Isolation and Genotypic Characterization of New Emerging Avian Reovirus Genetic Variants in Egypt

Round 1

Reviewer 1 Report

The research question is very important. ARV is one of the main pathogens of commercial and backyard chickens and it is spreading to different places of the World. Noteworthy, a high prevalence of ARV infections has recently been reported in many Egyptian farms, even in vaccinated poultry flocks, with consequences for this meat production chain. The manuscript reports the ARV detection and molecular characterization of some reoviruses isolated from birds with growth retardation / arthritis (40%) and increased mortality (5-7% on average). The study revealed co-circulation of different ARV genotypes in Egyptian broilers and breeders flocks and highlighted the prevalence of pathogenic variants inducing disease. The findings also demonstrate that circulating strains differ from the vaccine strains that have been granted licenses in Egypt.

The manuscript deserves to be published. The Introduction presents the scientific background, the Methods are the recommended ones according previous studies, the Results are very interesting and the Discussion section compare the main findings with other important studies in the field. However, I think the authors have to perform a major revision to improve it as presented below:

General: it is necessary to standardize the ARV acronym (it was wrongly abbreviated as AVR many times).

Abstract: Please review the sentence “The samples were screened for avian reovirus using real-time PCR, which resulted in the identification of fifteen ARV isolates (20%)” (lines 24-25). I suggest to correct to “ARV was screened using real-time PCR and fifteen positive samples were detected (20%)”. The eleven ARV isolates were obtained after incubation in ECE. 

Introduction: Please review the proteins encoded by genome segments in the second paragraph, since they are not divided into only three categories (lines 50-51). Remember that the ARV enzymes are also proteins. In the final part of that same paragraph, an update in the cited references would be welcome. There are some recent phylogenetic classification articles of ARVs (I included some of them at the end of this letter). Although there is still no more definitive phylogenetic classification for ARV genotypes, these studies revise previously proposed classifications and also report the classification of the samples compared in the results (“with close similarity to a recent Brazilian strain, Canadian virus isolates D2 and D10, and an isolate from China (ARV-strain SDYT-2020), whom assigned as cluster-V” – lines 207-209).

Methods: the assays for molecular characterization should be revised to avoid misleading and unnecessary information (topic 2.5). For example, what is a “strongly positive” sample? The result must be positive or negative. The authors probably have more other information, so report them (as for example the limit Ct value to include the sample in the sequencing study). It is also not necessary to provide detailed information, as for example:  (a) “Negative controls were PCR master mix, primers, and PCR grade water”, (b) “Specific PCR products were identified using 1.5 stained gel with ethidium bromide against 100 bp Plus DNA Ladder Gene RulerTM (Fermentas), using a Biometra® Compact electrophoresis device (Analytik, Jena) and a Biometra® gel documentation system (UV star 312nm - Biometra Laboratories, Milan, Italy). “ I suggest removing them!

Results: please also revise the sentence reporting “weak positive findings in the RT-qPCR test” (line 195), according I have already mentioned above. It would be also very important to standardize the names of all ARVs in the phylogenetic tree. Please use the same format you used for the ARVs sequenced in the study (Reo/Egypt/Broiler/ID), so it is possible to see the country name in the phylogenetic tree.

Discussion: This section compares the results with some references in the field. However, the text is basically repeating the findings without a more deep analysis of the impact of the study in comparison with other recent phylogenetic studies. Therefore I suggest the authors to improve it, mainly in two key points: (a) to compare the phylogenetic classification with other recent studies (added in the end); (b) to discuss the ARV genome (remember it is segmented) and the consequences for this virus evolution and diversification (also with the immunization consequences).         

In my opinion, the article will be more complete and will have a higher scientific impact with these corrections.

References:

De Carli S, Wolf JM, Gräf T, Lehmann FKM, Fonseca ASK, Canal CW, Lunge VR, Ikuta N. Genotypic characterization and molecular evolution of avian reovirus in poultry flocks from Brazil. Avian Pathol. 2020 Dec;49(6):611-620. doi: 10.1080/03079457.2020.1804528. Epub 2020 Sep 14. PMID: 32746617.

Kovács E, Varga-Kugler R, Mató T, Homonnay Z, Tatár-Kis T, Farkas S, Kiss I, Bányai K, Palya V. Identification of the main genetic clusters of avian reoviruses from a global strain collection. Front Vet Sci. 2023 Jan 12;9:1094761. doi: 10.3389/fvets.2022.1094761. PMID: 36713877; PMCID: PMC9878682.

Palomino-Tapia V, Nickel L, Schlegel B, Mitevski D, Inglis T, Abdul-Careem MF. Review of Viral Arthritis in Canada. Avian Dis. 2022 Dec;66(4):452-458. doi: 10.1637/aviandiseases-D-22-99997. PMID: 36715479.

