Piscine Orthoreovirus-1 (PRV-1) Has Been Present in Chilean Salmon Aquaculture since at Least 1994

Round 1

Reviewer 1 Report

 Manuscript fishes-2313821 describes the detection of a salmon virus (PRV-1) in cultured fish of Chile nearly two decades before disease association with this virus was documented. Although this work presents limited new data, it is a significant finding for filling in gaps for our understanding the global spread of PRV and also in understanding historical variations of virulence for this virus and thus putatively worthy of publication. However, my major criticism is a lack of confirmatory support for the single PRV-1 detection and appropriate endogenous controls to ensure non-false negative results for all other tests.  

I agree with the authors that the rather robust qPCR Ct for the single detection appears genuine. But unfortunately, the authors currently have little evidence to counter the following hypothesis challenge: What evidence is there that the positive result presented is not a false positive? I would very much like to see confirmatory testing be undertaken in either one or both the following forms: (1) Show that PRV can be detected in an adjacent FFPE slice from the same block where the single positive detection was obtained, and/or (2) show that a different portion of the PRV genome can also be amplified from the same block/sample as positive result (e.g., use different PCR primers to target an alternative genome segment). This will require more lab work, however the effort/time required should be minimal and it would drastically improve the scientific validity of the current finding. If these subsequent tests cannot verify repeated detection of PRV-1, then the authors will need to amend their presentation to reflect the uncertainty of the current finding.  

I have a few additional recommendations for improving the presentation and technical accuracy of the manuscript:

Title

Ln2: please delete “, but not PRV-2 or PRV-3,” from the title for specific accuracy. The fact that these PRV genotypes were not detected in 42 preserved blocks is not sufficient evidence to say that these two genotypes were not present in Chilean aquaculture in the 1990’s. It might ultimately be true, but this study does not prove it.

Abstract

Ln21: I recommend deleting this last sentence. This sentence is fine in the manuscript discussion/conclusion but is unnecessary here in my opinion and the sentence before it is a much stronger sentence to finish on.

Introduction

Ln32-35: This sentence is confusing. I suggest using words rather than commas for easier reading. Specifically, something like “PRV-1 is associated with HSMI in Atlantic salmon, Salmo salar (2) and lowered hematocrit in chinook salmon, Oncorhynchus tshawytscha (3) and coho salmon, Oncorhynchus kisutch (4); PRV-2 is associated with erythrocytic inclusion body syndrome (EIBS) in coho salmon (5), and PRV-3 is associated with HSMI-like disease with anemia in rainbow trout, Oncorhynchus mykiss (6) and brown trout, Salmo trutta (7).”

Ln 45: I don’t understand “at seawater farm level”. I think “and net-pens” might be more accurate wording. Please check and clarify as needed.

Ln52-54: I don’t understand this question and I highly recommend changing it into a statement instead. Please clarify.

Ln54-56: Rather than summarizing the study, please amend this statement to describe the aim(s) of the study. Laying out the aims/objectives is a critical component to the introduction and currently is not explicitly stated.

Materials and Methods

Ln59-60: This statement requires some expansion. It should be stated who (i.e., what group/agency) took these original samples and for what purpose. Additionally, pleas clarify what “the X region” of Chile is.

Ln61:  delete “and shipped” as this is unnecessary.

Ln69: I suggest starting the sentence with “For this study, an hsCRC…” as it appears this scoring matrix was specifically developed here. If it has been used previously then please also provide a citation.

Ln78-83:  This table has a lot of redundant text that could be removed for clean ease of reading. I suggest removing “tissue surface” from all table cells which could be defined once in the legend. Additionally, “Relative ponderation” should be changed to “Relative weighting” or “Relative proportion” for correct English accuracy.

Ln102: Define what ISAV stands for at first use.

Results

Ln122-123: ISAV is missing from this legend. Additionally, I suggest removing the word “negative” throughout the table and replace it with “ND” for Not Detected or “No Ct” for No amplification above a preset threshold and define their meaning in the legend for all qPCR results.  For the PRV IHC, use either “+” or “-“. This will clean up the table presentation considerably. Additionally, it should be noted which samples are from Atlantic salmon and which are from Coho in this table.

Ln125: This states that the sample was “negative” for PRV by IHC, but in Table 2 there is a “+” in the IHC column indicating a positive. Please correct as appropriate.

Ln139: change “sequence would correspond” to “sequence most likely corresponds” for specific accuracy, since no sequence was obtained in this study.

