The Myotis chiloensis Guano Virome: Viral Nucleic Acid Enrichments for High-Resolution Virome Elucidation and Full Alphacoronavirus Genome Assembly
Round 1
Reviewer 1 Report
In the manuscript entitled “The Myotis chiloensis guano virome: viral nucleic acid enrichment for high resolution virome elucidation and full alphacoronavirus genome assembly”, S Aguilar Pierlé et al described a viral enrichment method which allowed them to get more than 90% taxa of viral origin from guano samples, from which they could characterize a new alphacoronavirus carried by the Myotis chiloensis bat.
The manuscript is well written, the need for epidemiological surveillance for potential zoonotic agents at the human-animal interface is particularly well explained. The authors have dealed with the issues of complex sample virome deep sequencing, and found a set of methods which in a body solved the problem.
This work is very interesting because most of the time, viral metagenomic studies of complex/environmental samples result in only a few percents of viral raw reads, and one can think that we are probably missing many things. So this work deserves attention, and more the authors have produced a new sequence that they could assemble as a full-length genome of an alphacoronavirus.
Some modifications are however necessary to improve the quality of the manuscript.
Major comment 1: The authors should specify several details of the viral enrichment protocol:
Viral particle concentration was reached with several concatenated centrifugations and treatment with DNAse/RNAse before the RNA extraction step.
- Specify the ratio PBS/guano in vol/vol because 1:5 is not clear, and the starting volume was never known
- Define what is the volume of the original sample from step 1
- After treatment DNAse/RNAse/Turbonuclease, EDTA and heat inactivated the samples or the enzymes ?
Major comment 2: The phylogenetic analysis should be improved
- The authors specified the alignment parameters but they did not tell us which tool was used for the alignment (Mega, Seaview, Mafft.. any other). The alignment is the starting point of any phylogenetic analysis and this step is very important. Mafft in line is very efficient.
- The phylogenetic tree was generated using the NJ method with the JC nucleotide substitution model, both of which are outdated. I strongly suggest the authors to use IQ-Tree tool web server, which automatically will select the best model and will generate a maximum likelihood tree with strong branch supports.
Minor comments
- Page 7/15, line 181: (Tables 3) should be replaced with Table 1
- Page 9/15, line 212: alphacoroniviruses should be alphacoronaviruses
- Page 11/15, line 238: aetacoronavirus should be betacoronavirus
- Page 1115, line 255: outside of the scope should be outside the scope
Author Response
To Editor VIRUSES
Santiago, 30rd of December, 2021
Dear Denisa Cimpian,
With the present letter we are submitting our revised manuscript “The Myotis chiloensis guano virome: viral nucleic acid enrichment for high resolution virome elucidation and full alphacoronavirus genome assembly." by, Sebastian Aguilar Pierlé, Gabriel Zamora, Gonzalo Ossa, Aldo Gaggero and Gonzalo P. Barriga. for publication in the journal Viruses.
During our previous submission, the reviewers did a constructive evaluation of our work and recommended considering it for publication pending major revisions. They recognized that this work will be of great interest to the Viruses readers, especially due to its enrichment strategies and its zoonotic implications.
Please find this new version which includes the modifications suggested by the reviewers .
Kind regards,
Gonzalo Barriga
Emerging Viruses Laboratory,
Virology Program,
Institute of Biomedical Sciences,
Faculty of Medicine,
Universidad de Chile
E-mail address: [email protected]
Reviewer: 1
Comments:
The authors should specify several details of the viral enrichment protocol:Viral particle concentration was reached with several concatenated centrifugations and treatment with DNAse/RNAse before the RNA extraction step.
R: Thank you for the comment, details have been added to the materials and methods section, please find the answers to your comments below.
Specify the ratio PBS/guano in vol/vol because 1:5 is not clear, and the starting volume was never known
R: Thank you very much for this comment, we started with 1 gram of feces resuspended it until it reached 5 ml of PBS, this explanation was corrected in the current version
Define what is the volume of the original sample from step 1
R: Thank you for the comment, the bat guano is solid, therefore it was necessary to resuspend, for this we used PBS, briefly, we weighed 1 gram of bat guano and resuspended it until total volume reached 5 mls of PBS.
After treatment DNAse/RNAse/Turbonuclease, EDTA and heat inactivated the samples or the enzymes?
R: Thank you for your question, after the concentration step, we performed enzyme treatments that required inactivation, for this, we added RNA shield. This reagent inactivates the enzymes in the sample. There was no EDTA usage or heat inactivation, this was a mistake on our part due to its usage in previous protocols. The manuscript has been modified accordingly.
