Green Synthesis of Silver Nanoparticles with Extract of Indian Ginseng and In Vitro Inhibitory Activity against Infectious Bursal Disease Virus^†

Round 1

Reviewer 1 Report

I have revised the manuscript, and here are the comments.
1.    Change the title to “Green Silver Nanoparticles Fabricated by Indian Ginseng Extract and their Inhibitory Effects against Infectious Bursal Disease Virus.”
2.    Brief the encouragement lines at the beginning of abstract
3.    Clear the aim of this study in the abstract and introduction
4.    Scientific names must be italic i.e., Withania somnifera in line 22; check through the manuscript
5.    Add some data to the abstract
6.    Line 41, add space between “by1995.”
7.    Add lines about the defects of chemical and physical synthesis of nanoparticles also the advantage of green synthesis
8.    Line 79, adjust “10 mL of methanol: chloroform: water:: 12: 5 : 3” to “10 mL of methanol : chloroform: water (12:5:3, v/v).
9.    Add space between numerical values and units in the manuscript
10.    Enhance Figure 3A and Figure 4A, Figure 5B and C
11.    Where the control for comparison, clear
12.    Pay attention to the discussion; it should be enhanced
13.    Enhance the conclusion
14.    Delete lines 305-335
15.    Enhance and update the references list, i.e., journal titles and page numbers

Author Response

Dear editor,

Although we have tried our best to revise the manuscript to accommodate the suggestions of the reviewers individually, a greater effort has been made towards coming up with a revised version that meets the consensus of the suggestions collectively.

Individual responses to the reviewer comments follow.

Thank you.

Authors

Author Response File: Author Response.docx

Reviewer 2 Report

Ganguly and colleagues investigated the inhibitory activity of green synthesis AgNPs with plant extract (Indian ginseng) against IBDV. The study has some scientific merit in that it uses a more sustainable and cost-effective method to synthesize AgNPs for use in antiviral therapy against IBDV. However, some issues must be addressed. Here are the points I've identified:

Methodology

1. Line 85-89: This procedure is not clear. Was the silver nitrate solution constant at 10 mM? And why didn't the authors use different silver concentrations? because different metal precursor concentrations might have a significant effect on the synthesis results.

Section 2.3: Characterization of nanoparticles

2. The silver particles were separated using repeated high-speed centrifugation. What are the percentage conversion and final particle concentration?

3. Was the FTIR analysis performed on solid or liquid samples?

4. TEM analysis; the authors should add the type of solution used for particle suspension in the method section.

Results and Discussion:

1. The authors should go into more detail about why the optimal concentration and volume ratio resulted in better biosynthesized AgNPs.

2. Figure 2 caption; The red curve should be 5 μg/mL (line 170).

3. The authors used FTIR to confirm the presence of MCW extract in the biosynthesized nanoparticles. However, the authors did not perform FTIR for the functional group of the plant extract for comparison. How did the authors come up with this result?

4. Line 183; correct the referencing format

5. Figure 4; The TEM micrograph is not clear, and the scale is not visible in the figure.

6. The authors stated in the method section that the particle suspension was sonicated before performing the TEM analysis (Line 105). In this regard, the authors cannot conclude that the AgNPs obtained in this study were monodisperse because the sample was physically broken up by ultrasonic, which resulted in deagglomerates and a more uniform particle size. Dynamic light scattering (DLS) should be used to analyze the dispersity index.

7. How did the authors determine AgNPs size distribution? Is it done with the software? This should be added to the method section.

8. The concentration of biosynthesized AgNPs in this study should be analyzed in future studies rather than just weight concentration.

Conclusion; too general. The authors should emphasize the most significant or important findings.

The manuscript's back matter should be modified in accordance with the journal's format. (author contribution, review board statement, consent statement, data availability and etc)

Please use the proper referencing style.

Author Response

Dear editor,

Although we have tried our best to revise the manuscript to accommodate the suggestions of the reviewers individually, a greater effort has been made towards coming up with a revised version that meets the consensus of the suggestions collectively.

Individual responses to the reviewer comments follow.

Thank you.

Authors

Author Response File: Author Response.docx

Reviewer 3 Report

The authors reported an efficient method for green-synthesizing silver nanoparticles with an extract of Withania somnifera roots. They further tested its effect against non-enveloped Infectious Bursal Disease virus in chicken embryo fibroblast. The authors showed that the synthesized silver nanoparticles reduced the viral infectivity, decreased the intracellular viral load, and inhibited the cytopathy. 

The research showed its novelty by using the silver nanoparticles into a non-enveloped type of virus, and thus shed light on its potential use in the infections by similar viruses if the same inhibitory mechanisms can be proved. However, it cannot be accepted by its current form as the following issues remain to be stressed:

1. For all experiments with CEF, the authors should provide the design, i.e. how many replicates were included in each group, did the error bar show SEM or SD, what comparison the p value was showing, how was the p value calculated (test method, ttest, ANOVA or Tukey), etc., in the legend. I noticed that in figure 5, some graphs have no error bar. If you did not do the replicates, please add it to your experiment to validate the data. Also, whenever there is a significance, an asterisk should be added in the figure and specify with which group it was compared to have a significance.

2. Please add the manufacturers' information for all the reagents/kits/assays used in your study, so others have a reference to reproduce your data if needed.

3. Figure 5b, please re-adjust the figures so the gel picture is not blocking the bar graph.

4. Line274-284, this is over interpreting the results. You tested out an efficient way of synthesizing the nanoparticles and found that the particles can efficiently inhibit virus replication and improve cytopathy upon IBDV virus infection. Nevertheless, you did not test/confirm the pathway it exerts the effect. The similarity in the structure or biochemical nature does not necessarily mean the similarity in molecular interactions. Thus, such interpretation or suggestion should not be mentioned unless extra evidence is provided.

5. Please provide a figure or table for all the comparisons you mentioned with a significant p value. It is not convincing to just give it as p<0.01/0.05 without showing the original analyses.

Author Response

Dear editor,

Although we have tried our best to revise the manuscript to accommodate the suggestions of the reviewers individually, a greater effort has been made towards coming up with a revised version that meets the consensus of the suggestions collectively.

Individual responses to the reviewer comments follow.

Thank you.

Authors

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have carefully processed all comments. The quality of the manuscript has increased significantly. I have no further comments.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have addressed all of the reviewers’ queries and revised the manuscript. Some general issues remained to be addressed.

Title: The authors should recheck the title. Is thereof a typo or was it added on purpose?

Line 152, 159, 183, 208, 211, 239, etc. Please do not italicize the word Figure. Recheck and correct all.

Figure 5a,b: AgNO₃ rather than AgNO3. Check and correct all.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors showed their efforts on modifications. However, one remaining issue is still the data presentation in the figures.

1. In figure 5, why you have stars on all graphs including the control??? Also, what is the difference between ** and ***, is it p<0.01 vs. p<0.001? You must specify that in the caption.

2. You mentioned in the text that all assays were done in triplicates, n=3 should also be shown in the caption, together with your analytical methods (e.g. ANOVA, Tukey, etc.). 

3. Why in figure 5b, the error bar only showed the upper level, while in 5c, both upper and lower level were displayed? Please be consistent with the way displaying the data. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx