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Open access
Published: 07 April 2025
Thermal inactivation spectrum of influenza A H5N1 virus in raw milk
Mohammed Nooruzzaman, Lina M. Covaleda, Pablo Sebastian Britto de Oliveira, Nicole H. Martin, Katherine J. Koebel, Renata Ivanek, Samuel D. Alcaine & Diego G. Diel
Nature Communications volume 16, Article number: 3299 (2025) Cite this article
...
Abstract
The spillover of highly pathogenic avian influenza (HPAI) H5N1 virus to dairy cows and shedding of high amounts of infectious virus in milk raised public health concerns. Here, we evaluated the decay and thermal stability spectrum of HPAI H5N1 virus in raw milk. For the decay studies, HPAI H5N1 positive raw milk was incubated at different temperatures and viral titers and the decimal reduction time values (D-values) were estimated. We then heat-treated HPAI H5N1 virus positive milk using different thermal conditions including pasteurization and thermization conditions. Efficient inactivation of the virus (5-6 logs) was observed in all tested conditions, except for thermization at 50 °C for 10 min. Utilizing a submerged coil system with temperature ramp up times that resemble commercial pasteurizers, we showed that the virus was rapidly inactivated by pasteurization and most thermization conditions. These results provide important insights into the efficacy of thermal conditions and food safety measures utilized in the dairy industry.
...
Discussion
The high tropism of HPAI H5N1 virus and its replication in milk secreting epithelial cells of the mammary gland in dairy cows, lead to high viral loads and shedding (from 104.4 to up 108.8 TCID50.mL−1) in milk6,8,16. Despite the broad use of milk pasteurization by the dairy industry, the high viral loads detected in milk from infected cows raised major public health concerns. Although it has been shown that both high (HPAI)- and low pathogenic avian influenza (LPAI) viruses can be inactivated by pasteurization of various poultry products including fat-free egg products, allantoic fluid, and plasma17, and more recently in the milk byproduct concentrated lactose18, in the beginning of the dairy outbreak there was no data demonstrating the efficacy of pasteurization on HPAI H5N1 virus in dairy products. Initial laboratory studies evaluating inactivation of HPAI in raw milk revealed that the most used FDA approved milk pasteurization condition (72 °C for 15 sec) markedly reduced viral loads in milk, however, low amounts of infectious virus were still detected in heat treated samples13,19. Here we evaluated the stability and determined the decay of HPAI H5N1 virus in raw milk stored at different temperatures over time and investigated the efficacy and kinetics of inactivation of HPAI H5N1 virus following the two FDA approved pasteurization- as well as various thermization (subpasteurization) conditions widely used by the dairy industry.
Results from our decay studies revealed long-term stability of HPAI H5N1 in raw clinical milk samples collected from HPAI H5N1 infected cows and HPAI-spiked raw normal milk stored at 4 °C (refrigeration temperature), with infectious virus being detected for up to 56 days (8 weeks) in these samples. These results corroborate findings of a recent study in which infectious HPAI H5N1 was recovered for up to 5 weeks from milk samples spiked with the virus13, highlighting the potential public health risk posed by consumption of raw milk and other raw milk derived products such as raw milk cheeses which must be aged for 60 days at 4 °C20 prior to human consumption. Survival of HPAI H5N1 virus in raw milk incubated at 4 °C for 56 days with residual titers of about 2.5-3.5 log EID50.ml−1 as shown here (Supplementary Fig. 3), underscore the need for further studies to determine whether the aging process efficiently inactivates HPAI H5N1 virus in raw milk cheeses. The decay studies performed here provide valuable information regarding the sensitivity of HPAI H5N1 to higher temperatures (20-37 °C). Incubation of raw clinical milk from infected cows as well as HPAI-spiked raw milk at 20 °C resulted in inactivation of the virus within 21 days (3 weeks), whereas rapid virus decay was observed at 30 °C (6 days, <1 week) and 37 °C (2 days) in spiked milk samples. Therefore, thermal treatment of milk from infected cows to temperatures between 30–37 °C, could potentially be utilized to inactivate HPAI H5N1 virus in raw milk prior to its disposal in affected farms. This would minimize the risk of environmental contamination and further virus spread.
