Characterization of the Genomics and Antigenicity of a Naturally Attenuated Gammacoronavirus Infectious Bronchitis Virus Strain in the Genotype GVI-1 Lineage

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript (viruses-4059558) entitled “Genomics and antigenicity characterization of a naturally attenuated gammacoronavirus infectious bronchitis virus strain of genotype GVI-1 lineage” describes the avirulent feature of IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain (abbreviated as YC_GVI-1) belonging to the genotype GVI-1. The authors determined the whole-genome sequence of the YC_GVI-1 strain. The authors estimated that this strain originates from the recombination of two IBVs, genotype GI-19 ck/CH/LSD/111218 strain [Gene accession number #KX364300] and genotype GVI-1 CK/CH/GX/HX [Gene accession number #PB817796]. It is interesting that the IBV YC_GVI-1 strain shows an avirulent phenotype and induces virus-neutralizing activity against the homologous genotype GVI-1 strain and the heterologous genotype GI-19 strain. This reviewer thinks this study provides a promising vaccine strain that can be used to develop a broad-spectrum attenuated live vaccine against IBV in the GVI-1 and GI-19 lineages, and is worth publication in the journal, Viruses.

However, before accepting the manuscript, this reviewer would like to raise several Major and Minor issues that the authors should address.

Major issues:

1. The sequence of the IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain is not provided as the DNA database accession number. It should be disclosed alongside the publication, as in the Instructions for Authors.
2. The use of the IBV strain name is confusing and uncommon because it is often used interchangeably with the DNA database accession number. The authors should respect the IBV strain name. Otherwise, please follow the Instructions for the Authors as described in “Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table”. The explanation in lines 95-96 is only exceptional.
3. Regarding the above comment, the IBV genotype/lineage name and the IBV strain name are used non-distinctively throughout the manuscript. Two names should be used separately to better identify the virus strain.
4. Abstract section: It is described in the Instructions for the Authors that “The abstract should be a total of about 200 words maximum”. However, the present abstract contains about 300 words and exceeds the phrase limit. It should be more concise.
5. Superscripts: Figures that should be shown exponentially are not shown that way throughout the manuscript.
6. Figure 2 caption: This reviewer does not find the figure caption for Figure 2B. Furthermore, captions for Figure 2c (lines 294-296) and 2d (lines 298-300) are redundant.
7. Figure 3: The results of whole genome sequencing of IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain are shown. However, this figure does not present any novel findings; it merely illustrates the IBV genome. This figure does not need to be included in the manuscript. One option is to show this figure as a supplemental figure. Even though information on tRNA and rRNA is not necessary. In addition, the manuscript states that the genome contains nine open reading frames: 1a, 1ab, S, 3a, 3b, E, M, 5a, 5b, and N (line 314). However, no identification on CDS in Figure 3a is done. Moreover, CDS sizes in Figure 3a and Figure 3B are confusing. Which one is S and which one is N in Figure 3A?
8. Figure 5: The authors show the deduced amino acid sequence of the S protein, and present the 3D model of the S protein with the mutation site. The presented result is promising. However, this mode is only a predicted model generated in silico using AlhaFold3 and PyMOL, and no direct evidence supporting it was provided. The reliability of this model is not guaranteed in the manuscript. This figure does not need to be included in the manuscript. One option is to show this figure as a supplemental figure. Even though the explanation, such as the protein model, was created with SWISS-MODEL (https://swissmodel.expasy.org/) using PDB file 6CV0, which represents the Cryo-EM structure of the IBV-S protein (Shang et al., 2018: doi.org/10.1371/journal.ppat.1007009), it is recommended.

Minner issues:

