Inactivation of avian influenza virus in raw milk kefir

Authors: Porto Fett, Anna; Alvarez Narvaez, Sonsiray; VINAYAMOHAN, POONAM - Oak Ridge Institute For Science And Education (ORISE); Harrell, Telvin; SHWANI, ABDULKARIM - Oak Ridge Institute For Science And Education (ORISE); Suarez, David; Luchansky, John

Submitted to: Food and Environmental Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/23/2026
Publication Date: 3/9/2026
Citation: Porto Fett, A.C., Alvarez Narvaez, S., Vinayamohan, P.G., Harrell, T.L., Shwani, A., Suarez, D.L., Luchansky, J.B. 2026. Inactivation of avian influenza virus in raw milk kefir. Food and Environmental Virology. https://doi.org/10.1007/s12560-026-09686-2.
DOI: https://doi.org/10.1007/s12560-026-09686-2

Interpretive Summary: The emergence of highly pathogenic avian influenza virus (HPAIV) H5N1 in U.S. dairy cattle in March of 2024 raised significant concerns about the safety of raw milk from infected cows. Although pasteurization effectively inactivates the virus from raw milk, there is a paucity of information on interventions to destroy this virus in products such as yogurt and kefir prepared from (raw) milk that has not been pasteurized. Safety and shelf-life of fermented (raw) milk products is achieved using a (commercial) starter culture to acidify the milk from a starting pH of ca. pH 6.5 to an endpoint pH of equal or lower than pH 4.6. Kefir is typically fermented for an extended time (about 36 h) and at lower temperature (about 25°C) to a final pH of 4.4. However, these parameters of the fermentation process may not be effective to inactivate HPAIV H5N1 if the virus were present in raw milk. Thus, we monitored the viability of two strains (H5N1 and H5N9) of low pathogenic avian influenza virus (LPAIV) during fermentation of kefir to assess the potential for public health consequences. Raw (unpasteurized) milk was inoculated with a commercial kefir starter culture and with LPAIV strain H5N1 or strain H5N9. Next, inoculated raw milk was fermented at 25°C for ca. 36 h to an average endpoint pH of pH 4.4. Results confirmed that fermentation of raw milk to produce kefir delivered an appreciable reduction (virus levels decreased by ca. 99.99% infectious virus particles) of both LPAIV strains from their initial inoculated levels in raw milk. Our findings establish that the public health risk associated with kefir prepared using LPAIV inoculated raw milk is markedly lowered when raw milk is fermented at 25°C to pH 4.4 over a ca. 36-h period using a commercial starter culture.

Technical Abstract: Both highly pathogenic avian influenza virus (HPAIV) and its viral RNA have been detected in raw milk during the ongoing outbreak in dairy cows. While pasteurization effectively inactivates the virus in milk, comparatively little has been published on AIV inactivation in fermented dairy products made from raw milk. We evaluated the viability of two isolates of low pathogenic avian influenza virus (LPAIV) during fermentation of kefir to assess the potential for viral persistence. Raw (unpasteurized) milk (ca. 3.0% fat) was inoculated with ca. 7.5 log10 CFU per mL of active commercial kefir starter culture. Next, raw milk (125 mL) was inoculated with 1.0 mL of either strain A/rgGyrfalconHAxPR8/2014 H5N1 or A/turkey/Wisconsin/1968 H5N9 to achieve an average initial level of ca. 5.0 log10 50% egg infectious doses (EID50) per mL. The inoculated raw milk (ca. pH 6.55 ± 0.05) was fermented at 25°C for ca. 36 h to an average endpoint pH of pH 4.44 ± 0.05. Results confirmed that fermentation of raw milk to produce kefir delivered an appreciable reduction of both LPAIV strains, that being ca. 4.1 or 3.1 log10 EID50 per mL decrease of strains H5N1 or H5N9, respectively, from their initial levels in raw milk. In the absence of a starter culture, due to fermentation by the indigenous flora the pH of the raw milk after incubation at 25°C for ca. 36 h also significantly decreased on average to ca. pH 5.3 and, levels of strains H5N1 and H5N9 were reduced by 4.1 and 3.5 log10 EID50 per mL, respectively. Our findings established that fermentation of raw milk (with or without added kefir starter culture) to lower than pH 5.3 over ca. 36 h at 25°C appreciably decreases titers (greater than 3.1 log10 EID50 per mL) of AIV strains H5N1 and H5N9 and, therefore, lowers risk associated with its consumption.