Impact of pH and temperature in dairy processing on the infectivity of H5N1 avian influenza viruses

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 (clade 2.3.4.4b) have crossed the species barrier and caused a mastitis-like infection in dairy cows. The high levels of infectious virus found in the milk raised considerable concerns about the safety of raw milk products. This study examined the effect of temperature and pH on the stability of HPAIV and low-pathogenic avian influenza viruses (LPAIV). We found that H5N1 HPAIV remained infectious in milk at 4°C for four weeks, with slow decreases at 21°C, and complete inactivation at 37°C after four weeks. H5N1 LPAIV was stable at 50°C for 30 minutes but inactivated at higher temperatures (55°C for 10 minutes, 60°C for 1 minute, or 72°C for 30 seconds). At pH levels between 6 and 10, the virus remained stable but was partially inactivated at pH 5.0 and completely inactivated at pH 4.0. During yogurt production, H5N1 LPAIV was completely inactivated when the pH reached 4.3. In cheese production, the lowest pH reached was between 5.0 and 5.3. When H5N1 LPAIV was incubated with soft and semi-hard cheese for one day at 4 °C, infectious virus titers decreased by 5.1 and 3.9 log ₁₀ , respectively. When H5N1 LPAIV was incubated with buffer adjusted to pH 5.0, infectious virus titer dropped by only 3.3 log ₁₀ , suggesting that, alongside pH, other processes of cheese ripening likely influence virus stability. In conclusion, H5N1 avian influenza viruses are largely inactivated during lactic acid fermentation of raw milk. Future studies will assess the required cheese ripening time for complete inactivation.