Hemagglutinin of Emerging Low Pathogenic Avian Influenza Viruses Underpins High Pathogenicity to Mammals

ABSTRACT The pathogenicity of emerging early zoonotic avian H7N9 and H3N8 low‐pathogenic avian influenza viruses (LPAIVs) in humans is significantly enhanced compared to that of their internal gene providers, H9N2 avian influenza viruses (AIVs), suggesting a pivotal role for surface HA or NA. Here, we generated several reassortant AIVs that combined HA, NA, or HA + NA from emerging zoonotic H7N9 or H3N8 LPAIV with internal H9N2 AIV genes and investigated the impact of HA and NA on the replication and pathogenicity of reassortants in human cells and mice. The crucial affected phase was determined by analyzing the receptor binding, viral adsorption, HA cleavage efficiency, viral endocytosis, and budding. We found that mice infected with the virus containing the early zoonotic H7N9 LPAIV HA, but not NA, exhibited high mortality, weight loss, severe lung damage, and increased viral load in the lungs. We found that HA substitution enhanced viral replication in human A549 cells and displayed dual sialic acid receptor binding ability. This substitution also facilitated viral attachment to mammalian cells and promoted endocytosis by enhancing HA0 cleavage efficiency; however budding was not affected. Additionally, HA from emerging zoonotic H3N8 LPAIVs elevate the pathogenicity of reassortants in mice. Together, our study revealed that HA of emerging zoonotic LPAIVs contributes to high pathogenicity in mammals by augmenting viral entry and causing lung injury, thereby highlighting HA with double receptor binding properties and HA cleavage efficiency as new markers for risk assessment of emerging zoonotic AIVs in the future.