1. Line 58-59, Introduction section: In general, IBV genome structure is introduced as 13 ORFs with the following order, 5’ UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3’ UTR. Are there no 4b-4c and 6b in the genome of IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain?
2. Line 66-68: Based on the entire S1 gene sequences, IBV is initially classified into six genotypes (GI to GVI), comprising 32 distinct lineages (GI-1 to GI-27, GII-1 to GII-2, GIV-1, GV-1, and GVI-1) by Valastro, V. et al, 2016 [doi:10.1016/j.meegid.2016.02.015]. The genotype GVII and the 33rd lineage were later proposed by researchers. Please cite the references correctly.
3. Lines 68-69: Please cite the reference(s) that describes GI-1, GI-7, GI-13, GI-19, GI-22, GI-28, GVI-1 are prevailing in China.
4. Line 91: Please explain the rule of description on A211S, like A211S (Alanine to Serine changes at the 211th amino acid, one-letter code) of the S1 protein.
5. Lines 98, 104, 119, 140, 141, 211, 214, 215, 216, 257, 277, 292, 293, 295, 299, and 406. The authors used the term chicken “embryo”. If the authors inoculate the IBV into the embryo in the embryonating hen egg, or isolate the embryo from the egg and incubate it, this reviewer has no comments. However, the authors inoculate the IBV into the allantoic fluid in the embryonating egg and incubate the egg in the incubator. The authors measure the virus titer in the allantoic fluid of the egg, but not in the embryonic extract. Considering the methods the authors used, the use of “embryo” is not suitable, but the use of “embryonating hen egg” is proper.
6. Lines 106-109 and Table 1: RNA extracted from allantoic fluid of embryonating eggs using TRizol is used for virus gene detection by specific PCR. The viral primer used in this study is listed in Table 1. IBV, NDV, AIV H5, AIV H9, ALV-A, and IBDV are RNA viruses, but ILTV and FAdV-4 are DNA viruses. TRizol-extracted RNA is not a suitable sample for detecting ILTV or FAdV-4. Are there ILTV and FAdV-4 mRNA in the allantoic fluid?
7. Lines 113, 264, and 273: Please check which electron microscopy the authors used in this study, transmission electron microscopy (TEM) or scanning electron microscopy (SEM). Moreover, please add an explanation of the staining method, such as negative staining with tungsten (W), molybdenum (Mo), or uranium (U).
8. Line 132: Please specify the species that was used to produce the antibody against IBV.
9. Line 141: Please confirm if the authors really incubated the embryos (or embryonating eggs?) under 5% CO₂.
10. Lines 165-170: Please clarify the alignment program that the authors used, such as ClustalW, Muscle, or MAFFT, using MEGA X.
11. Line 224. Please cite the reference (s) on the CHO expression system.
12. Line 226: Please clarify which enzyme is conjugated with the anti-chicken second antibody.
13. Line 340, Figure 4A: Please show the bootstrap values based on 1000 replicates in each branch of the phylogenetic tree.
14. Lines 344-347, Figure 4B: Please clarify at which base homology analysis of IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain and eight representative strains of different genotypes was calculated, nucleotide or deduced amino acid base? In addition, how about the homology of 4b-4c and 6b CDSs?
15. Line 352-356: Please describe clearly in the manuscript that 10 unique amino acid substitutions, V39L, A109V, G119R, K131E, L185S, F389L, V485M, L942I, A957V, and V982I are located in the S1 or S2 protein.
16. Lines 357-359: Please explain that the S protein is composed of the S1 and S2 proteins, and that the Spike antigen is formulated with the trimeric structure of S1 and S2 proteins on the envelope.
17. Line 380 and 382: Please clarify that the anti-IBV antibody is IgG specific, like the anti-IBV IgG antibody.
18. Line 289: Please confirm if “NC50” is correct. I suppose it is NT50 along with Figure 6D. Please check if the “5.27” time is accurate. I assume “5.28” because 2⁴ (84.4)/ 2⁴ (16)=5.275.
19. Line 396: Please check if “(1.56±1.53)*10-8” is correct, because the mean value of 4.1 x 10^-9, 8.28 x 10^-9,71 x 10^-8 and 3.80 x 10^-8, as indicated in Figure 6E, is 1.537x10^-8.
20. Line 396, Figure 6B: Please show the vertical axis like ELISA value (S/P).
21. Line 396, Figures 6C and 6D: Please explain the criteria for positive and negative images. A faint band is observed in the dilution group, even though the authors judged it to be neutralized.
22. Line 399, Figures 6E and 6F: Please explain what the horizontal axis means. Serum dilution used for the primary antibody? Moreover, please show clearly that several amounts of S protein (0.125-1.0 µg/mL) are coated on the ELISA well.
23. Line 491: Please check if “the neutralizing titer” can be replaced with NT50 along with Figure 6D.
24. Line 524, In the Institutional Review Board Statement section: The authors should disclose the approval number for studies involving humans or animals according to the Instructions for Authors. 
25. Line 526, Data Availability Statement section: The authors are suggested to follow MDPI Research Data Policies (https://www.mdpi.com/ethics) according to the Instructions for Authors.
26. Lines 530-594, References section: Please follow the format as described in the Instructions for Authors. All authors should be shown. The authors do not abbreviate the author's name with “et al.”

Author Response

Major issues:

1. The sequence of the IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain is not provided as the DNA database accession number. It should be disclosed alongside the publication, as in the Instructions for Authors.

