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. 2026 Sep 23;373:199807. doi: 10.1016/j.virusres.2026.199807

Genetic Diversity of Clade 2.3.4.4b H5Nx high pathogenicity Avian Influenza Viruses detected in Korea during the 2025–2026 winter season and pathogenicity of H5N1 and H5N9 viruses

Yunyueng Jang 1, Ra Mi Cha 1, Min-Ji Park 1, Jong-Min Kim 1, Eui Hyeon Lim 1, Gyeong-Beom Heo 1, Se-Hee An 1, Bina Lee 1, Hyun-Ji Seo 1, Kwang-Nyeong Lee 1, Youn-Jeong Lee 1, Eun-Kyoung Lee 1,⁎
PMCID: PMC13675638  PMID: 42772571

Highlights

  • •

    During 2025-2026, clade 2.3.4.4b H5N1, H5N6, and H5N9 HPAIVs were detected in Korea, causing 62 H5Nx HPAI outbreaks in poultry, and 63 cases in wild birds.

  • •

    Seven H5N1 and three H5N9 genotypes identified in poultry, among which 25G2 of H5N1 was the dominant genotype.

  • •

    Most genotypes were reassortants containing gene segments derived from East Asian clade 2.3.4.4b H5N1 HPAIVs since 2022 and LPAIVs from wild birds.

  • •

    H5N1 and H5N9 viruses showed high infectivity in chickens with 103.3 and 103.2 EID50 of LD50 values, respectively.

  • •

    H5N1 virus caused rapid duck mortality, whereas H5N9 virus caused asymptomatic infection with efficient transmission.

Keywords: Clade 2.3.4.4b, H5N1, H5N6, H5N9, High pathogenicity avian influenza, Genotype, Pathogenicity, Korea

Abstract

Clade 2.3.4.4b H5N1, H5N6, and H5N9 high pathogenicity avian influenza viruses (HPAIVs) were detected in poultry and wild birds during the 2025–2026 winter season in Korea. During this period, 62 H5Nx HPAI outbreaks occurred in poultry, and 63 cases were reported in wild birds. Genome constellation analysis revealed substantial genotype diversity. Most H5N1 and H5N9 genotypes were reassortants containing gene segments derived from East Asian clade 2.3.4.4b H5N1 HPAIVs since 2022 and low-pathogenicity avian influenza viruses from wild birds. Notably, the European H5N1 genotype EA-2024-DI.2.1, which spread rapidly across Europe in 2025, and its reassortants were identified in Korea. The novel poultry genotypes were also detected in wild birds, supporting wild bird to poultry spillover, whereas one genotype detected in previous season was identified in small-scale poultry farms. H5N1 (C588) of major 25G2 genotype in poultry and H5N9 (C722) of N9-G2 genotype showed high infectivity in chickens with 103.3 and 103.2 EID50 of median lethal dose (LD50) values, respectively. The predominance of the 25G2 genotype in poultry may have been influenced by efficient infection and virus introduction into high-density poultry production areas, potentially facilitating horizontal transmission under field conditions. In young ducks, H5N1 caused rapid mortality, whereas H5N9 induced limited clinical signs without mortality. Both viruses were transmitted to all contact ducks, increasing the risk of transmission to poultry. Despite intensified surveillance and early detection efforts to limit virus spread, H5Nx HPAIV outbreaks occurred during the 2025–2026 winter season. These findings highlight the need for continued surveillance, together with rapid genomic and pathogenic characterization of newly introduced viruses.

1. Introduction

Since the emergence of the A/goose/Guangdong/1/1996 (Gs/GD) lineage in 1996, H5 high pathogenicity avian influenza viruses (HPAIVs) have diversified into multiple phylogenetically distinct hemagglutinin (HA) clades (WHO/OIE/FAO, 2014). Since 2014, clade 2.3.4.4 H5Nx HPAIVs have circulated globally, with clade 2.3.4.4b viruses predominating in recent years (Graziosi, Lupini, Catelli and Carnaccini, 2024). During 2020–2022, clade 2.3.4.4b H5N8 viruses circulated widely in poultry and wild birds, after which H5N1 HPAIVs became the predominant H5 subtype worldwide (Lewis et al., 2021; Tian et al., 2023). Multiple H5Nx subtypes, including H5N1, H5N2, H5N5, and H5N6, have emerged through reassortment with low-pathogenicity avian influenza viruses (LPAIVs) in wild birds (Cha et al., 2026; Fusaro et al., 2024; King et al., 2022; Nishiura et al., 2024).

Clade 2.3.4.4b H5N1 HPAIVs have continued to circulate worldwide, accompanied by the emergence of diverse reassortants, increased environmental persistence, and ongoing concerns regarding zoonotic spillover (Fusaro et al., 2024; EFSA et al., 2025a; EFSA et al., 2025b). Since 2021, the European EA-2020-C genotype of H5N1 has disseminated across Asia, Africa, and the Americas through intercontinental spread. This genotype has been detected in the Russian Far East, East Asia, and the United States, where it has undergone sustained regional circulation and reassortment with LPAIVs (Cui et al., 2022; Panova et al., 2025). In Europe, the EA-2024-DI genotype of H5N1 viruses was widely detected during the 2024–2025 season (EFSA et al., 2025a). A new sublineage of the EA-2024-DI genotype, DI.2.1, was first identified in Poland in September 2025 and rapidly spread across Europe, becoming the predominant genotype. This genotype was detected in wild birds, including common cranes, during mass mortality events and was also associated with widespread poultry outbreaks (Günther et al, 2026). Additional H5Nx viruses, including H5N5 and sporadic H5N2 reassortants, were also identified (EFSA et al., 2025b; Günther et al, 2026). In North America, H5N1 viruses were introduced in late 2021 and rapidly reassorted with local avian influenza viruses (Kandeil et al., 2023). The reassortant genotype D1.1 spread across multiple North American flyways and was identified in wild birds, poultry, mammals, and humans (Caserta et al., 2024; EFSA et al., 2026; Scotch et al., 2026). North American H5N1 viruses also disseminated into South America, causing multispecies outbreaks (Leguia et al., 2023). The sustained and widespread circulation of clade 2.3.4.4b H5Nx HPAIVs remains a major concern for animal and public health.

