Natalia Pessoa, VIDO/University of Saskatchewan

Natalia Pessoa (a), Nathalie Berube (a), Lauren Aubrey (a,b), Ulises Barron-Castillo (a,c), Leslie Macas Jacome (a,b), Jill Trann (a,c), Jill Van Kessel (a), Bryce Warner (a), Antonio Facciuolo (a,c) and Yan Zhou (a,c) (a) Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, SK, S7N 5E3, Canada (b) Vaccinology and Immunotherapeutics Program, School of Public Health, University of Saskatchewan, Saskatoon, SK, S7N 5E3, Canada (c) Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5E3, Canada

Fighting & Responding

Highly pathogenic avian influenza (HPAI) H5N1 was detected in dairy cattle in the United States in 2024, representing the first reported identification of this virus in bovines. Since then, the virus has spread to other mammalian species, including humans. These events highlight the capacity of HPAI H5N1 to adapt to new hosts, raising significant public health concerns due to the increased risk of reassortment and the acquisition of mammalian-adaptive traits that could facilitate human-to-human transmission. According to Canadian regulations, HPAI H5N1 viruses are classified as Risk Group 3 (RG3) pathogens, requiring working with live virus exclusively in containment level 3 (CL3) laboratories. However, work in CL3 facilities is labor-intensive and limits experimental flexibility. Therefore, validated inactivation protocols that allow downstream work under containment level 2 (CL2) conditions are essential. In this study, we evaluated multiple inactivation protocols for A/dairy cattle/Texas/24-008749-002/2024 (H5N1) across diverse sample types. Buffer AVL combined with 95% ethanol was tested for virus inactivation in infected cell culture supernatants, milk, blood, urine, and allantoic fluid. Buffer RLT with 70% ethanol was assessed using infected cell pellets, spiked milk and various bovine tissue samples. Heat treatment and Triton X-100 were evaluated for serum and milk samples. Finally, β-propiolactone, a widely used agent for vaccine virus inactivation, was tested to inactivate allantoic fluid and cell culture supernatant. All evaluated protocols inactivated H5N1 virus effectively, enabling safe handling under CL2 conditions. In addition, we determined the limit of detection (LOD) of the RT-qPCR assay used to confirm viral inactivation.