Ln140: There are two things that in my opinion are needed to help support this statement. First, as discussed above, secondary confirmation for the single PRV-1 detection needs to be done either by targeting an alternative genome segment or independently detecting PRV RNA in a second block section. Second, would be to run an endogenous control showing that extractions and general RNA integrity in the blocks is sufficient to support reasonable levels of detection by running a qPCR to detect a salmon housekeeping gene like EF1a or Beta-actin or similar. There are a lot of negative qPCR results presented (209 to be exact). Currently there is no evidence to say that any of these targets (other than PRV-1) would have amplified correctly even if the sample was infected. An endogenous positive control would go a long way in alleviating my concerns that the extractions and qPCR assays were all run appropriately and that the results are not false negatives.

Author Response

Reviewer 1

R: Thank you very much for the constructive criticism. In fact, we agree with all your comments, and we know that not having obtained an S1 or M2 sequence to confirm is a weakness of the study. However, we are convinced that it is timely information that adds to the knowledge of viral diseases in salmon aquaculture.

 The results were what they were, and we cannot control for that. Unfortunately for us, the amount of tissue in each FFPE block was very sparse and we have no more material left to reanalyze. However, what we do provide in this second version of the manuscript is the fact that when we did a two-step PCR to try to obtain material for the M1 and M2 sequences, we also did the two-step PCR for L1 as a control, so these results confirm the PRV-1 (L1) result obtained previously by the one-step PCR. We have included a detailed protocol regarding the execution of two-step PCR for S1, M2 and L1 segments.

We are a routine diagnostic laboratory in Chile, so all our PCRs include the necessary controls and, of course, EL1a as an endogenous extraction control. All this information has been included in this new version of the manuscript for your consideration.

Attached is a final version of our manuscript with all changes marked in bold.

Title

Ln2: please delete “, but not PRV-2 or PRV-3,” from the title for specific accuracy. The fact that these PRV genotypes were not detected in 42 preserved blocks is not sufficient evidence to say that these two genotypes were not present in Chilean aquaculture in the 1990’s. It might ultimately be true, but this study does not prove it.

R: OK it is done.

Abstract

Ln21: I recommend deleting this last sentence. This sentence is fine in the manuscript discussion/conclusion but is unnecessary here in my opinion and the sentence before it is a much stronger sentence to finish on.

R: OK it is done.

Introduction

Ln32-35: This sentence is confusing. I suggest using words rather than commas for easier reading. Specifically, something like “PRV-1 is associated with HSMI in Atlantic salmon, Salmo salar (2) and lowered hematocrit in chinook salmon, Oncorhynchus tshawytscha (3) and coho salmon, Oncorhynchus kisutch (4); PRV-2 is associated with erythrocytic inclusion body syndrome (EIBS) in coho salmon (5), and PRV-3 is associated with HSMI-like disease with anemia in rainbow trout, Oncorhynchus mykiss (6) and brown trout, Salmo trutta (7).”

R: OK it is done.

Ln 45: I don’t understand “at seawater farm level”. I think “and net-pens” might be more accurate wording. Please check and clarify as needed.

 R: OK it is done.

Ln52-54: I don’t understand this question and I highly recommend changing it into a statement instead. Please clarify.

R: OK it was deleted.

Ln54-56: Rather than summarizing the study, please amend this statement to describe the aim(s) of the study. Laying out the aims/objectives is a critical component to the introduction and currently is not explicitly stated.

R: OK it is done.

Materials and Methods

Ln59-60: This statement requires some expansion. It should be stated who (i.e., what group/agency) took these original samples and for what purpose. Additionally, pleas clarify what “the X region” of Chile is. 

R: OK it is done. L56-L59: This is how it reads now: “Samples were fixed in 10% formalin and subsequently processed for standard histopathological diagnosis in the laboratory of the Instituto de Patología Animal, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile.”

Ln61:  delete “and shipped” as this is unnecessary.

R: OK it is done.

Ln69: I suggest starting the sentence with “For this study, an hsCRC…” as it appears this scoring matrix was specifically developed here. If it has been used previously then please also provide a citation.

R: OK it is done.

Ln78-83:  This table has a lot of redundant text that could be removed for clean ease of reading. I suggest removing “tissue surface” from all table cells which could be defined once in the legend. Additionally, “Relative ponderation” should be changed to “Relative weighting” or “Relative proportion” for correct English accuracy.

