Pathogenicity in chickens and ducks of clade 2.3.4.4b H5N6 and H5N3 high pathogenicity avian influenza viruses in South Korea.

High pathogenicity avian influenza viruses (HPAIVs) of the A/Goose/Guangdong/1/96 lineage continue to diversify through reassortment, generating various H5Nx subtypes. In South Korea, H5N6 HPAIVs were introduced in wild birds and poultry during the 2023-2024 winter season, and the novel reassortant H5N3 HPAIV was first identified in wild birds in late 2024. Herein, we evaluated the pathogenicity and transmissibility of two recently emerged clade 2.3.4.4b H5 HPAIV subtypes-A/duck/Korea/D449/2023(H5N6) [D449(H5N6)] and A/northern pintail/Korea/WF369-1/2024(H5N3) [WF369-1(H5N3)]-in specific pathogen free chickens and commercial ducks. In chickens, the mean death time of the D449(H5N6) and WF369-1(H5N3) groups was 2.6 and 2.2 days, respectively, and the median chicken lethal dose (cLD50) of both viruses was calculated to be 104.5 median egg infectious dose (EID50). In contact chickens, D449(H5N6) achieved 100% transmission, whereas no transmission was observed in chickens exposed to WF369-1(H5N3), suggesting the rapid mortality (MDT 2.2 days) likely limited the window for horizontal transmission. Ducks inoculated with D449(H5N6) showed distinct clinical signs, including respiratory signs, which correlated with elevated viral titers in the lung and trachea. In contrast, ducks inoculated with WF369-1(H5N3) exhibited neurological signs, with viral replication in all internal organs. In addition, D449(H5N6) showed significantly higher infectivity with a median bird infectious dose (BID50) of <102 EID50, while the BID50 for WF369-1(H5N3) was 105 EID50. These strain-specific differences in virulence and transmissibility provide relevant information for early detection of viruses and disease control strategies in poultry. Our findings underscore the need for continued investigation to offer additional insight for field-level risk assessments.