Virus infection and the host response involve a complex interplay of host and viral networks in which many viruses attempt to subvert host cell processes to increase the efficiency of virus infection, and likewise the host employs a number of responses to generate an anti-viral state . Coronavirus (CoV) infection can cause alterations in the transcription and translation patterns, cell cycle, cytoskeleton, and apoptosis pathways of the host cell . The trachea and kidney are the primary target organs of IBV, investigation of the proteomic changes in these tissues after IBV infection in ovo helps to elucidate the IBV-host interaction and the pathogenic mechanisms of IBV. In this study, proteomic methods coupled with real-time RT-PCR and western blotting were applied to identify the differentially expressed proteins in trachea and kidney tissues of IBV-infected and mock-infected chicken embryos. We now attempt to interpret the possible functional roles of some proteins identified during IBV infection in ovo.
In our study, one of the major findings was that the abundances of some cytoskeletal proteins including TPM1 and MYLPF were decreased in the IBV-infected group. Their alterations were also confirmed at the mRNA level by real-time RT-PCR. Tropomyosin belongs to the family of actin-binding proteins that serves important functions in microfilament stabilization, regulation of microfilament branching, actin polymerization, and intracellular transport . Myosins are a large superfamily of motor proteins that are involved in movement along actin filaments, the development of myriad cells, targeted organelle transport, endocytosis, chemotaxis, cytokinesis, and signal transduction . Similar result was observed in the IBV-infected cells by using SILAC technique [17, 18]. Changes in cytoskeleton proteins have been reported in other virus infection in vitro, including infectious bursal virus , H9N2 avian influenza virus , respiratory syncytial virus , and SARS-associated CoV . During the process of virus infection, particularly in the stages of virus entry and virus budding, the cytoskeletal network of the host cell is involved in the transport of viral components within the cell. Moreover, some viral proteins can interact with the cytoskeletal transport machinery, such as actin-binding proteins or actin, and induce rearrangements of cytoskeletal filaments so that they can utilize them as tracks or push them aside when they represent barriers . In the present study, several actin-binding proteins, including TPM1 and MYLPF, their abundance were found to be decreased in the IBV-infected group, which suggests that IBV may also manipulate the host cytoskeletal network for its own infectious processes and replication.
It is well known that Ca2+ is one of the most universal and versatile signaling molecule, and involved in almost every aspect of cellular processes. The Ca2+ plays important roles in virus entry, viral gene expression, posttranslational processing of viral proteins, and the maturation and release of virions. Viruses can utilize host cellular Ca2+ and Ca2+-binding proteins to create a tailored cellular environment that meets their own demands for the replication cycle . In this study, the level of expression of some calcium ion-binding proteins, including Calbindin-D28 k, annexin A1, annexin A2, annexin A5, and annexin A6 were altered after IBV infection in ovo. Calbindin-D28 k is a cytosolic calcium-binding protein that facilitates 1, 25 (OH)2D3 dependent transcellular calcium transport. It was also observed to protect against apoptosis in different cell types [26, 27]. In this study, its abundance was remarkably increased in the IBV-infected group, which suggests that IBV might specially utilize calbindin-D28 k to perturb the cellular Ca2+ homeostasis and Ca2+-signaling network for its own benefit. Annexins are a family of structurally related proteins that bind phospholipids and cellular membranes in a calcium-dependent manner . Annexin A2 has been shown to take part in the initiation of membrane fusion in exocytosis, membrane trafficking, regulation of cell proliferation and apoptosis, and stabilization of membrane-associated protein complexes with the actin cytoskeleton [29, 30]. In addition, Annexin A2 can promote the entry of human immunodeficiency virus (HIV) into monocyte-derived macrophages , and it was also identified to be a potential receptor for respiratory syncytial virus on human epithelial cells . Annexin A2 on the lung epithelial cell surface was recognized by SARS-associated CoV spike domain 2 antibodies and identified as an autoantigen . Annexin A5 was found to be involved in cytomegalovirus infection  and influenza virus infection . Annexin A1 plays a critical role in a variety of cellular processes such as proliferation, differentiation, and apoptosis . Its abundance was shown to be increased in HepaRG cells infected with hepatitis B virus (HBV) in vitro
, and fish cells infected in vitro with infectious pancreatic necrosis virus . Changes in the abundance of some annexins family proteins also were identified in IBV-infected DF-1 cells by Edward Emmott and co-workers . In current study, the abundance of annexin A1, annexin A5, and annexin A6 were all decreased in the IBV-infected group. For annexin A2, two spots were identified in kidney tissue, the abundance of one spot was increased, and another spot was decreased. Of these, the decrease of annexin A5 was confirmed by real-time RT-PCR and western blotting analysis. These data suggested that they may play special roles during IBV infection or replication.
