Genetic Characterization of Avian Influenza H14 Viruses in Guangdong, China: Evidence of Intercontinental Transmission and Reassortment in the East Asian–Australasian Flyway

H14 avian influenza viruses (AIVs) are rare, with limited data on their circulation in mainland China. This study investigated the ecological origins and zoonotic potential of H14 viruses in southern China. Active surveillance was conducted in Guangdong (2022–2025). The isolates underwent full-genome sequencing and phylogenetic analysis. Receptor binding specificity for avian- and human-type receptors was evaluated using a solid-phase assay. In vitro replication was assessed in avian, mammalian, and human cell lines. In vivo pathogenicity was evaluated in mice. In this study, four H14 AIVs (one H14N6, two H14N7, and one H14N8) were isolated. We identified a novel H14 transmission network involving Mareca penelope and multiple shorebird species (Tringa, Himantopus), suggesting a multihost dispersal mechanism. Phylogenetically, the Guangdong isolates formed a distinct subclade within the Eurasian lineage, closely related to strains from Russia and Cambodia. Importantly, the three isolates (SZ3742/H14N6, ZJ3523/H14N7, and SZ3536/H14N8) exhibited dual receptor-binding specificity, recognising both avian-type (α-2,3) and human-type (α-2,6) sialic acids. Replication in human A549 cells was strain-dependent: SZ3742/H14N6 reached a peak titre comparable to that in DF-1 cells but lower than that in MDCK cells, whereas ZJ3523/H14N7 and SZ3536/H14N8 reached lower peak titres than in both reference cell lines. All three tested isolates replicated transiently at low titers in mouse lungs with limited viral pathogenicity, despite reduced fitness in human A549 cells. Molecular characterization revealed key mammalian adaptation markers and a typical HA cleavage site motif (PDKQTR↓GLF). This study provides the first evidence of H14 AIV circulation in Guangdong Province, China. The virus demonstrates a broad host range in migratory birds and possesses genetic markers associated with mammalian adaptation, highlighting the need for increased surveillance in the East Asian‒Australasian Flyway.