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Review
Peer-Review Record

Biocontrol of the Common Carp (Cyprinus carpio) in Australia: A Review and Future Directions

by Kenneth A McColl * and Agus Sunarto
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Submission received: 22 April 2020 / Revised: 21 May 2020 / Accepted: 29 May 2020 / Published: 2 June 2020
(This article belongs to the Special Issue Biology and Control of Invasive Fishes)

Round 1

Reviewer 1 Report

Summary.  The authors have re-organized and expanded their review of issues related to CyHV-3 biocontrol of carp in Australia.  The revised review is well organized and the additional information increases the scope of the discussion, resulting in a more informative overview.  Table 1 provides a concise presentation of knowledge gaps identified by the authors and is a valuable supplement to the text (as is Table 2).  I have only a couple of comments.

  1. A Table of Contents would be useful.
  2. Lines 219-222: When one test tests positive and another test tests negative on the same sample why is it a false positive especially when one test (ie cPCR) is known to be less sensitive? Was there evidence of cross contamination in the negative control samples processed in parallel with the test samples to corroborate the false positive interpretation of the results by the authors?
  3. Lines 249 to 251: Virus isolation and histopathology are generally considered much less sensitive than qPCR-based diagnostic assays for detection of virus. Further, virus isolation is not considered by the OIE to be a reliable diagnostic method for KHVD.  It is likely that below specific virus titers, qPCR-based tests will be positive on same fish samples that test negative by virus isolation and/or histopathology.  I wonder if the authors have considered including a serology approach (e.g. one that shows whether a virus-exposed NTS fish population has or has not generated a measurable antibody response to the virus)?  In this case, a positive serology result would be considered a correlate of infection.
  4. Lines 350: The authors could consider briefly elaborating on what they mean by a “virome study” (e.g. Are they suggesting to conduct a next generation sequence analyses study with a similar number of carp i.e. >800 carp?). Perhaps they should also consider a large-scale surveillance study conducted using a qPCR test for detection of CyHV-3 in MDB carp.

Author Response

Thank you Referee # 1 for your positive comments, and high scoring of our manuscript. In response to your comments:
1. We have now inserted a Table of Contents as requested
2. Lines 219-222 (with the various additions, including the Table of Contents, now become Lines 261-264): as noted in our responses to the first round of reviews, if a qPCR result was a genuine positive, then we would expect multiple copies of the specific terminase mRNA to be transcribed from a replicating virus. This would then increase the sensitivity of the RT-PCR. As demonstrated in the original publication [16], all samples were collected from moribund or freshly-dead fish (within 24 hrs of death), and viral mRNA was shown to persist in kidney or gill for up to 24 hrs in most cases. Yes, Table 2 in [16] shows that there was some contamination of negative control samples, and, when tested with the RT-PCR, these were indeed negative.
3. Lines 249 to 251 (now Lines 292-293): we agree entirely with the Referee about the limitations in sensitivity of virus isolation (VI) and histopathology. However, because we work in a high-security laboratory that specializes in the diagnosis of exotic disease incursions, we recognize the critical importance of multiple diagnostic approaches in some situations. In this case, VI and histopathology would simply be used to corroborate the PCR results. During the course of our work, we have often considered the use of a serological test, but have always been dissuaded by the fact that, in most cases, serological responses in individual fish are relatively insensitive, although they may be useful at a population level. Nevertheless, it is likely that, if the virus is to be released in Australia, a serological test will be implemented at some stage.
4. Line 350 (now Line 396): at the Referee’s suggestion, we have now included a very brief explanation of a ‘virome study’. When, and if, a virome study is conducted it may also be possible to conduct a qPCR study (similar to the nested PCR study already conducted).

Author Response File: Author Response.pdf

Reviewer 2 Report

I commend the authors on their effort. Congratulation on the competent compilation of information about this sensitive subject. Is good that unknown information is highlighted for managers to understand that the use of this virus still lacks more information to be validated.

Author Response

Thank you Referee # 2 for your generous response, and for recognising, as we do, that further work needs to be conducted.

Author Response File: Author Response.pdf

Reviewer 3 Report

 

This is a very interesting review article dealing with the biocontrol subject of Cyprinus carpio. The multidisciplinary approach of this review is an added value. I would like to highlight the deep bibliographical revision combining scientific and technical works that the authors have done.  

