Highly Pathogenic Avian Influenza in Cattle: Approaches for Inactivating a Risk-Group 3 Virus for Safe CL2 Laboratory Practices

In March 2024, H5N1 highly pathogenic avian influenza (HPAI) was detected in Texas dairy cattle and has since spread to over 300 herds in the United States. Historically, H5N1 transmission to humans has occurred because of contact with infected birds, and over 800 cases have been reported since 2003, with a mortality rate of 52%. Sustained human-to-human transmission of H5N1 has not been observed. HPAI requires physical containment and operational practices be completed in a containment level 3 (CL3) laboratory. To safely bring samples containing inactivated HPAI to CL2 laboratories for further analysis, we tested methods of inactivation for downstream RNA extraction or antibody response assays. Samples containing A/dairy cattle/Texas/24-008749-002/2024 (H5N1 cattle virus) destined for RNA extraction were incubated with buffer AVL from the Qiagen QIAamp Viral RNA Mini Kit with 95% ethanol, or buffer RLT from the Qiagen RNeasy Mini Kit with 70% ethanol. Samples tested for antibody assays, serum and milk containing HPAI, were incubated with 0.5% v/v Triton X-100 at 60℃. We found buffer AVL with 95% ethanol inactivated H5N1 cattle virus in supernatant from infected cells, milk, blood and urine. Buffer RLT with 70% ethanol completely inactivated H5N1 cattle virus in infected cell pellet, spiked milk, blood, urine and tissue. Finally, incubation with 0.5% v/v Triton X-100 followed by a 30-minute heat treatment at 60℃ completely inactivated H5N1 cattle virus in whey and serum. This work is essential for allowing the safe transfer of HPAI samples produced in CL3 to lower containment laboratory for downstream analyses.

Laruen Aubrey, Ulises Barron, Nathalie Berube, Natalia Pessoa, Leslie Macas, Jill Trann, Andrew Gentes, Jill Van Kessel, Bryce Warner, Antonio Facciuolo and Yan Zhou

Avian influenza in mammals, pandemic preparedness, and one health