Clade 2.3.4.4b H5Nx HPAIVs have been repeatedly introduced into Korea by migratory wild birds during successive winter seasons. During the 2021–2022 winter season, clade 2.3.4.4b H5N1 HPAIVs were identified in wild birds and poultry farms and were classified into six genotypes (Cha et al., 2023). During the 2022–2023 winter season, H5N1 viruses were genetically related to Eurasian and North American HPAIVs, and 21 genotypes were identified (Cha et al., 2025). More recently, multiple H5Nx subtypes have been detected in Korea during consecutive winter seasons (Cha et al., 2026). During the 2023–2024 winter season, two H5N1 genotypes and one H5N6 genotype co-circulated, with H5N6 as the predominant subtype (Cha et al., 2026). During the 2024–2025 winter season, a novel H5N3 virus was detected in northern pintail and H5N1 viruses were also detected in wild birds, followed by H5N1 HPAI outbreaks in poultry (Cha et al., 2026; Si et al., 2025). In October 2025, H5N1 was detected in common teal, followed by the first detection of H5N9 HPAIV in Korea (Si et al., 2026).

Pathogenicity studies in poultry have shown that genetic differences among H5Nx HPAIVs are associated with distinct patterns of virulence, virus shedding, tissue tropism, and transmission (Bordes et al., 2024; Lee et al., 2025; Pantin-Jackwood et al., 2023; Seekings et al., 2023). In chickens, H5 HPAIVs typically cause acute systemic infection and high mortality, whereas ducks show a wide range of clinical manifestations following H5 HPAI infection, ranging from asymptomatic infection to severe disease (Bordes et al., 2024; Graziosi, Lupini, Catelli and Carnaccini, 2024; Wang et al., 2021). Recent H5N3 and H5N6 viruses detected in Korea were highly virulent in chickens and showed efficient horizontal transmission in ducks despite differences in clinical outcomes (Jang et al., 2026). Therefore, evaluating the pathogenicity and transmissibility of newly detected HPAIVs is important for assessing their potential impact on poultry production and interspecies transmission.

During the 2025–2026 winter season, H5N1, H5N6, and H5N9 HPAIVs were detected concurrently in Korea. To better understand the epidemiology of these outbreaks and assess their potential implications for poultry and interspecies transmission, we analyzed the genetic characteristics of H5Nx HPAIVs detected during the 2025–2026 winter season in Korea, and evaluated the pathogenicity and transmissibility of two representative H5N1 and H5N9 viruses in chickens and ducks.

2. Materials and methods

2.1. Viruses

During the 2025–2026 HPAI outbreak season, clade 2.3.4.4b H5Nx HPAIVs were isolated from 62 poultry outbreaks by the Animal and Plant Quarantine Agency (APQA), Republic of Korea, including H5N1 and H5N9 viruses (Supplementary Table 1). In addition, 63 HPAI cases caused by H5N1, H5N6, and H5N9 viruses were detected in wild birds through surveillance conducted by the APQA and the National Institute of Wildlife Disease Control and Prevention (NIWDC) (https://www.mcee.go.kr/niwdc/web/index.do?menuId=50). Eight wild bird isolates were available to the APQA in this study (Supplementary Table 1). Three additional cases were identified from environmental samples, including soil and feathers, collected from rivers and reservoirs in the vicinity of affected farms during epidemiological investigations conducted by the APQA. Wild bird species were identified using a mitochondrial DNA-based barcoding assay of fecal samples, as previously described (Lee et al., 2010).

A/chicken/Korea/C588/2025(H5N1) (H5N1[C588]), representing the predominant 25G2 genotype in poultry, and A/chicken/Korea/C722/2025(H5N9) (H5N9[C722]), representing the N9-G2 genotype and the first H5N9 subtype identified in poultry in Korea, were selected as representative viruses for pathogenicity and transmissibility studies in chickens and ducks. The viruses were propagated and titrated in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs. Virus titers were calculated as the median egg infectious dose (EID50) using the Reed–Muench method.

2.2. Sequencing

A total 73 of H5Nx HPAIV isolates available to the APQA, were sequenced, including 62 from poultry, 8 from wild birds, and 3 from environmental samples. Viral RNA was extracted from H5Nx HPAIVs using the Patho Gene-spin DNA/RNA Extraction Kit (iNtRON Biotechnology, Seoul, Republic of Korea) according to the manufacturer’s instructions. Viral gene segments were amplified by reverse transcription polymerase chain reaction (PCR), and the purified PCR products were used to prepare sequencing libraries using the Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA). Whole-genome sequencing was performed using the MiSeq next-generation sequencing platform (Illumina). Genome sequences were assembled and analyzed using CLC Genomics Workbench (Qiagen, Germantown, MD, USA) and BioEdit. The genome sequences generated in this study were deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database (Supplement Table 1).