Comments for author File: Comments.docx

Author Response

Author's Reply to the Review Report (Reviewer 1)

Comment 1:General: it is necessary to standardize the ARV acronym (it was wrongly abbreviated as AVR many times).

Author’s comments: Done

Comment 2: Abstract: Please review the sentence “The samples were screened for avian reovirus using real-time PCR, which resulted in the identification of fifteen ARV isolates (20%)” (lines 24-25). I suggest to correct to “ARV was screened using real-time PCR and fifteen positive samples were detected (20%)”. The eleven ARV isolates were obtained after incubation in ECE.

Author’s comments: Done. Rephrased

Comment 3: Introduction: Please review the proteins encoded by genome segments in the second paragraph, since they are not divided into only three categories (lines 50-51). Remember that the ARV enzymes are also proteins.

Author’s comments: Done. The data mentioned in the introduction of the current manuscript resembled to recent data published up to 2023

Comment 4: In the final part of that same paragraph, an update in the cited references would be welcome.

Author’s comments: Recent references were added.

 Tang, Y.; Lin, L.; Sebastian, A.; Lu, H. Detection and characterization of two co-infection variant strains of avian orthoreovirus (ARV) in young layer chickens using next-generation sequencing (NGS). Scientific reports 2016, 6, 1-11.

Kovács E, Varga-Kugler R, Mató T, Homonnay Z, Tatár-Kis T, Farkas S, Kiss I, Bányai K, Palya V. Identification of the main genetic clusters of avian reoviruses from a global strain collection. Front Vet Sci. 2023 Jan 12;9:1094761.

Comment 5: There are some recent phylogenetic classification articles of ARVs (I included some of them at the end of this letter). Although there is still no more definitive phylogenetic classification for ARV genotypes, these studies revise previously proposed classifications and also report the classification of the samples compared in the results (“with close similarity to a recent Brazilian strain, Canadian virus isolates D2 and D10, and an isolate from China (ARV-strain SDYT-2020), whom assigned as cluster-V” – lines 207-209).

Author’s comments: Numerous categorization strategies based on serotyping, partial S1 genotyping using roman numerals (Used in our study according to Ayalew et al., 2016)

Comment 6: Methods: the assays for molecular characterization should be revised to avoid misleading and unnecessary information (topic 2.5). For example, what is a “strongly positive” sample? The result must be positive or negative. The authors probably have more other information, so report them (as for example the limit Ct value to include the sample in the sequencing study).

Author’s comments: Samples of Ct value more than 29 were excluded from sequencing.

Comment 7: It is also not necessary to provide detailed information, as for example:  (a) “Negative controls were PCR master mix, primers, and PCR grade water”, (b) “Specific PCR products were identified using 1.5 stained gel with ethidium bromide against 100 bp Plus DNA Ladder Gene RulerTM (Fermentas), using a Biometra® Compact electrophoresis device (Analytik, Jena) and a Biometra® gel documentation system (UV star 312nm - Biometra Laboratories, Milan, Italy). “ I suggest removing them!

Author’s comments: Removed

Comment 8: Results: please also revise the sentence reporting “weak positive findings in the RT-qPCR test” (line 195), according I have already mentioned above.

Author’s comments: rephrased. line186

 Comment 9: It would be also very important to standardize the names of all ARVs in the phylogenetic tree. Please use the same format you used for the ARVs sequenced in the study (Reo/Egypt/Broiler/ID), so it is possible to see the country name in the phylogenetic tree.

Author’s comments: Sequences submitted in genbank database in 2 batches. The 1st was named Reo/Egypt/Broiler/GIZA..... for 9 samples of cluster IV. The the next submession was for the other two sequences of cluster V, named; Reo/Egypt/Broiler/D6366/.

Discussion: This section compares the results with some references in the field. However, the text is basically repeating the findings without a more deep analysis of the impact of the study in comparison with other recent phylogenetic studies. Therefore I suggest the authors to improve it, mainly in two key points:

Comment 10: (a) to compare the phylogenetic classification with other recent studies (added in the end);

Author’s comments: Added

 Comment 11: (b) to discuss the ARV genome (remember it is segmented) and the consequences for this virus evolution and diversification (also with the immunization consequences).  

Author’s comments: Added

In my opinion, the article will be more complete and will have a higher scientific impact with these corrections.

References:

De Carli S, Wolf JM, Gräf T, Lehmann FKM, Fonseca ASK, Canal CW, Lunge VR, Ikuta N. Genotypic characterization and molecular evolution of avian reovirus in poultry flocks from Brazil. Avian Pathol. 2020 Dec;49(6):611-620. doi: 10.1080/03079457.2020.1804528. Epub 2020 Sep 14. PMID: 32746617.

