Chairs: Aoife Mulry and David Mark

Avian infectious bronchitis virus (IBV) is a highly contagious Gammacoronavirus that causes significant economic losses to the poultry industry through respiratory disease, reduced egg production, and increased susceptibility to secondary infections. Tracheal organ cultures (TOCs) are a physiologically relevant ex vivo model for IBV, representing the natural infection site and supporting growth of field strains that fail in conventional culture. However, TOCs present technical challenges for high-resolution imaging of virus-host interactions. To overcome these challenges, embedding, sectioning, and immunofluorescence protocols were optimised to visualise IBV proteins in infected TOCs. Refinements improved signal specificity and reduced background. Initial blocking with 0.5% BSA/PBS was inconsistent, showing non-uniform blocking and antibody adherence issues. Implementing 0.1% fish gelatin/PBS as the blocking agent reduced non-specific binding and improved signal clarity. Parallel experiments in chicken kidney cells (CKCs) provided complementary data comparing ex vivo (TOC) and in vitro (CKC) systems. Immunofluorescence analysis revealed viral protein distribution patterns: spike, envelope, and membrane proteins localised to cilia and perinuclear cytoplasm in TOCs, but to the plasma membrane in CKCs. Nucleocapsid protein and dsRNA were observed in perinuclear foci in both systems. Notably, beta-tubulin labelling showed loss of cilia in infected TOCs, visualising ciliostasis and epithelial damage caused by IBV in the trachea. These optimised methods establish a foundation for detailed investigation of IBV protein trafficking and localisation in a physiologically relevant model, while capturing virus-induced pathology in respiratory epithelium. These findings support future studies on IBV pathogenesis and potential strategies against this economically important avian pathogen.