Major comment 2: The phylogenetic analysis should be improved
The authors specified the alignment parameters but they did not tell us which tool was used for the alignment (Mega, Seaview, Mafft.. any other). The alignment is the starting point of any phylogenetic analysis and this step is very important. Mafft in line is very efficient.
R: We have specified the tool used for the alignment.
The phylogenetic tree was generated using the NJ method with the JC nucleotide substitution model, both of which are outdated. I strongly suggest the authors to use IQ-Tree tool web server, which automatically will select the best model and will generate a maximum likelihood tree with strong branch supports.
R: We thank the reviewer for his suggestions, we have used the IQ-Tree tool to generate the tree included in the revised version of the manuscript, all sections have been updated accordingly.
Reviewer 2 Report
Summary:
This study investigates the application of a novel enrichment strategy in guano bats (native Chilean species Myotis chioensis) virome extraction and proves that this method shows better coverage on detected viral sequences as compared to previous studies without viral enrichment. The study also manages to identify a new bat alphacoronavirus responsible for the mortality of suckling piglets from the guano bats. The author’s observation for the viral enrichment will benefit other researchers that have low sample volumes for metagenomics studies.
Comments:
Line 6: Affiliation for 1 is incomplete.
Line 5 and 15 – 17: The correspondence for the paper is unclear. Please place a * on all the responsible correspondence
Some minor mistakes were detected in the Introduction.
Line 61: Please include reference
Line 82: … distributed insectivorous bat species, we characterized …
Line 83: … virome of M. chiloensis. We monitored a bat …
Line 87: … animal industry challenge. Here we described a new bat …
Line 89: … further analysed. We also highlight the importance …
Material & Methods did not clearly state whether the sample from guano bats (2 detected in 2019 from Location C) was pooled or individually sequenced for Illumina. Please state it clearly. Some other mistakes are as below.
Line 96: (Figure 1) instead of (figure 1)
Line 145: … newly assembled M. chiloensis alphacoronavirus 1 was aligned …
The presentations of results need to be improved as the figures do not fully represent the studies. Suggest including comparison studies of enriched and not enriched guano bat samples for a comprehensive study. As I am not able to see how much the identified sequences had improved without comparison from the enriched and not enriched samples. Figure 4 will need to be shown in a complete tree for the phylogenetic tree. Table 1 is not necessary and can be written in text format or included in supplementary table.
Line 181: There is no Table 3 in the manuscript. Please include the table back in the manuscript.
Line 183: Figure 2 need to align to the left. In my opinion, Figure 2 is not necessary for this manuscript unless there is a comparison with another 4 studies used to compare with this study.
Line 210: … Thanks to this sequence a Phylogeny tree was built using 56 related sequences and the newly identified bat viral genome (Table S1).
Discussions from the authors are weak and lack references. Discussion on viral enrichment is lacking.
Author Response
To Editor VIRUSES
Santiago, 30rd of December, 2021
Dear Denisa Cimpian,
With the present letter we are submitting our revised manuscript “The Myotis chiloensis guano virome: viral nucleic acid enrichment for high resolution virome elucidation and full alphacoronavirus genome assembly." by, Sebastian Aguilar Pierlé, Gabriel Zamora, Gonzalo Ossa, Aldo Gaggero and Gonzalo P. Barriga. for publication in the journal Viruses.
During our previous submission, the reviewers did a constructive evaluation of our work and recommended considering it for publication pending major revisions. They recognized that this work will be of great interest to the Viruses readers, especially due to its enrichment strategies and its zoonotic implications.
Please find this new version which includes the modifications suggested by the reviewers .
Kind regards,
Gonzalo Barriga
Emerging Viruses Laboratory,
Virology Program,
Institute of Biomedical Sciences,
Faculty of Medicine,
Universidad de Chile
E-mail address: [email protected]
Reviewer: 2
Comments:
Line 6: Affiliation for 1 is incomplete.
R: Thank you for you comment, the current version has included the complete affiliation for 1
Line 5 and 15 – 17: The correspondence for the paper is unclear. Please place a * on all the responsible correspondence
R: Thank you for this comment, this information has been included in the current version.
Some minor mistakes were detected in the Introduction.
R: Thank you for the comment, we have addressed each of the issues below
Line 61: Please include reference
R: We thank the reviewer for this comment, the reference was included in the current version
Line 82: … distributed insectivorous bat species, we characterized …
R: This was modified in the current version of the manuscript.