Response：The full genome sequence of YC_GVI-1 is provided in the supplementary materials. we have already submitted it in the past two days, GenBank accession number is PX767077 in line 105-106

2. The use of the IBV strain name is confusing and uncommon because it is often used interchangeably with the DNA database accession number. The authors should respect the IBV strain name. Otherwise, please follow the Instructions for the Authors as described in “Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table”. The explanation in lines 95-96 is only exceptional.

Response：The YC_GVI-1 strain was first defined in the abstract (see line 20). Furthermore, this study represents a continuation of prior work, in which the strain was previously characterized and designated as YC_GVI-1 in published research(Xiong T, Xie H, Li L, Liang S, Huang M, Yu C, Zhuang T, Liang X, Liu D, Chen R. Prevalence, Genotype Diversity, and Distinct Pathogenicity of 205 Gammacoronavirus Infectious Bronchitis Virus Isolates in China during 2019-2023. Viruses. 2024 Jun 7;16(6):930. doi: 10.3390/v16060930.). To ensure nomenclatural consistency across studies, we retain the designation YC_GVI-1 in the current manuscript.

3. Regarding the above comment, the IBV genotype/lineage name and the IBV strain name are used non-distinctively throughout the manuscript. Two names should be used separately to better identify the virus strain.

Response：This manuscript has been thoroughly checked and revised throughout. The term "lineage(s)" has been changed to "genotype(s)" in lines 99 175, 181...

4. Abstract section: It is described in the Instructions for the Authors that “The abstract should be a total of about 200 words maximum”. However, the present abstract contains about 300 words and exceeds the phrase limit. It should be more concise.

Response：The abstract has been rephrased and condensed to 200 words （line 16-33）.

5. Superscripts: Figures that should be shown exponentially are not shown that way throughout the manuscript.

Response：Has been modified.

6. Figure 2 caption: This reviewer does not find the figure caption for Figure 2B. Furthermore, captions for Figure 2c (lines 294-296) and 2d (lines 298-300) are redundant.

Response：Has been added and modified in line 311-314

7. Figure 3: The results of whole genome sequencing of IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain are shown. However, this figure does not present any novel findings; it merely illustrates the IBV genome. This figure does not need to be included in the manuscript. One option is to show this figure as a supplemental figure. Even though information on tRNA and rRNA is not necessary. In addition, the manuscript states that the genome contains nine open reading frames: 1a, 1ab, S, 3a, 3b, E, M, 5a, 5b, and N (line 314). However, no identification on CDS in Figure 3a is done. Moreover, CDS sizes in Figure 3a and Figure 3B are confusing. Which one is S and which one is N in Figure 3A?

Response：This figure provides a systematic summary of the whole genome sequencing results. The nine identified open reading frames (ORFs) exhibit differences in genomic position and size compared to previously reported GVI-1 strains (e.g., MW791835.1), thereby offering meaningful comparative insights. Therefore, we respectfully maintain that this result should be retained in the manuscript, and we hope the reviewers will consider this justification.

This sentence (The sizes and genomic positions of the nine open reading frames (ORFs) are presented in Figure 3B) was added in line 338-339 "

8. Figure 5: The authors show the deduced amino acid sequence of the S protein, and present the 3D model of the S protein with the mutation site. The presented result is promising. However, this mode is only a predicted model generated in silico using AlhaFold3 and PyMOL, and no direct evidence supporting it was provided. The reliability of this model is not guaranteed in the manuscript. This figure does not need to be included in the manuscript. One option is to show this figure as a supplemental figure. Even though the explanation, such as the protein model, was created with SWISS-MODEL (https://swissmodel.expasy.org/) using PDB file 6CV0, which represents the Cryo-EM structure of the IBV-S protein (Shang et al., 2018: doi.org/10.1371/journal.ppat.1007009), it is recommended.

Response：Dear Reviewer, Thank you for your valuable comments on Figure 5 and the 3D model of the S protein. We fully understand your concerns regarding the reliability of the model and the level of evidence supporting it. After careful consideration, we would like to retain this figure in the main text of the manuscript for the following reasons:

• Utilization of reliable prediction and visualization tools

AlphaFold3, an innovative artificial intelligence model developed by DeepMind, has achieved unprecedented accuracy in predicting the structures of proteins and other biomolecular complexes. It has been widely used in protein structure prediction.

• Academic value and research significance of the model

The 3D model of the S protein shown in Figure 5 was generated through computational prediction using AlphaFold3 and PyMOL. Although it is an in silico (computer simulation) result, it provides important structural biology clues regarding the spatial distribution of mutation sites and conformational changes, which are crucial for understanding the functional regions of the protein. This model can visually present the potential impact of mutations on the protein structure, which is beneficial for subsequent experimental design and mechanism discussion.

