Characterization of SNX5 in Orange-Spotted Grouper (Epinephelus coioides) during In Vitro Viral Infection

Round 1

Reviewer 1 Report

Title

-        A term “in vitro” should be added. Response to viral infection was perform on cell line only?

Overall

-        Scientific writing can be improved.

 

Introduction

 

Line 57-58: Recent studies from what animal or human?

 

M&M

2.2

- Do all the primers require same annealing temp?

- What is the amplicon size for each primer pair?

- In Table 1, please adjust the layout for primer sequence. Page layout is not consistence.

 

2.4

Please provide details on animal use protocol, for example, number of fish, euthanasia protocol, ethical statement, husbandry, etc.

 

Please explain primer detail used in this section.

 

2.5 Line134

How many replications for each time point?

 

2.8

- This section should be revised.

- What statistical tool did you use? And on which parameters?

 

Results

Line 179 “the highest identity 179 compared with Epinephelus coioides was 98.56% for Epinephelus lanceolatus”

 

Please revise this sentence. Also, Epinephelus coioides is not the first mention, should it be E. coioides (also check this format throughout the MS) and please add common name for Epinephelus lanceolatus.

 

Table 2 can be added as a supplementary table. Accession No. submitted in the present study can be added in the text.

 

Table 3 have not been mentioned in the text. Table 3 can also be included as a supplementary material.

 

3.2

Why do you need to study the tissue distribution of SNX5? Please state the reason.

“in ten tissues of healthy Epinephelus coioides,” This sentence should be in M&M.

Did you do statistical analysis on tissue distribution?

 

3.4

“EcSNX5 or EcSNX5 siRNA were transfected into.” Transfected into….what?

Figure 5

What is the difference between “control” and “vector” please provide explanation in the legend?

I suggest the authors to integrate Fig5 A B D E as one.

Figure 7&8 may be combined as one figure. Put the same gene together. Also, please briefly explain “vector” and “control” (18S rRNA?).

 

Discussion

-        Please add some discussion regarding Characteristic of orange-spotted grouper EcSNX5 found in the present study.

-        “In the head kidney, the level of expression was the highest, Closely followed by the heart” In Abstract, you put heart before head kidney, please rearrange.

-        Please describe why EcSNX5 is highly expressed in heart, head kidney, …..any other organ. Is this similar to other fish species than zebrafish.

-        Line 306 “induced After stimulation”, capital letter.

-        Could you please add some discussion regarding the up-down-up-down expression pattern, and why the peak was observed 4 hours after infection?

-        Please add some brief explanation of how CP and RdRp gene work or facilitate the disease progression.

-        Please explain briefly what the mechanism is or what happen after these immune-related genes (IRF1, IRF3, IRF7, MX1, ISG15, ISG56, MDA5, TRIF, IL6, IL8, IL-1β and TNF-α) were suppressed.

Conclusion

-        Please give a short summary about benefit and further application of the understanding of SNX5 roles in orange-spotted grouper

Author Response

Response to Reviewer 1 Comments

 

Point 1: Title

A term “in vitro” should be added.

 

Response 1:

Thanks for your valuable suggestion. The title has been revised. （Line 3）

 

Point 2: Overall

-        Scientific writing can be improved.

 

Response 2:

Thanks for your valuable suggestion. The manuscript has been checked and modified.

 

Point3: Introduction 

Line 57-58: Recent studies from what animal or human?

 

Response3: 

Thanks for your valuable suggestion. The corresponding description has been modified. （Line 66-67）

 

Point4: 2.2

- Do all the primers require same annealing temp?

- What is the amplicon size for each primer pair?

- In Table 1, please adjust the layout for primer sequence. Page layout is not consistence.