Line 83: … virome of M. chiloensis. We monitored a bat …
R: This modification was included in the current version of the manuscript.
Line 87: … animal industry challenge. Here we described a new bat …
R: This modification was included in the current version of the manuscript.
Line 89: … further analysed. We also highlight the importance …
R: This modification was included in the current version of the manuscript.
Material & Methods did not clearly state whether the sample from guano bats (2 detected in 2019 from Location C) was pooled or individually sequenced for Illumina. Please state it clearly. Some other mistakes are as below.
R: Thank you for your comment, we pooled the 2019 samples before illumina sequencing, the idea was to obtain a viral overview from the bat colony. This was specified in the current version.
Line 96: (Figure 1) instead of (figure 1)
R: This modification was included in the current version of the manuscript.
Line 145: … newly assembled M. chiloensis alphacoronavirus 1 was aligned …
R: This modification was included in the current version of the manuscript.
The presentations of results need to be improved as the figures do not fully represent the studies. Suggest including comparison studies of enriched and not enriched guano bat samples for a comprehensive study. As I am not able to see how much the identified sequences had improved without comparison from the enriched and not enriched samples. Figure 4 will need to be shown in a complete tree for the phylogenetic tree. Table 1 is not necessary and can be written in text format or included in supplementary table.
R: We thank the reviewer for his comments. It should be noted that the reported studies were chosen due to the fact that they dealt with similar sampling methods/sources and sample treatment for a common goal. All studies used enrichment methods, otherwise it would be nearly impossible to identify viral sequences in such samples, this is yet another reason why we thought the studies were comparable to ours. In fact one of the studies (Salmier A et al.) used a robot in addition to centrifugation/filtration, similarly to our study. The enrichment methods employed by each study have been added to Table 1 and are now further discussed in the manuscript. We believe that with this additional information the Table is now useful for the reader and could be included in the main manuscript. Finally, a full version of Figure 4 is now included.
Line 181: There is no Table 3 in the manuscript. Please include the table back in the manuscript.
R: There was a confusion on our part with Table 1, the manuscript has been modified accordingly.
Line 183: Figure 2 need to align to the left. In my opinion, Figure 2 is not necessary for this manuscript unless there is a comparison with another 4 studies used to compare with this study.
R: We thank the reviewer for his comment. We believe that this figure provides additional detail in terms of viral enrichment performance for different microbes present in this complex sample and is of interest to further support the strategy’s success. Although we agree that a comparison with the other studies would be interesting, said studies did not go into that level of detail.
Line 210: … Thanks to this sequence a Phylogeny tree was built using 56 related sequences and the newly identified bat viral genome (Table S1).
R: This modification was included in the current version of the manuscript.
Discussions from the authors are weak and lack references. Discussion on viral enrichment is lacking.
R: Thank you for your comment, we have included additional elements in terms of discussing different viral enrichment strategies and how they compare to the one used in this study.
Reviewer 3 Report
This manuscript describes the use of pan virus family RT-PCR for identification of bat guano samples with coronavirus and/or paramyxovirus RNA, combined with a novel enrichment strategy and sequencing for viral genome characterization. Although this technique and genetic information is interesting, the manuscript appears to be hastily written and is missing information. For example, there is no Table 2 or Table 3, and although Table 3 is mentioned in the text, Table 2 is completely omitted. Two of the supplemental tables are not formatted in an understandable way, for example, there are no column headings for Table S3 and one tab on this table is labeled but has no content. Table 1 last entry is missing relevant information. Tables are mentioned out of order in the text. Other aspects of the manuscript are vague such as the program used for phylogenetic analysis and read mapping. Bioinformatic Analysis section mentions “Genomics Workbench” for QC but does not cite the company (CLC, Qiagen) or if it was used for other aspects of the analysis. The authors need to spend some more time and effort to make this manuscript complete and coherent, including making sure all figures and tables mentioned in the text are present, have understandable content, and are labeled/described thoroughly. Wording and spelling should also be checked.
Author Response
To Editor VIRUSES
Santiago, 30rd of December, 2021
Dear Denisa Cimpian,
With the present letter we are submitting our revised manuscript “The Myotis chiloensis guano virome: viral nucleic acid enrichment for high resolution virome elucidation and full alphacoronavirus genome assembly." by, Sebastian Aguilar Pierlé, Gabriel Zamora, Gonzalo Ossa, Aldo Gaggero and Gonzalo P. Barriga. for publication in the journal Viruses.