Minner issues:

1. Line 58-59, Introduction section: In general, IBV genome structure is introduced as 13 ORFs with the following order, 5’ UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3’ UTR. Are there no 4b-4c and 6b in the genome of IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain?

Response：The full genome sequence of YC_GVI-1 was assembled using MW815494.1 (CK CH TJ1904) as the reference strain. Its ORFs are in the following order: polyprotein 1a, 1b, S, 3a, 3b, E, M, 5a, 5b, and N proteins.

2. Line 66-68: Based on the entire S1 gene sequences, IBV is initially classified into six genotypes (GI to GVI), comprising 32 distinct lineages (GI-1 to GI-27, GII-1 to GII-2, GIV-1, GV-1, and GVI-1) by Valastro, V. et al, 2016 [doi:10.1016/j.meegid.2016.02.015]. The genotype GVII and the 33rd lineage were later proposed by researchers. Please cite the references correctly.

Response：Has been added and modified in line 60-63

3. Lines 68-69: Please cite the reference(s) that describes GI-1, GI-7, GI-13, GI-19, GI-22, GI-28, GVI-1 are prevailing in China.

Response：Two references was added in line 66 and in References

[10] Xiong T, Xie H, Li L, et al. Prevalence, Genotype Diversity, and Distinct Pathogenicity of 205  Gammacoronavirus Infectious Bronchitis Virus Isolates in China during 2019-2023. Viruses. 2024;16(6).

[11] Zeng Z, Yao L, Feng H, et al. Genetic and pathogenic characteristics of a novel recombinant GI-19 infectious  bronchitis virus strain isolated from northeastern China. Poult Sci. 2025;104(4):104985

4. Line 91: Please explain the rule of description on A211S, like A211S (Alanine to Serine changes at the 211th amino acid, one-letter code) of the S1 protein.

Response：(Alanine to Serine changes at the 211th amino acid) was added in line 100

5. Lines 98, 104, 119, 140, 141, 211, 214, 215, 216, 257, 277, 292, 293, 295, 299, and 406. The authors used the term chicken “embryo”. If the authors inoculate the IBV into the embryo in the embryonating hen egg, or isolate the embryo from the egg and incubate it, this reviewer has no comments. However, the authors inoculate the IBV into the allantoic fluid in the embryonating egg and incubate the egg in the incubator. The authors measure the virus titer in the allantoic fluid of the egg, but not in the embryonic extract. Considering the methods the authors used, the use of “embryo” is not suitable, but the use of “embryonating hen egg” is proper.

Response：I respectfully maintain a different perspective, considering the term "embryo" to be more appropriate than "embryonating hen egg" in this context. This usage is consistent with established terminology in previously published studies. We kindly request that this phrasing be retained.

6. Lines 106-109 and Table 1: RNA extracted from allantoic fluid of embryonating eggs using TRizol is used for virus gene detection by specific PCR. The viral primer used in this study is listed in Table 1. IBV, NDV, AIV H5, AIV H9, ALV-A, and IBDV are RNA viruses, but ILTV and FAdV-4 are DNA viruses. TRizol-extracted RNA is not a suitable sample for detecting ILTV or FAdV-4. Are there ILTV and FAdV-4mRNA in the allantoic fluid?

Response：In the experimental design, we have clearly classified ILTV and FAdV-4 as DNA viruses and did not include them in the PCR system for RNA virus detection. Therefore, the RNA samples extracted by TRizol are not suitable for the detection of these two viruses.

7. Lines 113, 264, and 273: Please check which electron microscopy the authors used in this study, transmission electron microscopy (TEM) or scanning electron microscopy (SEM). Moreover, please add an explanation of the staining method, such as negative staining with tungsten (W), molybdenum (Mo), or uranium (U).

Response：transmission electron microscopy (TEM) and negative staining with tungsten (W)

8. Line 132: Please specify the species that was used to produce the antibody against IBV.

Response：Mouse monoclonal antibody was added in line 145

9. Line 141: Please confirm if the authors really incubated the embryos (or embryonating eggs?) under 5% CO₂.

Response：Under 5% CO2 has been deleted line154

Lines 165-170: Please clarify the alignment program that the authors used, such as ClustalW, Muscle, or MAFFT, using MEGA X.

Response：“and subsequently aligned using ClustalW”was added in line 176

10. Line 224. Please cite the reference (s) on the CHO expression system.

Response：The reference was added.