 

Response4:

Thanks for your valuable suggestion. The annealing temperature of EcSNX5-ORF-F/R was 57℃, and the annealing temperature of all fluorescent quantitative primers was consistent at 60℃.The amplicon sizes for each primer pair were listed in the Table 1. The layout for primer sequence had been adjusted. （Line 115-117）

 

Point5: 2.4

Please provide details on animal use protocol, for example, number of fish, euthanasia protocol, ethical statement, husbandry, etc. Please explain primer detail used in this section.

 

Response5:

Thanks for your valuable suggestion. Details on animal use protocol had been added to the article. （Line378-382）

EcSNX5-ORF-F/R is the primer for amplification of EcSNX5 ORF. The pcDNA-EcSNX5-F/R is the primer for construction of eukaryotic expression vector. And the rest of the primers are used for qRT-PCR. （Line 115-116）

 

Point6: 2.5 Line134

How many replications for each time point?

Response6:

Thanks for your valuable suggestion. Three replicates were set up at each time point. （Line 144-145）

 

Point7: 2.8

- This section should be revised.

- What statistical tool did you use? And on which parameters?

Response7:

Thanks for your valuable suggestion. It has been consolidated into a table. （Line 184-185）

 

Point8: Line 179 “the highest identity 179 compared with Epinephelus coioides was 98.56% for Epinephelus lanceolatus”

 Please revise this sentence. Also, Epinephelus coioides is not the first mention, should it be E. coioides (also check this format throughout the MS) and please add common name for Epinephelus lanceolatus. Table 2 can be added as a supplementary table. Accession No. submitted in the present study can be added in the text. Table 3 have not been mentioned in the text. Table 3 can also be included as a supplementary material.

Response8:

Thanks for your valuable suggestion. The corresponding description has been modified in revised manuscript. （Line 190-194） Table 2 and Table 3 had been added as supplementary tables.

Point9:

Why do you need to study the tissue distribution of SNX5? Please state the reason.

“in ten tissues of healthy Epinephelus coioides,” This sentence should be in M&M.

Did you do statistical analysis on tissue distribution?

Response9: Tissue distribution can provide reference for the function of EcSNX5. “in ten tissues of healthy Epinephelus coioides,” had been added in M&M. （Line 128） The statistical analysis on tissue distribution is shown in Figure 3. Significant differences are indicated by different letters. （Line 210-212）

 

Point10: 3.4

“EcSNX5 or EcSNX5 siRNA were transfected into.” Transfected into….what?

Figure 5

What is the difference between “control” and “vector” please provide explanation in the legend?

I suggest the authors to integrate Fig5 A B D E as one.

Figure 7&8 may be combined as one figure. Put the same gene together. Also, please briefly explain “vector” and “control” (18S rRNA?).

Response10:

Thanks for your valuable suggestion. “EcSNX5 or EcSNX5 siRNA were transfected into.” Transfected into GS cells. （Line 220）The corresponding description has been modified as follow: Vector: Gene expression in GS cells transfected with pcDNA-3.1. Control: Gene expression in GS cells transfected with negative control siRNA. （Line 229-232）Figure5 A B D E had been consolidated into Figure 4. （Line 228）Figure 7&8 had been combined as one figure. （Line 259-265）

 

Point11: Please add some discussion regarding Characteristic of orange-spotted grouper EcSNX5 found in the present study.

Response11:

Thanks for your valuable suggestion. The description regarding characteristic of orange-spotted grouper EcSNX5 found in the present study had been added. （Line295-297）

 

Point12:   “In the head kidney, the level of expression was the highest, Closely followed by the heart” In Abstract, you put heart before head kidney, please rearrange.

 

Response12:

Thanks for your valuable suggestion. The abstract has been revised. （Line27）

 

Point13: Please describe why EcSNX5 is highly expressed in heart, head kidney, …..any other organ. Is this similar to other fish species than zebrafish.

 

Response13:

Thanks for your valuable suggestion. The corresponding description has been modified in revised manuscript. （Line299-304）There are no other studies related to the tissue distribution of SNX5 in other fishes.

