Volatile Organic Compounds Induced upon Viral Infection in Cell Culture: Uniform Background Study with Use of Viruses from Different Families

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is an interesting work. However, the results are inconclusive as to specific VOC compounds that could serve as reliable biomarkers of viral infection. This is because the same compounds appeared in both the treatment and control samples at similar concentrations. Below are just a few specific suggestions:

1. Line 43-45: "Different...detecting VOCs." Include some references to support this. One suitable reference is https://doi.org/10.1016/j.microc.2025.113700.

2. Line 153: "Fifferent classes of arganic compounds". Correct the typos here.

3. Figure 4: Include the percentage of the total variance in the original dataset that is explained by each specific principal component PC1 and PC2. Eg. PC1 (XX%).

Author Response

We thank the reviewer for the constructive evaluation of our manuscript. Indeed, our initial hypothesis assumed that viruses from different families would generate distinct, virus-specific VOC biomarkers following infection. However, as the reviewer correctly noted, our results showed that the same VOCs were present in both infected and control samples, for some the concentration differed.

1. Line 43-45: "Different...detecting VOCs." Include some references to support this. One suitable reference is https://doi.org/10.1016/j.microc.2025.113700.

Response:

Thank you for the comment, the reference was added to the manuscript.

 

2. Line 153: "Fifferent classes of arganic compounds". Correct the typos here.

 

Response: The typos have been corrected.

 

 

3. Figure 4: Include the percentage of the total variance in the original dataset that is explained by each specific principal component PC1 and PC2. Eg. PC1 (XX%).

Response:

Thank you for your valuable input. The figure has been amended to show all the data points (not only averages) and a subfigure for an additional principal component has been included.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents studies of cell cultures' response to viral infection by emission of volatile components. Studies were performed for evolution during three days using established chemical analysis technologies.
The results may be interesting; however, I have some objections, mainly to the way they were presented and discussed.

Lines 43-45 and 58-60 enumeration is repeated, and in my opinion could be aggregated into one paragraph.

Figure 1B shows that the chosen black color of bars for 24 h is the same as the whiskers, so they are not visible. What are the units of the y-axis?

Why is a table presented as Figure 1C? I can understand that Subfigures A and B were merged into one figure as they are related. But this table seems to show different results by different experimental methods. Even A and B, in my opinion, could be presented as separate figures.

Second column from the left of Fig.1C, should it be 72h?

How was the score in Fig.1C determined?

Line 153 should be Different.

Lines 153-157, the identified chemical components are listed in the text. In my opinion, a table with the list of chemical compounds, their abundance/concentration for various treatments, chemical formulas, etc., should be presented for the readers. As I know, the journal does not impose a limit on pages. But if the authors prefer, it can be provided as supplementary materials. However, personally, I would prefer to have it in the manuscript.

Figure 2, I'm not convinced that the chosen heatmap is the best way of presenting these results. Reading the color scale is always difficult. Probably even a linear or bar chart would be more appropriate. What else, probably using this chart, is it possible to compare chemical components for various viruses at a specified time moment. However, it is impossible to compare the time evolution of the concentration of a given component. Such data are placed in different subcharts, but what is more awkward, various scales of colors are used for subfigures. That is an important limitation since time trends of chemical components are discussed in the manuscript.

Lines 167-178, the statistical significance of some results is mentioned. The statistical analysis should be presented with numerical values, and the size of the effect should be presented.

It is not evident from the description what was tested for statistical significance, differences between various treatments at a given time moment, or evolution over time and changes over time. It seems that both types of results are discussed, but I'm not sure what and when statistical tests were used, what the results are, and the sizes of effects.

Figure 4, in lines 392-394, it is confirmed that replications were used, and the experiment does not present results of a single measurement. So why is only one dot for each treatment presented? What is there no data for EVH-1 24h? Probably the authors wanted to present only averages, and for them, the trend is more pronounced. Averages could be presented, but individual observations are necessary.

Also, I think that the choice of method for differentiation of the time of samples, by the size of markers, is confusing. It suggests that there is some measure presented. Better would be the usage of different symbols (circles, squares, triangles, etc.). However, I understand that the authors can have a different opinion.

What results were transformed by PCA? Concentrations for all chemical components?

It should be described how the GC-MS results were scaled to obtain concentrations.

 

Author Response

Reviewer 2

The manuscript presents studies of cell cultures' response to viral infection by emission of volatile components. Studies were performed for evolution during three days using established chemical analysis technologies.
The results may be interesting; however, I have some objections, mainly to the way they were presented and discussed.

 

Lines 43-45 and 58-60 enumeration is repeated, and in my opinion could be aggregated into one paragraph.

 

Response: We appreciate the reviewer’s observation. However, in our view, these two enumerations address related but distinct points, and merging them would reduce clarity. For this reason, we believe that the current structure is the most transparent and informative for the reader.