R: OK it is done.

Ln102: Define what ISAV stands for at first use.

R: OK it is done.

We have left the RNA extraction part from the FFPE block as a single section (L84) and included a part for "one-step qPCR" (L-104) and another for "two-step qPCR" (L-123).

L109-L121: This is how it reads now: “The RT-qPCR conditions for detection of each pathogen consisted in adding a master mix of 7.5 µl buffer, 0.15 µl 100 mM DTT, 0.225 µl diluted ROX solution, 300 nM each primer and 400 nM probe to 2 µl total RNA. Tubes were incubated for 10 min at 50°C to perform reverse transcription, followed by a denaturation step of 3 min at 95°C and 40 cycles of 3 sec at 95°C and 10 sec at 60°C. All qPCR assays were performed in duplicate. A positive control (RNA or DNA specific to the tested pathogen), a negative control without RNA or DNA and negative extraction control were also included in every run. All qPCR runs for the pathogens tested were accompanied by the expression of Atlantic salmon reference gene as endogenous extraction control (EF1a). Cycling threshold (Ct) values were recorded up to a maximum of 40 Ct. A Ct below the cut-off point was considered positive, and negative (NoCt) otherwise. The cut-off points for PRV-1, -2, and -3, ISAV, and EL1A assays was 35.00 and for P. salmonis was 33.01.”

L124-L147: This is how it reads now: “PRV-1 positive RNA extract was used as template for cDNA synthesis. Approximately 1 µg total RNA was reverse transcribed using the PrimeScript™ RT Reagent kit with gDNA Eraser (TAKARA) according to the manufacturer’s instructions. Specific primers were used for the S1 and M2 segments described by Kibenge et al. (18), and for the L1 segment described by Palacios et al. (2) were used. Briefly, the RT conditions consisted in the addition of a master mix of 2 µl 5X gDNA Eraser Buffer and 1 µl gDNA Eraser for each tube containing 1 µg total RNA (10 µl total volume). Tubes were incubated for 2 min at 42°C to erase gDNA. The reverse transcription proceeded immediately on the same tubes adding a master mix of 4 µl 5X PrimeScript Buffer, 1µl PrimeScript RT Enzyme Mix I and 4 µl specific primers (total volume 20 µl). Tubes were incubated 15 min at two different annealing temperatures (42°C and 50°C) followed by a 5 sec incubation at 85°C. The PCR products were confirmed by electrophoresis in 1% agarose gels and visualized with ethidium bromide staining. To confirm the results obtained by one-step RT-qPCR, we performed a qPCR for the L1 segment of PRV-1 using the KAPA Probe Fast qPCR kit Mastermix (2X) Universal. Briefly, a master mix consisting in 7,5 µl Buffer, 0,3 µl 50X ROX, 400 nM specific L1 primers and 200 µl L1 probe was added to 2 µl 1:10 diluted cDNA (total volume 15 µl). The PCR cycling conditions consisted in an incubation of 3 min at 95°C followed by 40 cycles of 3 sec at 95°C and 20 sec at 60°C. For the PCR stage of segments S1 and M2 the Platinum™ Hot Start PCR Master Mix (2X) was used. The liquid handling system created a master mix using 25 µl Platinum Hot Start PCR 2X Master Mix 200 nM each primer (either for S1 or M2, respectively) and 5 µl cDNA template to a total volume of 50 µl. The PCR cycling conditions consisted in an initial denaturation of 2 min at 94°C followed by 40 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C.” 

Results

Ln122-123: ISAV is missing from this legend. Additionally, I suggest removing the word “negative” throughout the table and replace it with “ND” for Not Detected or “No Ct” for No amplification above a preset threshold and define their meaning in the legend for all qPCR results.  For the PRV IHC, use either “+” or “-“. This will clean up the table presentation considerably. Additionally, it should be noted which samples are from Atlantic salmon and which are from Coho in this table.

R: OK it is done. We have included this sentence in the methodology, and it now reads as follows: “Cycling threshold (Ct) values were recorded up to a maximum of 40 Ct. A Ct below the cut-off point was considered positive, and negative otherwise. The cut-off points for PRV-1, -2, and -3, and ISAV assays was 35.0 and for P. salmonis was 33.01.”

Ln125: This states that the sample was “negative” for PRV by IHC, but in Table 2 there is a “+” in the IHC column indicating a positive. Please correct as appropriate. 

