Long Non-Coding RNA Encoded by Infectious Bronchitis Virus Facilitates Viral Replication via Direct Interaction with G3BP2 and Expression Regulation of a Novel Host MicroRNA

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting manuscript aimed to identify a host protein (G3BP2) that specifically interacts with the lncRNA encoded by infectious bronchitis virus (IBV). The introduction is interesting and provides relevant background information; however, the main objective of the study is not clearly stated. In addition, I recommend considering some minor comments in the remaining sections in order to improve the overall clarity of the study:

Introduction:

• In the last paragraph I suggest clearly describing the objective of the study.

Materials and Methods:

• Please provide the website of the bioinformatics and statistical software used, as well as the date of access to the software.
• I suggest describing in the statistical analysis section all variables analyzed and the groups used in each phase of the experiment.
• I believe it would be useful to explain the experimental design in more detail.

Results:

• Figure 2 shows results of the GO term enrichment analysis and the KEGG pathway analysis; however, the description of these analyses does not appear in the Materials and Methods section.
• Figure 5 shows results from the validation of the interaction between G3BP2 and IBV-lncRNA b; however, no interaction analysis is mentioned in Materials and Methods.

Discussion:

• I recommend mentioning briefly at the end of the first paragraph how the main results of the study helped to accomplish the objective or test the scientific hypothesis.
• I suggest not mentioning the figures in this section because they have already been described in the Results section.
• The results of the enrichment analysis do not appear to be discussed, nor are any previous studies cited to support these findings. Please review this.
• I suggest mentioning at the end if there were some limitations of the study to meet the objective or test the scientific hypothesis.

Conclusions:

• In this section, I recommend an initial concluding sentence that briefly explains the significance of the study findings.
• I suggest including at the end some recommendations for future work.

Author Response

Please find our point-by-point responses to all comments in the attached response letter.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Summary 

Overall, this manuscript presents an interesting and generally well-supported mechanism linking IBV-lncRNA to host antiviral regulation (via G3BP2 and a miRNA axis). The study would be strengthened by several minor revisions that improve clarity and reproducibility, including consistent miRNA naming/expression-direction statements, complete RT-qPCR parameters, consistent cell culture conditions, and harmonized statistical reporting (SD vs SEM). These issues appear addressable by textual and figure-legend edits without requiring major new experiments.

Minor Revision Comments 

1. Unify miRNA nomenclature and clarify an apparent expression-direction inconsistency.
   The Simple Summary/Abstract refers to novel-340, while the Introduction refers to miR-340 and states it is upregulated upon IBV infection. However, the Results describe novel-340 as gradually decreasing over the infection time course. Please ensure consistent naming throughout (miR-340 vs novel-340) and reconcile whether the miRNA is increased or decreased during infection (and under which condition, e.g., rIBV vs rIBV-C27107G).

2. RT-qPCR cycling conditions are incomplete (missing annealing temperature).
   The RT-qPCR protocol states “annealing for 1 min” but does not provide the annealing temperature. Please specify the annealing temperature and clarify whether 72°C is used only for extension.

3. Cell culture medium description is inconsistent and should be corrected for reproducibility.
   H1299 cells are described as maintained in RPMI-1640 (Section 2.1), but the transfection section states cells were cultured in DMEM. Please confirm which medium was used for H1299 and revise the manuscript accordingly (or explain if the medium was changed at specific steps).

4. Statistics reporting should be consistent across Methods and figure legends, and appropriate for time-course analyses.
   The Methods report data as mean ± SEM and use one-way ANOVA, while Figure 1 reports mean ± SD. Please standardize SD vs SEM throughout and ensure the statistical approach matches the time-course design (or clearly state how comparisons were performed at each time point).

5. Minor editing: remove duplicated sentence in virus titration description.
   There is a repeated sentence (“Viral titers were determined by plaque assay. Viral titers were determined by infecting …”). Please delete the duplication for clarity.

6. Clarify terminology and labeling for “ncRNA” vs “IBV-lncRNA.”
   The manuscript uses “ncRNA” to describe the recombinant virus product and states the mutant “does not produce ncRNA.” Elsewhere, groups/controls are labeled as “ncRNA group” and “anti-ncRNA control group.” Please define explicitly whether “ncRNA” always refers to IBV-lncRNA in this manuscript, and consider renaming groups/controls consistently (e.g., “IBV-lncRNA” / “anti-IBV-lncRNA”).

7. Minor grammar correction.
   In the Introduction, “IBV-lncRNA directly interact with G3BP2” should be corrected to “directly interacts.”

Recommendation: Minor revision.

Author Response

Please find our point-by-point responses to all comments in the attached response letter.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The objective of this manuscript was to investigate the functional role of IBV-lncRNA during infectious bronchitis virus infection. While the manuscript is interesting, the statistical analyses are incomplete because multiple range tests were not performed, and in some cases, a t-student test should be performed instead of an analysis of variance (ANOVA). Furthermore, the figures should include labels or asterisks to facilitate interpretation and identify significant differences.

In addition, the authors should consider the following points and suggestions, as well as answer some questions.

L.12. Infectious...
L.14. discovered...
L.25. Include the meaning of UTR.
L.72-86. An introduction typically ends with the manuscript's objective, not with results and conclusions. Please check and adjust.
L.92. fetal bovine serum (FBS).
L.148. Include the catalog number.
L.158. Table 2 and 3.
L.203. Include the meaning of PBS, RIPA and PMSF. Check that the meaning of the abbreviations has been included.
L.215. And why was not a multiple range test performed to identify statistically different treatments or groups? Some comparisons require a t-test. Please review.
L.227. What statistical test was performed to confirm significant differences? It would be helpful to indicate with asterisks in the figure 1 if there were differences in the titles.
L.229. Delete "the titer approximately".
L.257. Please complete the statistical analysis with a multiple range test such as Tukey's. Include the letters to identify whether or not there are significant differences.
L.284. And how is this identified in the figure? What statistical test did the authors perform?
L.296. Perform the missing statistical analysis to determine whether or not there are significant differences.
L.301. Perform the missing statistical analysis to determine whether or not there are significant differences.
L.347. Are the expression levels significantly lower? Perform the missing statistical analysis to determine whether or not there are significant differences.
L.363. It would be good to include the statistical analysis.
L.377-378. How is a negative correlation identified? It is assumed that all values ​​increase over time. Perform the missing statistical analysis to determine whether or not there are significant differences.
L.421. Perform the missing statistical analysis to determine whether or not there are significant differences.
L.443. Figure 2 and 5?
L.549. Improve the conclusion according to the results of this research

Comments for author File: Comments.pdf

Author Response

Please find our point-by-point responses to all comments in the attached response letter.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors considered each of the points suggested in the round of reviews.