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bioRxiv · 10.1101/2025.06.18.660327

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

Moreno, A.·Pongolini, S.·Merialdi, G.·Cattoli, G.·Terregino, C.·Santini, N.·Benedetti, S.·Loli Piccolomini, L.·Padovani, A.·Rosamilia, A.·Alborali, G. L.·Daminelli, P.
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Abstract

BackgroundThe 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 potential zoonotic transmission through the consumption of unpasteurized milk and raw-milk dairy products. While inactivation studies exist for pasteurized milk, data on virus persistence during the manufacture of raw-milk cheeses remain scarce. AimTo evaluate the survival and inactivation of avian influenza viruses (AIV), including both low pathogenic (LPAIV, H1N1) and highly pathogenic (HPAIV, H5N1) strains, during the production and ripening of Grana-type hard cheeses made from raw cows milk. MethodsExperimental cheesemaking was conducted using raw milk artificially contaminated with A/duck/Italy/281904-2/06 (H1N1; 107.75 EID50/mL) or A/duck/Italy/326224-2/22 (H5N1 clade 2.3.4.4b; 106.75 EID50/mL). Cheeses were produced in accordance with Parmigiano Reggiano production standards and ripened for 30 days at 5-6 {degrees}C. Viral presence was assessed in finished cheeses by inoculation on SPF embryonated chicken eggs (ECE), hemagglutination (HA) assay, and monoclonal antibody-based ELISA. ResultsNo infectious virus was detected in any cheese sample produced from contaminated milk following two blind passages in SPF-ECE. Both HA and ELISA tests yielded negative results, indicating complete inactivation of the virus. ConclusionThis study demonstrates that the traditional Grana-type cheese production process-- including curd cooking, acidification, and ripening--effectively inactivates both LPAIV and HPAIV, even at high contamination levels. These findings support the microbiological safety of hard cheeses made from raw milk with regard to AIV, contributing to risk assessment and food safety policies during avian influenza outbreaks.

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Moreno, A., Pongolini, S., Merialdi, G., Cattoli, G., Terregino, C., Santini, N., Benedetti, S., Loli Piccolomini, L., Padovani, A., Rosamilia, A., Alborali, G. L., Daminelli, P.. 2025-06-18. Inactivation of Influenza A Viruses (H1N1, H5N1) During Grana-Type Raw Milk Cheesemaking: Implications for Foodborne Transmission Risk. https://doi.org/10.1101/2025.06.18.660327

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