Celine Tan, University of Toronto

Celine Tan1,2, Sophie-Marie Aicher1,3, Jigme Yangden1,2, Jonathon D. Kotwa 1,2, Lauren Crawshaw4, Nathalie Bastien5, Yohannes Berhane6, Jeff Bowman4, Arinjay Banerjee3, Samira Mubareka1,2 1Department of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada 2Sunnybrook Research Institute, Toronto, ON, Canada 3Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK, Canada 4Wildlife Research and Monitoring Section, Ontario Ministry of Natural Resources, Peterborough, ON, Canada 5National Microbiology Laboratory, Winnipeg, MB, Canada 6National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB, Canada

Fighting & Responding

Highly Pathogenic Avian Influenza A (HPAI) viruses of clade 2.3.4.4b (H5N1 and H5N5) have caused outbreaks in livestock and wildlife species worldwide. While species such as red foxes and raccoons exhibit severe clinical signs of infection, other species such as pigs and coyotes display fewer clinical signs. Little is known about the innate immune mechanisms that contribute to differences in tolerating infections in these hosts. More importantly, advancements in studying host-virus interactions are hampered by the lack of molecular tools for non-model species. Here, we generated upper respiratory tract 2D cellular models on air-liquid interface (ALI) from red fox, raccoon, pig, and coyote nasal brushings. We demonstrated full differentiation of our models with beating cilia and mucus formation after 21 days. We showed immunocompetence in fox cells via dsRNA ligand stimulation and probed for innate immune gene expression with custom oligonucleotides. Preliminary infections using A/British_Columbia/PHL-2032/2024 (H5N1) at MOIs 0.1 and 1 show higher replication in fox than raccoon cells with slightly more cytopathic effect in fox cells. We are now performing immunofluorescent staining to evaluate sialic acid expression, and cellular composition from tight junctions and tubulin expression. These novel ALI models enable us to analyse antiviral responses to HPAI infection across clinically susceptible and tolerant mammalian hosts. We will generate RNA-Sequencing datasets comparing HPAI infections in our four species and identify immune response patterns associated with disease susceptibility and tolerance. This will provide critical insight into host determinants of HPAI pathogenesis and establish molecular resources for non-model species.