Table 1.

Pathogenicity and transmissibility of H5N1(C588) and H5N9(C722) in chickens and ducks.

Virus Host IVPI Dose
(EID50/0.1mL)		 Mortality (%) MDT HI titera
(log2, mean±SD) (positive no.)		 cELISAa
(positive no.)		 LD50
(EID50
/0.1mL)		 BID50b
(EID50
/0.1mL)
C588
(H5N1)		 Chicken 2.98 106 5/5 (100) 2.4 NT NT 3.3 -
105 5/5 (100) 2.1 NT NT
104 4/5 (80) 4.6 - (0/1) 0/1
103 1/5 (20) 4 - (0/4) 0/4
Contact 1/3 (33) 5 0/2 0/2
Duck NT 106 5/5 (100) 3.9 NT NT < 2 < 2
104 5/5 (100) 4.3 NT NT
102 3/5 (60) 5.7 5.5±0.5 (2/2) 2/2
Contact 3/3 (100) 4.3 NT NT
C722
(H5N9)		 Chicken 3.0 106 5/5 (100) 2.4 NT NT 3.2 -
105 5/5 (100) 2.6 NT NT
104 5/5 (100) 2.6 NT NT
103 2/5 (40) 4 - (0/3) 0/3
Contact 3/3 (100) 4 NT NT
Duck NT 106 0/5 (0) - 5±2 (4/5) 5/5 - 3
104 0/3c (0) - - 0/3 3/3
102 0/5 (0) - - 0/5 0/5
Contact 0/2c (0) - - 0/2 2/2
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SPF chickens and commercial ducks were inoculated intranasally with serial dilutions of H5N1(C588) or H5N9(C722). Abbreviations: IVPI, intravenous pathogenicity index; LD50, median lethal dose; MDT, mean death time; NT, not tested; BID50, bird infectious dose.

a

Seroconversion was confirmed by the HI assay and cELISA in surviving birds at 14 days post infection.

b

Infection was determined by a positive serologic response.

c

Died of accidental causes unrelated to infection

2.3. Phylogenetic analysis and genome constellation

Phylogenetic analysis was performed using the HA and neuraminidase (NA) gene sequences of H5Nx HPAIVs. Reference sequences were obtained from the EpiFlu database of GISAID and aligned with the sequences generated in this study. HA phylogenic tree dataset included HA gene sequences from H5Nx HPAI viruses detected in Eurasia and North America (2020–2026) (Supplementary Table 4). NA phylogenic tree dataset included N1 and N9 gene sequences from HxN1 viruses detected in Eurasia and North America (2020-2026) and HxN9 viruses detected in Eurasia and America (1995-2025) (Supplementary Table 4). Obtained sequences were reduced using the Cluster Database at High Identity with Tolerance (CD-HIT). Maximum-likelihood phylogenetic trees were constructed using MEGA version 12.1 with 1,000 bootstrap replicates. A monophyletic cluster was defined as having a bootstrap support value > 70%.

Genome constellation analysis was performed by phylogenetic analysis of each gene segment, as previously described (Cha et al., 2026). Briefly, phylogenetic trees were constructed using RAxML version 8.2.12 with a gamma distribution and the general time-reversible model. Reference datasets for the internal gene segments (PB2, PB1, PA, NP, M, and NS) sequences, collected from January 1, 2016, to March 15, 2026 (accessed on 16 March 2026), was obtained from the GISAID and GenBank databases, and reduced using the CD-HIT. Genotypes were classified according to phylogenetic tree topology, and a nucleotide sequence identity > 97% with a bootstrap support value > 70% was considered indicative of the same phylogenetic group. Genome constellations were determined based on the combination of phylogenetic groups assigned to the eight viral gene segments.

2.4. Animal experiments

Five-week-old SPF chickens (n = 72) and 2-week-old ducks (n = 52) were used for pathogenicity and transmissibility experiments. Before inoculation, serum samples from all birds were confirmed to be negative for antibodies against avian influenza viruses using a competitive enzyme-linked immunosorbent assay (cELISA; BIONOTE, Hwaseong-si, Republic of Korea). The birds were housed in negative-pressure, high-efficiency particulate air–filtered animal isolators within a biosafety level 3 facility. Water and feed were provided ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of the APQA (approval no. 2024-1458).

The pathogenicity of the viruses was assessed using the intravenous pathogenicity index (IVPI) test according to WOAH guidelines. Briefly, 10 SPF chickens were inoculated intravenously with 0.1 mL of a 1:10 dilution of infectious allantoic fluid containing either H5N1(C588) or H5N9(C722), and clinical signs were monitored for 10 days. The IVPI was calculated according to WOAH standard procedures. To determine the median chicken lethal dose (LD50), chickens were divided into four groups (n = 5 per group) and inoculated intranasally with 10-fold serial dilutions of H5N1(C588) or H5N9(C722) at 106, 105, 104, or 103 EID50/0.1 mL. To determine the median bird infectious dose (BID50) in ducks, three groups (n = 5 per group) were inoculated intranasally with each virus at 106, 104, or 102 EID50/0.1 mL. The LD50 and BID50 values were calculated using the Reed–Muench method.