 

Point14: Line 306 “induced After stimulation”, capital letter.

 

Response14:

Thanks for your valuable suggestion. The corresponding description has been modified in revised manuscript. （Line 312）

 

Point15: Could you please add some discussion regarding the up-down-up-down expression pattern, and why the peak was observed 4 hours after infection?

Response15:

Thanks for your valuable suggestion. The corresponding description has been added in revised manuscript. （Line 304-310）

 

Point16: Please add some brief explanation of how CP and RdRp gene work or facilitate the disease progression.

 

Response16:

Thanks for your valuable suggestion. The corresponding description has been added in revised manuscript. （Line 318-323）

 

Point17: Please explain briefly what the mechanism is or what happen after these immune-related genes (IRF1, IRF3, IRF7, MX1, ISG15, ISG56, MDA5, TRIF, IL6, IL8, IL-1β and TNF-α) were suppressed.

 

Response17:

Thanks for your valuable suggestion. The corresponding description has been added in revised manuscript. （Line 337-341）

Point18: Conclusion

Please give a short summary about benefit and further application of the understanding of SNX5 roles in orange-spotted grouper.

Response18:

Thanks for your valuable suggestion. The corresponding description has been added in revised manuscript. （Line 357-364）

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

General comments

            In the past years cell transfection and heterologous gene expression have allowed to assess the role of an increasing number of proteins on the replication of fish viruses. On this regard, the study of Wu et al. follows a similar approach and experimental design like many other works in the field (not a huge degree of novelty here). The protein under study (SNX5) seems to be relevant to nodavirus (RGNNV) infection of grouper, with the experimental evidence pointing to a SNX5 enhancing effect on NNV infection. The precise step in the virus life cycle that benefits from SNX5 function is not entirely clear. That could be the weak point in this paper, when trying to explain the positive effect on viral replication through SNX5 modulation of autophagy and apoptosis: apoptosis is usually a cell antiviral mechanism, and NNV has been reported to induce autophagy to assist its replication. How SNX5, an inhibitor of autophagy as well as an inducer of apoptosis could possibly enhance NNV replication is hard to comprehend. Thus, it is likely that SNX5 may be enhancing viral replication by other means: here, the inhibition of the expression of immune-related genes by SNX5 is supported by the experimental data.

Other remarks:

Figure 1 could go to supplementary materials.

Figure 5. The figure caption feels incomplete.

Figure 6. I believe there should be pictures of control non-infected cells in this figure.

1.- SNX5 expression in different tissues/organs (Fig. 4A): why not examine tissues from NNV-infected fish? I believe that is relevant to the study.

2.- SNX5 expression in GS cells after NNV infection (Fig. 4B). The up/down/up/down pattern was not explained. Was the experiment repeated to verify this odd pattern.

3.- Co-localization of SNX5 and NNV-CP proteins (Fig. 6) It seems clear that both proteins are cytoplasmic, but I´m not so sure that an actual subcellular co-localization has been proven (also, only one or two cells are shown).

Author Response

Response to Reviewer 2 Comments

 

Point 1: Title

A term “in vitro” should be added.

 

Response 1:

Thanks for your valuable suggestion. The title has been revised. （Line 3）

 

Point 2: Overall

-        Scientific writing can be improved.

 

Response 2:

Thanks for your valuable suggestion. The manuscript has been checked and modified.

 

Point3: Introduction 

Line 57-58: Recent studies from what animal or human?

 

Response3: 

Thanks for your valuable suggestion. The corresponding description has been modified. （Line 64-66）

 

Point4: 2.2

- Do all the primers require same annealing temp?

- What is the amplicon size for each primer pair?

- In Table 1, please adjust the layout for primer sequence. Page layout is not consistence.