R: OK it is done.

Ln139: change “sequence would correspond” to “sequence most likely corresponds” for specific accuracy, since no sequence was obtained in this study.

R: OK it is done.

Ln140: There are two things that in my opinion are needed to help support this statement. First, as discussed above, secondary confirmation for the single PRV-1 detection needs to be done either by targeting an alternative genome segment or independently detecting PRV RNA in a second block section. Second, would be to run an endogenous control showing that extractions and general RNA integrity in the blocks is sufficient to support reasonable levels of detection by running a qPCR to detect a salmon housekeeping gene like EF1a or Beta-actin or similar. There are a lot of negative qPCR results presented (209 to be exact). Currently there is no evidence to say that any of these targets (other than PRV-1) would have amplified correctly even if the sample was infected. An endogenous positive control would go a long way in alleviating my concerns that the extractions and qPCR assays were all run appropriately and that the results are not false negatives.

R: We have included this information throughout the manuscript.

L143-L148: This is how it reads now: “One of the 42 samples (2.4%) of the heart samples examined were positive for PRV-1 by one-step RT-qPCR (average Ct, 28.08, < 35.00) and two-step qPCR (average Ct, 32.15, < 35.00) (Figure 2). Therefore, we confirm the presence of PRV-1 in independent qPCR reactions in different parts of the FFPE block and the loss of sensitivity of two-step PCR by about 4 orders of magnitude.”

Moreover, we have included a Figure 2 to show the amplification plot and view the plate layout of the two-step qPCR confirming positive results for L1 and negative for S1.

L86-L92: The size of the L1 segment amplified by qPCR primers and probes is small (79 bp) (2), but the fragments to amplify the S1 and M2 segments with sequencing primers are as large as 1081 and 2179 bp, respectively (18). This situation, associated with the fact that the genetic material available in the sample was probably fragmented, degraded (Ct 28.22 for the EL1a reference gene) and/or chemically modified after having been stored for 3 decades prior to this analysis, could explain the negative results for large fragments in the S1 and M2 segments.

L186-L193: This is how it reads now: “The size of the L1 segment amplified by qPCR primers and probes is small (79 bp) (2) but the fragments to amplify the S1 and M2 segments with sequencing primers are as large as 1081 and 2179 bp, respectively (18). This situation, added with the fact that the genetic material available in the sample was probably fragmented, degraded (average Ct 33.22 for reference gene EL1a, but < 35.00) and/or chemically modified after having been stored for 3 decades prior to this analysis, could explain not achieving large amplicons in S1 and M2, and obtaining a considerably smaller amplicon in L1.”

Reviewer 2 Report

The Manuscript deals with the analysis of the heart of some Chilean Salmon farmed species of the presence of the Piscine orthoreovirus-PRV virus (which affects the heart and skeletal muscle dramatically). Heart and skeletal muscle inflammation (HSMI) is caused by (PRV). The analyses have been conducted in 30-year-old formalin-fixed/paraffin-embedded specimens. RT-PCR and immunohistological analyses have evidenced the presence of PRV of subtype 1 instead of subtypes 2 and 3 that are already detected in EU farms. The authors conclude that some embryos coming from the EU have brought the pathogen to Chile’s farm. Formalin-fixed paraffin-embedded tissues could be a valuable resource from a pathogen diagnosis perspective, thus in principle, this study is interesting. However,  it presents some critical points. Firstly must be considered as descriptive communication and not an article but a short paper. Secondly, the data can be not sufficiently trustworthy, because of the combination of the factors that generally can give artefacts of data: fixation procedure/time of permanence/procedure of the extraction. Furthermore, the presentation of the data is not suitable for publication.In particular:

1)      Figure 1. Histopathological changes in the heart of Atlantic salmon farmed in Chile between 1992 and 1999. Mild focal mononuclear infiltrate (fmi) and mild focal cardiomyocyte degeneration (fcd) in ventricular stratum spongiosum (H&E, 40X bar 50 µm).

The figure is not of good quality. Furthermore, no-difference with other samples analysed has been shown.

2)      Table 2. Results of hsCRC, IHC PRV and RT-qPCR for PRV-1, PRV-2, PRV-3, and P. salmonis in each FFPE sample analyzed (individual fish).  

The table should be synthesized in both the results and the captions. i.e mononuclear cells [i.e. macrophages, MØ)]; negative: (-), etc.