During our previous submission, the reviewers did a constructive evaluation of our work and recommended considering it for publication pending major revisions. They recognized that this work will be of great interest to the Viruses readers, especially due to its enrichment strategies and its zoonotic implications.
Please find this new version which includes the modifications suggested by the reviewers .
Kind regards,
Gonzalo Barriga
Emerging Viruses Laboratory,
Virology Program,
Institute of Biomedical Sciences,
Faculty of Medicine,
Universidad de Chile
E-mail address: [email protected]
Reviewer 3
This manuscript describes the use of pan virus family RT-PCR for identification of bat guano samples with coronavirus and/or paramyxovirus RNA, combined with a novel enrichment strategy and sequencing for viral genome characterization. Although this technique and genetic information is interesting, the manuscript appears to be hastily written and is missing information.
For example, there is no Table 2 or Table 3, and although Table 3 is mentioned in the text, Table 2 is completely omitted. Two of the supplemental tables are not formatted in an understandable way, for example, there are no column headings for Table S3 and one tab on this table is labeled but has no content. Table 1 last entry is missing relevant information. Tables are mentioned out of order in the text. Other aspects of the manuscript are vague such as the program used for phylogenetic analysis and read mapping. Bioinformatic Analysis section mentions “Genomics Workbench” for QC but does not cite the company (CLC, Qiagen) or if it was used for other aspects of the analysis. The authors need to spend some more time and effort to make this manuscript complete and coherent, including making sure all figures and tables mentioned in the text are present, have understandable content, and are labeled/described thoroughly. Wording and spelling should also be checked.
R: We thank the reviewer for his comments. We have addressed the issues with the tables in the manuscript. Table S3 has now column headings. It should be noted that the tool used to generate the taxonomic units described in Table S3 does not provide such headings. Considering this we numbered the levels of taxonomic classification achieved with the first 5 clearly defined as: 1 Order, 2 Family, 3 Genus, 4 Species, 5 Strain and the remainder levels as additional resolution achieved with further iterations of the analysis (this is reiterated in the key of the table). The phylogenetic analyses have been further explained. We have done our best to thoroughly revise the full manuscript for it to be more complete and coherent.
Round 2
Reviewer 1 Report
Reviewer comments to authors
The authors have modified their manuscript accordingly to the following reviewer’s comments:
- they add details in the viral enrichment paragraph in the Material and Methods section,
- they specified which samples were pooled before the library preparation
- they added discussion about enrichment strategies
- they displayed a full tree in Figure 4 as requested
- they experimented a phylogenetic tree reconstruction by using IQTree web server, and they made the output files available for review.
However there is a huge problem with the tree (Figure 4) which is not consistent at all, the major clades of alpha- and beta-coronaviruses are not separated enough, and notably the beta-CoVs including SARS-CoV and SARS-CoV-2 are embedded within the alpha-CoVs.
Specific comments and suggestions will follow, it’s obvious that the phylogenetic analysis absolutely needs to be improved before the manuscript can be accepted for publication.
Specific comments
- By looking at the consensus tree given by the IQtree server, the tips labels were identified (accession numbers were searched in Genbank then the label was replaced with the name as given by the authors in the manuscript). Once done, the IQtree tree was compared with the one in Figure 4 and there I could not find the new sequence of M. chiloensis alphacoronavirus, which is absent from this IQTree output. Not easy to check the location of the new strain..
- By looking at the log file, one can see that all references have high levels of gap/ambiguity and most of them failed instead of passing, that is anomalous. Just above, the log file shows that the alignment length is 34785 columns, whereas the longer reference sequence is 31357 bases (see Table S1). It seems that the alignment was not checked for ambiguously aligned regions which must be deleted, and maby neither checked for the 5’ and 3’ ends which need to be cut properly, before doing the phylogenetic tree analysis.
- The authors kept short reference sequences (8 references have lengthes between 405 and 919 nucleotides, one is 2780 nucleotides, and 4 are about 15000 bp). This is not suitable when doing maximum likelihood analyses, the method authorizes indels and or gaps (if some sequences are shorter) but not at this level. The phylogenetic analysis using IQTree was suggested to get a picture based on the complete genome sequences. (In the first version of the manuscript, the neighbor-joining method allowed to degap the alignment automatically. As a consequence, the analysis originally proposed by the authors very probably targeted only approximatively 400 nucleotides and not the complete genome.)
