Proteomics Analysis Of Chicken Trachea And Kidney After Infection By Infectious Bronchitis Coronavirus

Coronaviruses can infect a broad range of host including human and animal, cause respiratory, gastrointestinal, and neurological disorder of varying severity. Since the spring of 2003, Coronaviruses were brought to the centre of attention by the appearance of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and caused high mortality. Research on the pathogenesis and pathogenicity of coronaviruses is the current focus of attention. Infectious bronchitis virus (IBV) is belonging to gamma coronavirus, which replicates primarily in a large range of epithelial cells of trachea, kidney, proventriculus, and oviduct of chicken, and probably endemic in all regions with intensive impact on poultry production. Previous studies demonstrated that some other coronaviruses infection have dramatic effects on the patterns of host protein expression. In order to understand the IBV-host interaction and the pathogenesis of IBV, in this study, we for the first time used proteomics techniques and other molecular biological methods to study the global protein expression change profiles of trachea and kidney from chicken embryos following IBV H120 vaccine strain infection in ovo, and the trachea and kidney tissues protein expression changes of chickens during different stages after infection in vivo with highly virulent IBV ck/CH/LDL/97I P5 strain and embryo-passaged, attenuated IBV ck/CH/LDL/97I P115 strain. Gene Ontology annotation analysis of differentially expressed proteins was performed according their molecular function, biological process, and subcellular location. The aim of this study is to investigate the interactions of IBV with its host, and the pathogenesis of IBV at the protein level.The changes of protein expression in trachea and kidney of chicken embryo at 72h after infection in ovo with IBV H120 vaccine strain were first analyzed using 2-DE coupled with MALDI-TOF/TOF MS method. Seventeen differentially expressed proteins from tracheal tissues and nineteen differentially expressed proteins from kidney tissues were identified successfully. These proteins mostly related to the cytoskeleton organization, binding of calcium ions, response to stress, anti-oxidative, energy metabolism, and macromolecular biosynthesis. Notably, some of the identified proteins have the ability of regulation apoptosis. Eleven altered proteins including annexin A1, annexin A2, annexin A5, tropomyosin 1 alpha, peroxiredoxin-1, Calbindin-D28k, heat shock protein beta-1, myosin light chain type 2, extracellular fatty acid-binding protein, TRIM27.2, and enolase alpha were analyzed further at the mRNA level using real-time PCR. The trends of the changes in their mRNA levels were similar to the patterns of change in their corresponding proteins on 2-DE gels. Western blot analysis further confirmed the changes of annexin A5 and HSPB1. It was also in consisting with results obtained by real-time PCR.We focus on study the global protein expression changes in trachea and kidney tissues of chickens during different stages after infection in vivo with highly virulent IBV ck/CH/LDL/97I P5 strain and embryo-passaged, attenuated IBV ck/CH/LDL/97I P115 strain using 2-DE and 2-DIGE coupled with MALDI-TOF/TOF MS method. In total, sixty-two differentially expressed proteins were identified and classified into several functional categories including cytoskeletal organization, macromolecule metabolism, anti-oxidative stress, stress response, acute phase response, signal transduction and ion transport. The dynamic transcriptional alterations of fourteen selected proteins including heat shock protein beta-1, annexin A2, annexin A5, tropomyosin alpha, vimentin, lamin A, manganese-containing superoxide dismutase, and phosphoenolpyruvate carboxykinase mitochondrial were analyzed using real-time PCR method. The majority mRNA level changed trends of these genes were consistent with the change patterns of their corresponding proteins in 2-DE and 2-DIGE gels. Western blot analysis further confirmed the expression changes of HSP beta-1, annexin A2, and annexin A5 obtained by proteomics method. Results demonstrated that some key proteins which were likely to be important in the host response to virus infection, including cytoskeleton organization, anti-oxidative stress, stress responses, and energy metabolism, were resulted in different expressed change patterns between infection with the highly virulent IBV ck/CH/LDL/97I P5 strain and its embryo-passaged, attenuated P115 stain. Additionally, these dramatic differences between P₅- and P₁₁₅-strain induced changes were mainly observed at the early stage of virus infection.In general, these results provide valuable insights into the interactions of IBV with its host, the investigations in pathogenesis of IBV, and screening of preventive and therapeutic medicine. Meanwhile, our study may provide a reference for related research of SARS-CoV and other coronaviruses.