To examine contact transmission, three naïve chickens or ducks were co-housed with the group inoculated with 106 EID50/0.1 mL at 8 h post-inoculation to minimize exposure to residual inoculum. Oropharyngeal (OP) and cloacal (CL) swab samples were collected from both inoculated and contact birds at 1, 2, 3, 4, 5, 6, 7, 10, and 14 days post inoculation (dpi). At 14 dpi, serum samples were collected from surviving birds and tested for antibodies using cELISA and HI assays with homologous antigens according to WOAH guidelines. Infection was defined as the detection of virus shedding at any sampling time point or the detection of antibodies at 14 dpi.

To assess viral replication in internal organs, three chickens and three ducks per virus were inoculated intranasally with 106 EID50/0.1 mL and euthanized at 3 dpi. At necropsy, 12 tissues were collected for virus titration: trachea, thymus, heart, lung, kidney, brain, pancreas, cecal tonsil, liver, spleen, muscle, and proventriculus. Swab samples and 10% (w/v) tissue homogenates were inoculated onto DF-1 cells, a chicken embryo fibroblast-derived cell line. Viral titers were determined using the Reed–Muench method and expressed as log10 TCID50/0.1 mL. Titers below the limit of detection were assigned a value of 1 log10 TCID50/0.1 mL.

3. Results

3.1. HPAIVs isolated during the 2025–2026 winter season

During the 2025–2026 winter season, clade 2.3.4.4b H5N1, H5N6, and H5N9 HPAIVs were detected in Korea. Between September 2025 and April 2026, 62 HPAI outbreaks were reported in poultry farms, affecting chickens (n = 39), ducks (n = 18), and minor poultry (n = 5; Korean native chicken, n = 1; quail, n = 3; and goose, n = 1). During the same period, 63 HPAI cases were detected in wild birds by the APQA (n = 8) and the NIWDC (n = 55) (Fig. 1). In addition, Three HPAI subtypes were detected concurrently in wild birds early in the season. H5N1 (n = 52) was the predominant subtype, followed by H5N9 (n = 10) and H5N6 (n = 1). In poultry, H5N1 (n = 57) and H5N9 (n = 5) outbreaks were identified. In addition, one H5N6 was detected exclusively in duck samples collected at epidemiologically linked farm during preventive culling (data not shown). In epidemiological investigation of HPAI affected farms, the APQA additionally isolated three HPAIVs from environmental samples, comprising two H5N1 and one H5N9 isolates. Most H5 HPAIV outbreaks in both poultry and wild birds occurred in the western region of Korea, which contains major migratory bird habitats and high-density poultry farming areas (Supplementary Fig. 1).

Fig. 1.

Fig. 1

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Weekly prevalence of H5Nx HPAIVs in poultry farms and wild birds in Korea during the 2025–2026 winter season. HPAIV subtypes are shown according to the date of virus isolation. During the 2025–2026 winter season, H5N1 and H5N9 viruses were detected in poultry farms (A), whereas H5N1, H5N9, and H5N6 viruses were detected in wild birds (B).

3.2. Genetic characterization

All isolates belonged to clade 2.3.4.4b and possessed a multibasic HA cleavage site motif. Most isolates contained the PLREKRRKR/GLF motif, whereas one isolate contained the PLRGKRRKR/GLF motif (Supplementary Table 1). Maximum-likelihood phylogenetic analysis was performed using the HA gene sequences of 73 H5Nx HPAIVs isolated by the APQA, including 62 from poultry, eight from wild birds, and three from environmental samples near (soil, n = 2; feathers, n = 1) collected during epidemiological investigations around affected farms (Supplementary Table 1). In addition, two previously reported H5Nx HPAIV sequences in this season were retrieved from a public database reported by Si et al. (Si et al., 2026). Based on HA gene phylogeny, the H5Nx HPAIVs detected during the 2025–2026 season were classified into three HA clusters (Fig. 2). Two clusters corresponded to the previously defined G2c and G2d subgroups of clade 2.3.4.4b circulating in East Asia, whereas the remaining cluster was related to European HA genes from viruses detected since 2025 (Takadate et al., 2024). Most H5N1 HA genes detected in Korea during the 2025–2026 season belonged to the G2c subgroup. Within this subgroup, the Korean H5N1 viruses clustered with the H5N1 viruses detected in Japan during the same season. All H5N9 HA genes also belonged to the G2c subgroup and were genetically more closely related to H5N1 and H5N6 viruses detected in East Asia during the 2024–2025 season. Four H5N1 viruses belonged to the G2d subgroup and clustered with Korean and Japanese viruses detected during the previous season, as well as the Japanese virus detected during the 2025–2026 season. The HA genes of remaining H5N1 viruses clustered with sublineage EA-2024-DI.2.1 of H5 HPAIVs detected in Europe since 2025.

Fig. 2.

Fig. 2

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Maximum-likelihood phylogenetic tree of the hemagglutinin (HA) genes of H5Nx highly pathogenic avian influenza viruses (HPAIVs) isolated in Korea during the 2025–2026 winter season. The tree was constructed using HA gene sequences from Korean H5Nx HPAIV isolates and reference viruses. The scale bar indicates the number of nucleotide substitutions per site. Bootstrap support values were calculated from 1,000 replicates, and only values >70% are shown. Filled triangles and circles represent poultry and wild bird isolates, respectively. Virus names shown in red, green, and blue indicate isolates from Korean poultry and wild bird, and environmental samples collected during the 2025–2026 winter season, respectively. Colored bars represent the eight gene segments, with each color indicating the closest donor lineage or related virus for the corresponding segment.