Remarkably, several stress response and anti-oxidative proteins were found to be changed significantly in the present study. HSPB1 is an important small heat shock protein (HSP) that is synthesized in response to a wide variety of stressful stimuli, including viral infection. It has diverse functions including chaperone activity, F-actin modulation, signal transduction, resistance to oxidant stress, regulation of translational initiation, and modulation of inflammation, inhibition of apoptosis, and cell differentiation and proliferation [38, 39]. Enhanced levels of HSPB1 and/or phosphoHSPB1 can promote nuclear transport of adenovirus in MK2-deficient cells . The abundance of HSPB1 has found to be increased in cells infected in vitro with H9N2 avian influenza virus , African swine fever virus , and infectious bursal disease virus . In contrast, its abundance was found to be decreased in cells infected in vitro with mumps virus  and porcine circovirus type 2 , which suggests that HSPB1 may play different roles in different virus infections or different stages of infection. PRDX1 is the most ubiquitously expressed member of the peroxiredoxin family, which is involved in anti-oxidative processes, cell differentiation and proliferation, immune responses, regulation of apoptosis, and as a molecular chaperone . PRDX1 participates in the apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway, and plays an inhibitory role in ASK1-induced apoptosis . Its abundance was shown to be decreased in peripheral blood mononuclear cell (PBMC) following CSFV infection in vivo
. In our study, the abundance of HSPB1 and PRDX1 were shown to be decreased after IBV infection in ovo by 2-DE and real-time RT-PCR methods. Furthermore, the change of HSPB1 expression was confirmed by western blotting. This alteration may allow the infected cells to be eliminated by apoptosis, or serve as a form of host defense against IBV infection.
Viral replication requires energy and macromolecular precursors derived from the metabolic network of the host. In the present results obtained using 2-DE, the abundance of some proteins which are associated with carbohydrate, amino acid, and lipid metabolic processes were found to be differentially changed. Enolase-1 is a key enzyme of glycolysis and gluconeogenesis that catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate . Its abundance was found to be changed in many virus infections, such as white spot syndrome virus , and porcine reproductive and respiratory syndrome virus . Phosphoenolpyruvate carboxykinase is another gluconeogenic enzyme; it catalyzes the GTP-driven conversion of oxaloacetate to phosphoenolpyruvate . Increased expression of proteins related to energy metabolism was also found in HIV-infected peripheral blood mononuclear cells , chicken spleen tissue infected with Marek's disease virus , and human cytomegalovirus-infected human fibroblasts . The abundance of L-lactate dehydrogenase B also found to be increased in IBV-infected cells by SILAC technique [17, 18]. Up-regulation of proteins related to energy metabolism may meet the requirement for a large burst of oxygen and energy during rapid virus replication, and also may result from an attempt by the host to keep up with the energy demand during viral infection. Ex-FABP is 21 kDa lipocalin that is involved in fatty acid transport and lipid metabolism. It may play an important role in the protection of cells against the toxic effects of the accumulation of fatty acids. The expression of Ex-FABP is enhanced greatly in response to inflammatory stimuli and other stress conditions [52, 53]. In this study, its abundance was significantly increased in tracheal tissue of IBV-infected chicken embryos, suggesting may serve as a response to the inflammation induced by IBV infection in ovo.
The abundance of several proteins which involved in the immune response and antigen processing and presentation were also observed to be changed in this study. TRIM protein is a member of a protein family that is based on a conserved domain architecture characterized by a RING finger domain, one or two B-box domains, a coiled-coil domain and a variable C-terminus. TRIM proteins are involved in a variety of cellular processes that include signal transduction, transcriptional regulation, cell proliferation, apoptosis, and immunity . Many TRIM proteins, such as TRIM5α, TRIM11, TRIM22, TRIM28, TRIM31, and TRIM62, have been found to display antiviral activity or to be involved in processes associated with innate immunity [55–57]. An extended gene map revealed that TRIM27.2 lies within a sub-region of the chicken MHC-B that affects infectious disease . The major histocompatibility complex (MHC) plays an important role in regulation of the immune response and antigen presentation. The transcription levels of the MHC class II-associated invariant chain and MHC class II β chain were observed to be increased in tracheal epithelial layers of chickens three days after infection with an attenuated IBV-Massachusetts strain . In current study, the abundance of TRIM27.2 and MHC class I antigen were increased remarkably following IBV infection in ovo. According to our knowledge, TRIM27.2 has never been found in other virus analysis so far. This change might be induced specially by IBV infection.