Nevertheless, I think that in some parts of the text it lost the scientific sound and the authors must take care of it. You should just expose evidence and propose hypotheses and future lines of research more than try to convince the reader of the benefits of using CyHV-3 as biocontrol. There is some lack of knowledge that ought to be highlighted in the manuscript, mainly in the summary.

The reading of the manuscript denotes that the authors have experience in the control of invasive alien species. They focus in a very realistic manner most of the aspects, but I miss the proposal of a contingency plan, if possible, in case the evolution of the virus in the wild doesn’t work as expected.

On the other hand, the content is well structured but it’s very extensive and for that reason, I think it’s necessary that you explain in the introduction to how the content is organized throughout the manuscript. In addition, you should write a clear objective for the work since although it’s indicated by the title and can be deduced from the reading, it should be explicit.

And now, if you allowed me, I would add some suggestions and comments about the manuscript.

  • Throughout the text, in some occasions, you use “Australian carp”. I know that carp is no native in Australia but for a no native English speakers this naming could be confusing. Do you think it’s possible to use “carp in Australia” as you use on occasions in the manuscript?

 

  • The summary should contemplate results obtained in this work, but in lines 18-20 you are referring to results obtained in other works. In addition to the optimism that you propose, you should highlight the lack of knowledge not only about the suitable broad-scale complementary control measure but the factors that are related to the virus, to the hosts and to environmental issues.

 

  • I miss some bibliographical references for some claims in the text, for example:
    • Lines 179 to 183
    • Line 301
    • Lines 418 to 422.
    • Lines 471 to 473
    • Lines 520 to 523
    • Lines 568 to 570
    • Lines 595 to 597

 

  • Line 88: I would propose: … information required to understand not only the carp biology and ecology in this country, but also …

 

  • I would divide the section 2.1 in two: the first one from line 95 to 120 with the following heading: Distribution models of carp in Australia and biomass estimates. And the second one from line 121 to 140 with the heading: genomic and transcriptomic map of carp in Australia. The title Carp biology is confusing (same in Table 1).

 

  • Lines 183 to 184: This claim “Importantly, this broad observation includes humans whose fears of infection can also be allayed by several observations” doesn’t provide anything interesting to this review, or one of your objectives is calm to the people? I think you ought to focus more on scientific data and information than on the feelings and emotions of the people.

 

  • Related to the information about the virus: safety, efficacy, epidemiology, transmissibility, and virulence, you should consider the publications of Bergmann et al (2020) and Soto et al (2020) in order to update and to discuss some points. For example, in lines 239 to 240, you state “Again, this ignores the fact that to be a carrier, a non-target native species must first be infected, a claim that has never been properly demonstrated…” and Bergmann et al (2020) state “Susceptibility to virus infection is defined as replication in the host with a large variation among individuals regarding the establishment of infection and development of disease (Sluijsm et al. 2017). In terms of KHV there is a large number of fish which can be infected by the virus, replicate it internally and release the agent infectious to common carp or koi but do not show any external clinical signs or mortality themselves”. On the other hand, Soto et al could provide some interesting information to understand the discrepancies between the different diagnostic methods.

 

Bergmann, S. M., Jin, Y., Franzke, K., Grunow, B., Wang, Q., & Klafack, S. (2020). Koi herpesvirus (KHV) and KHV disease (KHVD) – a recently updated overview. Journal of Applied Microbiology. https://0-doi-org.brum.beds.ac.uk/10.1111/jam.14616

Soto, E., Tamez-Trevino, E., Yazdi, Z., Stevens, B.N., Yun, S., Martínez-López, B., & Burges, J. (2020) Non-lethal diagnostic methods for koi herpesvirus in koi Cyprinus carpio. Dis Aquat Org. 138: 195–205, 2020 https://0-doi-org.brum.beds.ac.uk/10.3354/dao03456

           

 

  • Lines 246 to 252: I suppose that this protocol is a monitoring plan required, as you say, for future susceptibility testing of NTS. It is a very interesting proposal in which sampling must be perfectly designed.

 

  • In section 2.5. You state that “the BBN identified river flow and water temperature as the two essential parameters determining the suitability of a habitat for adult and sub-adult carp, and both were rated as medium to high for most habitats throughout the study period”… I think you should consider that these two parameters determine the spawning carp migration and you must take it in mind not only to know the best place to apply the treatment but the best moment because as you claim, it’s better with the maximum density of carps, isn`t it? On the other hand, It would be fine if you could provide the approximate rank of temperatures to which you refer.