 

 

Figure 1B shows that the chosen black color of bars for 24 h is the same as the whiskers, so they are not visible. What are the units of the y-axis?

 

Response: We thank the reviewer for this helpful remark. The color of the 24-hour bars in Figure 1B has been changed to ensure that the whiskers are clearly visible.

 

Regarding the axis labeling: Figure 1B is a column plot, not an x–y scatter plot. Therefore, the x-axis contains categorical labels (virus names and time points), while the y-axis represents the measured values of OD.

Why is a table presented as Figure 1C? I can understand that Subfigures A and B were merged into one figure as they are related. But this table seems to show different results by different experimental methods. Even A and B, in my opinion, could be presented as separate figures.

 

Response: We thank the reviewer for this observation. It is correct that Figure 1B represents results obtained using a different experimental assay than the images shown in Figure 1A, and that Figure 1C contains averaged viral titers from two independent experiments performed on samples used for chromatographic analyses. Thus, panel C indeed reflects results generated by a different method.

However, all three datasets describe complementary aspects of the same biological question, namely the comparison of infection kinetics between different viruses. Presenting these elements together in a single figure follows a common convention, allowing the reader to visualize the overall dynamics of infection in one place and facilitating direct comparison across methods and virus types. Since other reviewers did not raise concerns regarding this layout, we believe that retaining the unified figure provides the clearest representation of the results.

 

Second column from the left of Fig.1C, should it be 72h?

 

Response: Thank you for your close attention, indeed it should be 72h.

 

 

How was the score in Fig.1C determined?

 

Response: The score presented in Fig. 1C was a visual assessment reflecting how strongly each infected cell line differed from the uninfected control. A score of 0 would corresponded to the absence of visible CPE. This type of evaluation cannot be quantified more objectively, because the cytopathic effects differ markedly between the viruses, as described in the Results section. The purpose of including this visual score was to illustrate how the observed CPE relates to the corresponding virus titers. This might be relevant for virologists who would like to interpret the results.

 

Line 153 should be Different.

 

Response: Thank you, it was corrected.

Lines 153-157, the identified chemical components are listed in the text. In my opinion, a table with the list of chemical compounds, their abundance/concentration for various treatments, chemical formulas, etc., should be presented for the readers. As I know, the journal does not impose a limit on pages. But if the authors prefer, it can be provided as supplementary materials. However, personally, I would prefer to have it in the manuscript.

 

Response:We thank the reviewer for this helpful suggestion. To accommodate this comment, as well as the remarks of Reviewer 3, we have added Table 1 to the main manuscript, which lists all identified chemical components together with their identification parameters.

However, for reasons of clarity and readability, we felt that including the full concentration data for every measurement within the main text would overly complicate the presentation. Therefore, all quantitative concentration values are now provided in Supplementary Material 1, where they can be reviewed in full detail.

 

We hope that this addition improves the manuscript’s transparency and usefulness to readers.

 

Figure 2, I'm not convinced that the chosen heatmap is the best way of presenting these results. Reading the color scale is always difficult. Probably even a linear or bar chart would be more appropriate. What else, probably using this chart, is it possible to compare chemical components for various viruses at a specified time moment. However, it is impossible to compare the time evolution of the concentration of a given component. Such data are placed in different subcharts, but what is more awkward, various scales of colors are used for subfigures. That is an important limitation since time trends of chemical components are discussed in the manuscript.

 

Response: We appreciate the reviewer’s thoughtful comments regarding the visualization of the VOC concentration data. We agree that the original heatmap layout—with multiple subfigures and differing color scales—made it difficult to compare temporal trends across viruses. Although heatmaps can be an effective way to communicate complex multivariate data, we acknowledge that the chosen format did not fully support clear interpretation.

In response to the reviewer’s suggestion, we have redesigned Figure 2 by unifying the color scale and presenting the data as one consolidated heatmap rather than three separate subfigures. This modification greatly improves consistency and allows the reader to more easily follow time-dependent trends for each chemical component across all virus treatments. We believe the revised figure now offers a clearer and more intuitive representation of the dataset.

 

 

Lines 167-178, the statistical significance of some results is mentioned. The statistical analysis should be presented with numerical values, and the size of the effect should be presented.

 

It is not evident from the description what was tested for statistical significance, differences between various treatments at a given time moment, or evolution over time and changes over time. It seems that both types of results are discussed, but I'm not sure what and when statistical tests were used, what the results are, and the sizes of effects.

 

Response:

We thank the reviewer for highlighting the need for clarity regarding our statistical analysis. Here, we provide a detailed explanation of the tests performed, the comparisons made, and the effect sizes. This information was added to the manuscript R1 (lines 194-204).