Specific suggestions
- Delete all the short reference sequences, especially since most of them do not seem very useful. Keep the complete genome sequences and the newly obtained genome sequence to do the alignment. As previously suggested, MAFFT in line is very efficient.
- Check the alignment quality, perform end-trimming and degap eventually. MEGA or G-Blocks in line can be used.
- Finally, submit the corrected alignment to IQTree server.
- Editing the consensus tree under FigTree and choosing to order nodes et to root by midpoint rooting would allow to get a very correct phylogenetic tree.
- Of course, it may be necessary to modify the comments depending the results that will be obtained with the new tree (end of page 6/12).
- The sequence of the new genome must be submitted as a fasta file in Genbank or another international database.
Other comments:
- page 9/12: the discussion on enrichment strategies has been pasted two times. Suppress
When looking at studies in the literature that attempted to enrich for similar samples as ours, we determined that our strategy achieved the highest level of enrichment when estimating the proportion of viral sequences. Studies that employed centrifugation, filtration and Nuclease treatments were below our level of enrichment [33-34] . In Paskey’s study of 2020, none of the aforementioned strategies were employed (or at least cited in the Materials and Methods), which could explain that this work reported the lowest level of enrichment. Interestingly, Salmier’s study of 2017 incorporated the use of a benchtop instrument for automated nucleic acid extraction, the NucliSENS easyMAG (Biomérieux). The instrument’s proprietary magnetic silica particle manipulation technology did not seem to impact viral nucleic acid enrichment, which is not surprising as it is not specifically targeted for this. Nucleic acid extraction methods utilized for these studies were similar except for the latter study. This suggests that this didn’t impact the level of enrichment. Our strategy included centrifugation, filtration, nuclease treatment and in addition to this, negative enrichment through probe hybridization to select for host and microbial nucleic acids in an automated benchtop instrument. It is likely that Paskey also used a similar approach (albeit, without automation), as he used a previous version of the Illumina kit we used, that likely includes probe negative enrichment. This is not specified in Table 1 as it is not explicit in the publication’s material and method’s section. Despite this, it achieved lesser viral enrichment. The key to our approach resides in miniaturization of the reaction which improves kinetics paired with highly precise manipulation of magnetic beads, improving all cleanup steps. Our study shows that enrichment of such complex samples for virome studies can be improved utilizing a combination of traditional techniques including centrifugation, filtration and nuclease treatment paired with molecular negative enrichment utilizing a mixture of probes. Automation, miniaturization and precise bead handling thanks to a patented magnetic tweezer further improve enrichment and impact the resolution of the obtained data.
- page 6/12, end of the page: alphacoroniviruses should be written alphacoronaviruses (As seen on Figure 4, the newly identified genome sequence is grouped with a nine different bat alphacoroniviruses...)
- page 8/12, Table 1, study of Salmier et al: nucelase treatment should be written nuclease treatment.
Author Response
To Editor VIRUSES
Santiago, 08th of January, 2021
Dear Denisa Cimpian,
With the present letter we are submitting our revised manuscript “The Myotis chiloensis guano virome: viral nucleic acid enrichment for high resolution virome elucidation and full alphacoronavirus genome assembly." by, Sebastian Aguilar Pierlé, Gabriel Zamora, Gonzalo Ossa, Aldo Gaggero and Gonzalo P. Barriga. for publication in the journal Viruses.
During our previous submission, the reviewers did a constructive evaluation of our work and recommended considering it for publication pending minor revisions. They recognized that this work will be of great interest to the Viruses readers, especially due to its enrichment strategies and its zoonotic implications.
As a minor point, the text box you included seems to have disturbed the formatting of the second page, so we tried to fix it to the best of our abilities.
Please find this new version which includes the modifications suggested by the reviewers.
Kind regards,
Gonzalo Barriga
Emerging Viruses Laboratory,
Virology Program,
Institute of Biomedical Sciences,
Faculty of Medicine,
Universidad de Chile
E-mail address: [email protected]
Reviewer 1 comments to authors
The authors have modified their manuscript accordingly to the following reviewer’s comments:
- they add details in the viral enrichment paragraph in the Material and Methods section,
- they specified which samples were pooled before the library preparation
- they added discussion about enrichment strategies
- they displayed a full tree in Figure 4 as requested
- they experimented a phylogenetic tree reconstruction by using IQTree web server, and they made the output files available for review.
R: Thank you for these comments.
However there is a huge problem with the tree (Figure 4) which is not consistent at all, the major clades of alpha- and beta-coronaviruses are not separated enough, and notably the beta-CoVs including SARS-CoV and SARS-CoV-2 are embedded within the alpha-CoVs.
