Antiviral Activity of Glucosyl Hesperidin Against Feline Calicivirus
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAntiviral Activity of Hesperidin glucoside against Feline Calicivirus, a Norovirus Surrogate by Choi et al. dela with antiviral potential of hesperidin in vitro. In general, the paper is written quite nicely, and it was an interesting topic to read about. I have several comments/questions/suggestions for Authors:
- My first comment would be on term "Hesperidin glucoside"- this is incorrect. It is either "hesperidin" or "hesperetin glucoside" (or "glycoside of hesperetin", more general, but still correct term. You have described it quite nicely from line 45 and further--- however, I must insist that you make the changes in ENTIRE manuscript, in order to be completely precise and clear.
- Second remark would be on type of testing of antiviral activity. In your paper, you have tested only one type of potential mechanism of activity (I will refer you to reference https://www.sciencedirect.com/science/article/abs/pii/S0944711308001128 with a short disclaimer, this is not my work, I am not Author or Co-Author of the paper, it a reference where I have first found these experiments determining mechanism-of-activity, and I find it quite illustrating). I kindly suggest to you that you add a brief comment, a sentence or two, regarding the mechanism of activity of your compound in terms of anti-noroviral activity.
- Minor comments- all abbreviations should be defined at first appearance. Avoid staring the sentences with abbreviations. Figures should be self-standing, thus all abbreviations and explanations should be given in figure titles.
- typo line 78- DoGenBio (Seoul, Lorea). All... please make the change.
- Additionally, I would strongly suggest adding a comment on SI. What is a generally accepted number for natural products? Make a comment on this in terms of your obtained results.
Looking forward in reading the corrected version of your paper. Kindest regards.
Author Response
Comment 1
My first comment would be on term "Hesperidin glucoside"- this is incorrect. It is either "hesperidin" or "hesperetin glucoside" (or "glycoside of hesperetin", more general, but still correct term. You have described it quite nicely from line 45 and further--- however, I must insist that you make the changes in ENTIRE manuscript, in order to be completely precise and clear.
Response 1
Dear Reviewer,
We deeply appreciate your comments and suggestions on the article. The answer to your question or suggestion is written below.
I’ll explain in detail about hesperidin glucoside.
Hesperidin is a natural flavonoid glycoside consisting of hesperetin and the disaccharide rutinose (rhamnosyl-glucose). In contrast, hesperidin glucoside is a modified derivative produced by enzymatically attaching an additional glucose moiety to hesperidin. It is different from hesperidin or hesperetin glucoside. The molecular weight of hesperetin, hesperidin, and hesperidin glucoside is MW 303, 609, and 773, respectively. The term "hesperidin glucoside" may be changed to the more commonly used "glucosyl hesperidin", to avoid confusion. Therefore, "hesperidin glucoside" has been changed to "glucosyl hesperidin" in the entire text.
Comment 2
Second remark would be on type of testing of antiviral activity. In your paper, you have tested only one type of potential mechanism of activity (I will refer you to reference https://www.sciencedirect.com/science/article/abs/pii/S0944711308001128 with a short disclaimer, this is not my work, I am not Author or Co-Author of the paper, it a reference where I have first found these experiments determining mechanism-of-activity, and I find it quite illustrating). I kindly suggest to you that you add a brief comment, a sentence or two, regarding the mechanism of activity of your compound in terms of anti-noroviral activity.
Response 2
The purpose of this study was to conduct basic research whether hesperidin glucoside exhibits antiviral activity. However, this study did not clarify whether the mechanism of action involves inhibition of viral adsorption/penetration, suppression of viral replication, or enhancement of host cell recovery. Need for future research on the mechanism of action is added in the text. (=> 209-214, reference 41,42)
Comment 3
Minor comments- all abbreviations should be defined at first appearance. Avoid staring the sentences with abbreviations. Figures should be self-standing, thus all abbreviations and explanations should be given in figure titles.
Response 3
Revised according to comments. (=> All figures, texts)
Comment 4
typo line 78- DoGenBio (Seoul, Lorea). All... please make the change.
Response 4
Revised according to comments. (=> Line 78)
Comment 5
Additionally, I would strongly suggest adding a comment on SI. What is a generally accepted number for natural products? Make a comment on this in terms of your obtained results.
Response 5
We thank the reviewer for this suggestion. While higher SI values are typically regarded as desirable in evaluating antiviral agents, a definitive threshold has not been established. An SI value of around 3 is generally considered insufficiently effective, but for natural products, it may be considered to have potential for development and may require further investigation. An explanation and reference for the SI values of natural products has been added to the text. It is also suggested that lower SI values may confer certain benefits, where they are often associated with enhanced water solubility and, consequently, improved bioavailability. (=> Line 215-223, reference 38,43).
Best Regards
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsIn this study, the authors tested cytotoxicity and antiviral efficacy of hesperidin glucoside (HG), particularly using Crandell-Rees feline kidney cells and feline calicivirus. It was observed that HG had a low cytotoxicity and a low antiviral efficacy at high concentrations of ~8mM. This manuscript may be improved by considering several issues as indicated below.
1. In this study, the authors did not study norovirus or human cells. So, it is confusing to mention about norovirus in this manuscript. It is recommended to eliminate description about norovirus from this manuscript.
2. In Figure 2, it was strange to see that all three replicates of experiment resulted in the cell viability of 100% without variation for many cases, because the value of (Test group absorbance)/(Negative control group absorbance) should fluctuate.
3. Although positive control was included in this study, negative control was not. It would be necessary to include negative control to show that the antiviral efficacy observed for HG at 8mM may be biologically relevant.
Author Response
Comment 1
In this study, the authors did not study norovirus or human cells. So, it is confusing to mention about norovirus in this manuscript. It is recommended to eliminate description about norovirus from this manuscript.
Response 1
Dear Reviewer,
We deeply appreciate your comments and suggestions on the article. The answer to your question or suggestion is written below.
Because there are no in vitro systems or in vivo animal models that are available for growing norovirus, feline calicivirus has been widely accepted as a surrogate of norovirus (e.g., Test using feline calicivirus was officially recognized by US EPA). Accordingly, feline calicivirus has been employed in many studies as a substitute for norovirus. However, in accordance with the reviewer’s comment, the term “norovirus” was eliminated from the title. (=> Line 3)
Comment 2
In Figure 2, it was strange to see that all three replicates of experiment resulted in the cell viability of 100% without variation for many cases, because the value of (Test group absorbance)/(Negative control group absorbance) should fluctuate.
Response 2
As cell viability was expressed as a percentage relative to the negative control, values very close to 100% were rounded in the figure. This may give the impression of no variation, although raw absorbance data showed minimal but consistent fluctuations.
Comment 3
Although positive control was included in this study, negative control was not. It would be necessary to include negative control to show that the antiviral efficacy observed for HG at 8mM may be biologically relevant.
Response 3
We thank the reviewer for this suggestion. In our study, untreated cells (without hesperidin glucoside treatment) served as a reference, which functionally represented the negative control. We have revised the text to describe this more explicitly. (=> Line 150) In future studies, we will include a dedicated negative control group to further strengthen the demonstration of biological relevance.
Best Regards
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors, thank you for your answers and revised manuscript. I will be recommending the paper for acceptance. Kindest regards.
