Distinct infectivity, pathogenicity, and adaptive evolution of clade 2.3.4.4 H5N2 avian influenza viruses in chickens...

High pathogenicity avian influenza virus (HPAIV) drives fulminant poultry epizootics with high mortality and significant production losses. In Taiwan, HPAIV clade 2.3.4.4c H5N2 has circulated since 2015 across different farms, providing an opportunity to assess strain-specific virulence over time. Four representative strains were examined: the index strain GS-2015 (January 2015) and three subsequent isolates-CK-2015 (December 2015), CK-2017 (2017), and CK-2018 (2018). Virulence was evaluated in the chicken model alongside genomic characterization. Overall, strains isolated after the index case exhibited higher mortality rates, shorter mean death times (MDTs), and lower minimal lethal doses compared with GS-2015. Marked variation in the 50 % bird lethal dose (BLD₅₀) was observed across isolates: the index case showed the lethality only at the highest infection dose of 10⁶ EID₅₀, whereas progressively lower BLD₅₀ values were determined for CK-2015 (10⁴), CK-2017 (10^3.77), and CK-2018 (10^3.5). Noticeably, CK-2018 was most infectious (lowest BID₅₀/BLD₅₀) yet produced the longest survival and most prolonged shedding. CK-2017 was intermediate, with higher splenic loads than CK-2018. Across all strains, oropharyngeal shedding preceded and exceeded cloacal shedding. On 3 dpi, the index strain consistently yielded the lowest viral loads across most organs, except the pancreas. Phylogenetic analysis indicated these four strains exhibited divergence toward different evolutionary directions. Comparative genomic analysis revealed progressive accumulation of non-synonymous substitutions across all gene segments, particularly in the viral polymerase complex, NS1, and surface glycoproteins. Several substitutions occurred in functional domains of NS1, suggesting fine-tuning of polymerase activity, antigenicity, and immune evasion. In conclusion, even within the same genotype, clade 2.3.4.4c H5N2 displayed marked heterogeneity, with strains circulating for longer periods showing virulence, survival rates, and extended viral shedding changes in chickens. This virulence-infectivity trade-off not only poses challenges for outbreak control in poultry but also reflects a broader evolutionary trend in which pathogenicity is balanced with transmissibility.