[30] Porta M, Pumarega J, Gasull M, et al. Individual blood concentrations of persistent organic pollutants and chemical  elements, and COVID-19: A prospective cohort study in Barcelona. Environ Res. 2023;223:115419.

11. Line 226: Please clarify which enzyme is conjugated with the anti-chicken second antibody.

Response：I am sure that enzyme is conjugated with the anti-chicken second

12. Line 340, Figure 4A: Please show the bootstrap values based on 1000 replicates in each branch of the phylogenetic tree.

Response：the bootstrap values has added in Figure 4A

13. Lines 344-347, Figure 4B: Please clarify at which base homology analysis of IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain and eight representative strains of different genotypes was calculated, nucleotide or deduced amino acid base? In addition, how about the homology of 4b-4c and 6b CDSs?

Response：“The nucleotide sequences homology analysis” has added in line 369.

14. Line 352-356: Please describe clearly in the manuscript that 10 unique amino acid substitutions, V39L, A109V, G119R, K131E, L185S, F389LV485M, L942I, A957V, and V982I are located in the S1 or S2 protein.

Response： has challged to “The YC_GVI-1 S protein contains 10 unique amino acid substitutions, specifically V39L, A109V, G119R, K131E, L185S, F389L, and V485M in the S1 subunit, and L942I, A957V, and V982I in the S2 subunit (Figure 5A).”in line 380-381

15. Lines 357-359: Please explain that the S protein is composed of the S1 and S2 proteins, and that the Spike antigen is formulated with the trimeric structure of S1 and S2 proteins on the envelope.

Response：“S protein is cleaved by host proteases to generate S1 and S2 subunits.”has added in line 372

16. Line 380 and 382: Please clarify that the anti-IBV antibody is IgG specific, like the anti-IBV IgG antibody.

Response：anti-IBV antibody has challge to anti-IBV IgG antibody in line 408

17. Line 289: Please confirm if “NC50” is correct. I suppose it is NT50 along with Figure 6D. Please check if the “5.27” time is accurate. I assume “5.28” because 2⁴(84.4)/ 2⁴ (16)=5.275.

Response：NC50 has challged to NT50.

2^6.4/2⁴=2^2.4 =5.28,so I am sure that “5.27” time is accurate

18. Line 396: Please check if “(1.56±1.53)*10-8” is correct, because the mean value of 4.1 x 10-9, 8.28 x 10-9,71 x 10-8 and 3.80 x 10-8, as indicated in Figure 6E, is 1.537x10-8.

Response：The binding affinities to the S1 proteins of YC_GVI-1 and JS96_GI-19 were (1.54±1.53)*10^-8 and (6.77±2.42)*10^-8, respectively ~4.30-fold higher for the homotypic S1 protein in line 419-421

19. Line 396, Figure 6B: Please show the vertical axis like ELISA value (S/P).

Response：

20. Line 396, Figures 6C and 6D: Please explain the criteria for positive and negative images. A faint band is observed in the dilution group, even though the authors judged it to be neutralized.

Response：A distinct single band is judged as positive, while a smear and no band are judged as negative.

21. Line 399, Figures 6E and 6F: Please explain what the horizontal axis means. Serum dilution used for the primary antibody? Moreover, please show clearly that several amounts of S protein (0.125-1.0 µg/mL) are coated on the ELISA well.

Response：“of the serum antibody concentration...”has challged to  “of the serum antibody mass concentration on the x-axis and the OD450 value on the y-axis.”

22. Line 491: Please check if “the neutralizing titer” can be replaced with NT50 along with Figure 6D.

Response：has been repalced.

23. Line 524, In the Institutional Review Board Statement section: The authors should disclose the approval number for studies involving humans or animals according to the Instructions for Authors.

Response：LL-G-20250201-01 has added in line 560

24. Line 526, Data Availability Statement section: The authors are suggested to follow MDPI Research Data Policies (https://www.mdpi.com/ethics) according to the Instructions for Authors.

Response：“The authors confirm that the data supporting the findings of this study are available within the article.”has added in line 562-563

25. Lines 530-594, References section: Please follow the format as described in the Instructions for Authors. All authors should be shown. The authors do not abbreviate the author's name with “et al.”

Response：has modified in line 568-670

Reviewer 2 Report

Comments and Suggestions for Authors

Overall, this is an interesting study characterizing the genetic, biological, and immunological features of an IBV "GVI-1“ variant, and demonstrated promising protection results against the prevalent strain of ”GI-19“ sublineage. However, major comments need to be addressed before consideration for acceptance.

