Approaches for inactivating highly pathogenic avian influenza H5N1 cattle isolate for safe containment level 2 laboratory practices.

In March 2024, highly pathogenic avian influenza (HPAI) H5N1 was detected in Texas dairy cattle and has since spread to over 500 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 to be completed in a containment level 3 (CL-3) laboratory. To safely bring samples containing inactivated HPAI to CL-2 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 (Qiagen) with 95% ethanol, or Buffer RLT (Qiagen) with 70% ethanol. Samples tested for antibody assays, serum, and milk containing HPAI were incubated with 0.5% vol/vol Triton X-100 at 60°C. We found that Buffer AVL with 95% ethanol inactivated H5N1 cattle virus in supernatant from infected cells, milk, blood, and urine. Buffer RLT with 70% ethanol inactivated H5N1 cattle virus in infected cell pellet, spiked milk, blood, urine, and tissue. Finally, incubation with 0.5% vol/vol Triton X-100 followed by a 30 minute heat treatment at 60°C completely inactivated the H5N1 cattle virus in whey and serum. This work is essential for allowing the safe transfer of H5N1 samples produced in CL-3 to lower containment laboratories for downstream analyses.IMPORTANCEHistorically, human infections from highly pathogenic avian influenza (HPAI) have occurred from contact with infected birds, with estimated mortality rates of 52%. Recently, this virus has spilled over into many mammalian species and has rapidly spread between dairy cattle herds in the United States, causing multiple human infections after exposure to infected cows. Characterization of this virus is imperative for reducing risk to humans. Work with live HPAI virus must be undertaken in containment level 3 facilities, which limits the amount and type of work that can be done due to time-consuming biosafety procedures and lack of equipment. In this article, we outline how to effectively inactivate HPAI to enable safe work in containment level 2 facilities and facilitate more efficient work on this pathogen.