On December 3, 2023, a suspected HPAI infection that caused white diarrhea, reduced feed intake, and increased deaths was reported in 39-day-old broiler ducks at a broiler duck farm (D448) in Goheung, South Korea (Figure 1). We detected matrix and H5 genes in the clinical samples from this index farm by real-time reverse transcription PCR. We determined the deduced amino acid sequence of the HA cleavage site of the H5 genes to be PLREKRRKR/GLF, which indicated high pathogenicity. For the NA gene, we detected both N1 and N6 genes in some flocks at the farm, at which the flocks were separated in different houses. We analyzed co-infection status at that farm by using whole-genome sequences of avian influenza viruses obtained from pooled oropharyngeal swab samples of 20 live ducks from each of 11 flocks using the Nanopore (Oxford Nanopore, https://nanoporetech.com) amplicon sequencing method (Appendix).

We found that 3 flocks, numbers 1, 4, and 5, were co-infected with H5N6 and H5N1 viruses, whereas the other 8 flocks were infected with H5N6 virus only (Figure 2). Analysis of the average coverage at each gene segment as percentage composition showed that birds in flock 4 had more viral reads of H5N1, whereas flocks 1 and 5 had more reads of H5N6 (Figure 2, panel B). We observed the same co-infection pattern in pooled cloacal swabs of flock 4 (data not shown). Because all the swabs from flocks were pooled at sample collection, no clear evidence was found supporting infection with the 2 viruses in a single bird. Because this farm was located very close to the south sea and seawall lake and had a relatively low level of biosecurity, we considered this farm susceptible to virus introduction by migratory birds (Figure 1, panel A).

We detected HPAI H5N6 virus (WA875) in Jeolla-do province in an apparently healthy wild mandarin duck, which we captured and sampled on December 4, 2023, for the active wild bird surveillance program. Two additional broiler-duck farms in the same province were found to be infected with H5N6 (D449) and H5N1 (D502) virus on December 5 and December 20, 2023 (Figure 1, panel A). We assessed the genetic relationships among the HPAI viruses by determining and comparing the complete genome sequences of A/duck/Korea/D448-N6/2023(H5N6), A/duck/Korea/D448-N1/2023(H5N1), A/duck/Korea/D449/2023(H5N6), A/mandarin duck/Korea/WA875/2023(H5N6), and A/duck/Korea/D502/2023(H5N1). Their sequences have been deposited in GISAID (https://www.gisaid.org; accession nos. EPI_ISL_18819959–61, EPI_ISL_18819826, and EPI_ISL_18819797).

The H5N6 viruses, D448-N6, D449, and WA875, showed high nucleotide sequence identities in all 8 genes among them (>99.8%). The sequences of their polymerase basic (PB) 1, hemagglutinin (HA), and matrix (M) genes were very close (99.53%–99.83%) to the respective genes of clade 2.3.4.4b HPAI H5N1 viruses isolated from wild birds in Japan and South Korea in 2022‒23. The 4 internal genes of the H5N6 viruses, PB2, polymerase acidic protein (PA), nucleoprotein (NP), and nonstructural protein (NS), were closely related to the respective genes found in the Eurasian low pathogenicity avian influenza (LPAI) viruses of diverse subtypes isolated from wild birds in 2020 and 2022. Their N6 genes appeared to be close to the poultry viruses isolated in East Asia in 2021 and 2022, although the nucleotide identities were relatively low (98.1%–98.62%) (Table 1). Of interest, the protein encoded by the N6 gene in the isolates from this study had a deletion of 12 aa residues at positions 58–69; this neuraminidase (NA) stalk deletion has been often observed in poultry-adapted viruses (11,12). From the avian influenza active surveillance program in South Korea during 2019–2023, N6 genes were detected only in LPAI viruses isolated from wild birds; we did not observe this NA stalk deletion among the analyzed viruses (data not shown).

We found no HPAI H5N6 viruses showing nucleotide similarities >98.5% in all of the 8 genes at once to the new H5N6 isolates in the public databases. However, a wild bird isolate from Japan (A/peregrine falcon/Saga/4112A002/2023, EPI_ISL_18740267) that was collected on December 6, 2023, was almost identical to the H5N6 Korean viruses (T. Hiono, pers. comm., email, 2024 Jan 11), suggesting that these emerged viruses spread coincidently throughout this winter in East Asia.

The nucleotide sequences of the coding regions of 2 poultry H5N1 viruses, D448-N1 and D502, were very similar (>99.0%) and were very closely related to the sequences of clade 2.3.4.4b H5N1 viruses circulating in Japan and Canada in 2023 (Table 2; Appendix Figures 1–5,7–9). Those clade 2.3.4.4b HPAI H5N1 viruses of diverse genotypes have been prevalent in Europe and North America (3) and had been introduced into South Korea during the epidemics of 2021–22 and 2022–23 (5,8). We did not detect significant mutations related to mammal adaptation or antiviral resistance in the newly isolated H5N6 and H5N1 HPAI viruses.

This study describes the simultaneous introduction of H5N1 virus and a new reassortant H5N6 HPAI virus of clade 2.3.4.4b into South Korea in 2023. Better understanding of this spatial and genomic dynamic requires enhanced and timely sharing of genetic information and international surveillance of HPAI and LPAI viruses in wild birds and poultry.