In March 2024, public health agencies in the United States reported highly pathogenic avian influenza (HPAI) virus of subtype A(H5N1) in dairy cattle, with virus detected in milk samples from symptomatic animals. Viral genetic fragments were subsequently found in pasteurized retail dairy products, which aroused concern about potential exposure through the human food supply. Heat treatment has been shown to reduce or eliminate infectious virus from both milk from infected cows and milk spiked with HPAI A(H5N1) virus. However, whether HPAI A(H5N1) virus in spiked milk from healthy cows replicates the properties of virus in milk from infected cows is unclear. We directly compared the stability of HPAI A(H5N1) virus in infected cows’ milk with that in spiked milk at 4°C (refrigeration temperature) and after heat treatment at 63°C (low-temperature, longer-duration pasteurization) or 72°C (high-temperature, shorter-duration pasteurization). (A list of the virus isolates is provided in Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org; additional details of the methods are also provided in the Supplementary Appendix.)
HPAI A(H5N1) virus in raw milk from infected cows stored at 4°C can remain infectious for at least 5 weeks, with a modest 2-log decline in titer. An extended analysis of raw milk samples from four different infected cows showed that infectious virus could be detected for up to 22 weeks at 4°C (Figure 1A), with decay rates only slightly greater than those in virus-spiked medium or phosphate-buffered saline (PBS) (Figure 1B). In contrast, when virus isolates from the same infected cows were spiked into raw milk from healthy cows, virus was undetectable within 2 weeks (Figure 1A). Similar decay (within 3 weeks) was observed for five other influenza viruses spiked into healthy cows’ milk, whereas stability was retained in PBS or medium (Fig. S1). These observations suggest that the stability of virus produced in the mammary gland is enhanced.
Heat treatment of milk from HPAI A(H5N1)–infected cows reduces virus titers to below detectable levels (5 min at 63°C) or by at least 4.5 log units (30 sec at 72°C). We have now compared heat inactivation at 63°C or 72°C in milk from infected cows and milk spiked with matched virus isolates. Although the two types of milk samples had similar virus titers before heat treatment (Fig. S2), virus in spiked milk decayed more rapidly at 63°C, with shorter half-lives in two of three samples (Figure 1C and 1D and Fig. S3). Differences were less pronounced at 72°C (Figure 1D and Fig. S4). These data suggest that virus produced in the mammary gland is more heat-stable than virus spiked into milk, particularly at lower pasteurization temperatures.
These findings show that HPAI A(H5N1) virus is more stable in milk from infected cows than in milk from healthy cows spiked with matched virus isolates, both during cold storage and after heat treatment. The mechanisms are not known but may involve protective factors in milk, such as fats, casein proteins, or debris. Although pasteurization has been shown to inactivate HPAI A(H5N1) virus, the persistence of infectious virus in raw milk highlights the potential risk of transmission through unpasteurized dairy products, both for humans and domesticated animals. Overall, our results suggest that, whenever possible, materials from infected animals should be used to examine the characteristics of avian HPAI A(H5N1) virus in dairy products.

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