Severe Respiratory Disease in Cynomolgus Macaques After Inhalation of Aerosolized A/Chile/25945/2023 (Clade 2.3.4.4b) H5N1 Avian Influenza Virus

The ongoing surge of clade 2.3.4.4b H5N1 viruses in avian species has resulted in numerous spillover events into mammalian species including dairy cows and humans, posing a potential pandemic threat of this H5N1 clade in human population. We have previously shown that inhalation of small-particle aerosols containing clade 1 H5N1 A/Vietnam/1203/2004 (VN04) by cynomolgus macaques triggered fever and severe respiratory disease and death, and that the severity of disease was dependent on the inhaled dose of virus. Here, we investigated whether a clade 2.3.4.4b virus isolate from a human case in Chile would trigger severe respiratory disease in cynomolgus macaques. Macaques were implanted with radiotelemetry devices to continuously monitor body temperature and infected with A/Chile/25945/2023 by small-particle aerosol. On days 2, 4, 7, 11, and 15 respiratory function was assessed using a head-out plethysmography chamber. At doses equivalent to the LD50 (5.1 log10 pfu) for VN04, two out of two macaques were febrile but only had mild-moderate respiratory disease unlike VN04 virus at this dose level. At 6 log10 pfu, macaques developed respiratory disease characterized by a sharp increase in respiratory rate with one of the two succumbing on day 7 post-infection. At 6.7 log10 pfu, two out of two macaques developed severe acute respiratory distress syndrome and succumbed on day 4 post-infection. Chest radiography and gross pathology indicated severe pneumonia in euthanized macaques. This demonstrates that akin to well-characterized VN04 infection, A/Chile/25945/2023 can be lethal in cynomolgus macaques through aerosolized virus exposure although a somewhat higher dose is required to achieve the same disease outcome and lethality. Together, our macaque model of clade 2.3.4.4b H5N1 virus would foster evaluation of potential medical countermeasures against this pandemic potential virus in a pathophysiologically relevant model to human severe influenza.