Inactivation of Influenza A Viruses (H1N1, H5N1) During Grana-Type Raw Milk Cheesemaking: Implications for Foodborne Transmission Risk

The detection of H5N1 highly pathogenic avian influenza virus (HPAIV) in lactating dairy cattle in the United States, with high viral titers in raw milk, has raised concerns about zoonotic transmission through unpasteurized milk and dairy products. While viral inactivation during pasteurization is documented, data on persistence in raw-milk cheeses remain limited. This study evaluated the survival of avian influenza viruses (AIVs), both low pathogenic (LPAIV, H1N1) and highly pathogenic (HPAIV, H5N1), during the production and ripening of Grana-type hard cheeses from raw cow's milk. Experimental cheesemaking was carried out with milk artificially contaminated with A/duck/Italy/281904-2/06 (H1N1; 10^7.75 EID₅₀/mL) or A/duck/Italy/326224-2/22 (H5N1 clade 2.3.4.4b; 10^6.75 EID₅₀/mL). Cheeses were manufactured under Parmigiano-Reggiano standards and ripened 30 days at 5-6 °C. Viral detection in finished cheeses was performed using inoculation in specific-pathogen-free embryonated chicken eggs (SPF-ECEs), hemagglutination (HA) assay, and monoclonal antibody-based ELISA. No infectious virus was detected in cheese samples after two blind passages in SPF-ECEs. Both HA and ELISA tests were negative, indicating complete viral inactivation. These results demonstrate that Grana-type cheese processing, including cooking, acidification, and ripening, effectively inactivates LPAIV and HPAIV. Findings support the microbiological safety of raw-milk hard cheeses regarding AIV, contributing to risk assessment and food safety policies.