 

  • Lines 407 to 410: I would like to know how you could explain the prediction about the seasonal losses mainly in immature carp (it’s interesting).

 

  • Lines 444 to 446: I would like to know how you can calculate that the chance of cross-species transmission is infinitesimally small. I think you ought to find the most suitable expression.

 

  • Lines 489 to 490: I can’t understand how you propose as a complementary measure to the biocontrol with CyHV-3 a more virulent strain of CyHV-3? That’s not complementary, that’s the same. I think you ought to talk about that in the section where you expose the information about the efficacy of the virus.

 

  • Lines 514 to 516: I have some doubts about the suggestion of releasing the virus during high-flow season because at this moment the density of fish decrease (that’s good to avoid the contact between native fish and birds and the virus, but it would avoid the transmission between carps unless the models abovementioned show the opposite).

 

  • Lines 539 to 452: I don’t know the Australian legislation, but I would like to know if the carps killed by this treatment are classified as “hazardous waste” and if there are some protocols to deal with them.

 

  • Lines 577 to 589: This information could be eliminated because is redundant with the information in the following summary, lines 590 to 597.

 

  • Lines 743 to 745: The citation should be corrected to be coherent with the rest of them.

 

  • In Table 1:
  • I would change “Carp biology in Australia” because it’s a little bit confusing (see comments about the name of section 2.1).
  • In “Safety of the virus” I would add the surveillance (monitoring) plan and the contingency plan.
  • In “Epidemiological modeling of virus release and spread” you repeat carp biology (I suppose that in a broad sense, but hydrology?, or you are mean ecology?) You could eliminate it and let the 4 points.
  • In “Broad-scale control measures to complement the virus” I don’t understand why you include the Marek’s disease model.
  • In “Social risks” don’t you think that the perception of the society could have changed after the coronavirus outbreak?

 

  • In Table 2 you could include other control measures as you mention in lines 455 to 458. I know that most of them aren’t a broad-scale control option (well, it depends on how they are applied).

