Genetic evolution and phylodynamic of the 2.3.4.4b clade of H5N1 AIVs from 2021 to 2024: spatiotemporal spread and pathogenicity of Chinese isolates

Since 2021, the novel reassorted 2.3.4.4b clade of H5N1 avian influenza viruses (AIVs) has shown a global transmission leading to significant harm to wild birds, poultry, and mammals in the natural environment. Here, we conducted correlation analyses and experiments to understand the genetic and biological characteristics of the 2.3.4.4b clade of H5N1 AIVs worldwide from 2021 to 2024. Phylogenetic analysis showed that 7,305 virus isolates worldwide experienced complex reassortment leading to 115 genotypes (2.3.4.4b G1-G115). Of these, G1, G3, G13, and G15 continue to spread and could subsequently become the dominant genotypes of the 2.3.4.4b clade H5N1 virus. G21, G28, G61, G75, and G89 genotypes spread widely and infect multiple hosts, and the G80 genotype infects a variety of mammals. Notably, G7, G13, G15, G78, and G80 genotypes can infect humans. Systematic geographical transmission analysis showed that the transmission route of Chinese isolates belonging to the G21 genotype followed Japan-South Korea-Vietnam-China. Viruses representing G21, G113, G114, and G115 are four new genotypes with 100% mortality and that replicated in the lungs and nasal turbinates of mice; QY6 (G114 genotype) also replicated in the spleen and brain. In summary, our findings provide significant insights into the global evolution and reassortment patterns of the current 2.3.4.4b clade H5N1 AIVs as well as the geographical transmission pathways of the G21 genotype virus. This highlights the necessity of implementing monitoring and active prevention measures.