Effect of Tilapia Parvovirus (TiPV) on Fish Health: An In Vitro Approach

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper by Kumar, Das and colleagues is interesting and well written, mainly described TiPV induces significant cytotoxicity in DRG cells, leading to severe cellular damage and alters host immune responses by modulating the expression of key immune genes, which may contribute to its virulence and persistence. While the data presented supports the authors’ conclusions, there are still some issues in the manuscript that need to be improved:

1. Lines 30-41: To make the Introduction in the paper more focused, it recommend streamlining the Introduction by removing sections that are not directly related to our specific topic. Since this paper addresses the situation of parvovirus in tilapia, it would be more effective to concentrate on descriptions pertinent to tilapia culture and diseases rather than the broader context of aquaculture diseases.
2. It recommend that the author carefully review and correctly cite all references, ensuring that the cited data matches the original literature. For instance, the nucleotide sequence 4269 (Lines 55-56) appears in Liu's work rather than Du's. Additionally, could you please verify if Line 61, Das,et al, 2024, accurately describes the first discovery of parvovirus in crocodiles?
3. Additionally, it suggest incorporating appropriate references in the descriptions related to lines 72-74, line 97......
4. Why did authors choose to use the second-generation TiPV strain for the test instead of the virus that has been consistently stable over multiple passages? This decision was made because the second-generation virus may still contain residual tilapia cell tissue, such as cell debris and other interfering substances, which could potentially affect the overall test results. Did the authors consider this factor? Are there alternative experiments that could better validate the appropriateness of using the second-generation tissue viruses?
5. Figure 2: Is it appropriate to use the term "treatment group" to identify the outcomes of the TiPV cell group? I believe it would be more accurate to refer to this as an infection group, since the focus is on the infection rather than a treatment.
6. Figure 3: Why did the author select these particular immune genes for the experiment following TiPV infection in fish, like ? Is there a specific rationale or relevant basis for this choice? Additionally, can the experimental results obtained by the authors provide insight into the specific route of TiPV infection?

Author Response

Dear Reviewer,

We sincerely thank you for your response and comments. We appreciate the thorough review and helpful suggestions, which we believe have improved the manuscript. We have considered and tried to address all the comments and suggestions you and the reviewer gave.

We have attempted to answer the comments systematically. Our response to the comments is included in the following paragraphs.

 

1. Lines 30-41: To make the Introduction in the paper more focused, it recommend streamlining the Introduction by removing sections that are not directly related to our specific topic. Since this paper addresses the situation of parvovirus in tilapia, it would be more effective to concentrate on descriptions pertinent to tilapia culture and diseases rather than the broader context of aquaculture diseases.

• We completely agree with the reviewer's comments. As suggested, we hve removed the paragraph and started introduction from tilapia aquaculture.

 

2. It recommend that the author carefully review and correctly cite all references, ensuring that the cited data matches the original literature. For instance, the nucleotide sequence 4269 (Lines 55-56) appears in Liu's work rather than Du's. Additionally, could you please verify if Line 61, Das,et al, 2024, accurately describes the first discovery of parvovirus in crocodiles?

•  We are really sorry for our mistake. We have revised the reference and included reference studied the parvovirus infection in crocodiles.

 

3. Additionally, it suggest incorporating appropriate references in the descriptions related to lines 72-74, line 97......

• As per the suggestions, we have included the references in the rwevised manuscript.

 

4. Why did authors choose to use the second-generation TiPV strain for the test instead of the virus that has been consistently stable over multiple passages? This decision was made because the second-generation virus may still contain residual tilapia cell tissue, such as cell debris and other interfering substances, which could potentially affect the overall test results. Did the authors consider this factor? Are there alternative experiments that could better validate the appropriateness of using the second-generation tissue viruses?

• We completely agreed with the reviewer's comments that residual tilapia cell tissue might interfere with results. To avoid this, the virus grown in the cell line was centrifuged, and the supernatant was filtered through a 0.2 μm filter to remove debris and other microbial particles. The content solution was again inoculated in a fresh cell line, and the whole process was repeated again. Later, the virus particle was used to infect the cell line and analysis (MTT assay and Gene expression).

 

5. Figure 2: Is it appropriate to use the term "treatment group" to identify the outcomes of the TiPV cell group? I believe it would be more accurate to refer to this as an infection group, since the focus is on the infection rather than a treatment.

• As suggested, we have revised the terminology treatment group with infected in Figure 2 and the manuscript.

 

6. Figure 3: Why did the author select these particular immune genes for the experiment following TiPV infection in fish, like ? Is there a specific rationale or relevant basis for this choice? Additionally, can the experimental results obtained by the authors provide insight into the specific route of TiPV infection?