Major comments: 

1. Nomenclature: The author stated that current nomenclature is based on S1 sequencing, why would the author would claim the variant a ”GVI-1“ based on whole genome sequencing?

2. Recombination events: In figure 4.C, the authors claimed that "PP817796.1 and KX364300.1 336 serving as its major and minor parental strains", but visually, PP817796.1 (blue) maintains high similarity (~near 1.0) across most of the genome, while KX364300.1 (red) shows a large drop in similarity in a region roughly around ~20–23 kb (by the axis). In order to claim the recombination event, you should show at least one of the following as a complement (preferably both):
(1) RDP4 event table: which methods supported the same event, breakpoint coordinates, p-values, and the assigned major/minor parents.
(2) Bootscan plot (SimPlot bootscanning, not just similarity plot) demonstrating a region-wise shift in phylogenetic affinity.

Also consider adding more sequences of reference strains for recombinant analysis to minimize bias.

3. Introduction and discussion: the prevalence of GI-19 in China should be highlighted and also please provide reasons why you're choosing GI-19 for the NA study and challenge study for a GVI-1 strain? Audience might have questions, like, is there GI-19 vaccine available, and why do you evaluate the efficacy of a strain from a different lineage?

 

Minor comments:

1. Figure 4B: since PP817796.1 is claimed the parent strain of YC_GVI-1, comparison betwen PP817796.1  YC_GVI-1 shoud be added.

 

Comments on the Quality of English Language

The overall language quality of the article is concerning, please consider refering to a native speaker or for language tool to edit the manuscript.

Author Response

Major comments:

1. Nomenclature: The author stated that current nomenclature is based on S1 sequencing, why would the author would claim the variant a ”GVI-1“ based on whole genome sequencing?

Response：Previous research based on the evolutionary analysis of the S1 gene sequence found that CK/CH/SC/YC_GVI-1-DK/LMB20210104 belongs to the GVI-1 genotype and was named YC_GVI-1. The relevant research has been published (Xiong T, Xie H, Li L, Liang S, Huang M, Yu C, Zhuang T, Liang X, Liu D, Chen R. Prevalence, Genotype Diversity, and Distinct Pathogenicity of 205 Gammacoronavirus Infectious Bronchitis Virus Isolates in China during 2019-2023. Viruses. 2024 Jun 7;16(6):930. doi: 10.3390/v16060930.). Given that YC_GVI-1 has weak virulence, this paper selects it as the research object to further study its genomic and immunogenic characteristics. The viral genome sequence is being uploaded to NCBI.

2. Recombination events: In figure 4.C, the authors claimed that "PP817796.1 and KX364300.1  serving as its major and minor parental strains", but visually, PP817796.1 (blue) maintains high similarity (~near 1.0) across most of the genome, while KX364300.1 (red) shows a large drop in similarity in a region roughly around ~20–23 kb (by the axis). In order to claim the recombination event, you should show at least one of the following as a complement (preferably both):
   (1) RDP4 event table: which methods supported the same event, breakpoint coordinates, p-values, and the assigned major/minor parents.

Response：Has added more sequences of reference strains for recombinant analysis and RDP4 event table in Figure 4C
(2) Bootscan plot (SimPlot bootscanning, not just similarity plot) demonstrating a region-wise shift in phylogenetic affinity. Also consider adding more sequences of reference strains for recombinant analysis to minimize bias.

Response：has added recombination breakpoint in Figure 4D

3. Introduction and discussion: the prevalence of GI-19 in China should be highlighted and also please provide reasons why you're choosing GI-19 for the NA study and challenge study for a GVI-1 strain? Audience might have questions, like, is there GI-19 vaccine available, and why do you evaluate the efficacy of a strain from a different lineage?

Response： Relevant supplementary content has been added in lines 81 to 91 in introduction. And the relevant discussion is also available in the "discussion" section, please refer to lines 493-504.

Minor comments:

1. Figure 4B: since PP817796.1 is claimed the parent strain of YC_GVI-1, comparison betwen PP817796.1and YC_GVI-1 shoud be added.

Response：In Figure 4B, the comparative analysis between PP817796.1 and YC_GVI-1 is already available, labeled as CK/CH/GX/HX (PP8177796.1), GVI-1. Apologies if the notation within the parentheses is not prominent enough.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for considering the suggestions and comments that I raised. I confirmed that some of the issues were carefully resolved by the authors, but I still have concerns about the authors' replies. This reviewer considers that they must be addressed before acceptance for publication.