Genotypes were designated by combining the season year with sequential numbers assigned according to the order of detection. Although the HA genes were phylogenetically divided into three clusters, all H5Nx HPAIVs shared > 97% nucleotide identity in the HA gene and were therefore analyzed as a single H5 group in this study. The H5N1 viruses detected during the 2025–2026 winter season were classified into 12 genotypes, including five detected exclusively in wild birds by the NIWDC (data not shown). The first reassortant H5N1 virus detected in wild bird was previously reported by Si et al., and subsequently designated as genotype 25G1 in this study (Si et al., 2026). Diverse H5N1 genotypes were also detected in wild birds throughout the season, including genotype 24G0, which had been detected during the previous season (Cha et al., 2026).

In poultry, H5N1 viruses were classified into six newly designated genotypes, 25G2–25G7, whereas genotype 24G1, previously reported during the 2024–2025 winter season, was detected again in a small-scale poultry farms (Fig. 2, Supplementary Table 2, Supplementary Fig. 2 and 3) (Cha et al., 2026). Genotypes 25G2–25G7 were subsequently identified throughout the season in poultry and wild birds. Notably, genotype 25G2 was the predominant genotype in poultry farms and continued to be detected until the end of the season, although it was identified in only a single wild bird sample. Genotypes 25G1, 25G2, 25G3, 25G4, and 25G6 were reassortants between East Asian H5N1 HPAIVs that emerged during the 2022–2023 winter season and internal genes derived from Eurasian LPAIVs in wild birds. Genotype 25G5 was classified as the European genotype EA-2024-DI.2.1 (DI.2.1). The DI.2.1 related genotype, 25G7, showed an identical genome constellation with 25G5, except for the PB2 and NP genes, which were derived from LPAIVs.

H5N9 viruses were classified into three genotypes, N9-G1, N9-G2, and N9-G3, according to the order of detection. The first H5N9 virus detected in wild bird has been reported previously, and designated as genotype N9-G1 (Si et al., 2026). All three H5N9 genotypes were detected in wild birds, whereas only N9-G2 and N9-G3 were identified in poultry. Among the H5N9 viruses, N9-G2 and N9-G3 shared the HA, NA, and M gene segments with N9-G1 but possessed distinct internal gene constellations. Following the detection of one H5N6 virus in wild birds by NIWDC, an H5N6 virus with the same genotype was identified in epidemiological samples (data not shown).

Overall, 16 genotypes (H5N1, n = 12; H5N6, n = 1; H5N9, n = 3) were identified among H5Nx HPAIVs through APQA and NIWDC surveillance. Of these, 10 genotypes (H5N1, n = 7; H5N6, n = 1; H5N9, n = 2) were detected in poultry, whereas 15 genotypes (H5N1, n = 11; H5N6, n = 1; H5N9, n = 3) were detected in wild birds.

Mutation analysis revealed multiple substitutions previously associated with host adaptation, viral replication, and virulence (Supplementary Table 3). Internal gene segments carried several substitutions associated with increased polymerase activity or replication in mammalian cells, including PB2-K389R, I495V, and V598T/I, PB1-D3V and D622G, and PA-N383D and N409S (Flores et al., 2024; Gao et al., 2019; Hu et al., 2017; Wen et al., 2018). Several of these markers were conserved across multiple genotypes, whereas PB2-I292V, HA-N154D, and NS1-D74N and L103F showed genotype-dependent distributions. Notably, PB2 E627K substitution, which is well known mammalian adaptation marker, was identified in two epidemiologically linked viruses of genotype 25G5, one isolated from a duck and the other from an environmental sample (wild duck feather) collected from reservoir in the vicinity of affected farm (Min et al., 2013; Kim et al., 2025) (Supplementary Table 1 and 3).

3.3. Pathogenicity and transmissibility

The pathogenicity and transmissibility of two HPAIVs, H5N1(C588) representing the predominant 25G2 genotype in poultry, and H5N9(C722), representing the first H5N9 isolate identified in poultry in Korea, were evaluated in SPF chickens and commercial ducks. H5N1(C588) and H5N9(C722) had IVPI values of 2.98 and 3.0, respectively, in chickens, classifying both viruses as HPAI according to WOAH criteria (Table 1). Chickens inoculated intranasally with 106 EID50 of H5N1(C588) developed clinical signs, including depression and greenish diarrhea, at 2–3 dpi, resulting in 100% mortality, with a mean death time (MDT) of 2.4 days. This virus transmitted to 1/3 contact chickens, and the LD50 was 103.3 EID50. All chickens inoculated with 106 EID50 of H5N9(C722) also died, exhibiting the same clinical signs and an MDT of 2.4 days. All contact chickens in the H5N9(C722) group died within 4 days, and the LD50 value was estimated to be 103.2 EID50. Seroconversion was not detected in the surviving chickens.

Viral shedding and tissue replication were evaluated in chickens inoculated with 106 EID50 of each virus (Fig. 3). In chickens infected with H5N1(C588), viral titers in OP swabs increased from 1 to 3 dpi, reaching a peak of 103.7 TCID50/0.1 mL. Viral titers in CL swabs peaked at 2 dpi (102.9 ± 1.0 TCID50/0.1 mL) and declined by 3 dpi. H5N1(C588) was detected in all examined organs from all chickens, with mean titers ranging from 103.7 ± 0.5 TCID50/0.1 mL in muscle to 105.7 ± 0.1 TCID50/0.1 mL in the lung (Supplementary Fig. 4). In chickens infected with H5N9(C722), viral titers in both OP and CL swabs increased from 1 to 3 dpi, reaching peak titers of 103.7 ± 0.4 TCID50/0.1 mL and 104.0 ± 0.6 TCID50/0.1 mL, respectively. H5N9(C722) was also detected in all organs collected from all chickens. Mean viral titers ranged from 103.3 ± 1.2 TCID50/0.1 mL in the brain to 105.3 ± 0.2 TCID50/0.1 mL in the trachea and 105.3± 0.4 TCID50/0.1 mL in the kidney, the organs with the highest viral titers.

