Viruses are obligatory intracellular pathogens that rely on the host cell for essentially all steps of their life cycle. Although coronaviruses use host proteins as part of their replication strategies, it has also become clear that the immune, metabolic, stress, cell cycling and other pathways are activated by infection. Determination, using genomics and proteomics, of the extent to which virus-host interaction is coronavirus-specific and organ-specific, will be of importance [14, 27]. Our previous study on the global profile of host protein alterations in response to IBV infection was focused on an in ovo infection model system . In this study, we applied a comparative proteomics technical platform for the first time to explore the abundance changed protein in trachea and kidney tissues from chicken infected in vivo with the highly virulent ck/CH/LDL/97I P5 strain of IBV and the embryo-passaged, attenuated ck/CH/LDL/97I P115 strain of IBV. Some identified proteins are likely to be important in the host response to virus infection, including cytoskeletal proteins, stress response proteins, and anti-oxidative proteins. Interestingly, the abundance of these proteins showed different change patterns with IBV strains differ in virulence, suggesting that some of these differences might be responsible for virulence, and consistent with our previous study which demonstrated the differences in pathology and virulence for these two different viruses [22, 28]. These results provide an overview of the proteome profile of the host in response to different virulent IBV infection in vivo.
The cytoskeletal network is a cellular scaffold system whose functions include maintenance of cellular shape, enabling of cellular migration, cell division, intracellular transport, signaling, and membrane organization. Some host cytoskeletal proteins have been reported as differentially altered by virus infection in quantitative proteomic studies [16–18]. Recently, study has revealed that several different cellular proteins involved in cell morphology and the cytoskeleton changed in abundance in IBV infected cells [19, 20]. IBV infection resulted in a number of changes to the nucleolus both in terms of gross morphology and protein content . Our results presented a tendency that the abundance of several cytoskeletal proteins showed increased with the degree of virulence getting strong, which is possibly due to the collapse and dispersal of the cytoskeleton in the IBV-infected cells, as demonstrated by other viruses . Vimentin is a major component of type III intermediate filaments that has been reported to be redistributed in cells around sites of virus replication and assembly during virus infection. For instance, infection with African swine fever virus (ASFV) can lead to the rearrangement of vimentin into a cage surrounding a virus factory, which may prevent movement of viral components into the cytoplasm and concentrate late structural proteins at sites of virus assembly . Specifically, vimentin was observed to be increased in abundance in the cytoplasmic proteome of IBV-infected cells [19, 20]. In our study, the abundance of vimentin was increased more strongly in the highly virulent P5 strain infected group, compared with the attenuated P115 strain infected group at 21 dpi. Given that vimentin can confer rigidity to domains of the cytoplasm, the vimentin cage may provide a physical scaffold to facilitate the construction of the virus factory. The stronger induction of vimentin could be a result of the large replication capacity of the highly virulent IBV strain. Tropomyosin belongs to the family of actin-binding proteins that serve important functions in microfilament stabilization, regulation of microfilament branching, actin polymerization, and intracellular transport. The abundance of alpha-tropomyosin was found increased in IBV-infected cell [19, 20]. In our study, the abundance of alpha-tropomyosin was increased much more strongly with highly virulent IBV ck/CH/LDL/97I P5 infection than with the attenuated P115 strain, it is likely that it help to orchestrate virus assembly, release and efficient cell-to-cell spread, also may due to the different virulence between IBV ck/CH/LDL/97I P5 and P115 strain. In addition, in our previous in ovo infection model , the abundance of some cytoskeletal including alpha-tropomyosin was decreased in the IBV-infected embryonic tissues, this difference need to be further investigated.
In both our previous  and current study, the abundance of annexin A2 and annexin A5 were found to be changed upon IBV infection. Annexins are a family of conserved proteins characterized by their ability to bind and order charged phospholipids in membranes, often in response to elevated intracellular calcium. These family members are involved specifically in a diverse range of cellular functions both inside the cell and extracellularly . Annexin A2 (ANXA2) can associate with actin filaments and mediates membrane trafficking and membrane-cytoskeletal interactions. It has been identified as an important host factor for several viruses and at different stages of their life cycle. ANXA2 is incorporated into cytomegalovirus and influenza virus particles, promotes the entry of virus, and plays a role in Human immunodeficiency virus -1 (HIV-1) assembly, Bluetongue virus (BTV) release, and hepatitis C virus (HCV) replication [33–40]. Annexin A5 (ANXA5) is involved in various intra- and extracellular processes including signal transduction, anti-inflammatory processes, membrane trafficking, and ion channel activity , it also acts to regulate blood coagulation, binding to and shielding exposed phospholipids and masking their pro-thrombotic properties . The expression of ANXA2 and ANXA5 has been found to be altered during other IBV infection system [19, 20]. In this study, the abundance of ANXA2 showed weaker increase at early stages of infection with the highly virulent IBV ck/CH/LDL/97I P5 strain than with the attenuated P115 strain. For ANXA5, the level of expression induced at 7 dpi with the highly virulent P5 strain of IBV was greater than that induced by the attenuated P115 strain. These trends were also validated by western blot analysis. Whether this difference between P5 and P115 infectioned groups is related with the virulence of IBV or various responses of the host cell deserves further study.