Author Response

Responses to Referee # 3

This is a very interesting review article dealing with the biocontrol subject of Cyprinus carpio. The multidisciplinary approach of this review is an added value. I would like to highlight the deep bibliographical revision combining scientific and technical works that the authors have done.
Thank you Referee # 3 for your compliments. Clearly, we differ with you on a number of important issues, but we are also in agreement on many aspects of our work. We appreciate your detailed comments, and, as you will see, we believe that many of your suggested changes have greatly improved this paper. Thank you for a very careful review.
Nevertheless, I think that in some parts of the text it lost the scientific sound and the authors must take care of it. You should just expose evidence and propose hypotheses and future lines of research more than try to convince the reader of the benefits of using CyHV-3 as biocontrol. There is some lack of knowledge that ought to be highlighted in the manuscript, mainly in the summary.
We have searched Referee # 3’s specific comments to find where “the text lost the scientific sound”. The only comment we could find was Lines 183 to 184 (see below) which we have addressed. We hope that the small changes to the Abstract now identify where the major deficiencies in our knowledge still remain.
The reading of the manuscript denotes that the authors have experience in the control of invasive alien species. They focus in a very realistic manner most of the aspects, but I miss the proposal of a contingency plan, if possible, in case the evolution of the virus in the wild doesn’t work as expected.
The absence of any mention of a “contingency plan” is a good observation. We have now added a paragraph in Section 3 (Lines 657-669) that we believe addresses the referee’s criticism.
On the other hand, the content is well structured but it’s very extensive and for that reason, I think it’s necessary that you explain in the introduction to how the content is organized throughout the manuscript. In addition, you should write a clear objective for the work since although it’s indicated by the title and can be deduced from the reading, it should be explicit.
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We hope that the addition of a Table of Contents (as suggested by Referee # 1) will demonstrate “how the content is organized”. We have added a final short sentence to the Introduction to clarify the objective of the paper.
Throughout the text, in some occasions, you use “Australian carp”. I know that carp is no native in Australia but for a no native English speakers this naming could be confusing. Do you think it’s possible to use “carp in Australia” as you use on occasions in the manuscript?
Good suggestion. We have searched the manuscript, and believe that we have now changed all instances of “Australian carp” to “carp in Australia”.
The summary should contemplate results obtained in this work, but in lines 18-20 you are referring to results obtained in other works. In addition to the optimism that you propose, you should highlight the lack of knowledge not only about the suitable broad-scale complementary control measure but the factors that are related to the virus, to the hosts and to environmental issues.
Because our paper is a review, it includes the results from 92 references, including the most recent studies. We’re not sure why the referee finds that unacceptable. We have, however, expanded the last sentence of the Abstract to comply with the referee’s suggestion that we highlight areas where knowledge is still deficient.
I miss some bibliographical references for some claims in the text, for example:
Lines 179 to 183 (now Lines 222-226): the point being made here is that the absence of references is the “compelling evidence”, and therefore, of course, there are no references that can be cited.
Line 301 (now Line 343): we have now included the same reference [34] as was cited on Line 193. This reference discusses why latency has not yet been demonstrated for CyHV-3.
Lines 418 to 422 (now Lines 464 to 467): presumably this refers to the statement that ‘behavioural fever’ “has not prevented mass mortalities of carp in thermally variable natural aquatic environments overseas”. Again, it is not possible to supply references for an event that has not been documented in a natural environment.
Lines 471 to 473 (now Lines 512 to 521): reference [76] covers all the information in this section, including up to Line 521. Perhaps it is the section from Lines 519 to 521 that is problematic? Initially we thought it important to raise the issue of whether the original work [76] was conducted on ‘sonless’ or ‘daughterless’ carp. However, we have now decided to delete that sentence, and allow the original investigators to correct the literature when convenient.
4
Lines 520 to 523 (now Lines 563 to 566): all comments in the paragraph beginning on Line 558 (“Beckett et al [82] conducted a comprehensive.......”) can be attributed to [82], except those related to prey-switching at the end of the paragraph (attributed to [83, 84, 85]).
Lines 568 to 570 (now Lines 611 to 613): all statements in Section 2.9 are attributed to [89].
Lines 595 to 597 (now Lines 640 to 642): all of the information in this section is attributed to [90]. However, perhaps we caused confusion through the use of a poorly constructed colloquialism (a ‘do nothing to control carp attitude’). We have now replaced those words with a simple statement (Lines 640 to 642).
Line 88 (now Lines 126-127): I would propose: … information required to understand not only the carp biology and ecology in this country, but also …
Yes, a good suggestion that has now been incorporated.
I would divide the section 2.1 in two: the first one from line 95 to 120 with the following heading: Distribution models of carp in Australia and biomass estimates. And the second one from line 121 to 140 with the heading: genomic and transcriptomic map of carp in Australia. The title Carp biology is confusing (same in Table 1).
This seems like a good suggestion. However, given that we have specifically mentioned in the introduction to Section 2 that “an understanding of the biology of the targeted pest species” is important, we would prefer to retain “2.1 Carp biology in Australia” as a Level 2 Heading, and then introduce the referee’s suggested headings as Level 3 Headings.