Fig. 3.

Fig. 3

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Viral titers in swabs collected from chickens and ducks inoculated with H5N1(C588) or H5N9(C722). Five specific pathogen–free (SPF) chickens and five commercial ducks were inoculated intranasally with 106 EID50/0.1 mL of H5N1(C588) or H5N9(C722). Oropharyngeal (OP; A and C) and cloacal (CL; B and D) swabs were collected from chickens (A and B) and ducks (C and D) at 1–7, 10, and 14 days post inoculation (dpi). Viral titers were determined using DF-1 cells and presented as mean ± standard deviation. The limit of detection was 1 log10 TCID50. NT, not tested because of 100% mortality.

Ducks inoculated with 106 EID50 of H5N1(C588) developed clinical signs, including greenish diarrhea, depression, head tremors, and immobility, beginning at 3 dpi, and exhibited 100% mortality with an MDT of 3.9 days (Table 1). Similar clinical signs were observed in ducks inoculated with 102 and 104 EID50 of H5N1(C588). The two surviving ducks in the 102 EID50 group were positive for antibodies by both the HI assay and cELISA, with HI titers of 32 and 64. The LD50 and BID50 values of H5N1(C588) were estimated to be < 102 EID50. In the transmission experiment, H5N1(C588) was transmitted to all contact ducks, resulting in 100% mortality by 5 dpi. All ducks inoculated with 106 EID50 of H5N9(C722) survived. In this group, only one duck exhibited mild respiratory signs and abnormal neck posture without depression or reduced feed intake. Deaths unrelated to H5N9(C722) infection occurred in the 104 EID50 and contact groups at 8 and 10 dpi, respectively. HI antibodies were detected in four of the five ducks in the 106 EID50 group, with HI titers ranging from 4 to 32. Seroconversion was confirmed by cELISA in the 104 EID50, 106 EID50, and contact groups. The BID50 of H5N9(C722) was calculated to be 103 EID50 based on cELISA results (Song et al., 2009).

Viral shedding and viral replication in internal organs were evaluated in ducks inoculated with 106 EID50 of each virus. In ducks inoculated with H5N1(C588), virus was detected in OP swabs from 1 to 5 dpi, reaching a peak titer of 103.9 ± 0.9 TCID50/0.1 mL at 4 dpi, before all ducks died (Fig. 3). Virus was also detected in CL swabs from 2 to 5 dpi, with a peak titer of 103.0 ± 2.0 TCID50/0.1 mL. At 3 dpi, viral replication was detected in all examined organs from all ducks, with the highest viral titer observed in the brain (105.5 ± 0.4 TCID50/0.1 mL). In ducks inoculated with H5N9(C722), virus was detected in OP swabs from 1 to 7 dpi, reaching a peak titer of 103.6 ± 0.6 TCID50/0.1 mL at 3 dpi. In CL swabs, H5N9(C722) was detected at low titers from 2 dpi through 10 dpi, with the highest titer (102.5 TCID50/0.1 mL) observed at 7 dpi. At 3 dpi, virus replication in internal organs varied among ducks (Supplementary figure 4). No virus was detected in the brain or muscle, whereas the trachea, thymus, and lung were positive in all ducks. The highest tissue viral titer was detected in the trachea, reaching 105.4 ± 0.9 TCID50/0.1 mL.

Gross lesions were observed in both chickens and ducks infected with H5N1(C588) and H5N9(C722), consistent with previous reports of H5 HPAIV infection in these species (Jeong et al., 2009; Kim et al., 2025; Meseko et al., 2023). Chickens infected with H5N1(C588) showed splenic necrosis and hepatic congestion. H5N9(C722) was associated with thymic and renal enlargement and hemorrhage, as well as splenic hemorrhage in chickens. In ducks, H5N1(C588) was associated with splenic congestion and hemorrhage, pancreatic necrotic foci, and hepatic hemorrhage. Ducks infected with H5N9(C722) showed thymic hemorrhage, splenic petechiae, and pancreatic necrosis.

4. Discussion

Clade 2.3.4.4b H5Nx HPAIVs have diversified through reassortment with LPAIVs from wild birds and have been repeatedly introduced into Korea through migratory flyways (Cha et al., 2026; Lee et al., 2025). In the present study, we characterized the genetic diversity of H5Nx HPAIVs detected during the 2025–2026 winter season. We evaluated the pathogenicity and transmissibility of two representative viruses, H5N1 and H5N9, in poultry to better understand the outbreak patterns associated with these viruses in Korea.

During the 2025–2026 winter season, H5Nx HPAIVs caused 62 outbreaks in poultry farms and were detected in 63 wild bird cases in Korea. H5N1 was the predominant subtype in both poultry and wild birds, accounting for 57 poultry outbreaks and 52 wild bird detections, whereas H5N6 and H5N9 were also detected concurrently. Notably, H5N9 HPAIV was identified in Korea for the first time. H5N9 viruses have been reported only sporadically in wild birds and poultry, including in France during 2015–2016 and in the United States in 2025 (Briand et al., 2017; WOAH, 2026). In Korea, H5N9 was first detected in wild birds in October 2025 and was subsequently identified in domestic poultry in December 2025. In neighboring Japan, H5N1 was detected in both domestic and wild birds during the same season, whereas H5N5 was additionally detected in wild birds (Authority et al., 2026). These findings indicate that the 2025–2026 season was characterized by the concurrent circulation of multiple H5Nx subtypes across East Asia.