• We screened several genes to investigate the effect of TiPV. However, only a few genes showed good amplification in the PCR and qPCR test runs. Hence, we selected the TLR, IL, MHC, CR and TNF genes. These genes provided basic information on inflammatory, receptor and health response during TiPV infection. Although it provided basic information on how TiPV modulates the immune response, further study is needed to identify key genes that interact with the virus during the infection process.

Reviewer 2 Report

Comments and Suggestions for Authors

This MS is devoted to the study of Tilapia parvovirus on fish cell culture. The authors obtained interesting data that indicate a significant pathological effect of parvovirus on fish farms. Although I have identified many mistakes and gave a number of recommendations, I nevertheless believe that after their correction this MS can be published in Microbiology Research journal

 

Line 1 “Brief Report” – May be Research Article?

Line 95-97. Since this paragraph is about in vitro models of the zebrafish, it would be more correct to insert the sentence in Line 95-97 after the sentence located in Line 46-49

Line 98-106. It is not clear, is it research conducted by the authors themselves in frame of current MS, or is it data obtained in previous studies by other researches. In addition, there is not any references to a research article. To avoid misunderstanding, please, correct these sentences, and set references if it is needed.

Line 118. The phrase “based on passive data collection during the outbreak period” is not clear, what do Authors mean? Please, clarify.

Line 123-124. Although the Authors pointed out that the samples were processed like by Austin and Austin (2016), but the Authors should briefly describe the procedure (in a separate chapter, or in a supplementary file).

Line 148-149. Please describe briefly the procedure for obtaining tilapia extracts

Line 155-156 “monitor background cytopathic effects” Please briefly give the list what cytopathic effects were used.

Line 169: Please set model, manufacture and Country of “A UV-visible spectrophotometer”

In Line 156 and Line 201 Authors used “at 28 °C.”, But in Line 195 – Authors used “at 37˚C”. Could the authors explain why they used different temperatures for incubation?

Line 248. After “days post-infection” set abbreviation “(dpi)”

Line 248-250. Please insert additional photos where this effect will be visible.

Line 266: Authors wrought about observation of “live/dead cells” in culture. But Authors used only MTT test, which allow only estimate metabolic activity of cells, but not “live/dead cells”. For calculation of “live/dead cells” Authors should use Annexin whit Propidium Iodide, for example. Authors cannot use “live/dead cells” term in MS, and have to rewrite current paragraph, to avoid misunderstanding.

Line 309-323. These sentons are discussion element. Please move its into Discussion section.

Figure 3. Please set housekeeping gene expression (β-actin). This will help to show the reliability of the increase in another genes expression.

Line 327-336. Please summarize the paragraph by inserting a brief (one sentence) summary of the data obtained in the current MS.

Line 337-340. Please, move these sentences in previous paragraph.

For sentence Line 344-347 set reference to Figure.

In the end of sentence (Line 348-351) set reference to Figure3

Line 355-367. In this paragraph the authors talk about “cellular viability assays” although they used the MTT test, which shows a decrease/increase cellular oxidoreductase enzymes activity. The MTT test only talks about assessing cell metabolic activity. A decrease in color during the MTT test does not directly indicate cell culture viability. Please, be careful when describing the MTT test. I recommend that authors speak more carefully about “cellular viability assays” and use more neutral terminology. If the authors would like to use the term "cellular viability assays", then additional tests with Propidium iodide and Annexin (for example) should be performed as markers of necrosis/apoptosis.

Line 368-369. Neither in the Materials and Methods nor in the Results sections, the authors provide information on how the "dead cell percentages" were calculated. I can propose that the authors calculated the concentration of cells in the experiment and in the control, and judged mortality based on this. If so, then this may indicate not only mortality, but also a decrease or complete absence of dividing cells. The authors should carefully rewrite this paragraph taking into account the above.

Author Response

Dear Reviewer,

We sincerely thank you for your response and comments. We appreciate the thorough review and helpful suggestions, which we believe have improved the manuscript. We have considered and tried to address all the comments and suggestions you and the reviewer gave.

We have attempted to answer the comments systematically. Our response to the comments is included in the following paragraphs.

 

1. Line 1 “Brief Report” – May be Research Article?

• As per the suggestion, the line 1 is revised.

 

2. Line 95-97. Since this paragraph is about in vitro models of the zebrafish, it would be more correct to insert the sentence in Line 95-97 after the sentence located in Line 46-49

• As suggested, we have revised the sentence in the manuscript.

 

3. Line 98-106. It is not clear, is it research conducted by the authors themselves in frame of current MS, or is it data obtained in previous studies by other researches. In addition, there is not any references to a research article. To avoid misunderstanding, please, correct these sentences, and set references if it is needed.

