A single-residue deletion in H9N2 hemagglutinin uncouples viral burden from disease severity

H9N2 avian influenza viruses are characterized by significant genetic diversity. Deletions within the hemagglutinin (HA) receptor-binding domain represent a notable, albeit rare, evolutionary trajectory. Here, we systematically characterized the multifaceted impact of a single-amino-acid deletion at residue 227 of HA (designated HA Δ227 ), a naturally occurring deletion within the 220-loop that overlaps a key antigenic epitope, using isogenic reverse genetics H9N2 viruses. Paradoxically, the HA Δ227 imposed a fitness cost and was associated with enhanced HA receptor-binding avidity, reduced apparent virion-associated NA activity, altered HA/NA functional balance, attenuated viral replication in vitro , and reduced respiratory shedding in vivo . Notably, despite this compromised replicative fitness, infection with the mutant virus was associated with an altered host immune response in chickens, characterized by elevated levels of interferon-γ (IFN-γ), interleukin-10 (IL-10), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1). These altered immune responses were associated with more severe pulmonary lesions, indicating a dissociation between disease severity and viral RNA load. Furthermore, the deletion increased the pH threshold for HA-mediated membrane fusion and substantially altered antigenic relationships. Collectively, our findings identify HA residue 227 as a functionally important site at which a single-residue deletion is associated with a phenotypic trade-off between replicative capacity and disease severity: reduced replicative fitness accompanied by altered inflammatory responses and greater tissue injury. This study highlights the potential phenotypic significance of a rare deletion in influenza virus HA and demonstrates that viral burden alone may not adequately predict disease severity.