For each time point (24, 48, and 72 hours post-infection), a separate one-way ANOVA was performed using only the samples collected at that specific time. Thus, virus-infected samples at 24 h were compared exclusively with the 24-h control group, the 48-h virus samples with the 48-h control group, and the 72-h virus samples with the 72-h control group. When the ANOVA indicated significant differences, Tukey’s HSD test was used to identify which virus types differed from the corresponding time-matched control.

Analysis of variance revealed that at 24 h post-infection, sample group (virus vs. control) had a significant effect on VOC profiles (F(15, 22.49) = 2.81, p = 0.013, partial η² = 0.652), with an observed noncentrality parameter of 42.11 and observed power of 0.934, indicating that VOC composition differed significantly between virus-infected and control samples even at this early stage. At 48 h post-infection, the effect of virus type on VOCs approached significance (F(36, 3.68) = 3.64, p = 0.121, partial η² = 0.973), with an observed noncentrality parameter of 131.21 and observed power of 0.437, reflecting a very large effect size. By 72 h post-infection, a significant overall effect of virus type on the combined VOC profiles was observed (F(30, 3.61) = 7.95, p = 0.036, partial η² = 0.985, observed power = 0.75), demonstrating that virus infection substantially altered VOC composition compared with controls, with a very large effect size.

 

 

Figure 4, in lines 392-394, it is confirmed that replications were used, and the experiment does not present results of a single measurement. So why is only one dot for each treatment presented? What is there no data for EVH-1 24h? Probably the authors wanted to present only averages, and for them, the trend is more pronounced. Averages could be presented, but individual observations are necessary.

 

Also, I think that the choice of method for differentiation of the time of samples, by the size of markers, is confusing. It suggests that there is some measure presented. Better would be the usage of different symbols (circles, squares, triangles, etc.). However, I understand that the authors can have a different opinion.

 

Response: Thank you for this observation. We now present data with a dot for each measurement. Our former decision to present averages was dictated by the plot readability. However, we believe that using the latter suggestion (ie. differentiating times by marker, not size), the resultant plot with all the data is sufficiently legible. Use of all the data also allows for much greater statistical power of PCA and provides for greater explained variance along the first principal components.

 

What results were transformed by PCA? Concentrations for all chemical components?

 

Response: Yes, these were the concentrations with the background (ie. empty medium concentrations) subtracted.

 

It should be described how the GC-MS results were scaled to obtain concentrations.

Response: Compound concentrations were determined using the internal standard (IS) method. For each sample, peak areas of the analytes were extracted from the GC-MS chromatograms and normalized by the peak area of the internal standard to correct for variations in injection volume and instrument response. The normalized ratios were then converted to absolute concentrations in µg per g of sample by multiplying by the known amount of internal standard added and dividing by the sample mass .

Mathematically, this can be expressed as:

Concentration (µg/g) = (Area compound/Area IS) ​​× mIS​ ÷ msample​

 

This was added to materials and method section.

Reviewer 3 Report

Comments and Suggestions for Authors

Before the manuscript can be considered for publication, the following comments and suggestions are respectfully offered to improve the clarity, scientific rigor, and overall quality of the work.

1. Page 3 Lines 95-96: The term “High-Sensitivity MS” is unclear. Please specify the exact mass-spectrometry technique being referred to and add appropriate references to support this section.

2. Page 11 Lines 403, 409, 410: “…1 mL min -1 .”. Correct the unit.

3. Results: Please include a table listing all VOCs identified in this study, along with their experimental LRIs, corresponding literature LRIs, and any additional identification parameters.

4. Figure 4: All replicates should be added to the PCA plot to make the visualization of the obtained data more reliable.

5. Page 9 Line 311: “...Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa...”. Put the scientific names in italics.

Author Response

Reviewer 3

Before the manuscript can be considered for publication, the following comments and suggestions are respectfully offered to improve the clarity, scientific rigor, and overall quality of the work.

Page 3 Lines 95-96: The term “High-Sensitivity MS” is unclear. Please specify the exact mass-spectrometry technique being referred to and add appropriate references to support this section.

 

Response: Thank you for the comment, we deleted the “High-Sensitivity MS” term as other used terms are more relevant for our research.

 

Page 11 Lines 403, 409, 410: “…1 mL min -1 .”. Correct the unit.

 

Response: Thank you. The unit was corrected.

 

Results: Please include a table listing all VOCs identified in this study, along with their experimental LRIs, corresponding literature LRIs, and any additional identification parameters.

 

Response:

The table was included (Table 1).

 

Figure 4: All replicates should be added to the PCA plot to make the visualization of the obtained data more reliable.

 

Response: Thank you for this suggestion. We have now replaced the plot to include all the replicates.

 

1. Page 9 Line 311: “...Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa...”. Put the scientific names in italics.

Response: Thank you. The bacteria species were put in italics.