Exploring the Interactions between Human microRNAs and the Ilheus Virus Genome

Round 1

Reviewer 1 Report

The work by Souza and Casseb focuses on interactions between human microRNAs and the genome of the Ilheus virus transmitted by mosquitoes. The authors take the challenge to unravel these interactions to facilitate the development of procedures for personalized antiviral interventions. The paper is quite well written although its clarity could be improved by adding some figures (for example data processing scheme). Below are my comments to the authors:

Major remarks:

(1) The discussion section looks like a second Introduction rather. It summarizes some information about miRNAs but does not discuss anything. I suggest removing this section from the paper and moving the most important information to the Introduction.

(2) In accordance with good practice and scientific ethics, authors should provide references to articles about the tools used in the analysis (at least where the tool is mentioned for the first time):

- "analysis using the DAVID tool," - a reference for DAVID

- "using the RNAfold software" - a reference for RNAfold

- "virus from NCBI" - a reference to NCBI

- "MUSCLE global alignment algorithm" - a reference to MUSCLE

- "bioinformatics tools DIANAtools and miRBase were used" - a reference to DIANAtools and miRbase

(3) Section "4.1. Database and alignment" describes the sources of data used in the analysis. However, not all the databases are mentioned here. In some previous sections the authors write about miRbase - why is it not mentioned and cited here???

Did the authors think about searching some other databases of viral genomes? There are some more, providing annotations that might be helpful in the analysis - see ViralZone https://viralzone.expasy.org/ (doi: 10.1093/nar/gkq901) or Virxicon https://virxicon.cs.put.poznan.pl/ (doi:10.1093/bioinformatics/btaa1066).

Can the authors explain their choice and add references to the other data sources (for example mentioned above)?

(4) Please, prepare and add the data processing scheme followed in the study. It would be very helpful for the readers to see a general workflow and then read the explanations of particular steps of processing.

Minor remarks:

(1) Abstract: "crucial sections of the Islet virus" - I think, the authors meant the Ilheus virus, not Islet

(2) "Their intrinsic versatility in modulating a spectrum of cellular processes 51 positions them pivotally within the regulatory framework governing fundamental phenomena, encompassing cell proliferation, differentiation, and immune responses [3,4]."

MicroRNAs are not just studied in reference to humans. They also play crucial roles in plants. I suggest adding a short note on this as well with example citations (e.g., doi: 10.1101/gad.1004402, 10.3390/molecules23061367, 10.1186/s12986-018-0305-8) 

(3) typos and small errors:

- "immune responses [3,4]. ," -> "immune responses [3,4]."

- "with the miRNAs regulating these pathways" -> "with miRNAs regulating these pathways"

- "structures of the miRNAs (Figure 1)." -> "structures of miRNAs (Figure 1)."

(3) typos and small errors:

- "immune responses [3,4]. ," -> "immune responses [3,4]."

- "with the miRNAs regulating these pathways" -> "with miRNAs regulating these pathways"

- "structures of the miRNAs (Figure 1)." -> "structures of miRNAs (Figure 1)."

Author Response

We thank you for the suggestions and they were inserted throughout the manuscript. Below are the answers to the requested items:

(1) The discussion section looks like a second Introduction rather. It summarizes some information about miRNAs but does not discuss anything. I suggest removing this section from the paper and moving the most important information to the Introduction.

Thank you for your suggestions. We have made revisions to the text to align it with your request. Unfortunately, due to submission guidelines, the discussion section is a mandatory component. Therefore, we have made adjustments to ensure that it aligns with your requirements.

(2) In accordance with good practice and scientific ethics, authors should provide references to articles about the tools used in the analysis (at least where the tool is mentioned for the first time)

(3) Section "4.1. Database and alignment" describes the sources of data used in the analysis. However, not all the databases are mentioned here. In some previous sections the authors write about miRbase - why is it not mentioned and cited here???

(4)Did the authors think about searching some other databases of viral genomes? There are some more, providing annotations that might be helpful in the analysis - see ViralZone https://viralzone.expasy.org/ (doi: 10.1093/nar/gkq901) or Virxicon https://virxicon.cs.put.poznan.pl/ (doi:10.1093/bioinformatics/btaa1066).

We adjusted what was requested throughout the text. All changes are marked in red.

We considered the Virxicon database to obtain viral miRNAs with potential interaction with the human genome, however, under the criteria pre-established in the study, we did not obtain data.

Can the authors explain their choice and add references to the other data sources (for example mentioned above)?

The chosen databases yielded the largest number of data for analysis using the adopted tools.