HA genes of clade 2.3.4.4b H5Nx HPAIVs belonging to the G2 group, which comprises the G2a–G2d subgroups, have been detected widely in Eurasia (Baek et al., 2021; Mine et al., 2024). Among these, the G2c and G2d subgroups have predominated in Korea and Japan since 2022 (Cha et al., 2025; Nishiura et al., 2025). H5 HPAIVs in the G2c subgroup have been closely associated with East Asian and Russian H5N1 lineages, whereas G2d subgroup viruses have been distributed more broadly across Asia, Russia, and North America (Isoda et al., 2022; Mine et al., 2024). During the 2025–2026 winter season, H5Nx HPAIVs detected in Korea and Japan were consistently classified into three HA phylogenetic clusters. Most Korean viruses clustered within the G2c subgroup, whereas Japanese H5Nx HPAIVs included a higher proportion of G2d subgroup viruses based on sequences available in the GISAID database. Together, these findings support repeated introductions into Korea and Japan through multiple, partially overlapping migratory flyways connecting northern breeding grounds with East Asian wintering sites, and continued circulation of certain clusters or genotypes in the region across multiple endemic periods (Lee et al., 2025; Takadate et al., 2026).

Genome constellation analysis revealed substantial genetic diversity during the 2025–2026 winter season. A total of 16 H5Nx HPAIV genotypes were identified through APQA and NIWDC surveillance, including 10 genotypes detected in poultry and 15 detected in wild birds. Nine genotypes were identified in both poultry and wild birds, supporting the spillover of viruses circulating in wild bird populations into domestic poultry. In contrast, genotype 24G1, detected until June 2025 during the previous winter season, was identified exclusively in poultry in this season, suggesting local persistence from the previous season (Cha et al., 2026). Nevertheless, reintroduction from undetected circulation in wild birds cannot be excluded, and further studies are required to better understand the origin and circulation of genotype 24G1.

Four novel H5N1 genotypes (25G2, 25G3, 25G4, and 25G6) and the H5N9 genotypes identified during the 2025–2026 season shared genomic backbones closely related to clade 2.3.4.4b H5N1 viruses that have circulated in East Asia since 2021 (Cha et al., 2023). In particular, the EA-2020-C genotype (designated 24G0 in our previous study) originated from clade 2.3.4.4b H5N1 HPAIVs circulating in Europe during 2021–2022 and has been repeatedly detected in East Asia since the 2022–2023 winter season (Cha et al., 2026; Lee et al., 2025; Nishiura et al., 2024). During the 2025–2026 winter season, the EA-2020-C genotype and its reassortants accounted for a large proportion of the H5Nx HPAIVs detected in Korea, indicating continued diversification of this genomic backbone through reassortment with co-circulating LPAIVs in wild birds. Based on GISAID data, four genotypes (24G0, 25G1, 25G3, and 25G7) were identified in Korea and Japan, whereas one additional genotype was detected exclusively among Japanese sequences. These findings suggest that continued reassortment in wild bird populations along migratory flyways, together with regional circulation within East Asia, contributed substantially to the genotype diversity observed in Korea.

From the middle of the 2025–2026 season, viruses carrying the DI.2.1 genomic backbone were identified in Korea. After detection of DI.2.1 in September 2025, this genotype spread rapidly across Europe and was subsequently detected in wild Anseriformes in Kazakhstan in October 2025 (EFSA et al., 2026). In the present study, genotype 25G5 was classified as DI.2.1, whereas genotype 25G7 represented a reassortant carrying the PB2 and NP gene segments derived from LPAIVs circulating in wild birds. The same genotype (25G7) was also detected in Japan in early 2026. Notably, two H5N1 viruses of genotype 25G5, detected in poultry and a wild bird feather sample, possessed the PB2 E627K substitution, which has been reported sporadically among DI.2.1 variant in Europe (EFSA et al., 2025b) (Supplementary Table 3). These findings indicate that EA-2024-DI.2.1 and its reassortants in Europe, Central Asia, and East Asia demonstrates their broad geographic distribution across Eurasia. Importantly, the earlier European-origin EA-2020-C genotype was introduced into East Asia and subsequently underwent regional circulation and reassortment with co-circulating LPAIVs (Cha et al., 2023; Cha et al., 2026; Takadate et al., 2024). Therefore, EA-2024-DI.2.1 may also contribute to further regional reassortment and circulation following its introduction into East Asian wild bird populations. Continued genomic surveillance is warranted to monitor its spread, persistence, and reassortment in wild birds and poultry.

The pathogenicity and transmissibility of the representative H5N1(C588) and H5N9(C722) HPAIVs were examined in chickens and ducks. H5N1(C588) belonged to genotype 25G2, the predominant genotype detected in poultry, whereas H5N9(C722) belonged to genotype N9-G2, which was associated with the first H5N9 outbreak in poultry during the 2025–2026 winter season. Both viruses were highly virulent in chickens, causing substantial mortality, elevated virus shedding titers, and systemic replication. The LD50 values of H5N1(C588) and H5N9(C722) were 103.3 EID50 and 103.2 EID50, respectively, which were lower than those reported for recent clade 2.3.4.4b H5Nx HPAIVs in East Asia, indicating efficient infection of chickens at a relatively low infectious dose (Cha et al., 2025; Jang et al., 2026). Although transmission dynamics are influenced by factors including receptor-binding affinity, viral shedding patterns, and environmental conditions, the introduction of these viruses may have facilitated horizontal transmission in high-density poultry production areas (Germeraad et al., 2019; Guinat et al., 2025; EFSA et al., 2026).

