First Isolation, Molecular Identification, and Phylogenetic Characterization of A3B5 Very Virulent Infectious Bursal Disease Virus in Pullets in Chile

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors suspected IBDV in a commercial layer production that had signs of severe pathology. They discovered the birds were infected with IBDV that had a very virulent (vv) phenotype clinically. When they characterized the virus molecularly, they found that it corresponded to a A3B5 genotype.

 

General points

The authors need to distinguish the IBDV they describe from the traditional very virulent (vv) viruses, as the traditional vv viruses belong to genogroup A3B2 by definition. Simply calling this virus a “very virulent” strain is not sufficient. I suggest the authors refer to it throughout the manuscript as “A3B5 vvIBDV” to distinguish it from the A3B2 vv IBDV strains, including in the title.

 

Specific points:

Line 69 and 73: Please update to 9 VP2 genogroups

Line 72: Please change “Re-arrangements” to “reassortment”

Line 77: 45 genotypes can be identified only if there are 9 segment A genogroups

Line 82: Please define cvIBDV

Line 91: Please change the word “sacrificed” to “humanely euthanized”

Line 106: Please explain if specific pathogen free (SPF) chicken embryos were used, if not, please mention if you performed any test to discard the presence of vertically transmitted pathogens or IBDV antibodies. Please mention the viral titer of the inoculum.

Line 107: please change “ reverse transcriptase” to “reverse transcription”

Line 154: Please add genotype A9 to the analysis

Figure 1: Please add scale bars to the images with lesions. Please also have an image of a bursa in isolation (not in the carcass), and include a bursa from an IBDV negative bird as a control

Figure 2:Please improve the image resolution and add an image of an uninfected embryo as a negative control

Figure 3: Lanes 6-11 might not be necessary.

Line 206: Provisional Genbank accession codes do not exist when checked. The manuscript should show definitive accession codes before publication.

Figures 4 and 5: Please provide information on the size of the sequences that were in the trees- for example, were they trimmed trees? Please also give the amino acids position in the genome that the trees are based upon. In each phylogeny there is the same strain code: 16124_CL_2024. Please make a distinction by adding the segment letter in each.Please also increase the resolution

Table 2: Please discuss why you chose to compare your sequences with those from Brazil and USA?

Table 3: this could be supplemental information.

Figure 6: Please add at least 3 more representative amino acid sequences for each genogroup including A9.

Line 295: Please discuss how a B5 genogroup strain could have been introduced in South America.

Did the authors search for other virulence markers in VP1, such as TDS/N?

Please discuss the impact of the virus adaptation in embryos and their subsequent molecular classification.

Please discuss how the A3B5 IBDV got into a commercial layer farm.

Please discuss the impact of detecting very virulent strains in Chile, such as governmental declarations, exportation bans, authorization of new autogenous vaccines, very virulent IBDV testing in live bird imports, etc.

Line 349: Please mention if your research was performed with the approval of the Institutional Animal Care and Use Committee (IACUC)

Reference 44: please add the link to the website software where it can be downloaded

 

Comments on the Quality of English Language

The manuscript requires an English language proficiency check.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.   2. General points Comment : The authors need to distinguish the IBDV they describe from the traditional very virulent (vv) viruses, as the traditional vv viruses belong to genogroup A3B2 by definition. Simply calling this virus a “very virulent” strain is not sufficient. I suggest the authors refer to it throughout the manuscript as “A3B5 vvIBDV” to distinguish it from the A3B2 vvIBDV strains, including in the title. Response: Thank you for pointing this out. We agree with this comment. We consider it important to include “A3B5 vvIBDV” in the title, but not in the entire document.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Line 69 and 73: Please update to 9 VP2 genogroups Response 1: Thank you for pointing this out. We agree with this comment.

Comments 2: Line 72: Please change “Re-arrangements” to “reassortment”

Response 2: Thank you for pointing this out. We agree with this comment.

 Comments 3: Line 77: 45 genotypes can be identified only if there are 9 segment A genogroups

 Response 3: Thank you for pointing this out. We agree with this comment.

 

 Comments 4: Line 82: Please define cvIBDV

 Response 4: Thank you for pointing this out. We agree with this comment.

 Comments 5: Line 91: Please change the word “sacrificed” to “humanely euthanized”

 Response 5: Thank you for pointing this out. We agree with this comment.