• As per the suggestion, we have revised the text and included appropriate references.

 

4. Line 118. The phrase “based on passive data collection during the outbreak period” is not clear, what do Authors mean? Please, clarify.

• We wanted to write ‘’based on questionnaires and passive data collection from fishers during the outbreak period’’; as suggested, we have revised the text.

 

5. Line 123-124. Although the Authors pointed out that the samples were processed like by Austin and Austin (2016), but the Authors should briefly describe the procedure (in a separate chapter, or in a supplementary file).

• As per the suggestion, we have revised the text and included a brief information in the supplementary file.

 

6. Line 148-149. Please describe briefly the procedure for obtaining tilapia extracts

• As per the suggestion we have included included a brief information in the supplementary file.

 

7. Line 155-156 “monitor background cytopathic effects” Please briefly give the list what cytopathic effects were used.

• As suggested, we have included the list of TiPV characteristics CPE in the revised text.

 

8. Line 169: Please set model, manufacture and Country of “A UV-visible spectrophotometer”

• As per the suggestions, we have included the details.

 

9. In Line 156 and Line 201 Authors used “at 28 °C.”, But in Line 195 – Authors used “at 37˚C”. Could the authors explain why they used different temperatures for incubation?

• We are extremely sorry for this typographical mistake. In all incubation conditions, we have kept 28 °C. Accordingly, the text has been modified.

 

10. Line 248. After “days post-infection” set abbreviation “(dpi)”

• As per the suggestions, we have included the details.

 

11. Line 248-250. Please insert additional photos where this effect will be visible.

• We are extremely sorry that we forgot to take pictures during the virus isolation in the Cell Line. The only pictures we have are of the virus infection process (different time points) in the cell lines (included in Figure 2).

 

12. Line 266: Authors wrought about observation of “live/dead cells” in culture. But Authors used only MTT test, which allow only estimate metabolic activity of cells, but not “live/dead cells”. For calculation of “live/dead cells” Authors should use Annexin whit Propidium Iodide, for example. Authors cannot use “live/dead cells” term in MS, and have to rewrite current paragraph, to avoid misunderstanding.

• We agreed with the reviewer's comments. As suggested, we have revised the manuscript and changed the word live/dead cells with viable and non-viable cells.

 

13. Line 309-323. These sections are discussion element. Please move its into Discussion section.

• As per the suggestions, we have revised the manuscript.

 

14. Figure 3. Please set housekeeping gene expression (β-actin). This will help to show the reliability of the increase in another genes expression.

• As suggested, we have revised the text and included secretion of the housekeeping β-actin gene in the revised figure and manuscript.

 

15. Line 327-336. Please summarize the paragraph by inserting a brief (one sentence) summary of the data obtained in the current MS.

• As per the suggestions, we have included a brief summary of the results in the revised paragraph.

 

16. Line 337-340. Please, move these sentences in previous paragraph.

• As suggested, we have revised the text.

 

17. For sentence Line 344-347 set reference to Figure.

• As per the suggestions, we have cited the figure in the revised text.

 

18. In the end of sentence (Line 348-351) set reference to Figure3

• As per the suggestions, we have cited the figure in the revised text.

 

19. Line 355-367. In this paragraph the authors talk about “cellular viability assays” although they used the MTT test, which shows a decrease/increase cellular oxidoreductase enzymes activity. The MTT test only talks about assessing cell metabolic activity. A decrease in color during the MTT test does not directly indicate cell culture viability. Please, be careful when describing the MTT test. I recommend that authors speak more carefully about “cellular viability assays” and use more neutral terminology. If the authors would like to use the term "cellular viability assays", then additional tests with Propidium iodide and Annexin (for example) should be performed as markers of necrosis/apoptosis.

• We completely agreed with the reviewer's comments. As suggested, we have revised the manuscript and changed the terminology in the text.

 

20. Line 368-369. Neither in the Materials and Methods nor in the Results sections, the authors provide information on how the "dead cell percentages" were calculated. I can propose that the authors calculated the concentration of cells in the experiment and in the control, and judged mortality based on this. If so, then this may indicate not only mortality, but also a decrease or complete absence of dividing cells. The authors should carefully rewrite this paragraph taking into account the above.

• Again, we are sorry for our wrong interpretation of MTT results; we have revised the text and presented our MTT results as viable and non-viable cells.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The changes improved the quality of the manuscript. I suggest the manuscript accepted for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

I have no any comments. The Authors take into account all comments all of my recommendations/comments and now MS in suitable for publication in Microbiology Research journal