In Table 1, the two headings suggested by the referee essentially separate naturally into ‘Knowns’ and ‘Unknowns’.
Lines 183 to 184 (now Lines 226 ff): This claim “Importantly, this broad observation includes humans whose fears of infection can also be allayed by several observations” doesn’t provide anything interesting to this review, or one of your objectives is calm to the people? I think you ought to focus more on scientific data and information than on the feelings and emotions of the people.
In any open forum on the potential use of CyHV-3 as a biocontrol agent, the question of human susceptibility is almost always raised (the mere mention of ‘herpesvirus’, even if it is a herpesvirus of carp, causes alarm among many people). So, yes, using scientific data and information to ‘calm’ people is very definitely one of the objectives of our work (and this review), and we consider it important to retain this statement.
5
Related to the information about the virus: safety, efficacy, epidemiology, transmissibility, and virulence, you should consider the publications of Bergmann et al (2020) and Soto et al (2020) in order to update and to discuss some points. For example, in lines 239 to 240, you state “Again, this ignores the fact that to be a carrier, a non-target native species must first be infected, a claim that has never been properly demonstrated…” and Bergmann et al (2020) state “Susceptibility to virus infection is defined as replication in the host with a large variation among individuals regarding the establishment of infection and development of disease (Sluijsm et al. 2017). In terms of KHV there is a large number of fish which can be infected by the virus, replicate it internally and release the agent infectious to common carp or koi but do not show any external clinical signs or mortality themselves”. On the other hand, Soto et al could provide some interesting information to understand the discrepancies between the different diagnostic methods.
Thank you, Referee # 3, for bringing these recent references to our attention. However, in our view they provide no new convincing evidence that CyHV-3 can infect non-carp species.
Ever since sensitive PCRs for detecting DNA became widely used in the early 1990s, the potential for contamination has been well-known. Diagnostic laboratories now take extraordinary precautions to avoid contamination, but, inevitably, there are always rare false-positive cases when using PCRs to detect DNA, especially when processing large numbers of samples. In addition, the very fine work of Yuasa et al [49] provided a clear demonstration that fish (in this case, goldfish) could act as fomites for CyHV-3 in co-habitation studies. Presumably, the virus attached to mucus on the gills or skin, but without any evidence of virus replication. In other words, the fish were not infected; they were simply fomites. We believe that these two explanations – contamination, and/or fomites – explain the results of most earlier work on the host-specificity of CyHV-3.
The critical advance that allowed us to recognize the true specificity of CyHV-3 was the development of an RT-PCR by Yuasa’s group for the identification of CyHV-3 mRNA from the terminase gene [46]. We utilized this RT-PCR in our work on the species-specificity of CyHV-3 [16] (which the recent paper by Bergmann et al (2020) failed to cite). In our work, we used a PCR for viral DNA to screen hundreds of samples from the species that were tested for their susceptibility to CyHV-3. Not surprisingly, despite taking as much care as possible, there were occasional positive results. However, every single ‘positive’ result was negative when the same sample was tested by Yuasa et al’s RT-PCR. In other words, there was no evidence of virus replication in any of the samples, implying that the positive PCR results were due to contamination or, in the case of gill samples, because the samples acted as a fomite. Therefore, we maintain our view that “Again, this ignores the fact that to be a carrier, a non-target native species must first be infected, a claim that has never been properly demonstrated…”.
This argument about the specificity of CyHV-3 has now been going on for some years. In 2018, we had a long on-line discussion with a reviewer for one of our papers [34], a
6
discussion that was eventually terminated by the Editor (who was, himself, an author of work on CyHV-3) in our favour. In our view, the simplest solution to the question of species-specificity of CyHV-3 would be for all future work to use the RT-PCR of Yuasa et al.
Lines 246 to 252 (now Lines 289-295): I suppose that this protocol is a monitoring plan required, as you say, for future susceptibility testing of NTS. It is a very interesting proposal in which sampling must be perfectly designed.
Yes, as mentioned in our last response, it is, we believe, the only way to resolve the impasse about the species-specificity of this virus. It is also important to note that it would not be sufficient to use any RT-PCR (or RT-qPCR). As Yuasa et al [46] demonstrated, attention must be given to the design of the primers. Poorly-designed primers, such as those used in [45], will only lead to continued confusion.
In section 2.5. You state that “the BBN identified river flow and water temperature as the two essential parameters determining the suitability of a habitat for adult and sub-adult carp, and both were rated as medium to high for most habitats throughout the study period”… I think you should consider that these two parameters determine the spawning carp migration and you must take it in mind not only to know the best place to apply the treatment but the best moment because as you claim, it’s better with the maximum density of carps, isn`t it? On the other hand, It would be fine if you could provide the approximate rank of temperatures to which you refer.
The Final Report on the modelling aspects of CyHV-3 as a potential biocontrol agent [64] is currently being converted to four separate manuscripts that will be submitted to peer-reviewed journals. It is probably best to wait for all the details of the work to appear in those future publications; our manuscript simply provides a brief summary of the results.
Lines 407 to 410 (now Lines 454-455): I would like to know how you could explain the prediction about the seasonal losses mainly in immature carp (it’s interesting).
Again, as this current review simply offers a brief summary of the (complex) modelling results, we believe it is better to await the future publications from the modelling work.