3)      One of the 42 samples (2.4%) of the heart samples examined were positive for PRV-1 by RT-qPCR (cycle threshold, CT, 28.08). However, this sample was negative by IHC for PRV.

This data indicate the possible artefacts in RT-PCR. The immunohistochemistry is maintained for a long time when is embedded in paraffin. The author's speculation about the paraffin blocks, like the degradation of the viral proteins and/or the nature of the samples, is not a plausible reason for the lack of detection. The most apparent is the RT-PCR artefact. To confirm this research lacks: use some samples frozen at the time (1993-94). Use paraffin blocks from pathological hearts of 2023 to compare the RT_PCR data (RT-PCR/IHC results)

Author Response

Reviewer 2

The figure is not of good quality. Furthermore, no-difference with other samples analysed has been shown.

R: The microphotography is of good quality; another issue is that the tissue changes are slight. In any case, we have included a picture from a normal heart like a control.

The table should be synthesized in both the results and the captions. i.e mononuclear cells [i.e. macrophages, MØ)]; negative: (-), etc.

R: Ok It is done.

This data indicates the possible artefacts in RT-PCR. The immunohistochemistry is maintained for a long time when is embedded in paraffin. The author's speculation about the paraffin blocks, like the degradation of the viral proteins and/or the nature of the samples, is not a plausible reason for the lack of detection. The most apparent is the RT-PCR artefact. To confirm this research lacks: use some samples frozen at the time (1993-94). Use paraffin blocks from pathological hearts of 2023 to compare the RT_PCR data (RT-PCR/IHC results)

We have left the RNA extraction part from the FFPE block as a single section (L82) and included a part for "one-step qPCR" (L-104) and another for "two-step qPCR" (L-123). Furthermore, we have focused the possible explanation for the negative IHC-PRV result on the low viral load in the tissues and not on the possible degradation of the proteins.

L109-L121: This is how it reads now: “The RT-qPCR conditions for detection of each pathogen consisted in adding a master mix of 7.5 µl buffer, 0.15 µl 100 mM DTT, 0.225 µl diluted ROX solution, 300 nM each primer and 400 nM probe to 2 µl total RNA. Tubes were incubated for 10 min at 50°C to perform reverse transcription, followed by a denaturation step of 3 min at 95°C and 40 cycles of 3 sec at 95°C and 10 sec at 60°C. All qPCR assays were performed in duplicate. A positive control (RNA or DNA specific to the tested pathogen), a negative control without RNA or DNA and negative extraction control were also included in every run. All qPCR runs for the pathogens tested were accompanied by the expression of Atlantic salmon reference gene as endogenous extraction control (EF1a). Cycling threshold (Ct) values were recorded up to a maximum of 40 Ct. A Ct below the cut-off point was considered positive, and negative (NoCt) otherwise. The cut-off points for PRV-1, -2, and -3, ISAV, and EL1A assays was 35.00 and for P. salmonis was 33.01.” 

L124-L147: This is how it reads now: “PRV-1 positive RNA extract was used as template for cDNA synthesis. Approximately 1 µg total RNA was reverse transcribed using the PrimeScript™ RT Reagent kit with gDNA Eraser (TAKARA) according to the manufacturer’s instructions. Specific primers were used for the S1 and M2 segments described by Kibenge et al. (18), and for the L1 segment described by Palacios et al. (2) were used. Briefly, the RT conditions consisted in the addition of a mastermix of 2 µl 5X gDNA Eraser Buffer and 1 µl gDNA Eraser for each tube containing 1 µg total RNA (10 µl total volume). Tubes were incubated for 2 min at 42°C to erase gDNA. The reverse transcription proceeded immediately on the same tubes adding a mastermix of 4 µl 5X PrimeScript Buffer, 1µl PrimeScript RT Enzyme Mix I and 4 µl specific primers (total volume 20 µl). Tubes were incubated 15 min at two different annealing temperatures (42°C and 50°C) followed by a 5 sec incubation at 85°C. The PCR products were confirmed by electrophoresis in 1% agarose gels and visualized with ethidium bromide staining. To confirm the results obtained by one-step RT-qPCR, we performed a qPCR for the L1 segment of PRV-1 using the KAPA Probe Fast qPCR kit Mastermix (2X) Universal. Briefly, a mastermix consisting in 7,5 µl Buffer, 0,3 µl 50X ROX, 400 nM specific L1 primers and 200 µl L1 probe was added to 2 µl 1:10 diluted cDNA (total volume 15 µl). The PCR cycling conditions consisted in an incubation of 3 min at 95°C followed by 40 cycles of 3 sec at 95°C and 20 sec at 60°C. For the PCR stage of segments S1 and M2 the Platinum™ Hot Start PCR Master Mix (2X) was used. The liquid handling system created a mastermix using 25 µl Platinum Hot Start PCR 2X Master Mix 200 nM each primer (either for S1 or M2, respectively) and 5 ul cDNA template to a total volume of 50 µl. The PCR cycling conditions consisted in an initial denaturation of 2 min at 94°C followed by 40 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C.” 