MnSOD is the primary antioxidant enzyme in the mitochondria that catalyzes the conversion of superoxide molecules to hydrogen peroxide and molecular oxygen and therefore forms one of the cell's major defense mechanisms against oxidative stress . Studies have reported that viral infections cause oxidative stress, which is associated with the activation of phagocytes and an increase in the release of reactive oxygen species (ROS) that play a positive modulatory role in immune activation, the inflammatory response, eradication of viral infections and immunity-induced cellular injury . The expression of MnSOD was altered during infection with some viruses, such as porcine reproductive and respiratory syndrome virus, influenza A virus, and SARS-CoV [45–47]. The abundance of the anti-oxidative protein MnSOD was increased much significantly in highly virulent IBV ck/CH/LDL/97I P5 infected group than the attenuated P115 strain infected group. The much amplitude increased production of MnSOD during highly virulent strain infection can affect the host cell pro-/anti-oxidant balance, which probably results in more significant immune activation and a stress response induced by reactive oxygen species. It is important in the eradication of viral infections and immune-induced cellular injury.
When comparing the protein profiles of groups infected with different strains, we found that the abundance of HSPB1, a well-known heat-shock protein, in highly virulent IBV ck/CH/LDL/97I P5 strain infected group was significantly higher than the embryo-passaged, attenuated P115 strain at the early stage of infection. Western blot and real-time RT-PCR further confirmed this alteration. HSPB1 has different cytoprotective roles, including acting as a molecular chaperone, maintaining the normal function of cells through interaction with and stabilization of the cytoskeleton, regulation of translational initiation, modulation of inflammation, inhibition of apoptosis, stimulation of innate and adaptive immune responses, and responding to a wide variety of stressful stimuli [48–50]. It is probably involved in all phases of the viral life cycle, including cell entry, virion disassembly, viral genome transcription, replication and morphogenesis. The abundance of HSPB1 was observed to be increased in IBV-infected Vero cells using stable isotope labeling with amino acids in cell culture (SILAC) . IBV infection induces cell cycle arrest at both S and G2/M phases and caspase-dependent apoptosis at late stages of the viral infection cycle [51–53]. Results presented in our study were probably due to the highly virulent virus triggering a markedly more robust inflammation and stress response. It also suggests that many important, and probably different events in IBV pathogenesis and immunology, such as the stress response, inflammation, and apoptosis, occur early in infection, and that these events may contribute to development of an appropriate immune response and the outcome of viral infection. In our previous study , the abundance of HSPB1 was found to be decreased after IBV infection in ovo. This difference in different infection model need to be further examined.
In this study, infection with both the highly virulent strain and the embryo-passaged, attenuated strain of IBV was accompanied by elevation of proteins related to energy metabolism. Viruses are obligate parasites that are completely dependent on their host's cellular metabolism for reproduction. Viral infection has been shown to modulate the levels of numerous host metabolic components in pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, pentose phosphate pathway, and macromolecular biosynthesis in order to redirect valuable resources to their own mass production. Studies have demonstrated that the rate of glycolysis in cells infected by Rous sarcoma virus, feline leukemia virus, and poliomyelitis virus was increased by as much as 370% . Glycolytic enzymes are involved not only in carbohydrate metabolism and increased ATP production, also take part in the induction of anti-oxidative stress in host cells and contribute to transcription of RNA virus genomes . The abundance of some proteins involved in glycolysis, such as aldose reductase, pyruvate kinase, alpha-enolase, and triosephosphate isomerase was found increased in IBV infected cell [19, 20]. Our study also showed that the abundance of some glycolytic enzymes, including phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate kinase, and alpha-enolase, were increased significantly in the early stage of infection with both the highly virulent and the attenuated IBV strain. These findings support the view that IBV infection probably triggers activation of host energy metabolic components via systemic or global mechanisms, to keep up with the energy demands of its own replication.
The post-translational modification (PTM) plays critical roles in cellular regulation. It has been reported that PTM and expression of highly related gene sequences can induce multiple protein spots in animals . An advantage of 2-DE is the monitoring of multiple forms of a protein species, which offers the opportunity to investigate the effects of the virus infection on protein modification. Our study revealed that several proteins were expressed by multiple spots in our study. Although we do not know at present how these multiple spots were generated, some of them were more likely due to post-translational modifications (PTMs), or highly related gene sequences coding isoforms .