Although the grade A pasteurized milk ordinance (PMO)21 requires that milk from sick animals is segregated on the farm, FDA and USDA found PCR positive shelf milk samples12, indicating that the virus may find its way into bulk raw milk making it into grocery stores. Importantly, follow up testing of these samples to determine virus infectivity have shown that no infectious virus was present in these PCR positive samples12, which is likely a result of pasteurization. While early laboratory studies have shown that heat treatment of HPAI H5N1 spiked milk samples to conditions that mimic FDA approved pasteurization conditions (63 °C for 30 min and 72 °C for 15 sec) results in drastic reduction in viral titers (about 4.5 log10 EID50), residual virus infectivity was still detected when these samples were subjected to the thermal treatment at 72 °C for 15 sec13,19. Results here, however, using raw milk from HPAI infected cows or raw milk spiked with HPAI H5N1 virus, show that the two FDA approved pasteurization conditions efficiently inactivated the virus resulting in up to 7.3 log EID50 reduction in virus infectivity. The discrepancies between our study and the two previous studies could be caused by differences in heat treatment methods or equipment. While the studies that showed residual infectivity of the virus were conducted only using thermocyclers to heat inactivate the virus, we used the thermocycler method and a submerged coil heating system, with the latter having the capability to record the sample temperature as it flows through and is incubated within the heating coils. This capability unequivocally demonstrates the efficiency of pasteurization and several thermization temperatures in inactivating HPAI H5N1 virus in raw milk. Notably, our findings using this controlled heating system corroborate and strengthens results of a study conducted by USDA and FDA which showed that HTST pasteurization (72 °C for 15 sec) in a lab-scale continuous flow pasteurizer efficiently inactivated HPAI H5N1 in milk22. Together, these findings demonstrate that pasteurization is highly effective in reducing the load of HPAI H5N1 virus in milk.
We extended our thermal inactivation studies to include four thermization conditions that are frequently used in dairy industry for production of raw milk cheeses14,15. Three out of four thermization conditions tested including 60 °C for 10 min, 63 °C for 22 s and 69 °C for 22 s resulted in inactivation of HPAI in raw milk. Thermization of raw milk at 50 °C for 10 min, however, only partially reduced HPAI H5N1 virus titers (0.83–2.42 log EID50 reduction). These results demonstrating lack of inactivation of the virus in raw milk at 50 °C and complete inactivation at 60 °C, led us to investigate virus decay in temperatures between 50–60 °C to determine the z-value of HPAI H5N1 in milk. Our results indicate that HPAI H5N1 genotype B3.13 virus had z-values of 9.93–10.33 °C in milk, which is slightly higher than z-values (4.58–4.69 °C) of a Korean H5N1 virus in chicken meat23. Together these results demonstrate that HPAI H5N1 is readily inactivated in raw milk subjected to thermal treatment at temperatures on and above 60 °C. This was confirmed by our inactivation kinetics studies. Based on the estimated D-value at 50 °C (D50°C = 7.19 min), it would take ~50.5 min to achieve a 5-log reduction in HPAI H5N1 virus loads in contaminated raw milk. A 10 °C increase in the treatment temperature to 60 °C resulted in rapid virus inactivation within 5 sec of treatment. While the inactivation was too fast to capture shorter-time intervals, the approximated D-value at 60 °C is 1.04 s (1/ (4.8 log EID50/5)), assuming a linear decay over 5 sec, highlighting the efficacy of thermal treatment in ensuring safety of milk and other dairy products derived from pasteurized milk. Additionally, several of the thermization conditions tested in our controlled experimental settings proved effective against HPAI H5N1 virus, suggesting the use of thermized milk treated at 60 °C and above for 15-20 sec or even at 54 °C and above for 15 min to produce cheeses would reduce the risk of human exposure to products with infectious HPAI H5N1 virus.
In summary, our study provides a comprehensive overview of the thermal inactivation spectrum of HPAI H5N1 virus in raw milk, demonstrating the efficacy of thermal treatment including thermization and pasteurization conditions on inactivation of HPAI H5N1 virus in milk.