R: L168-L172: This is how it reads now: “One of the 42 samples (2.4%) of the heart samples examined were positive for PRV-1 by one-step RT-qPCR (average Ct, 28.08, < 35.00) and two-step qPCR (average Ct, 32.15, < 35.00) (Figure 2). Therefore, we confirm the presence of PRV-1 in independent qPCR reactions in different parts of the FFPE block and the loss of sensitivity of two-step PCR by about 4 orders of magnitude.”

L186-L193: This is how it reads now: “The size of the L1 segment amplified by qPCR primers and probes is small (79 bp) (2), but the fragments to amplify the S1 and M2 segments with sequencing primers are as large as 1081 and 2179 bp, respectively (18). This situation, added with the fact that the genetic material available in the sample was probably fragmented, degraded (average Ct 33.22 for reference gene EL1a, but < 35.00) and/or chemically modified after having been stored for 3 decades prior to this analysis, could explain not achieving large amplicons in S1 and M2, and obtaining a considerably smaller amplicon in L1.”

Attached is a final version of our manuscript with all changes marked in bold.

Reviewer 3 Report

 In the present study, the authors want to evaluate the presence/absence of PRV-1 in formalin-fixed, paraffin-embedded Atlantic salmon heart tissues (FFPE) cultured in Chile during 1992 and 1999. In general, it is quite interesting, but before the manuscript is accepted, some minor issues need to be revised.

1. Title: Piscine Orthoreovirus PRV(-1), but not PRV-2 or PRV-3, has been present in Chilean salmon aquaculture since at least 1994. ”.” should be deleted.

2. Key words: Atlantic does not need to be bold.

3. Forty two (42) cardiac tissue samples preserved as formalin-fixed, paraffin-embedded tissue blocks for about 30 years up to the time of this study were used. Can you provide a figure of the extracted RNA results?

4.  How different are PRV-1, PRV-2 and PRV-3?

5. Figure 1. Histopathological changes in the heart of Atlantic salmon farmed in Chile between 1992 and 1999. There is only one picture, so it is necessary to specify which year the sample is from?

Author Response

R: Thank you very much for the constructive criticism. Attached is a final version of our manuscript with all changes marked in bold. At the same time, we are providing a point-by-point responses to your comments here:

1. Title: Piscine Orthoreovirus PRV(-1), but not PRV-2 or PRV-3, has been present in Chilean salmon aquaculture since at least 1994. ”.” should be deleted.

A: OK it is done. We have changed the title to one that is a little more direct. This is how it reads now: “Piscine Orthoreovirus-1 (PRV-1) has been present in Chilean salmon aquaculture since at least 1994.”

2. Key words: Atlantic does not need to be bold.

A: OK it is done.

3. Forty two (42) cardiac tissue samples preserved as formalin-fixed, paraffin-embedded tissue blocks for about 30 years up to the time of this study were used. Can you provide a figure of the extracted RNA results?

A: OK it is done. We have included a Figure 2 to show the amplification plot and view the plate layout of the two-step qPCR confirming positive results for L1 and negative for S1.

4. How different are PRV-1, PRV-2 and PRV-3?

A: OK it is done.

5. Figure 1. Histopathological changes in the heart of Atlantic salmon farmed in Chile between 1992 and 1999. There is only one picture, so it is necessary to specify which year the sample is from?

A: OK it is done. This sample corresponds to the year 1994. It is the qPCR PRV-1 positive sample.

Attached is a final version of our manuscript with all changes marked in bold.

Round 2

Reviewer 1 Report

The authors have sufficiently addressed my concerns to the point where I see no reason not to publish this manuscript. I think it is a worthwhile contribution to the scientific literature for understanding PRV historic distributions and transmissions/evolution.

