BACKGROUND METHODOLOGY RESULTS conclusion Differential Proteomics of Bursal Tissues of Chickens Infected with Highly Pathogenic H5N1 Avian Influenza virus Vinod RMT Balasubramaniam1, Iekhsan Othman1, Abdul R Omar2 and Sharifah S Hassan1 1.Virus-Host Interaction Group, Infectious Disease Laboratory (MR3), School of Medicine and Health Sciences, Monash University, Sunway Campus, 46150 Sunway, Malaysia 2. Institute of Bioscience, University Putra Malaysia, UPM Serdang, Selangor, 43400, Malaysia MONASH UNIVERSITY BACKGROUND Highly Pathogenic Avian Influenza (HPAI) Virus (H5N1) is a member of the Orthomyxoviridae family of negative-stranded, segmented RNA viruses and the involvement of the bursa of Fabricius (one of the central lymphoid organ in chickens essential to the ontogenetic development of adaptive immunity in chickens) during influenza infections poultry is still unresolved. In this study, we explored the proteomes of bursal tissue proteins of chickens infected with H5N1 virus, A/chicken/Malaysia/5858/2004 H5N1using two-dimensional gel electrophoresis (2-DE) combined with LC-ESI-Q-TOF tandem mass spectrometry (MS/MS). METHODOLOGY RESULTS Inoculation of live HPAI H5N1 into SPF chickens and isolation of infected bursal tissues 24h post infection 3 10 3 10 M pH M pH 10 10 9 9 Protein extraction & BCA analysis 8 8 7 5 6 7 5 6 1 2 1 3 4 2 3 4 Rehydration of sample in 24cm GE IPG strips CONTROL INFECTED Fig. 2: 2D gel picture showing the differentially expressed proteomes between control and infected bursa In Gel Trypsin digestion, LC MS/MS and peptide sequence analysis Spot No Spot ID/SWISS PROT ACC. NO. Matched Peptide Coverage % 1 2 3 4 5 6 7 8 9 10 apolipoprotein A (P08250) proactivator polypeptide(O13035) chromatid cohesion protein PDS5(Q5F3V3) small ubiquitin-related modifier(Q5ZHQ1) myosin associated proteins(F1NWJ9) peptidyl-prolylcis-trans isomerase(P24367) calcium transporter (Q9IAL7) actin (P68139) heat shock protein 70 (P08106) eukaryotic translation initiation factor (Q5ZLX2) 7 12 8 9 5 11 10 6 4 29 40 27 39 15 19 30 25 20 IEF for a total of total of 70 kV·h and equilibration of strips with 10 mg/mL of DTT and 40 mg/ mL of iodoacetamide in sample buffer Silver staining Table 1: Some of the protein s that have been found to be differentially regulated in chicken bursal tissues after infection with HPAI H5N1 conclusion 2DGE using Ettan Dalt 6 (GE HEALTHCARE) To the best of our knowledge, we have performed the first comparative analysis of the proteome changes in bursal tissues of chickens infected with H5N1. The identification of these proteins might provide insights towards the pathogenesis of H5N1 infection in chickens and the role played by bursa in the development of immunity and antiviral activity. Fig. 1: Schematic diagram showing the methodology of the entire experiment PRESENTED IN 6th AOHUPO CONGRESS, MAY 5th to 7th 2012, Beijing China