The unresolved points that this reviewer pointed out as Major issues in the first round-review:

1. DNA database accession number or the nucleotide sequence should be disclosed alongside the publication, as in the Instructions for Authors. The authors replied that the full genome sequence of YC_CV-1 had already been submitted and received a GenBank accession number [PX767077]. Unfortunately, this reviewer cannot find it using this accession number. Moreover, I cannot see the full genome sequence of YC_CV-1 even though the authors told us to provide it in the supplementary materials. It should be provided in an easily open format, such as a text file.
2. The use of the IBV strain name is confusing and uncommon because it is often used interchangeably with the DNA database accession number. This reviewer agrees with the consistent naming of materials, calling IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 as YC_CV-1. However, I cannot agree with the use of reference CK/CH/FJ/202005 and CK/CH/GX/HX strains calling MW791835.1 (lines 22, 24, 195, 355, 340,345, 350, 372, 383, 384, 484, 498, and 521) and PP817796.1 (lines 24, 196, 345, 350, 372, 383, 385, 485, 499, 518, and 522) without definition.  The authors should follow the “Instructions for the Authors” as described in “Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table”.
3. Figure 3: The results of whole genome sequencing of IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain are shown. I withdraw the retraction of Figures 3 and agree to include them in the manuscript. However, I still think information on tRNA, rRNA, and other is not necessary. Nine coding frames (CDS)s: 1a, 1b, S, 3a, 3b, E, M, 5a, 5b, and N are shown in Figure 3B. However, no identification of CDS in Figure 3A is done. Please show them in Figure 3A.
4. Figure 5: The authors show the deduced amino acid sequence of the S protein, and present the 3D model of the S protein with the mutation site. The presented result is promising. However, this mode is only a predicted model generated in silico using AlhaFold3 and PyMOL, and no direct evidence supporting it was provided. This reviewer agrees to retain Figure 5, which shows the in silico modeling. However, if this model is a prediction, it does not fit the results section because the authors do not provide any real evidence supporting it at present. This reviewer would like to suggest moving Figure 5 from the Results section to the Discussion section and presenting it as the reliable model predicted by the software.

In addition to the above, this reviewer still has minor concerns.

The unresolved points that this reviewer pointed out as Miner issues in the first round-review:

1. Table 1: The viral primer used in this study is listed. IBV, NDV, AIV H5, AIV H9, ALV-A, and IBDV are RNA viruses, but ILTV and FAdV-4 are DNA viruses. TRizol-extracted RNA is not a suitable sample for detecting ILTV or FAdV-4. The authors replied that ILTV and FAdV-4 were classified as DNA viruses and were not included in the PCR system for RNA virus detection. However, the manuscript states, “Conventional PCR was applied to screen exogenous viruses with primer sequences provided in Table 1 (lines 117-118),” with the method described only for RNA extraction (lines 115-117). No DNA extraction method is provided in the manuscript.
2. Lines 122, 272, and 281: The authors replied that transmission electron microscopy (TEM) and negative staining with tungsten (W) were used. If TEM was used, please correct the scanning electron microscopy (SEM) in lines 272 and 281.
3. Line 414, Figures 6E and 6F: Regarding the horizontal axis, the authors reply that it means the serum antibody mass concentration. Mathematically, 10⁰, 10^-2, and 10^-4 are defined as 1, 0.01, and 0.0001, but log (0), log (-1), and log (-4) are undefined. There is no negative output from a log function.
4. Lines 548-650, References section: Please follow the format as described in the Instructions for Authors. All authors should be shown. The authors do not abbreviate the author's name with “et al.” (Please check refs 3, 4, 11, 16, 18, 33, 34, and 39)
5. In addition to the above issues, please check one minor point. PP8177”7”96.1 in lines 178, 193, and 196, if they are PP817796.1.

Author Response

Reviewer 1

Major comments:

1. DNA database accession number or the nucleotide sequence should be disclosed alongside the publication, as in the Instructions for Authors. The authors replied that the full genome sequence of YC_CV-1 had already been submitted and received a GenBank accession number [PX767077]. Unfortunately, this reviewer cannot find it using this accession number. Moreover, I cannot see the full genome sequence of YC_CV-1 even though the authors told us to provide it in the supplementary materials. It should be provided in an easily open format, such as a text file.

Response：We have not requested a specific release date for your sequence data. Therefore, Our record(s) will be released to the public database once they are processed. And we contact the GenBank annotation staff to release the sequences to the public database we submitted as soon as possible.

The full genome sequence of YC_CV-1 has been re-supplemented in the supplementary materials.