Kovács E, Varga-Kugler R, Mató T, Homonnay Z, Tatár-Kis T, Farkas S, Kiss I, Bányai K, Palya V. Identification of the main genetic clusters of avian reoviruses from a global strain collection. Front Vet Sci. 2023 Jan 12;9:1094761. doi: 10.3389/fvets.2022.1094761. PMID: 36713877; PMCID: PMC9878682.

Palomino-Tapia V, Nickel L, Schlegel B, Mitevski D, Inglis T, Abdul-Careem MF. Review of Viral Arthritis in Canada. Avian Dis. 2022 Dec;66(4):452-458. doi: 10.1637/aviandiseases-D-22-99997. PMID: 36715479.

Author Response File: Author Response.docx

Reviewer 2 Report

This study find new ARV clusters in broiler flocks in Egypt based on the partial segment analysis of S1 gene. Meanwhile, the commercial vaccines seemed to be inadequte to protect the broilers. However, there are a few concerns as follow:

1. S1 gene is used for genotyping in this study, are there any  diversities in other genes related to protection? and how these major genes changed comparing to S1133?

2. Could S1133 protect against the challenge of new isolates? A cross protection or cross VN protection study is necessary.

3. There are different cluster nomenclature, such as cluster 4,  cluster-IV, genotype 4. Please unify them.

4. Word such as AVR and ARV, Genebank (GenBank) should be unified or checked. Addtional, Line 43, 85-90 of should be 85-90%, Line 121,50 buffered glycerin should be 50%. Line 173, .best models should be Best models.

5. Figure 2 should be more clear in high resolution.  Figure 3, Genotype cluster 1-6 should be unified with the text in this study.

Comments for author File: Comments.pdf

Author Response

Author's Reply to the Review Report (Reviewer 2)

1. S1 gene is used for genotyping in this study, are there any diversities in other genes related to protection? And how these major genes changed comparing to S1133?

Authors comments: Sequencing include only partial sequencing of S1 gene (σC). The strains under study had low sequence similarity percent (47–51%) when compared with various commercial vaccines belonging to genotypic cluster-I (e.g., strain S1133).

2. Could S1133 protect against the challenge of new isolates? A cross protection or cross VN protection study is necessary.

Author’s comments: We have no enough fund or facilities. But further work will be done later including pathogenicity and S1133 vaccine evaluation. Our work includes only genetic relation with vaccine strains used in Egypt.

3. There are different cluster nomenclature, such as cluster 4,  cluster-IV, genotype 4. Please unify them.

Author’s comments: Done all over the manuscript

4. Word such as AVR and ARV,

Author’s comments: Done all over the manuscript

5. Genebank (GenBank) should be unified or checked.

Author’s comments: Changed all over the manuscript

6. Addtional, Line 43, 85-90 of should be 85-90%,

Author’s comments: Done

7. Line 121, 50 buffered glycerin should be 50%.

Author’s comments: Done

8. Line 173, .best models should be Best models.

Author’s comments: Done

9. Figure 2 should be clearer in high resolution. 

Author’s comments: Done

10. Figure 3, Genotype cluster 1-6 should be unified with the text in this study.

Author’s comments: Done

11. The yellow color highlighted words.

Author’s comments: Revisions were done all over the manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I have already reviewed the first submission. I think the manuscript deserves to be published. The Introduction presents the scientific background, the Methods are the recommended ones according previous studies, the Results are very interesting and the Discussion section compare the findings with some studies in the field. However, I realized that the manuscript needed some major modifications, so I suggested to the authors that they do a major revision to improve it.

In this new version (R1), they have made some of the necessary modifications in the manuscript. However the manuscript still has to be further improved in two points:

Results: I have already mentioned that “Please use the same format you used for the ARVs sequenced in the study (Reo/Egypt/Broiler/ID), so it is possible to see the country name in the phylogenetic tree.” So the authors have to change all other IDs to this same format (Reo/”country”/”chicken type”/ID) in the phylogenetic tree. It would be even better if each sequence have the year of isolation too. It is important to compare the sequences from different countries and years directly in the phylogenetic tree.   

Discussion: I have mentioned before that “This section compares the results with some references in the field. However, the text is basically repeating the findings without a more deep analysis of the impact of the study in comparison with other recent phylogenetic studies. Therefore I suggest the authors to rewrite the Discussion in two key points: (a) to compare the phylogenetic classification with other recent studies (added in the end); (b) to discuss the ARV genome! The complete RNA genome have different segments with very important consequences for ARV  evolution (frequently recombination and mutation) and diversification into lineages and sub-lineages.” These two topics were not adequately answered, so I suggest an additional effort to work on them to improve the whole Discussion.

Comments for author File: Comments.docx

Author Response

The tree was updated according to recent studies (2022-2023) and unified for naming. Also strains name was unified all over the draft.

Discussion was re-edited and updated according to the recent studies sent here. Detailed analysis also reported including recombination events.

Author Response File: Author Response.docx