R: Thanks to the adjustments suggested by the reviewer this is no longer the case, beta-CoVs segregate with other beta.
Specific comments and suggestions will follow, it’s obvious that the phylogenetic analysis absolutely needs to be improved before the manuscript can be accepted for publication.
R: We thank the reviewer for his suggestions an improved version of the phylogenetic tree was included in the current version
Specific comments
- By looking at the consensus tree given by the IQtree server, the tips labels were identified (accession numbers were searched in Genbank then the label was replaced with the name as given by the authors in the manuscript). Once done, the IQtree tree was compared with the one in Figure 4 and there I could not find the new sequence of M. chiloensis alphacoronavirus, which is absent from this IQTree output. Not easy to check the location of the new strain.
R: We have renamed the new sequence so that it is easy to identify now in the IQTree output, we will label it appropriately (accession number) once submission is complete.
- By looking at the log file, one can see that all references have high levels of gap/ambiguity and most of them failed instead of passing, that is anomalous. Just above, the log file shows that the alignment length is 34785 columns, whereas the longer reference sequence is 31357 bases (see Table S1). It seems that the alignment was not checked for ambiguously aligned regions which must be deleted, and maby neither checked for the 5’ and 3’ ends which need to be cut properly, before doing the phylogenetic tree analysis.
- The authors kept short reference sequences (8 references have lengthes between 405 and 919 nucleotides, one is 2780 nucleotides, and 4 are about 15000 bp). This is not suitable when doing maximum likelihood analyses, the method authorizes indels and or gaps (if some sequences are shorter) but not at this level. The phylogenetic analysis using IQTree was suggested to get a picture based on the complete genome sequences. (In the first version of the manuscript, the neighbor-joining method allowed to degap the alignment automatically. As a consequence, the analysis originally proposed by the authors very probably targeted only approximatively 400 nucleotides and not the complete genome.)
R: See below.
Specific suggestions
- Delete all the short reference sequences, especially since most of them do not seem very useful. Keep the complete genome sequences and the newly obtained genome sequence to do the alignment. As previously suggested, MAFFT in line is very efficient.
- Check the alignment quality, perform end-trimming and degap eventually. MEGA or G-Blocks in line can be used.
R: Thank you for your comment, the short sequences have been eliminated and the alignment revised.
- Finally, submit the corrected alignment to IQTree server.
R: The corrected alignment was submitted to the IQTree server.
- Editing the consensus tree under FigTree and choosing to order nodes et to root by midpoint rooting would allow to get a very correct phylogenetic tree.
R: We have not had the time to adjust in FIgTree, however we made adjustments in the CLC tool.
- Of course, it may be necessary to modify the comments depending the results that will be obtained with the new tree (end of page 6/12).
R: We have adjusted the manuscript in accordance with the new tree.
- The sequence of the new genome must be submitted as a fasta file in Genbank or another international database.
R: Thank you for this comment, all sequences have been submitted to the SRA and the genome sequence is being submitted to genbank.
Other comments:
- page 9/12: the discussion on enrichment strategies has been pasted two times. Suppress
When looking at studies in the literature that attempted to enrich for similar samples as ours, we determined that our strategy achieved the highest level of enrichment when estimating the proportion of viral sequences. Studies that employed centrifugation, filtration and Nuclease treatments were below our level of enrichment [33-34] . In Paskey’s study of 2020, none of the aforementioned strategies were employed (or at least cited in the Materials and Methods), which could explain that this work reported the lowest level of enrichment. Interestingly, Salmier’s study of 2017 incorporated the use of a benchtop instrument for automated nucleic acid extraction, the NucliSENS easyMAG (Biomérieux). The instrument’s proprietary magnetic silica particle manipulation technology did not seem to impact viral nucleic acid enrichment, which is not surprising as it is not specifically targeted for this. Nucleic acid extraction methods utilized for these studies were similar except for the latter study. This suggests that this didn’t impact the level of enrichment. Our strategy included centrifugation, filtration, nuclease treatment and in addition to this, negative enrichment through probe hybridization to select for host and microbial nucleic acids in an automated benchtop instrument. It is likely that Paskey also used a similar approach (albeit, without automation), as he used a previous version of the Illumina kit we used, that likely includes probe negative enrichment. This is not specified in Table 1 as it is not explicit in the publication’s material and method’s section. Despite this, it achieved lesser viral enrichment. The key to our approach resides in miniaturization of the reaction which improves kinetics paired with highly precise manipulation of magnetic beads, improving all cleanup steps. Our study shows that enrichment of such complex samples for virome studies can be improved utilizing a combination of traditional techniques including centrifugation, filtration and nuclease treatment paired with molecular negative enrichment utilizing a mixture of probes. Automation, miniaturization and precise bead handling thanks to a patented magnetic tweezer further improve enrichment and impact the resolution of the obtained data.