2. The use of the IBV strain name is confusing and uncommon because it is often used interchangeably with the DNA database accession number. This reviewer agrees with the consistent naming of materials, calling IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 as YC_CV-1. However, I cannot agree with the use of reference CK/CH/FJ/202005(1)and CK/CH/GX/HX(PP817796.1) strains calling MW791835.1 (lines 22, 24, 195, 355, 340,345, 350, 372, 383, 384, 484, 498, and 521) and PP817796.1 (lines 24, 196, 345, 350, 372, 383, 385, 485, 499, 518, and 522) without definition.  The authors should follow the “Instructions for the Authors” as described in “Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table”.

Response：The entire manuscript has been revised consistently for uniformity. Please see the sections marked in red font.

 

3. Figure 3: The results of whole genome sequencing of IBV CK/CH/SC/YC_GVI-1-DK/LMB20210104 strain are shown. I withdraw the retraction of Figures 3 and agree to include them in the manuscript. However, I still think information on tRNA, rRNA, and other is not necessary. Nine coding frames (CDS)s: 1a, 1b, S, 3a, 3b, E, M, 5a, 5b, and N are shown in Figure 3B. However, no identification of CDS in Figure 3A is done. Please show them in Figure 3A.

Response：The coding sequences (CDS) in Figure 3A have been annotated by the sequencing service company. non-essential genomic features such as tRNA and rRNA have been removed to improve clarity and focus on relevant coding regions in Figure 3B.

4. Figure 5: The authors show the deduced amino acid sequence of the S protein, and present the 3D model of the S protein with the mutation site. The presented result is promising. However, this mode is only a predicted model generated in silico using AlhaFold3 and PyMOL, and no direct evidence supporting it was provided. This reviewer agrees to retain Figure 5, which shows the in silico modeling. However, if this model is a prediction, it does not fit the results section because the authors do not provide any real evidence supporting it at present. This reviewer would like to suggest moving Figure 5 from the Results section to the Discussion section and presenting it as the reliable model predicted by the software.

Response：We concur with the reviewer's recommendation to retain Figure 5. The corresponding description of the results has been relocated to the Discussion section to better align with the interpretive context of the predicted structural model in 501-509.

 

Minner comments:

1. Table 1: The viral primer used in this study is listed. IBV, NDV, AIV H5, AIV H9, ALV-A, and IBDV are RNA viruses, but ILTV and FAdV-4 are DNA viruses. TRizol-extracted RNA is not a suitable sample for detecting ILTV or FAdV-4. The authors replied that ILTV and FAdV-4 were classified as DNA viruses and were not included in the PCR system for RNA virus detection. However, the manuscript states, “Conventional PCR was applied to screen exogenous viruses with primer sequences provided in Table 1 (lines 117-118),” with the method described only for RNA extraction (lines 115-117). No DNA extraction method is provided in the manuscript.

Response：Has added the sentence “Meanwhile, viral DNA in the allantoic fluid was extracted using a Viral DNA Extraction Kit (OMEGA Bio-Tek, Inc. Georgia, USA).” in line 120-122. 

 

2. Lines 122, 272, and 281: The authors replied that transmission electron microscopy (TEM) and negative staining with tungsten (W) were used. If TEM was used, please correct the scanning electron microscopy (SEM) in lines 272 and 281.

Response：Has modified to TEM

 

3. Line 414, Figures 6E and 6F: Regarding the horizontal axis, the authors reply that it means the serum antibody mass concentration. Mathematically, 100, 10-2, and 10-4 are defined as 1, 0.01, and 0.0001, but log (0), log (-1), and log (-4) are undefined. There is no negative output from a log function.

Response：Figures 6E and 6F are drawn with reference to the literature. And “...with the logarithm of the serum antibody mass concentration on the x-axis” has challenged into “...with the logarithm of the serum antibody concentration (µg/mL) from -4 to 4 (equivalent to 0.0001µg/mL ~ 1000µg/mL) on the x-axis” in line 425-426.

 

4. Lines 548-650, References section: Please follow the format as described in the Instructions for Authors. All authors should be shown. The authors do not abbreviate the author's name with “et al.” (Please check refs 3, 4, 11, 16, 18, 33, 34, and 39)

Response：Has modified.

 

5. In addition to the above issues, please check one minor point. PP8177”7”1 in lines 178, 193, and 196, if they are PP817796.1.

Response：Has modified in line182, and two other places were replaced with CK/CH/GX/HX.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

None. All comments addressed.

Comments on the Quality of English Language

The overall language quality of the article is concerning, please consider refering to a native speaker or for language tool to edit the manuscript.

Author Response

The overall language has been rechecked and rewritten.