 

Comments 6: Line 106: Please explain if specific pathogen free (SPF) chicken embryos were used, if not, please mention if you performed any test to discard the presence of vertically transmitted pathogens or IBDV antibodies. Please mention the viral titer of the inoculum.

Response 6: Thank you for pointing this out. We agree with this comment.

Comments 7: Line 107: please change “ reverse transcriptase” to “reverse transcription”

Response 7: Thank you for pointing this out. We agree with this comment.

 

Comments 8: Line 154: Please add genotype A9 to the analysis

Response 8: Thank you for pointing this out. We agree with this comment.

 

Comments 9: Figure 1: Please add scale bars to the images with lesions. Please also have an image of a bursa in isolation (not in the carcass), and include a bursa from an IBDV negative bird as a control.

Response 9: Thank you for pointing this out. We agree with this comment. There is not negative control because these are birds that arrive as part of routine diagnosis.

 

Comments 10: Figure 2: Please improve the image resolution and add an image of an uninfected embryo as a negative control.

Response 10: Thank you for pointing this out. We agree with this comment.

 

 

Comments 11: Figure 3: Lanes 6-11 might not be necessary.

Response 11: We consider that they are necessary since the analyses are done with both segments.

 

Comments 12: Line 206: Provisional Genbank accession codes do not exist when checked. The manuscript should show definitive accession codes before publication.

Response 12: Thank you for pointing this out. We agree with this comment. We requested the immediate release of the databases.

 

Comments 13: Table 2: Please discuss why you chose to compare your sequences with those from Brazil and USA?

Response 13: Thank you for pointing this out. We agree with this comment.

Comments 14: Table 3: this could be supplemental information.

Response 14: Thank you for pointing this out. We consider that tables 3 and 4 should be included in the main text.

Comments 15: Figure 6: Please add at least 3 more representative amino acid sequences for each genogroup including A9.

Response 15: Thank you for pointing this out . We consider it unnecessary to add more sequences because the objective of identifying residues associated with very virulent strains in our sequence was met. Genogroup 9 was added.

 

Comments 16: Line 295: Please discuss how a B5 genogroup strain could have been introduced in South America.

Response 16: Thank you for pointing this out. We agree with this comment.

Comments 17: Did the authors search for other virulence markers in VP1, such as TDS/N?

Response 17: Thank you for pointing this out. This will be discussed in the broader characterization that we are working on.

Comments 18: Please discuss how the A3B5 IBDV got into a commercial layer farm.

Response 18: Thank you for pointing this out. We agree with this comment.

Comments 19: Please discuss the impact of the virus adaptation in embryos and their subsequent molecular classification.

Response 19: Thank you for pointing this out. We believe that there is no relevance because the virus was identified and classified prior to viral isolation.

Comments 20: Please discuss the impact of detecting very virulent strains in Chile, such as governmental declarations, exportation bans, authorization of new autogenous vaccines, very virulent IBDV testing in live bird imports, etc.

Response 20: Thank you for pointing this out. We agree with this comment.

Comments 21: Line 349: Please mention if your research was performed with the approval of the Institutional Animal Care and Use Committee (IACUC).

Response 21: Thank you for pointing this out.  In this study, all data analyzed were collected as part of routine diagnosis.

Comments 22: Reference 44: please add the link to the website software where it can be downloaded

Response 22: Thank you for pointing this out. We agree with this comment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

First Isolation, molecular identification, and phylogenetic characterization of VVIBDV in Pullets in Chile by Cadiz et al is a well written manuscript. There are minor corrections before acceptance for publication

Line 166. I think "No evident lesions" should be replaced by "No other significant lesions"

Figure 7 didn't print out for me to analyze as it is cited in line 265.

References:  #22 has a format issue with journal name

 

Author Response

Response to Reviewer 2
1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.   2. General points First Isolation, molecular identification, and phylogenetic characterization of VVIBDV in Pullets in Chile by Cadiz et al is a well written manuscript. There are minor corrections before acceptance for publication
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: Line 166. I think "No evident lesions" should be replaced by "No other significant lesions" Response 1: Thank you for pointing this out. We agree with this comment. Comments 2: Figure 7 didn't print out for me to analyze as it is cited in line 265. Response 2: Thank you for pointing this out. There was an error in this part, Fig. 7 does not exist.

 Comments 3: References:  #22 has a format issue with journal name

 Response 3: Thank you for pointing this out. We agree with this comment.

 

Author Response File: Author Response.pdf