pH-based inactivation of highly pathogenic H5N1 virus in unpasteurized milk

An outbreak of highly pathogenic avian influenza H5N1 was confirmed in dairy cattle in March 2024 and subsequently spread to dairy herds throughout the United States, resulting in spillover cases in humans and other animals. Milk produced by infected dairy cattle, which has been shown to harbor high viral titers, poses a potential threat to peridomestic wildlife around dairies due to lack of treatment and disposal in wastewater systems that are often open to the environment. Here, we investigated a method for inactivating H5N1 virus in unpasteurized milk, utilizing the irreversible conformational change of the hemagglutinin (HA) glycoprotein induced by low pH. The pH needed for complete inactivation of three distinct influenza A viruses was determined for subsequent use in acidifying milk. The use of common, readily available acidification solutions like vinegar demonstrated that a 3% final concentration of vinegar (pH 3.6) induced complete inactivation of H5N1 in unpasteurized milk after 60 min. Our results suggest that pH-based inactivation of H5N1 in unpasteurized milk is a quick, feasible strategy that could be used on dairy farms prior to disposal of contaminated milk, thus reducing the risks of environmental contamination and exposure of animals around farms. IMPORTANCE: Dairy farms experiencing outbreaks of highly pathogenic avian influenza H5N1 generate large volumes of milk with high titers of infectious H5N1 virus. While milk from sick cows does not enter the consumer supply chain, disposal of this untreated, contaminated milk through on-site wastewater systems still poses a significant risk for environmental contamination and exposure of peridomestic wildlife, potentially leading to spillover events. A simple method for inactivation of virus in milk prior to disposal could mitigate this risk. We show that lowering the pH of virus-laden milk with commercially available vinegar can neutralize dairy cattle H5N1 virus within 1 h, providing a quick and effective strategy for potential use on dairies to inactivate the virus in milk before disposal.