Abstract
Part I: Proteomics Analysis of Avian Reovirus Infected Host Cells to Identify Candidate Proteins Involved in Virus Infection Mechanisms. Avian reoviruses (ARVs) are members of Reoviridae family, which are non-enveloped RNA viruses with 10 double-stranded RNA as genome content. The viruses are important pathogens of birds that are mainly transported through fecal-oral route, causing considerable economic losses in the poultry industry, and thus understanding more about the viral infection mechanism would be beneficial to the poultry industry. Proteomic analysis is a good technique to identify a whole view of the protein quantity differences in the host cell pre- and post-infection by ARV. To acquire the whole regulatory view of ARV to the host cell, immunofluorescence microscopy was first used to identify the cytoskeleton structure differences caused by ARV infection. The MTT and AlamarBlue assays were performed to verify the cell viability during the infection process. The 2D-DIGE, followed by MALDI-TOF MS analysis, was then used to identify the proteins up- or down-regulated during the ARV infection process. These proteins were classified by different functions and locations. Validation of 2D-DIGE was carried out by Western blot analysis. The immunofluorescence microscopy showed the arrangement, but not the quantity of cytosol F-actin, was changed after ARV infection. The AlamarBlue assay assured the unchanged cell viability. The 2D-DIGE and MALDI-TOF MS analysis identified 87 proteins with expression level regulated during ARV infection process. Among these proteins, peroxiredoxin 6 (PRDX6), Phosphoglycerate mutase 1 (PGAM1), Proteasome activator complex subunit 3 (PSME3), Annexin A5 (ANXA5), peroxiredoxin 4 (PRDX4), 60 kDa heat shock protein (HSP60), Small ubiquitin-related modifier 3 (SUMO3), and Voltage-dependent anion channel 2 (VDAC2) were used in the validation performed with Western blot analysis. The functions of these proteins and the possible regulatory mechanism in the infection process were then being discussed. We performed the first analysis of the proteome of chicken fibroblast embryo cells (DF-1) infected with avian reovirus. A series of differentially expressed cellular proteins associated with cytoskeleton components, regulators of apoptosis, energy metabolism, signal transduction, and ubiquitin-proteasome pathways were identified. These data provides the fundamental understanding of processes during ARV infection and facilitates probing the candidates for viral molecular pathogenesis studies. Part II:□Exogenous Chicken Cytokine Interleukin-1β□Enhanced Avian Reovirus Infection in Host Cells. The pro-inflammatory cytokine Interleukin-1β (IL-1β) is one of the members of IL-1 family and is belonged to the primary antiviral responses in virus-infected cells. The signal transduction pathway caused by IL-1β involved the recruitment of MyD88 to the IL-1RAcP and the down-stream activation of IRAKs and TRAF6 proteins, which would finally induce the activation of transcription factors such as NF-κB and AP-1. Because the enhanced IL-1β activity was found in culture supernatant of avian reovirus (ARV) infected cell and the IL-1β over expression related symptom arthritis syndrome is the primary effect in ARV strain S1133 infected chicken, it is implied that the chicken IL-1β□may play an important role on ARV infection processes. In this study, we found that ARV infection was enhanced through treatment of exogenous chicken cytokine IL-1β to the cultured chicken cells. The increases of viral titers, viral protein quantities and viral RNA quantities were found while the cells were pretreated by chicken IL-1β before infected by ARV, and the level of increases were depend upon the quantities of added exogenous IL-1β proteins. Overexpression of the down-stream adopter protein MyD88 further proved the IL-1β enhancement. The possible applications of the infection-regulatory role of IL-1β were also discussed.