(4) Please, prepare and add the data processing scheme followed in the study. It would be very helpful for the readers to see a general workflow and then read the explanations of particular steps of processing.

We constructed a new figure to better explain our methods. We also created an item for this (item 4.5)

Minor remarks:

• (1) Abstract: "crucial sections of the Islet virus" - I think, the authors meant the Ilheus virus, not Islet
• (2) "Their intrinsic versatility in modulating a spectrum of cellular processes 51 positions them pivotally within the regulatory framework governing fundamental phenomena, encompassing cell proliferation, differentiation, and immune responses [3,4]."
• MicroRNAs are not just studied in reference to humans. They also play crucial roles in plants. I suggest adding a short note on this as well with example citations (e.g., doi: 10.1101/gad.1004402, 10.3390/molecules23061367, 10.1186/s12986-018-0305-8) 
• (3) typos and small errors:
• - "immune responses [3,4]. ," -> "immune responses [3,4]."
• - "with the miRNAs regulating these pathways" -> "with miRNAs regulating these pathways"
• - "structures of the miRNAs (Figure 1)." -> "structures of miRNAs (Figure 1)."

All changes were made to the manuscript and are marked in red

Reviewer 2 Report

The author wants to investigate the potential interaction between human miRNA and ILHV genome. By using database searching and sequence alignment, the author identifies several miRNA potential works on ILHV genome. The author only gives the prediction without validate any of the miRNA function, I do not think it is qualified for publication at this stage.

Several major concern:

1. The author only use published bioinformatic tools to identify the miRNA, no improvement can be found by using this approach.

2. I strongly recommend author to do some validation using the identified miRNA, for example using qPCR to validate the knock down efficiency

3. the second structure is one of the potential influence factors, have author search the database about the abundance or the localization of the miRNA, more information will help the author to give a rank for the miRNA sequence, after that the author could validate the top hit and claim they provide a universal platform to identify potential miRNA for ILHV genome

The language is fine.

Author Response

Thank you for your contributions. Below we answer the questions:

1. The author only use published bioinformatic tools to identify the miRNA, no improvement can be found by using this approach.

While it's true that utilizing published bioinformatic tools forms the basis of our study, the novelty and significance of our work lie in the specific context of the Ilheus virus. By employing these tools in a targeted manner, we aim to identify miRNA interactions unique to the Ilheus virus genome. Our focus on this specific virus differentiates our research, contributing valuable insights that can inform subsequent experimental analyses. The specialized nature of our investigation within the realm of the Ilheus virus sets the stage for potential discoveries that could be pivotal for understanding its intricate interactions with human miRNAs.

2. I strongly recommend author to do some validation using the identified miRNA, for example using qPCR to validate the knock down efficiency

Certainly, your suggestion will be taken into serious consideration. The experimental validations, including the use of qPCR to confirm the knockdown efficiency of the identified miRNAs, are essential steps to enhance the credibility of our findings. We intend to conduct these validations in future studies, utilizing the data presented in this article and adopting an in vitro approach. This will allow us to thoroughly validate the computational predictions and strengthen the scientific rigor of our research. Your recommendation aligns with our commitment to ensuring the robustness and reliability of our results in subsequent investigations.

3. the second structure is one of the potential influence factors, have author search the database about the abundance or the localization of the miRNA, more information will help the author to give a rank for the miRNA sequence, after that the author could validate the top hit and claim they provide a universal platform to identify potential miRNA for ILHV genome

We answer the questions in the text, in item 4.4

Round 2

Reviewer 1 Report

The authors substantially improved the manuscript, especially in the part concerning a discussion of their results. One thing that is still missing in some places is citations of the appropriate literature. Whenever the third-party method, tool, or algorithm is mentioned, they should refer to the respective paper - this is how it is followed in high-quality scientific papers and top journals. So, for example here:
"We considered the Virxicon database for obtaining viral miRNAs with potential 239 interaction with the human genome; however, according to the pre-established criteria in 240 the study, we did not obtain data." the citation to the work about Virxicon should be cited (see Kudla et al., 2020;  doi:10.1093/bioinformatics/btaa1066)

No comments here.

Author Response

Thank you for your comment and we have inserted your comment on line 243.

Reviewer 2 Report

The author has addressed most of my concerns, significantly improving the quality of the paper. However, I would strongly encourage the inclusion of some experimental data in the paper. Nevertheless, considering it as a technology paper, the current version may also be acceptable.

Author Response

We appreciate the suggestion. We will carry out in vitro experimental analysis in other works of the group