Lines 444 to 446 (now Lines 493): I would like to know how you can calculate that the chance of cross-species transmission is infinitesimally small. I think you ought to find the most suitable expression.
Yes, perhaps we have resorted to hyperbole with this statement, and we are happy to substitute ‘very’ for ‘infinitesimally’. The statement is based on the work of our collaborator,
7
Professor Eddie Holmes, whose work on herpesviruses [67, 68] has shown that spill-over events or host-jumps occur on time-scales of millions of years, and, when they occur, they are invariably into taxonomically closely-related species. This, to us, suggests that the chance of cross-species transmission is remote (which is consistent with observations on MYXV, also a DNA virus, in Australia over the past 60 yrs).
Lines 489 to 490 (now Lines 537 ff): I can’t understand how you propose as a complementary measure to the biocontrol with CyHV-3 a more virulent strain of CyHV-3? That’s not complementary, that’s the same. I think you ought to talk about that in the section where you expose the information about the efficacy of the virus. ‘Complementary’ may be defined as: “combining in such a way as to enhance or emphasize the qualities of each other or another” (Google). In our view, this is precisely what might happen with a more virulent strain of CyHV-3. In Australia, rabbit haemorrhagic disease virus (RHDV) was considered to be complementary to MYXV, and then, more recently, the new strains of RHDV (such as RHDV2) are considered to complement RHDV. RHDV2 is antigenically and genetically distinct from RHDV, and can cause disease in RHDV-vaccinated rabbits and in wild rabbits previously exposed to RHDV. To that extent, RHDV2 is complementary to RHDV. We believe the same argument may apply to new strains of CyHV-3 (although we have not yet been able to test that hypothesis).
Lines 514 to 516 (now Lines 563 ff): I have some doubts about the suggestion of releasing the virus during high-flow season because at this moment the density of fish decrease (that’s good to avoid the contact between native fish and birds and the virus, but it would avoid the transmission between carps unless the models abovementioned show the opposite).
Yes, we agree with Referee # 3’s observation. In this review, we are simply reporting the results from recent studies, many of which are yet to reach the refereed literature. In Lines 565 ff, we actually question the practicality of the proposed option, and we suggest alternative strategies that might be used.
Lines 539 to 452 (now Lines 587 ff): I don’t know the Australian legislation, but I would like to know if the carps killed by this treatment are classified as “hazardous waste” and if there are some protocols to deal with them.
Again, in this review we are simply reporting the results from recent studies, many of which are yet to reach the refereed literature. We can’t specifically answer the question of whether or not dead carp are classified as “hazardous waste”. However, given that the work
8
reported in [87] constituted part of the NCCP Final Report to the Federal Govt of Australia, and that no decision has yet been made on the use of CyHV-3 as a biocontrol agent in Australia, it is likely that no decisions have been made by Govt on how to classify mass kills of carp due to CyHV-3. Silva et al [87] produced recommendations on how to deal with large fish kills in various waterways, and Tilley et al [88] then discussed different methods of disposal of the dead and moribund fish.
Lines 577 to 589 (now Lines 627 ff): This information could be eliminated because is redundant with the information in the following summary, lines 590 to 597.
Excellent suggestion, thank you. We have deleted the dot-points (Lines 627 to 639) because, as the Referee mentions, they are repeated in the final paragraph.
Lines 743 to 745 (now Lines 804-807): The citation should be corrected to be coherent with the rest of them.
We believe this comment refers to the format of Reference # 35. We will actually need the advice of the journal Editor on how to format this reference.
In Table 1:
I would change “Carp biology in Australia” because it’s a little bit confusing (see comments about the name of section 2.1). Given our positive response (p. 4 of this document) to the Referee’s earlier comment about Section 2.1, we would like to suggest that, because ‘modelling’ and ‘genomics’ have now been listed as sub-sections, they do actually fit comfortably under ‘Carp biology in Australia’. Is this reasonable?
In “Safety of the virus” I would add the surveillance (monitoring) plan and the contingency plan. We are not sure what ‘surveillance (monitoring) plan’ refers to. If it relates to the detection of potential cross-reactive viruses in carp that might confer resistance, then that is already covered in the Table under ‘Efficacy of the virus’ (PCR and virome surveys). With regard to a ‘contingency plan’, we have added a comment in Section 3. However, given what has been learned from the rabbit precedent in Australia, we are already planning for the use of complementary measures with CyHV-3 (as identified in Table 1).
In “Epidemiological modeling of virus release and spread” you repeat carp biology (I suppose that in a broad sense, but hydrology?, or you are mean ecology?) You could eliminate it and let the 4 points. Yes, very good point. We have done as the Referee suggests.
In “Broad-scale control measures to complement the virus” I don’t understand why you include the Marek’s disease model. As discussed in Section 2.7, we suggest that new, more virulent strains of CyHV-3 may evolve in response to the use of CyHV-3 vaccines (just as they
9
did for Marek’s disease virus). However, reference to the ‘Marek’s disease model’ may be too obscure, so we have replace it with a more direct summary.
In “Social risks” don’t you think that the perception of the society could have changed after the coronavirus outbreak? Good point. We have now included an appropriate entry under “Unknowns” for this category.
In Table 2 you could include other control measures as you mention in lines 455 to 458 (now Lines 502 to 505). I know that most of them aren’t a broad-scale control option (well, it depends on how they are applied). Yes, all of these methods have been tried in Australia, and they will still have a role in the future, but definitely not as ‘broad-scale’ controls.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

