Differential analysis of IBV-infected primary chicken embryonic fibroblasts and immortalized DF-1

ABSTRACT Infectious bronchitis virus (IBV), the causative agent of infectious bronchitis, is responsible for major economic losses in the poultry industry worldwide. While IBVs can usually be passaged in primary chicken embryonic fibroblasts (CEFs), most of the wild ones cannot adapt to passaged cell lines. In this study, the wild strain CK/CH/MY/2020 was used to infect primary CEF and immortalize DF-1 CEF cells. Results indicated that IBV was able to cause lesions and pass onto CEF, but not DF-1. Indeed, the virus could enter DF-1 cells and synthesize the associated structural gene but could not assemble into complete viral particles for release. Furthermore, transcriptome sequencing analysis showed significant differences in gene expression between CEF and DF-1 cells after viral infection, although the corresponding antiviral responses could be activated in both cell types. The biggest difference was in terms of the amino acid biosynthesis pathway and the cytokine receptor interaction pathway, which were significantly and specifically activated in CEF. This could actually explain why intact viruses can be assembled but not in DF-1. In addition, SLBP and P2RX7 affect the replication of IBV’s structural genes to some extent. Overall, IBV can enter CEF and DF-1 cells, but the complex intracellular cytokine interactions affect the assembly and release of viral particles. The insight will be useful for the study of IBV through in vitro transmission and pathogenesis. IMPORTANCE Infectious bronchitis virus (IBV) is responsible for high morbidity and mortality as well as substantial economic losses worldwide. Transcriptome sequencing of IBV-infected chicken embryonic fibroblast and DF-1 cells revealed that the virus elicits antiviral immunity in cells after viral infection, but IBV cannot activate DF-1 cells to produce sufficient amounts of viral structures to assemble into complete virions, which may be caused by the interactions between cytokines. The study of IBV cellular adaptations is important for vaccine development and investigation of the pathogenesis of IBV.

The topic of the manuscript is interesting, which will be useful for the study of IBV through in vitro transmission and pathogenesis.Unfortunately, there are some questions for the author to be published on Microbiology Spectrum.1.RNA-Seq.Huge sequencing datasets or raw data must also be deposited, e.g. as a NCBI BioProject and the accession number provided.Please, read the Information for authors guide for further information.2.The Figures is too blurry and need to be replaced.3.The English proofreading by a native speaker with professional knowledge (especially the discussion part) is required.
IBV is a gamma-coronavirus.Unlike other coronaviruses which could infect different cell lines, while the virus can easily propagate in chicken embryos and primary cell cultures such as CEK cells, adaptation of IBVs on immortalized cell lines is another story.In the manuscript, Yang and colleagues performed transcriptome analysis on IBV-infected CEF and DF-1 cells, and revealed immune-related genes SLBP and P2RX7 can affect replication of the viral structural genes, which provides an interesting point in the research of in vitro pathogenesis of IBVs, since the IBV strain used in the current research can only infect CEF cells.However, the methods part is quite rough.For instance, how was WB performed?Any Ab information?How did IBV in-vitro infection was performed since the timing of virus adsorption and proliferation was quite tricky?Lack of these information has increased the difficulty for the readers to understand the manuscript.Other minor points required to be addressed are as follows.5, Line 208-211: Although these two genes were not included in the key..., they are considered to be important due to their highly significant differential expression as well as ... 6, Any verification for the RNA-seq results? 7, Line 429: References is misspelled.

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Dear Editor, Dear reviewers
Thank you for your letter dated October 9.We were pleased to know that our work was rated as potentially acceptable for publication in Journal, subject to adequate revision.We thank the reviewers for the time and effort that they have put into reviewing the previous version of the manuscript.Their suggestions have enabled us to improve our work.
Based on the instructions provided in your letter, we uploaded the file of the revised manuscript.Accordingly, we have uploaded a copy of the original manuscript with all the changes highlighted by using the red typeface.Appended to this letter is our point-by-point response to the comments raised by the reviewers.The comments are reproduced and our responses are given directly afterward in a different color (red).We would like also to thank you for allowing us to resubmit a revised copy of the manuscript.

Reviewer comments:
Reviewer #1 (Comments for the Author): 1.RNA-Seq.Huge sequencing datasets or raw data must also be deposited, e.g. as a NCBI BioProject and the accession number provided.Please, read the Information for authors guide for further information.
The raw date has been uploaded to NCBI under the number SUB13934948.5, Line 208-211: Although these two genes were not included in the key..., they are considered to be important due to their highly significant differential expression as well as ... I apologize for not being able to understand this part of your question.This part of the question refers to the fact that the two genes selected for validation were not the genes with significant differences in fig3, because when reviewing the genes in question, I noticed that they had antiviralrelated effects in other studies.6, Any verification for the RNA-seq results?I have added a validation process related to RNA-seq results.(line 380-390 and Table 2) 7, Line 429: References is misspelled.line417

2.The Figures is too blurry
The spelling error has been corrected, thank you very much for your careful guidance!8. How did IBV in-vitro infection was performed since the timing of virus adsorption and proliferation was quite tricky?
In vitro culture of viruses has been added to the methodology.(line 343-

349)
November 30, 2023 1st Revision -Editorial Decision Re: Spectrum02402-23R1 (Differential analysis of IBV-infected primary chicken embryonic fibroblasts and immortalized DF-1) Dear Dr. Xin Yang: Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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Sincerely, Frederick S. Kibenge Editor Microbiology Spectrum Reviewer #1 (Comments for the Author): No.
1, Fig 1: Arrows pointing to lesions is required.2, Fig 2: Scale bar is missing.3, Fig 7: Name of the groups is required to be modified.4, Line 104, 108: Fig 2 is related to the description of the two paragraphs.
and need to be replaced.All images in the article have been re-uploaded with clarity guaranteed!3.The English proofreading by a native speaker with professional knowledge (especially the discussion part) is required.I have sought out a professional editing agency for English touch-ups.Thank you for your correction.Reviewer #2 (Comments for the Author): 1, Fig 1: Arrows pointing to lesions is required.The lesions have been indicated in the figure by red boxes 2, Fig 2: Scale bar is missing.After adjusting the clarity of the image the scale is now visible, if it still doesn't work I will re-upload it, thank you for your patience.3, Fig 7: Name of the groups is required to be modified.Protein expression of SLBP and P2RX7 and effects on viral structural protein expression.(line 556) 4, Line 104, 108: Fig 2 is related to the description of the two paragraphs.Relevant comments have been added in lines 102 and 108 I have linked the diagrams to the text in the sections you mentioned, inserting them in the appropriate places.(lines 102 (fig2(a) and lines 108 (fig 2(b)).