 

Response4:

Thanks for your valuable suggestion. The annealing temperature of EcSNX5-ORF-F/R was 57℃, and the annealing temperature of all fluorescent quantitative primers was consistent at 60℃.The amplicon sizes for each primer pair were listed in the Table 1. The layout for primer sequence had been adjusted. （Line 114-116）

 

 

Point5: 2.4

Please provide details on animal use protocol, for example, number of fish, euthanasia protocol, ethical statement, husbandry, etc. Please explain primer detail used in this section.

 

Response5:

Thanks for your valuable suggestion. Details on animal use protocol had been added to the article. （Line390-394）

EcSNX5-ORF-F/R is the primer for amplification of EcSNX5 ORF. The pcDNA-EcSNX5-F/R is the primer for construction of eukaryotic expression vector. And the rest of the primers are used for qRT-PCR. （Line 114-115）

 

Point6: 2.5 Line134

How many replications for each time point?

Response6:

Thanks for your valuable suggestion. Three replicates were set up at each time point. （Line 146-147）

 

 

Point7: 2.8

- This section should be revised.

- What statistical tool did you use? And on which parameters?

Response7:

Thanks for your valuable suggestion. It has been consolidated into a table. （Line 188）

Point8: Line 179 “the highest identity 179 compared with Epinephelus coioides was 98.56% for Epinephelus lanceolatus”

 Please revise this sentence. Also, Epinephelus coioides is not the first mention, should it be E. coioides (also check this format throughout the MS) and please add common name for Epinephelus lanceolatus. Table 2 can be added as a supplementary table. Accession No. submitted in the present study can be added in the text. Table 3 have not been mentioned in the text. Table 3 can also be included as a supplementary material.

Response8:

Thanks for your valuable suggestion. The corresponding description has been modified in revised manuscript. （Line 194-197） Table 2 and Table 3 had been added as supplementary tables.

Point9:

Why do you need to study the tissue distribution of SNX5? Please state the reason.

“in ten tissues of healthy Epinephelus coioides,” This sentence should be in M&M.

Did you do statistical analysis on tissue distribution?

Response9: Tissue distribution can provide reference for the function of EcSNX5. “in ten tissues of healthy Epinephelus coioides,” had been added in M&M. （Line 129） The statistical analysis on tissue distribution is shown in Figure 3. Significant differences are indicated by different letters. （Line 214-215）

 

Point10: 3.4

“EcSNX5 or EcSNX5 siRNA were transfected into.” Transfected into….what?

Figure 5

What is the difference between “control” and “vector” please provide explanation in the legend?

I suggest the authors to integrate Fig5 A B D E as one.

Figure 7&8 may be combined as one figure. Put the same gene together. Also, please briefly explain “vector” and “control” (18S rRNA?).

Response10:

Thanks for your valuable suggestion. “EcSNX5 or EcSNX5 siRNA were transfected into.” Transfected into GS cells. （Line 225-226）The corresponding description has been modified as follow: Vector: Gene expression in GS cells transfected with pcDNA-3.1. Control: Gene expression in GS cells transfected with negative control siRNA. （Line 236-239）Figure5 A B D E had been consolidated into Figure 4. （Line 234-235）Figure 7&8 had been combined as one figure. （Line 270-276）

 

Point11: Please add some discussion regarding Characteristic of orange-spotted grouper EcSNX5 found in the present study.

Response11:

Thanks for your valuable suggestion. The description regarding characteristic of orange-spotted grouper EcSNX5 found in the present study had been added. （Line307-309）

 

Point12:   “In the head kidney, the level of expression was the highest, Closely followed by the heart” In Abstract, you put heart before head kidney, please rearrange.

 

Response12:

Thanks for your valuable suggestion. The abstract has been revised. （Line 28）

 

Point13: Please describe why EcSNX5 is highly expressed in heart, head kidney, …..any other organ. Is this similar to other fish species than zebrafish.

 

Response13:

Thanks for your valuable suggestion. The corresponding description has been modified in revised manuscript. （Line 315-316）There are no other studies related to the tissue distribution of SNX5 in other fishes.