Author Response

Many thanks for the constructive criticism and valuable review to improve the quality of the manuscript.

Reviewer 2 Report

The Manuscript deals with the analysis of the heart of some Chilean Salmon farmed species of the presence of the Piscine orthoreovirus-PRV virus since 1994. The analyses have been conducted in 30-year-old 10% formalin-fixed/paraffin-embedded specimens. RT-PCR and immunohistology analyses have evidenced the presence of PRV of subtype 1 (only one sample!) instead of subtypes 2 and 3 that are already detected in EU farms. The authors conclude that some embryos coming from the EU have brought the pathogen to Chile’s farm. The authors present a review form of MS.

I am still perplexed about the data presented, where only one sample resulted positive for the virus. The histological pictures do not be sufficiently. 

1) In the reviewed version the authors have inserted a new picture in figure 1. However, the new picture (control without the virus, by RT-PCR analysis) is in a different magnification and does not show real differences between the samples, i.e. the macrophages (perhaps also mast cells). The authors should show a good version of pictures with Masson’s  staining that can show the myofibres in the cells and the connective tissue

2) Table 1 and Table 2 are poor presentations (too much to write on. In particular, table 2 should be in supplemental data and substituted with graphic histograms and statistical reports.  

3) Another question is that only one sample has revealed the positivity for the PRV1 (ID.40). My initial question was:  "are the authors sure that this positivity is not an artefact? " And if the answer is “yes we are sure”, the study should be only a short communication because the data are really poor.

I suggest the author reconsider the organisation/presentation of the data and make new histological analyses to be sure of their affirmations.

Author Response

With all due respect reviewer 2, you are assuming that the results should be different from the ones exposed, why? we are talking about 17 years before an outbreak of HSMI, we are showing that 30 years ago there was very low prevalence of PRV1, low tissue viral load and mild microscopic lesions. Our suggestion is that you evaluate the case considering the epidemiological situation of PRV - HSMI worldwide and not just take a "figure". The situation has biological plausibility and is demonstrated in the results and discussion.

We suggest that reviewer 2 consider a better understanding of the epidemiological situation of PRV-HSMI in his evaluation, not only in Chile, but also in other salmon farming countries. The molecular results are very specific. We cannot control the fact that we have only found one positive sample out of 42. 

Regarding your comment on Figure 1A, it is not a problem, we have included the microphotography with the same 40X objective.

Reviewer 3 Report

The manuscript has met the requirement of publication.

Author Response

Many thanks for the constructive criticism and valuable review to improve the quality of the manuscript.

Round 3

Reviewer 2 Report

1. The authors have ignored my suggestion to improve the quality of the tables (too much written inside). In my opinion, they should look at the paper without hurrying and prepare a manuscript for the level of Fishes journal.
2. Table 1 and Table 2 are poor presentations (too much to write on. In particular, table 2 should be in supplemental data and substituted with graphic histograms and statistical reports. 
3. Moreover, the references are written not in line with the Fishes journal. They should be changed as reported above (I have picked up a reference from an article already published):
4. He, S.W.; Wang, G.H.; Yue, B.; Zhou, S.; Zhang, M. TO17: A teleost antimicrobial peptide that induces degradation of bacterial nucleic acids and inhibits bacterial infection in red drum, Sciaenops ocellatus. J. Fish. Shellfish Immunol. 2018, 72, 639–645. [Google Scholar] [CrossRef]

Author Response

R: Thank you reviewer 2 for your constructive suggestions.

The authors have ignored my suggestion to improve the quality of the tables (too much written inside). In my opinion, they should look at the paper without hurrying and prepare a manuscript for the level of Fishes journal. Table 1 and Table 2 are poor presentations (too much to write on. In particular, table 2 should be in supplemental data and substituted with graphic histograms and statistical reports. 

R: We have improved the content of the tables and have included Table 2 as a Supplementary Table 1.

Moreover, the references are written not in line with the Fishes journal. They should be changed as reported above (I have picked up a reference from an article already published):

He, S.W.; Wang, G.H.; Yue, B.; Zhou, S.; Zhang, M. TO17: A teleost antimicrobial peptide that induces degradation of bacterial nucleic acids and inhibits bacterial infection in red drum, Sciaenops ocellatus. J. Fish. Shellfish Immunol. 2018, 72, 639–645. [Google Scholar] [CrossRef]

R: The editorial team will correct the references if the manuscript is accepted.