I would like to give thanks for consider some of my suggestions in this last version, although the number of the lines mentioned in the cover letter doesn’t match with the number of lines of the manuscript, then I hope to have been working in the last one.

I would like to recognize again to the authors the effort in this review with a multidisciplinary approach.

Related to the summary, I agree with the authors that in a review the results of all the references consulted must be included, but in the summary only the authors’ results, after contrasting and discussing the content of the references, should appear. That is, the sentence “Brief results are included from recent studies on: the modelling of release and spread of the virus; the ecological and social concerns associated with virus release; and, the restoration benefits that might be expected following carp control” should be excluded, especially when the authors recognize in the cover letter that “The Final Report on the modelling aspects of CyHV-3 as a potential biocontrol agent [64] is currently being converted to four separate manuscripts that will be submitted to peer- reviewed”.

 

On the other hand, related to the bibliographical references, I always expect in a scientific paper that most of the statements, especially the critical ones, are supported by scientific bibliography even they are “compelling evidences”. That’s the explanation why I asked for it when I find that it’s necessary. Related to this, I think is necessary too, to specify in the text the information that comes from Technical Reports that are going to be submitted to peer- reviewed and published.

Regarding to the contingency plan, I was referring to a plan to take once an identified risk occurs, in this case, that the virus be harmful to non-target species. Are there some measures to take? However, the authors are referring to a plan to reach the goal of control the carps.

Finally, I think the authors should be more explicit related to some issues. For example, if one of the objectives is using scientific data and information to “calm” people, why don’t include this objective in the introduction. Anyway, I think that with a clear and objective explanation and discussion of all the data as the authors are perfectly doing expressions as “Importantly, this broad observation includes humans whose fears of infection can also be allayed by several observations” are no need.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.


Round 1

Reviewer 1 Report

First of all, I would like to remark that the subject is very interesting and that you show a deep knowledge of it from a virologist perspective. 

In the first part, up to point 4 "Responses to criticisms about the use of CyHV-3 as a biocontrol agent" it looks like a review article, but from that point, it looks like more a discussion between Boutier et al, Kopf et al, and the authors, where you just refute their concerns.

I would say that in this second part, some statements are more emotional than scientific, moreover, they don't contribute to the review. 

Furthermore, you propose indirectly to biocontrol the carp, appealing to optimism, even when some of the statements are based on hypotheses and speculations and when you recognize the necessity of more studies. 

All the information that you present here is very valuable for a risk analysis or assessment. 

I think you could do a better review changing the mood, being more objective, more scientific.   

Reviewer 2 Report

This article focuses a very relevant topic and approach a problem that needs to tackled. The authors present a well written text, well supported by literature. The major problem I see with this article is the sheer amount of open questions that the authors refer to future publications for potential answers. I thus recommend this article to be published, after being updated and reformulated, after theses “future” publications and reports are available and that this information is incorporated into this work, effectively closing these open questions that may affect the overall conclusions of the study. Although the authors present a very compelling case, I think these issues should be approached with the utmost care, and so we must harvest all the possible information to perform a correctly informed judgement.

Reviewer 3 Report

Summary. The authors provide an overview of their requirements for a potential biocontrol virus (i.e. safety and efficacy) and then respond to criticisms on the proposed use of CyHV-3 as a biocontrol agent within a broader integrated pest management plan for carp in Australia. The review is well written and within the scope of the journal. Specific comments are provided below. I will refer to co-existing species as non-target species (NTS) throughout the review.