 

Point14: Line 306 “induced After stimulation”, capital letter.

 

Response14:

Thanks for your valuable suggestion. The corresponding description has been modified in revised manuscript. （Line 324）

 

Point15: Could you please add some discussion regarding the up-down-up-down expression pattern, and why the peak was observed 4 hours after infection?

Response15:

Thanks for your valuable suggestion. The corresponding description has been added in revised manuscript. （Line 316-322）

 

Point16: Please add some brief explanation of how CP and RdRp gene work or facilitate the disease progression.

 

Response16:

Thanks for your valuable suggestion. The corresponding description has been added in revised manuscript. （Line 330-335）

 

Point17: Please explain briefly what the mechanism is or what happen after these immune-related genes (IRF1, IRF3, IRF7, MX1, ISG15, ISG56, MDA5, TRIF, IL6, IL8, IL-1β and TNF-α) were suppressed.

 

Response17:

Thanks for your valuable suggestion. The corresponding description has been added in revised manuscript. （Line 349-353）

Point18: Conclusion

Please give a short summary about benefit and further application of the understanding of SNX5 roles in orange-spotted grouper

Response18:

Thanks for your valuable suggestion. The corresponding description has been added in revised manuscript. （Line 369-376）

 

 

Round 2

Reviewer 2 Report

No further comments

Author Response

Response to Reviewer 2 Comments

 

Point 1: The precise step in the virus life cycle that benefits from SNX5 function is not entirely clear.  That could be the weak point in this paper, when trying to explain the positive effect on viral replication through SNX5 modulation of autophagy and apoptosis: apoptosis is usually a cell antiviral mechanism, and NNV has been reported to induce autophagy to assist its replication.  How SNX5, an inhibitor of autophagy as well as an inducer of apoptosis could possibly enhance NNV replication is hard to comprehend.  Thus, it is likely that SNX5 may be enhancing viral replication by other means: here, the inhibition of the expression of immune-related genes by SNX5 is supported by the experimental data.

Response 1:

Thanks for your valuable suggestion. The corresponding description has been added in revised manuscript and marked with yellow shades. (Line 351-355)

 

Point 2: Figure 1 could go to supplementary materials.

Response 2:

Thanks for your valuable suggestion. Figure 1 has been removed from the article and placed in the supplementary material.

 

Point3: Figure 5. The figure caption feels incomplete.

Response3: 

Thanks for your valuable suggestion. The figure caption of Figure 5 have been supplemented. (Line 229-232)

 

Point4: Figure 6. I believe there should be pictures of control non-infected cells in this figure.

Response4:

Thanks for your valuable suggestion. Pictures have been added as requirement. (Line 245)

 

Point5: SNX5 expression in different tissues/organs (Fig. 4A): why not examine tissues from NNV-infected fish? I believe that is relevant to the study.

 

Response5:

Thanks for your valuable suggestion. In this study, we investigated the roles of Ec-SNX5 during viral infection in vitro, so we just examined the gene expression in GS cells. The obtained data will provide a reference for in vivo study and which will be our interest in future research.

 

Point6: SNX5 expression in GS cells after NNV infection (Fig. 4B). The up/down/up/down pattern was not explained. Was the experiment repeated to verify this odd pattern.

Response6:

Thanks for your valuable suggestion. Three rereading experiments were performed. The corresponding description has been added in revised manuscript and marked with yellow shades. (Line 304-310)

 

Point7: Co-localization of SNX5 and NNV-CP proteins (Fig. 6) It seems clear that both proteins are cytoplasmic, but I´m not so sure that an actual subcellular co-localization has been proven (also, only one or two cells are shown).

Response7:

Thanks for your valuable suggestion. In our experiments, all pictures we obtained showed the same results for the colocalization of CP and EcSNX5. So we believed that the data is available to support the conclusion.

Author Response File: Author Response.pdf