HA thermostability mutations S84F, G167N, and D168N potentiate H9N2 virus transmission in a warming environment

Avian influenza A (H9N2) viruses are widespread in poultry and occasionally infect mammals, contributing genes to other emerging strains. The factors that allow these viruses to withstand higher temperatures are not well understood. In this study, we used high-temperature selection and deep mutational scanning of a saturated H9N2 hemagglutinin (HA) mutant library. We identified three HA mutations, S84F, G167N, and D168N, that increase viral thermostability. Viruses with these mutations survived better at elevated temperatures. In transmission experiments with chickens, the S84F mutation allowed waterborne spread at a temperature that blocked transmission of the wild-type virus. The combination of all three mutations was associated with evidence of airborne transmission in one of two experimental replicates. These results link HA thermostability to environmental persistence and transmission potential under thermal stress, which may affect the ecology of H9N2 viruses.