...
https://www.nature.com/articles/s41467-025-58219-1
Open access
Published: 07 April 2025
Thermal inactivation spectrum of influenza A H5N1 virus in raw milk
Mohammed Nooruzzaman, Lina M. Covaleda, Pablo Sebastian Britto de Oliveira, Nicole H. Martin, Katherine J. Koebel, Renata Ivanek, Samuel D. Alcaine & Diego G. Diel
Nature Communications volume 16, Article number: 3299 (2025) Cite this article
...
Abstract
The spillover of highly pathogenic avian influenza (HPAI) H5N1 virus to dairy cows and shedding of high amounts of infectious virus in milk raised public health concerns. Here, we evaluated the decay and thermal stability spectrum of HPAI H5N1 virus in raw milk. For the decay studies, HPAI H5N1 positive raw milk was incubated at different temperatures and viral titers and the decimal reduction time values (D-values) were estimated. We then heat-treated HPAI H5N1 virus positive milk using different thermal conditions including pasteurization and thermization conditions. Efficient inactivation of the virus (5-6 logs) was observed in all tested conditions, except for thermization at 50 °C for 10 min. Utilizing a submerged coil system with temperature ramp up times that resemble commercial pasteurizers, we showed that the virus was rapidly inactivated by pasteurization and most thermization conditions. These results provide important insights into the efficacy of thermal conditions and food safety measures utilized in the dairy industry.
...
Discussion
The high tropism of HPAI H5N1 virus and its replication in milk secreting epithelial cells of the mammary gland in dairy cows, lead to high viral loads and shedding (from 104.4 to up 108.8 TCID50.mL−1) in milk6,8,16. Despite the broad use of milk pasteurization by the dairy industry, the high viral loads detected in milk from infected cows raised major public health concerns. Although it has been shown that both high (HPAI)- and low pathogenic avian influenza (LPAI) viruses can be inactivated by pasteurization of various poultry products including fat-free egg products, allantoic fluid, and plasma17, and more recently in the milk byproduct concentrated lactose18, in the beginning of the dairy outbreak there was no data demonstrating the efficacy of pasteurization on HPAI H5N1 virus in dairy products. Initial laboratory studies evaluating inactivation of HPAI in raw milk revealed that the most used FDA approved milk pasteurization condition (72 °C for 15 sec) markedly reduced viral loads in milk, however, low amounts of infectious virus were still detected in heat treated samples13,19. Here we evaluated the stability and determined the decay of HPAI H5N1 virus in raw milk stored at different temperatures over time and investigated the efficacy and kinetics of inactivation of HPAI H5N1 virus following the two FDA approved pasteurization- as well as various thermization (subpasteurization) conditions widely used by the dairy industry.
Results from our decay studies revealed long-term stability of HPAI H5N1 in raw clinical milk samples collected from HPAI H5N1 infected cows and HPAI-spiked raw normal milk stored at 4 °C (refrigeration temperature), with infectious virus being detected for up to 56 days (8 weeks) in these samples. These results corroborate findings of a recent study in which infectious HPAI H5N1 was recovered for up to 5 weeks from milk samples spiked with the virus13, highlighting the potential public health risk posed by consumption of raw milk and other raw milk derived products such as raw milk cheeses which must be aged for 60 days at 4 °C20 prior to human consumption. Survival of HPAI H5N1 virus in raw milk incubated at 4 °C for 56 days with residual titers of about 2.5-3.5 log EID50.ml−1 as shown here (Supplementary Fig. 3), underscore the need for further studies to determine whether the aging process efficiently inactivates HPAI H5N1 virus in raw milk cheeses. The decay studies performed here provide valuable information regarding the sensitivity of HPAI H5N1 to higher temperatures (20-37 °C). Incubation of raw clinical milk from infected cows as well as HPAI-spiked raw milk at 20 °C resulted in inactivation of the virus within 21 days (3 weeks), whereas rapid virus decay was observed at 30 °C (6 days, <1 week) and 37 °C (2 days) in spiked milk samples. Therefore, thermal treatment of milk from infected cows to temperatures between 30–37 °C, could potentially be utilized to inactivate HPAI H5N1 virus in raw milk prior to its disposal in affected farms. This would minimize the risk of environmental contamination and further virus spread.