Specific comments.

Safety. a) Lines 110 to 113. The authors present evidence from the literature to support their view that CyHV-3 is specific for carp and thus does not pose a biological threat to co-existing species. For example, the authors indicated that following exposure of NTS to CyHV-3-infected carp, there was 1) low prevalence of CyHV-3 positive NTS, 2) no clinical signs of disease in NTS and 3) low virus loads in NTS. The authors interpreted these results as evidence supporting the absence of an infection (or based on their use of “supposedly” perhaps implying cross contamination??). In this case, the method of virus exposure was natural and the tissues evaluated in the reference cited were all internal tissues (i.e. kidney, spleen, liver, intestine and brain). Detection of low copy numbers of CyHV-3 nucleic acid at low prevalence in a number of different species suggests that CyHV-3 was present at detectable levels at some point. If not here, then when does virus presence imply an infection? How do the authors explain the transmission of virus from NTS to naïve carp in the cohabitation experiment reported in the same paper (i.e. reference 30). An alternative interpretation in this case is that the virus can establish an infection (albeit inefficiently) in NTS and that further investigation is required to better understand what, if any, effects this might have on the dynamics of CyHV-3 in an ecosystem.    

It might be useful for the authors to define what they mean by infection or identify their minimum criteria for infection – at least as it pertains to CyHV-3 as a biocontrol agent for carp. This would provide a starting point for a discussion about what constitutes an infection by this virus.

The authors should also consider presenting an infection (or virus presence) threshold above which is an unacceptable risk and below which is an acceptable risk to the ecosystem if CyHV-3 was used for biocontrol of carp. These suggestions establish a framework for present and future discussions.

 

b) Lines 121 to 134 & 267 to 270. The authors are using the negative results of a RT-conventional PCR (cPCR) test as evidence that CyHV-3 does not establish an infection in NTS. The implication is that the presence of replicating virus is a critical component for establishing that a fish is infected with CyHV-3 (which speaks to my suggestion in the paragraph above). In the reference cited (i.e. ref 9), the NTS were pre-screened with a qPCR assay and 11% of the samples tested positive with Ct = 37.8±1.9. None of these samples tested positive with the RT-cPCR test (which would have provided evidence of a replicating virus). The conclusion drawn by the authors is that CyHV-3 was present (??) but not replicating. Typically, qPCR tests have a higher (i.e better) analytical sensitivity than conventional PCR tests. As such, it would be quite unlikely that the RT-cPCR would be able to detect the low virus loads associated with Ct values ≥35. So, do the negative results mean that the detected nucleic acid was from a virus that is not replicating or do they mean that the virus is replicating but at levels below the limit of detection of the RT-cPCR test (at that time)?  

The authors should discuss this possibility and how it fits into their framework of acceptable/unacceptable risk. Further investigation of what happens to the virus detected in NTS fish is required – do these fish eventually test negative? If NTS that test positive for CyHV-3 are stressed, does clinical disease develop or does the virus load increase? As I mentioned above, long term in vivo investigations are required to develop a more comprehensive understanding of what happens to CyHV-3 detected in NTS. I am not suggesting that the authors do this for this review, rather I see this as a knowledge gap that should be addressed by the larger scientific community.

Efficacy. Lines 158 to 160. The authors indicate that efficacy of a biocontrol virus is determined by its transmissibility and virulence. Again defining these terms in the context of a biocontrol virus would be really helpful to facilitating the present and future discussions. For example, what are the authors using as a proxy for virulence? I am assuming cumulative mortality but I would like to know what the authors are thinking.

As the authors know, viral traits of virulence and transmission are intricately linked to each other, the host and the environment. Perhaps the authors should consider presenting evidence about the relative susceptibility of Australia’s carp population to CyHV-3 isolates – particularly to the one or more isolates that may be selected to use as the biocontrol viruses. Within this context (ie host resistance), the authors should also discuss any surveillance-type evidence from qPCR tests establishing that Australia’s carp populations are free of CyHV-3 (if this is available). Unfortunately the molecular testing results reported in reference 67 were obtained using conventional PCR assays which are less sensitive than qPCR tests.

Looking forward, are there any plans for a large-scale experiment(s) to be conducted in a closed system outdoors to establish proof of concept for efficacy? If so, what would this look like, what factors would be taken into consideration etc?

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