R: Thank you for your comment, the duplicated paragraph was suppressed, we apologize for this mistake.
- page 6/12, end of the page: alphacoroniviruses should be written alphacoronaviruses (As seen on Figure 4, the newly identified genome sequence is grouped with a nine different bat alphacoroniviruses...)
R: Thank you for your comment, we corrected the mistyped word.
- page 8/12, Table 1, study of Salmier et al: nucelase treatment should be written nuclease treatment.
R: Thank you for your comment, we corrected the mistyped word.
Reviewer 2 Report
There are still some mistakes in the revised manuscript:
- Consider using updated papers for referencing. For example, there are a lot of updated papers on cross-species transmission in bats for rabies, henipaviruses and coronaviruses.
- Spelling error: alphacoroniviruses change to alphacoronavirus
- Spelling error: aetacoronavirus change to betacoronavirus
- Figure 4: Outgroup for the tree is not clearly stated. There is a need to add the bootstrap value to each node of the tree. The naming of each taxon is not standardized, please follow the specification by the journal.
- Alphacoronaviruses of bats origin found in the same clade identified in this study also include Kenya if according to the highlighted clade in Figure 4. Please include in the text.
- Discussion added for enrichment studies were repeated. Please remove the repeated text in Discussions.
Author Response
To Editor VIRUSES
Santiago, 08th of January, 2021
Dear Denisa Cimpian,
With the present letter we are submitting our revised manuscript “The Myotis chiloensis guano virome: viral nucleic acid enrichment for high resolution virome elucidation and full alphacoronavirus genome assembly." by, Sebastian Aguilar Pierlé, Gabriel Zamora, Gonzalo Ossa, Aldo Gaggero and Gonzalo P. Barriga. for publication in the journal Viruses.
During our previous submission, the reviewers did a constructive evaluation of our work and recommended considering it for publication pending minor revisions. They recognized that this work will be of great interest to the Viruses readers, especially due to its enrichment strategies and its zoonotic implications.
As a minor point, the text box you included seems to have disturbed the formatting of the second page, so we tried to fix it to the best of our abilities.
Please find this new version which includes the modifications suggested by the reviewers.
Kind regards,
Gonzalo Barriga
Emerging Viruses Laboratory,
Virology Program,
Institute of Biomedical Sciences,
Faculty of Medicine,
Universidad de Chile
E-mail address: [email protected]
Reviewer 2 comments to authors
There are still some mistakes in the revised manuscript:
- Consider using updated papers for referencing. For example, there are a lot of updated papers on cross-species transmission in bats for rabies, henipaviruses and coronaviruses.
R: Thank you for your comment, we have added the following references
Benavides JA, Valderrama W, Recuenco S, Uieda W, Suzán G, Avila-Flores R, Velasco-Villa A, Almeida M, Andrade FAG, Molina-Flores B, Vigilato MAN, Pompei JCA, Tizzani P, Carrera JE, Ibanez D, Streicker DG. Defining New Pathways to Manage the Ongoing Emergence of Bat Rabies in Latin America. Viruses. 2020 Sep 8;12(9):1002. doi: 10.3390/v12091002. PMID: 32911766; PMCID: PMC7551776.
Latinne A, Hu B, Olival KJ, Zhu G, Zhang L, Li H, Chmura AA, Field HE, Zambrana-Torrelio C, Epstein JH, Li B, Zhang W, Wang LF, Shi ZL, Daszak P. Origin and cross-species transmission of bat coronaviruses in China. Nat Commun. 2020 Aug 25;11(1):4235. doi: 10.1038/s41467-020-17687-3. PMID: 32843626; PMCID: PMC7447761.
Wang LF, Anderson DE. Viruses in bats and potential spillover to animals and humans. Curr Opin Virol. 2019 Feb;34:79-89. doi: 10.1016/j.coviro.2018.12.007. Epub 2019 Jan 18. PMID: 30665189; PMCID: PMC7102861.
Shi J, Sun J, Hu N, Hu Y. Phylogenetic and genetic analyses of the emerging Nipah virus from bats to humans. Infect Genet Evol. 2020 Nov;85:104442. doi: 10.1016/j.meegid.2020.104442. Epub 2020 Jul 3. PMID: 32622923.