In ducks, the two viruses showed markedly different pathogenicity. H5N1 caused severe neurological signs and rapid mortality, even at the lowest inoculation dose. The virus was detected in all examined internal organs, with the highest viral titers observed in the brain, suggesting that systemic infection and neurotropism likely contributed to the severe clinical signs (Supplementary Fig. 4). The severe outcomes of H5N1 infection may also have been influenced by age-dependent factors, as younger ducks have been reported to exhibit higher mortality following HPAIV infection (Jang et al., 2020). In contrast, H5N9 caused limited clinical signs without mortality, although viral shedding in CL swabs was detected through 10 dpi (Fig. 3). Despite these differences in pathogenicity, both viruses were transmitted to all contact ducks. Previous studies of clade 2.3.4.4b H5Nx HPAIVs have also shown efficient transmission in ducks, with strain-dependent differences in pathogenicity (Lee et al., 2025). Collectively, these findings suggest that infected ducks may facilitate virus circulation within duck populations, thereby increasing the risk of transmission to poultry.

Genotype 25G2 was the predominant genotype detected in poultry during the 2025–2026 season, accounting for 30 outbreaks. However, it was identified in only one environmental sample collected during an epidemiological investigation of migratory bird habitats. This outbreak pattern differed from those observed during previous seasons in Korea, in which the predominant genotypes were generally similar between wild birds and poultry (Cha et al., 2023; Cha et al., 2025; Cha et al., 2026). The predominance of genotype 25G2 in poultry may have been influenced by higher infectivity under field conditions, particularly in layer farms located in high-density poultry farming areas where HPAI outbreaks were concentrated. However, introduction through environmental contamination associated with wild birds cannot be excluded, and the potential influence of sampling bias resulting from spatial and temporal variations in bird migration and surveillance should also be considered (Hayes et al., 2025; Llanos-Soto, Yaffy, Pavlak and Ivanek, 2025).

This study revealed multiple introductions of genetically diverse H5 viruses during the 2025–2026 winter season and demonstrated their relatively high infectivity in chickens. Epidemiological investigations of concurrent outbreaks, together with virus detection and genetic analyses, helped identify potential sources of virus introduction in the affected regions and provided evidence of viral contamination in the surrounding environment. Despite enhanced surveillance and early detection efforts to limit virus spread, H5 HPAI outbreaks still occurred in poultry farms. These findings highlight the importance of continued surveillance in wild birds and poultry, coupled with the rapid genomic and pathogenic characterization of newly emerging viruses, to support the implementation of effective avian influenza control disease.

Funding

This work was supported by the Animal and Plant Quarantine Agency (APQA) of the Ministry of Agriculture, Food, and Rural Affairs of the Republic of Korea [grant B1543418-2026-27-0101].

Generative AI statement

The authors declared that generative AI was not used in the creation of this manuscript.

Ethics declaration

This study was conducted in accordance with the following guidelines for animal welfare and/or reporting: local legislation and institutional requirements. This study was approved by the Animal and Plant Quarantine Agency. (Approval No. 2024-1458).

CRediT authorship contribution statement

Yunyueng Jang: Writing – review & editing, Writing – original draft, Project administration, Conceptualization. Ra Mi Cha: Validation, Resources, Investigation, Funding acquisition. Min-Ji Park: Visualization, Investigation, Data curation. Jong-Min Kim: Visualization, Investigation, Data curation. Eui Hyeon Lim: Visualization, Investigation. Gyeong-Beom Heo: Validation, Software, Resources. Se-Hee An: Validation, Resources. Bina Lee: Resources, Methodology. Hyun-Ji Seo: Validation, Resources. Kwang-Nyeong Lee: Supervision, Conceptualization. Youn-Jeong Lee: Supervision, Project administration. Eun-Kyoung Lee: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

We thank the Animal and Plant Quarantine Agency (APQA), Ministry of Agriculture, South Korea, for their support.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2026.199807.

Appendix. Supplementary materials

mmc1.docx (2MB, docx)
mmc2.xlsx (32.9KB, xlsx)

Data availability

The genome sequence data generated in this study are available in the GISAID EpiFlu database under the accession numbers listed in Supplementary Table 4.

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Further readings

  1. Jang Y., Seo S.H. Age-Dependent Lethality in Ducks Caused by Highly Pathogenic H5N6 Avian Influenza Virus. Viruses. 2020;12(6):591. doi: 10.3390/v12060591. [DOI] [PMC free article] [PubMed] [Google Scholar]
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  3. Lee D.H., Bertran K., Kwon J.H., Swayne D.E. Evolution, global spread, and pathogenicity of highly pathogenic avian influenza H5Nx clade 2.3.4.4. J. Vet. Sci. 2017;18(S1):269. doi: 10.4142/jvs.2017.18.S1.269. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (2MB, docx)
mmc2.xlsx (32.9KB, xlsx)

Data Availability Statement

The genome sequence data generated in this study are available in the GISAID EpiFlu database under the accession numbers listed in Supplementary Table 4.

Articles from Virus Research are provided here courtesy of Elsevier

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