Novel Reassortant H9N2 Avian Influenza Viruses with Dual Receptor-Binding Capacity and Evidence of Direct Mammalian Infectivity Circulating in Northeast China Live Poultry Markets.

H9N2 low-pathogenic avian influenza viruses (LPAIV) represent an ongoing zoonotic threat due to their enzootic circulation in poultry, reassortment capacity, and increasing human transmission events. This study characterized three H9N2 isolates recovered from apparently healthy poultry in a Changchun live poultry market (September-November 2022) that exhibited unprecedented genetic and phenotypic characteristics indicating enhanced zoonotic risk. Phylogenetic analysis showed a complex mosaic genome combining segments from four distinct lineages: HA from the BJ/94-like lineage (human-associated), PB1/NP/NS from the F98-like lineage, NA from the FJ/30-C-like branch, and PB2/M genes from the G1-like lineage. Bayesian molecular clock analysis estimated the most recent common ancestor at February 2022, with HL55 and HL56 diverging by May 2022, indicating rapid local viral evolution. All isolates retained hallmark LPAIV characteristics (monobasic HA cleavage site, zero intravenous pathogenicity index in chickens). However, receptor-binding assays demonstrated a critical divergence among the isolates: while HL45 exhibited exclusive avian α2-3 receptor preference, both HL55 and HL56 retained strong avian receptor binding while additionally showing measurable affinity for human α2-6 receptors-a dual-binding phenotype associated with enhanced zoonotic potential. Most significantly, the HL55 isolate successfully infected BALB/c mice without prior adaptation, causing transient upper respiratory tract replication, moderate weight loss (~9.2%), and mild disease without mortality or systemic dissemination. These findings demonstrate that the direct mammalian infectivity of this specific mosaic H9N2 lineage adds to the growing body of evidence regarding the zoonotic potential of contemporary H9N2 variants. The presence of known mammalian-adaptation markers (PB2 A588V, NA stalk deletion, HA position 226 leucine), combined with demonstrated dual receptor-binding capacity and inherent mammalian infectivity, underscores the accelerated evolutionary trajectory of H9N2 viruses toward increased zoonotic competence. These findings warrant intensified surveillance in live poultry markets, comprehensive antigenic characterization of emerging variants, and enhanced biosecurity measures to mitigate the risk of spillover events and potential pandemic emergence.