Emergence of a Distinct Evolutionary Cluster of Clade 2.3.4.4b H5 Viruses in China during 2024-2025 with Rapid Evolution and Antigenic Divergence

Clade 2.3.4.4b H5 viruses have undergone continuous evolution since their emergence in China, but the extent of genetic and antigenic diversification among recently circulating viruses remains unclear. Here, we characterised 18 H5 viruses isolated through routine surveillance in China during 2024-2025, together with publicly available clade 2.3.4.4b H5 sequences collected since 2020. Phylogenetic analysis revealed that these isolates formed a distinct evolutionary cluster within clade 2.3.4.4b, designated the G2-derived emerging cluster (G2-EC), which emerged around mid-2024 and exhibited clear genetic divergence from previously circulating H5N1 viruses. G2-EC viruses accumulated a characteristic set of hemagglutinin amino acid substitutions, most of which were located in antigenic regions. Across the clade 2.3.4.4b H5 virus population, the estimated mean HA evolutionary rate was higher during 2024-2025 than during preceding periods. These genetic changes were accompanied by marked antigenic divergence from the Re14 vaccine strain, with 4- to 32-fold reductions in HI titres. Genomic analysis revealed acquisition of PB1 segment from a distinct genetic background in G2-EC H5N1 viruses, followed by the emergence of H5N6 viruses sharing a highly similar genetic backbone but carrying a distinct NA segment, indicating sequential reassortment. Animal experiments showed that representative G2-EC viruses retained high pathogenicity and efficient direct-contact transmission in SPF chickens, while exhibiting relatively limited lethality in mice. Collectively, these findings document a recent evolutionary transition of clade 2.3.4.4b H5 viruses in China, characterised by genetic and antigenic divergence accompanied by sequential reassortment, underscoring the need for continued genomic and antigenic surveillance.