Although the grade A pasteurized milk ordinance (PMO)21 requires that milk from sick animals is segregated on the farm, FDA and USDA found PCR positive shelf milk samples12, indicating that the virus may find its way into bulk raw milk making it into grocery stores. Importantly, follow up testing of these samples to determine virus infectivity have shown that no infectious virus was present in these PCR positive samples12, which is likely a result of pasteurization. While early laboratory studies have shown that heat treatment of HPAI H5N1 spiked milk samples to conditions that mimic FDA approved pasteurization conditions (63 °C for 30 min and 72 °C for 15 sec) results in drastic reduction in viral titers (about 4.5 log10 EID50), residual virus infectivity was still detected when these samples were subjected to the thermal treatment at 72 °C for 15 sec13,19. Results here, however, using raw milk from HPAI infected cows or raw milk spiked with HPAI H5N1 virus, show that the two FDA approved pasteurization conditions efficiently inactivated the virus resulting in up to 7.3 log EID50 reduction in virus infectivity. The discrepancies between our study and the two previous studies could be caused by differences in heat treatment methods or equipment. While the studies that showed residual infectivity of the virus were conducted only using thermocyclers to heat inactivate the virus, we used the thermocycler method and a submerged coil heating system, with the latter having the capability to record the sample temperature as it flows through and is incubated within the heating coils. This capability unequivocally demonstrates the efficiency of pasteurization and several thermization temperatures in inactivating HPAI H5N1 virus in raw milk. Notably, our findings using this controlled heating system corroborate and strengthens results of a study conducted by USDA and FDA which showed that HTST pasteurization (72 °C for 15 sec) in a lab-scale continuous flow pasteurizer efficiently inactivated HPAI H5N1 in milk22. Together, these findings demonstrate that pasteurization is highly effective in reducing the load of HPAI H5N1 virus in milk.
We extended our thermal inactivation studies to include four thermization conditions that are frequently used in dairy industry for production of raw milk cheeses14,15. Three out of four thermization conditions tested including 60 °C for 10 min, 63 °C for 22 s and 69 °C for 22 s resulted in inactivation of HPAI in raw milk. Thermization of raw milk at 50 °C for 10 min, however, only partially reduced HPAI H5N1 virus titers (0.83–2.42 log EID50 reduction). These results demonstrating lack of inactivation of the virus in raw milk at 50 °C and complete inactivation at 60 °C, led us to investigate virus decay in temperatures between 50–60 °C to determine the z-value of HPAI H5N1 in milk. Our results indicate that HPAI H5N1 genotype B3.13 virus had z-values of 9.93–10.33 °C in milk, which is slightly higher than z-values (4.58–4.69 °C) of a Korean H5N1 virus in chicken meat23. Together these results demonstrate that HPAI H5N1 is readily inactivated in raw milk subjected to thermal treatment at temperatures on and above 60 °C. This was confirmed by our inactivation kinetics studies. Based on the estimated D-value at 50 °C (D50°C = 7.19 min), it would take ~50.5 min to achieve a 5-log reduction in HPAI H5N1 virus loads in contaminated raw milk. A 10 °C increase in the treatment temperature to 60 °C resulted in rapid virus inactivation within 5 sec of treatment. While the inactivation was too fast to capture shorter-time intervals, the approximated D-value at 60 °C is 1.04 s (1/ (4.8 log EID50/5)), assuming a linear decay over 5 sec, highlighting the efficacy of thermal treatment in ensuring safety of milk and other dairy products derived from pasteurized milk. Additionally, several of the thermization conditions tested in our controlled experimental settings proved effective against HPAI H5N1 virus, suggesting the use of thermized milk treated at 60 °C and above for 15-20 sec or even at 54 °C and above for 15 min to produce cheeses would reduce the risk of human exposure to products with infectious HPAI H5N1 virus.
In summary, our study provides a comprehensive overview of the thermal inactivation spectrum of HPAI H5N1 virus in raw milk, demonstrating the efficacy of thermal treatment including thermization and pasteurization conditions on inactivation of HPAI H5N1 virus in milk.
...
https://www.nature.com/articles/s41467-025-58219-1