude the virus fitness. In Uruguay, we limited our analysis to genetic variability because we do not have facilities to culture the virus but recognize the need for these studies. Despite the above limitations, we believe that our research results contribute to understanding indels origin and prevalence by providing new insights into the genome variability and dynamics of accessory ORFs within SARS-CoV-2 lineages. Editor comments As pointed out by the reviewer, while the technical analysis and writing are done very well, there is an overabundance of speculative discussion in the manuscript. It would take much more data and analysis to untangle the combined effect of viral fitness, population genetics, and public health measures on the spread and prevalence of particular SARS-CoV-2 lineages in this pandemic. We appreciate that these hypotheses were presented sensibly, but politely request that the discussions from line 280 onwards be scaled back to strike a more balanced tone. Please also address the other points raised by the reviewer below. Answer: We thank the editor for the positive comments. Following the suggestions, we reduced the most speculative part of the discussion and moderated some sentences' tone (highlighted in yellow). 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. Answer: References were checked and modified accordingly. Additional changes Typos and spelling mistakes were corrected throughout the manuscript. References suggested by the reviewers were included, and figures and tables were checked https://doi.org/10.1371/journal.pone.0263563.r002
24 Jan 2022 Decision Letter - Herman Tse, Editor Consecutive deletions in a unique Uruguayan SARS-CoV-2 lineage evidence the genetic variability potential of accessory genes PONE-D-21-32425R1 Dear Dr. Perez, 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, Herman Tse Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0263563.r003
Formally Accepted
8 Feb 2022 Acceptance Letter - Herman Tse, Editor PONE-D-21-32425R1 Consecutive deletions in a unique Uruguayan SARS-CoV-2 lineage evidence the genetic variability potential of accessory genes Dear Dr. Pérez: 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. Herman Tse Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0263563.r004

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