Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV, Decapod Penstylhamaparvovirus 1) in Commodity Red Claw Crayfish (Cherax quadricarinatus) Imported into South Korea

Round 1

Reviewer 1 Report

Ln32: It is misleading for authors to make a conclusion basing on only PCR in dead animal without signs associated that they are susceptible. IHHNV sequences have been reported in apparently health animals before. Indeed, ref. 24 from where the authors picked their primers is very clear.

Introduction: silent on the virology and only mentions infectious type. Its important that authors give a brief virology and position infectious types in the broader classification scheme of the virus being studied.

Lns 74-80: I find the description of the sample analysis plan inaccurate. The pooling and extraction of NAs was not the best approach for such a small sample size they had. I am imagining the size of 6mgs! The over pooling could have affected the results through the dilution effect

Lns: 88-92: What was the criteria for selecting the samples for sequencing?

Fig. The gel image looks to have been artificially enhanced given the differences in intensity of the ladder! If the analyzed samples were 7, why did authors select only 2 for sequencing? They should have included all the seven since the coast difference cannot be used as justification.

Conclusion: Its not proper to include references in the conclusion. This section should concentrate on the synthesized take home message from the findings and implication on science.

Author Response

Please see the attached file.

Thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

 

 

Lines 63-66: ".... Our results confirm the presence of infectious-type IHHNV in 65 C. quadricarinatus, suggesting that this species is

susceptible to the virus".

-This assumption is logical. But, on the other side, asymptomatic carriership is also a possibility. the term "susceptible" is vague. But, as no associated disease was described - this is speculative. Remember that speculations are to be avoided,

 

Lines 141-150:  "The IHHNV sequences obtained in this study exhibited high similarity (>96% iden-141 tity) with non-structural protein 1 of the reference IHHNV strain (decapod penstyldenso-142 virus 1, NC_039043.1) …………. clustered closely"

• Why do the authors think that a 380 nucleotide sequence suffices for drawing conclusions? A study by Tang and Lightner showed variations in all ORFs. This should e discussed/ n
• Also, the legend of Fig 2 should address the issue of the 380 bp.

 

 

 

Author Response

Please see the attached file.

Thank you.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript entitled “Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV, Decapod penstyldensovirus 1) in Commodity Red Claw Crayfish (Cherax quadricarinatus) Imported into South Korea” presents novel information on the occurrence of pathogenic infectious agents in commodity crayfish, and is of potential interest to aquaculture and fisheries; it is well structured and sufficiently clear, with overall good English level.

Nevertheless, a few changes are needed to improve the quality of the manuscript:

Page 2, line 50: more information concerning the viral agent under study (e.g. single stranded DNA virus..., target tissues/organs... etc. etc.) should be added.

Page 2, lines 64-66: these lines belong to discussion and conclusions; here the authors should only state the aim of the study.

Page 2, line 82: “DNA was amplified by PCR using primers IHHNV-389F/R”. Furthermore, in this paragraph the authors should indicate what is the target of these primers and what did they use as positive control.

Page 2, line 88: the authors should remove the primers names before the word PCR (here and throughout the manuscript). Why samples of only two batches and only one type of tissue were sequenced?

Page 3, Table 1: why is the line 20-002 in bold and underlined?

Page 3, line 116: what positive control was used?

Page 4, line 119: “Vial DNA was not detected in 1/10 batches...” I assume that the authors refer to the 20-006 batch, which is not shown in Figure 1. In such case, the authors should either remove this reference to Figure 1 or provide a new picture including the negative batch. Another option could be referring to Table1.

Page 4, line 128: how about the gills? The positivity of gill tissues should also be discussed.

Page 4, line 136: the authors should delete “PCR” before primer set and use the full name of the primers.

Page 4, line 148: the authors should discuss the occurrence of infectious and non infectious endogenous types.

Page 5, line 170: a reference to freshwater crustacean aquaculture/fisheries would be more appropriate.

Page 6, line 179: cultured

Author Response

Please see the attached file.

Thank you.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

1. Why did the authors decide to sequence only 2 of the 8 amplified samples? This is important because we need to be sure that all the amplicons could be sequences and provide proof of the reliability of the amplification method used. 2. Did the authors submit their sequences to the gene bank? Where are the accession numbers? This provides for quality assurance and reliability of the sequences generated. This is particularly important given the small length of the sequences that were generated and their reliability.  

Author Response

Please see the attached file. Thank you for the helpful advice.

Author Response File: Author Response.docx