Experimental Dengue Virus Type 4 Infection Increases the Expression of MicroRNAs-15/16, Triggering a Caspase-Induced Apoptosis Pathway

Round 1

Reviewer 1 Report

In this manuscript, Casseb et al. performed a DENV4 infection time-course in hepatocyte cell lines, examining levels of miR-15, -16, NS1 and caspase cleavage (apoptosis). The authors found that, as expected, NS1 expression increased over time. In addition, miR-15 and -16 expression increased as did caspase cleavage, indicating DENV4 infection causes hepatocyte apoptosis. This is an interesting finding; however, the study could have been more rigorous. For example, analyzing Bcl-2 expression and microscopy analysis of cells for apoptosis at the same time points as in the graphs. Another major issue was that the rationale for the study was unclear. Because there are other viral proteins expressed, the authors cannot attribute effects to NS1. General comment are listed below.

Line 50: define they (as is, it is referring to the genome).

Paragraph starting on line 53 missing multiple references.

Reference 8 on line 63 is inappropriate; a more general review of miRNA is required.

Reference 10 on line 70 is inappropriate; a more general review of miRNA is required.

It is unclear why the Introduction contains details regarding miR processing and mechanisms of function, but no information regarding the interplay between miRs and viral infection. The Introduction also lacks a rationale for the study: why miR-15 and -16, and why NS1?

In the Methods 2.5, it is unclear how pGEM Easy can be used to measure extract amount.

Also, in the Methods it is not clear what “experimental samples” are (supernatant- or cell-derived?). This information is important, as the reduction in caspase activity would be consistent with loss of cells in the culture due to apoptosis at later stages in the time course (if cells/cell extracts are the experimental samples).

Based on the above, no microscopy is available for the times reported in the graphs. DAPI should be/should have been included to indicate cells undergoing apoptosis, or a specific apoptosis marker could have been used.

Line 106: “We had previously written about how the comparative CT method was used to measure the expression.” Missing reference.

For all figures, details beyond the title are needed to have a figure legend.

Line 153, “the level of miRNA-15 slowly went up…” is not consistent with the graph, which shows not much difference between 24 and 48h then a substantial increase at 72h.

For the Discussion:

The authors should state whether miR-15 and -16 were identified in the previous Casseb et al. study (or any other transcriptome-based studies).

References are need for statements in the paragraph starting on line 205.

On line 230, “This suggests that NS1 can initiate this apoptosis pathway.” Is erroneous. There are not experiments addressing this possibility in the study, and there is no correlation between NS1 and caspase cleavage in figure 5.

The authors included Yu et al. for the relationship between miR-15 and Bcl-2; however, the more extensive set of studies regarding this relationship was not mentioned. It is surprising that Bcl-2 transcript levels were not evaluated in this study.

Are references 25 and 27 really appropriate for the effects of miR-15 and -16 (line 224)?

Line 245: “pro-inflammatory drugs” is incorrect terminology; the authors probably mean pro-inflammatory cytokines.

In the Conclusion miR-15 may not be an effective disease marker as its change in expression was weak (~2-fold).

Author Response

Dear,
We appreciate your suggestions. We have introduced changes to the text for better understanding.
Emphasize that we carry out a language revision using specialized professionals.
Unfortunately, we cannot express Bcl-2 as suggested at this time. However, we have relevant data in our article to be considered for publication in this esteemed journal.

Reviewer 2 Report

In this manuscript, Casseb and colleagues investigated the correlation between dengue virus infection and the expression profile of NS1 protein including miRNAs 15 & 16 in HepG2 and Huh 7.5 cells. The authors presented data on viral replication by measuring genome copies using RT-qPCR. They also quantified microRNA and apoptosis using RT-qPCR and Caspase kit respectively. They concluded that DENV4 infection significantly increased apoptosis and miRNAs 15 and 16 expressions.

I have concerns with the overall presentation of the manuscript. The manuscript is poorly written with sparse details about the reported data. A native English Language speaker needs to go through and edit this report.

 

Figures: The authors need to include more detailed figure legends. Lines should be drawn to join the bars that are being compared. The specific statistical analysis used for each figure should also be added as well as the number of biological experimental replicates. Additionally, Figure 4C needs additional control images such DAPI-stained nucleus to indicate the presence of cells and secondary antibody-stained cells to show the specificity of the primary antibody.

 

Finally, the authors need to write a more comprehensive result section which includes the rationale for each experiment and concluding remarks for the reported data.  

Author Response

Dear,
We appreciate the suggestions. We carry out an extensive review of the English language by a native English Language speaker.
Figure legends were revised and adjusted, and verification of the topics contained in the text was carried out.
Unfortunately, due to time and lack of reagents, we cannot include additional controls in Figure 4C.

Round 2

Reviewer 1 Report

The authors have addressed some comments to improve the body of the manuscript. Surprisingly, the introduction was not modified to provide a rationale for the study. "They" (previously line 50; now line 158) must still be changed to a more descriptive word or phrase. They added references for the overview of NS1; however ref. 7 appears to have nothing to do with NS1. The authors added some details to the figure legends; however, the legends could still be improved (e.g., how were the cells infected in figure 1; what MOI was used?). Tukey post-hoc test instead of Tukey post-test.

Most significant in the re-submission is why the authors did not address at least some experimental issues with the study. While important microscopy experiments missing from the study may have required some effort, it should have been trivial to obtain Bcl-2 PCR primers for analysis of specimens the authors presumably have.

Author Response

Thanks for the comments. They were of great importance for the improvement of the manuscript. Below we explain the questions raised.

 

The authors have addressed some comments to improve the body of the manuscript. Surprisingly, the introduction was not modified to provide a rationale for the study. "They" (previously line 50; now line 158) must still be changed to a more descriptive word or phrase. They added references for the overview of NS1; however ref. 7 appears to have nothing to do with NS1. The authors added some details to the figure legends; however, the legends could still be improved (e.g., how were the cells infected in figure 1; what MOI was used?). Tukey post-hoc test instead of Tukey post-test.

We appreciate the comments and have made changes to the manuscript to meet your requests.

 

Most significant in the re-submission is why the authors did not address at least some experimental issues with the study. While important microscopy experiments missing from the study may have required some effort, it should have been trivial to obtain Bcl-2 PCR primers for analysis of specimens the authors presumably have.

Unfortunately, due to logistical and financial issues in the last year, it was impossible to carry out this analysis. We hope for your understanding. Due to a problem with the device in which the original tissues were stored and the RNAs, it made the analysis of BCL-2 by RT-qPCR or by immunohistochemistry unfeasible.

Reviewer 2 Report

The authors present important data in this report.

The current version is a significant improvement over the previous version of the manuscript. However, the results section still needs a little bit of work. This sections is sparsely reported and more details need to be included for clarity. For example,

1. The authors need to include the rationale for each experiment while describing their data. They should also include at least a concluding sentence at the end of each subsection. This will add meaning to the description of the subtopics. 

2. Figure legends should be placed below the figures and not above. The figure legends should include more details. This will enable readers to gain a clear understanding of reported data just by reading figure legends.

Author Response

Thanks for the comments. They were of great importance for the improvement of the manuscript. Below we explain the questions raised.

 

1. The authors need to include the rationale for each experiment while describing their data. They should also include at least a concluding sentence at the end of each subsection. This will add meaning to the description of the subtopics. 

We appreciate your comments and have made changes to the manuscript to meet your requests.

 

2. Figure legends should be placed below the figures and not above. The figure legends should include more details. This will enable readers to gain a clear understanding of reported data just by reading figure legends.

We changed the legend in position and text, inserting more information like infected cells and MOI.