Proteomics analysis of differentially expressed proteins in chicken trachea and kidney after infection with the highly virulent and attenuated coronavirus infectious bronchitis virus in vivo

Abstract

Background: Infectious bronchitis virus (IBV) is first to be discovered coronavirus which is probably endemic in all regions with intensive impact on poultry production. In this study, we used two-dimensional gel electrophoresis (2-DE) and two-dimensional fluorescence difference gel electrophoresis (2-DIGE), coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS), to explore the global proteome profiles of trachea and kidney tissues from chicken at different stages infected in vivo with the highly virulent ck/CH/LDL/97I P5 strain of infectious bronchitis virus (IBV) and the embryo-passaged, attenuated ck/CH/LDL/97I P115 strain.

Results: Fifty-eight differentially expressed proteins were identified. Results demonstrated that some proteins which had functions in cytoskeleton organization, anti-oxidative stress, and stress response, showed different change patterns in abundance from chicken infected with the highly virulent ck/CH/LDL/97I P5 strain and those given the embryo-passaged, attenuated P115 stain. In addition, the dynamic transcriptional alterations of 12 selected proteins were analyzed by the real-time RT-PCR, and western blot analysis confirmed the change in abundance of heat shock proteins (HSP) beta-1, annexin A2, and annexin A5.

Conclusions: The proteomic alterations described here may suggest that these changes to protein expression correlate with IBV virus' virulence in chicken, hence provides valuable insights into the interactions of IBV with its host and may also assist with investigations of the pathogenesis of IBV and other coronavirus infections.

Figure 1

The viral load in each sample was quantified using real-time RT-PCR. The average viral copy number (VCN) per g tissue of each group was calculated. Error bars indicate standard error of the mean, and dpi represent days post-inoculation.

Figure 2

Representative 2-DE maps of tracheal tissues from the IBV ck/CH/LDL/97I P₅-infected group, P₁₁₅-infected group and control group. (A) 4 dpi; (B) 7 dpi; (C) 14 dpi; (D) 21 dpi. Protein samples were separated on 13 cm pH 3-10 linear IPG strips, followed by SDS-PAGE, and stained with Coomassie Blue R-350. The images were analyzed using Image Master 2D Platinum 6.0 software. The differentially expressed protein spots identified were marked with circles and labeled with the respective Match ID listed in Tables 1 and 2.

Figure 3

Representative 2-DIGE maps of kidney tissues from the IBV ck/CH/LDL/97I P₅-infected group, P₁₁₅-infected group and control group. (A) 4 dpi; (B) 7 dpi; (C) 14 dpi; (D) 21 dpi. Protein samples labeled with fluor were separated on 24 cm pH 3-10 linear IPG strips, followed by SDS-PAGE, gels were scanned on a Typhoon 9400 scanner, and image analysis was performed with Ettan™ DeCyder Software version v6.5. The protein spots identified were marked with circles and labeled with the respective Match ID listed in Tables 3 and 4.

Figure 4

Gene Ontology annotation analysis of differentially expressed proteins according to their cellular component (A) and biological process (B). This classification was produced on the basis of an analysis using the GOSlimViewer tool at the Agbase database http://www.agbase.msstate.edu/. The results were compared between trachea and kidney, P5-infected and P115-infected group, and different day's post-inoculation.

Figure 5

Transcript analysis of the 12 proteins differentially expressed in trachea (A) and kidney (B) of chickens infected with the IBV ck/CH/LDL/97I P₅ and ck/CH/LDL/97I P₁₁₅ strains by real-time RT-PCR. Total RNA extracts were prepared from the trachea and kidney of chickens in the control, P₅-infected, and P₁₁₅-infected group's at all four time points. Data represent means of three biological replicates per group. Error bars indicate standard error. Samples were normalized with the expression of the 18S ribosomal RNA gene. For symbols indicating different genes, refer to Tables 1, 2, 3, and 4.

Figure 6

(A) Western blot analysis of heat shock protein beta-1 (HSPB1) and annexin A2 (ANXA2) in trachea tissue, annexin A5 (ANXA5) in kidney tissue of chickens infected with the IBV ck/CH/LDL/97I P₅ and ck/CH/LDL/97I P₁₁₅ strain. (B) Protein band density was analyzed with the BandScan software. Beta-Actin was used as the internal control. Mean values ± SE were calculated from three independent samples. AU, arbitrary units.