Highly pathogenic avian influenza viruses (HPAIVs) of the H5 subtype pose a significant threat to global poultry industries and public health. Since their initial emergence in poultry in 1996 as the goose/Guangdong lineage (gs/GD), followed by the first human cases in 1997, H5N1 viruses have undergone continuous evolution and have caused significant economic losses in the poultry industry. Over decades, reassortment events between H5 HPAIVs and low pathogenicity avian influenza viruses have led to the emergence of clade 2.3.4.4 H5Nx, particularly novel H5N6 and H5N8 viruses in Eurasia. In recent years, outbreaks of H5N1 HPAIVs within the 2.3.4.4b clade, beginning in late 2020, have resulted in substantial poultry and wild bird mortality across various regions worldwide. Notably, these H5Nx viruses have demonstrated an expanded ability to infect a range of mammalian species, including humans, cats, red foxes, grey seals, raccoons, Chilean dolphins, and, most recently, dariy cows. This ongoing evolution of the H5N1 virus poses a significant risk to both animal and human populations.

During routine surveillance of live poultry markets (LPMs) in Guangdong, China, winter 2023, novel reassortant H5N1 HPAIVs were detected in cloacal swab samples collected from asymptomatic geese in Foshan city. RT-qPCR targeting the matrix protein, supplemented with an in-house designed TaqMan probe for H5 gene detection, was used to screen the samples as previously described. H5N1 HPAIV was detected in 16.6% of goose samples, but no positive results were found in chicken samples. Eleven viruses from the positive goose samples were sequenced, revealing high homology (>99%) across all eight segments. The representative strain, A/Goose/Guangdong/1189/2023 (H5N1) (GD1189), (GenBank, No. 5844-5851), was selected for further analysis.

The sequence analysis and BLAST results suggested that all strains possessed a multibasic HA cleavage site (PLREKRRKR/GLF), and shared significant genetic similarity with H5N1 HPAIVs previously detected in wild birds in South Korea and Japan since 2022. Notably, GD1189 exhibited close genetic relatedness to A/water/Tottori/NK1220-1/2022 (H5N1) and A/environment/Kagoshima/KU-G4/2022 (H5N1) (GISAID, No. EPI3077291 and EPI2789597), which were the most closely related HA sequences identified in the GISAID(Supplementary Table 1). Importantly, the HA of GD1189 exhibits a high degree of amino acid homology (99.12%) with a human H5N1 virus discovered in Jiangsu Province, China (Supplementary Table 2).

Phylogenetic analysis of the GD1189 revealed a unique genetic composition, suggesting a complex evolutionary history. The HA gene belonged to the 2.3.4.4b evolutionary branch (Figure 1A), closely related to strains prevalent in Eurasia in recent years, including those detected in wild waterfowl. Similarly, the NA gene also clustered with Eurasian strains (Supplementary Figure 1). While five internal genes (PB2, PB1, NP, M and NS) clustered with H5N1 subtype strains (Supplementary Figure 2), the PA gene diverged and grouped with avian influenza strains of the H6N6 and H6N2 subtypes that circulating in southern China (Figure 1B). Notably, the M gene, encoding the matrix protein involved in viral assembly, clustered with European H5N1 subtype strains, while the remaining five internal genes formed a cluster with Asian H5N1 subtype strains. These findings suggest that GD1189 circulating in southern China might have originated from a European H5N1 strain introduced to Asia by migratory birds, followed by reassortment with local H6N6 subtype strains in Guangdong province, southern China. This complex reassortment event may have contributed to the unique genetic composition observed in GD1189 and highlights the potential for ongoing evolution and adaptation of H5N1 viruses in avian populations.