- Spelling error: alphacoroniviruses change to alphacoronavirus
R: Thank you for your comment, we corrected the mistyped word.
- Spelling error: aetacoronavirus change to betacoronavirus
R: Thank you for your comment, it was corrected the mistyped word
- Figure 4: Outgroup for the tree is not clearly stated. There is a need to add the bootstrap value to each node of the tree. The naming of each taxon is not standardized, please follow the specification by the journal.
R: Thank you for your comment, bootstrap values are included in the tree and in Supplementary file 1, we have adhered to the journal’s specifications in terms of taxon naming.
- Alphacoronaviruses of bats origin found in the same clade identified in this study also include Kenya if according to the highlighted clade in Figure 4. Please include in the text.
R: Thank you for your comment, this was corrected in the current new version
- Discussion added for enrichment studies were repeated. Please remove the repeated text in Discussions.
R: Thank you for your comment, the duplicate paragraph was suppressed.
Reviewer 3 Report
Although the authors did respond to reviewer comments, the manuscript in its current form needs additional information added and text revision. For example, about half of the new paragraph added to discussion section is duplicated (repeated verbatim) text within the same paragraph. There was not significant improvement on the quality or labelling of the figures or the description of the bioinformatic analysis. For example, for the comment “Bioinformatic Analysis section mentions “Genomics Workbench” for QC but does not cite the company (CLC, Qiagen) or if it was used for other aspects of the analysis.” The authors simply added “(CLC, Qiagen) “ in the text but did not add further text describing if this program was used for the entire analysis (read mapping, de novo assembly). The Figures and Table 1 are still poorly labeled, for example, there is no description accompanying Figure 3 such as the program used to generate it or why there are the different number of various sized circles arranged in the order presented. The last row of Table 1 publication is incomplete for author and other publication information. The methods described for phylogenetic analysis included bootstrap analysis but no bootstrap numbers were shown on the phylogenetic tree.
Author Response
To Editor VIRUSES
Santiago, 08th of January, 2021
Dear Denisa Cimpian,
With the present letter we are submitting our revised manuscript “The Myotis chiloensis guano virome: viral nucleic acid enrichment for high resolution virome elucidation and full alphacoronavirus genome assembly." by, Sebastian Aguilar Pierlé, Gabriel Zamora, Gonzalo Ossa, Aldo Gaggero and Gonzalo P. Barriga. for publication in the journal Viruses.
During our previous submission, the reviewers did a constructive evaluation of our work and recommended considering it for publication pending minor revisions. They recognized that this work will be of great interest to the Viruses readers, especially due to its enrichment strategies and its zoonotic implications.
As a minor point, the text box you included seems to have disturbed the formatting of the second page, so we tried to fix it to the best of our abilities.
Please find this new version which includes the modifications suggested by the reviewers.
Kind regards,
Gonzalo Barriga
Emerging Viruses Laboratory,
Virology Program,
Institute of Biomedical Sciences,
Faculty of Medicine,
Universidad de Chile
E-mail address: [email protected]
Reviewer 3 comments to authors
Reviewer comments to authors 3
Although the authors did respond to reviewer comments, the manuscript in its current form needs additional information added and text revision. For example, about half of the new paragraph added to discussion section is duplicated (repeated verbatim) text within the same paragraph. There was not significant improvement on the quality or labelling of the figures or the description of the bioinformatic analysis. For example, for the comment “Bioinformatic Analysis section mentions “Genomics Workbench” for QC but does not cite the company (CLC, Qiagen) or if it was used for other aspects of the analysis.” The authors simply added “(CLC, Qiagen) “ in the text but did not add further text describing if this program was used for the entire analysis (read mapping, de novo assembly). The Figures and Table 1 are still poorly labeled, for example, there is no description accompanying Figure 3 such as the program used to generate it or why there are the different number of various sized circles arranged in the order presented. The last row of Table 1 publication is incomplete for author and other publication information. The methods described for phylogenetic analysis included bootstrap analysis but no bootstrap numbers were shown on the phylogenetic tree.
R: We thank the reviewer for the additional comments. The duplicated paragraph has been suppressed. We also have made an effort to further improve the quality of the labelling of the figures and further explain usage of bioinformatic tools. We have also modified the labelling of Figure 3 and Table 1. The bootstrap numbers were already included in Supplementary file 1 and are now included in the tree.
