Since 2020, high pathogenicity avian influenza H5Nx viruses of clade 2.3.4.4b have become enzootic in Europe, causing recurrent epidemic waves characterized by extensive reassortment events. Here, we describe the emergence of a single high-fitness genotype (EA-2024-DI) that has driven two consecutive waves, evolving into distinct sub-lineages. While its circulation is ongoing, during the 2025-2026 wave it caused an unprecedented number of cases in wild birds. Using phylodynamic analyses of a large dataset of genomic sequences, we compared the spatial diffusion and host transmission pattern of the EA-2024-DI sub-lineages across the three most recent epidemic waves (2023-2024, 2024-2025 and 2025-2026). We show that the genotype has persisted over time and has spread primarily through wild Anseriformes, but with a marked change in the transmission patterns between the different waves and a shift in the epicenter from Eastern to Central Europe, the latter having emerged as an important hub for virus diffusion throughout Europe. Our results reveal a recent increase in the frequency of viruses from wild and domestic mammals carrying mutations enhancing virus replication in mammalian hosts, highlighting the importance of proactive monitoring of this group of hosts to better understand its role in the virus ecology and evolution.

In autumn 2025, highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus, genotype EA-2024-DI.2.1, caused systemic infections leading to a mass mortality event among the western migrating subpopulation of Eurasian cranes (Grus grus) in Germany. Gregarious behavior at feeding and resting sites likely promoted rapid viral spread within the population.

Zoos with avian populations are vulnerable to incursions of the high pathogenicity avian influenza virus (HPAIV) H5Nx due to the free-range husbandry, bird population density and shared open water areas between zoo birds and wild waterfowl. An outbreak of HPAIV H5N1, clade 2.3.4.4b, genotype EA AB, at the Zoological Garden Karlsruhe, Germany, in 2022, was managed applying legal restriction measures but exceptionally exempting culling orders for RT-qPCR-positive but clinically healthy birds. A critical factor in the zoo’s response was the implementation of a segregation concept approved in advance. The entire bird population could be rapidly separated into epidemiological housing units (epUs) cared for by separate staff. A total of 79 birds initially tested RT-qPCR-positive, but only 21 (26.6%) clinically diseased birds had to be euthanized or succumbed. Seroconversion amounted to 94.8% of the remaining 58 birds. Extensive RT-qPCR investigations of 3,634 samples confirmed infections remained confined to three initially infected epUs out of a total of 25. Spread of virus in the infected epUs was limited to 3 weeks after segregation. In the cohort of infected pelicans, surviving individuals remained seropositive with elevated levels of H5-specific antibody titers for the following 2 years suggesting ongoing protection. The described science-based control measures rested on a legally binding yet interpretive statement regarding the pertinent animal health legislation are exemplary for managing an outbreak of high pathogenicity avian influenza (HPAI) in zoos.

Global SARS-CoV-2 surveillance based on consensus genome sequences has enabled unprecedented tracking of viral spread and evolution but provides limited resolution of intra-sample diversity and limited sensitivity for the reliable detection of recombinant genomes. Here, we analyzed 3,360 SARS-CoV-2 genomes generated through regional surveillance in northeastern Germany (Mecklenburg–Western Pomerania) between 2022 and 2025. Reconstruction of local spatio-temporal variant dynamics revealed substantial lineage diversity, regional deviations from global circulation patterns, and periods of intense co-circulation favoring co-infection and recombination. To systematically identify recombinant genomes, we implemented a two-stage framework combining high-sensitivity consensus-based screening (REcombination BARcode detector REBAR) with targeted raw-read validation. Among 61 candidate recombinants, 21 were supported by read-level evidence, including 17 previously unrecognized genomes. Recombinant breakpoints clustered in functionally relevant genomic regions, particularly around ORF1b and the spike gene, indicating non-random patterns of genome exchange. Despite frequent co-infections, only a small subset of recombinants showed evidence of onward transmission, suggesting that successful establishment represents a major constraint on recombinant SARS-CoV-2 evolution. Our findings demonstrate that recombination-aware genomic surveillance complements routine consensus genome surveillance by resolving intra-sample diversity and recombinant diversity while remaining scalable for population-level implementation.

The global emergence of the avian influenza virus (AIV) H5N1 clade 2.3.4.4b since 2016 has caused substantial losses in wild bird and poultry populations, along with heightened risks of transmission to humans and other mammals. Vaccination of poultry has been a key strategy to curb the virus’s spread and mitigate its socioeconomic impact. This report describes an outbreak of high pathogenicity avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in a flock of 15,000 brown layer chickens (170 days old), all of which had received a four-dose vaccination regimen with H5N1/H5N8 commercial vaccines at 17, 50, 100, and 125 days of age. Despite this vaccination history, H5N1 infection was confirmed approximately seven weeks post-vaccination. H5N1 infection was confirmed by RT-qPCR, virus isolation, and full genome sequencing covering all eight gene segments, followed by phylogenetic and molecular analyses. Clinical signs included reduced feed intake, decreased egg production, and a cumulative mortality rate of 35% over 52 days. Hemagglutination inhibition (HI) testing with various H5 antigens revealed inconsistent antibody titers (geometric mean: 4.0 to 9.1 log2). Genetic analysis of the full-length HA and NA gene sequences further revealed strong similarity to contemporaneous H5N1 clade 2.3.4.4b strains circulating in Egypt, with multiple mutations in the HA head domain, particularly near immunogenic epitopes and receptor binding sites. These findings highlight the limitations of current vaccination strategies under conditions of antigenic mismatch and complex immunization schedules, emphasizing the need for improved vaccine matching and continuous molecular surveillance. To improve outbreak management in poultry, enhanced vaccination protocols, stringent biosecurity measures, and rigorous monitoring practices are critical.

Abstract Facing the emergence of highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in birds and its circulation in dairy cattle, rapid and reliable assays to detect HPAIV infection in humans are needed in diagnostic laboratories worldwide. We adapted and evaluated the performance of a molecular influenza A subtyping assay for the detection of A(H1N1)pdm09, A(H3N2), A(H5) and a pan-influenza A target on a high-throughput, fully automated platform. Previously published target primers and probes (Panning et al., Terrier et al., and the World Health Organization) were modified for target inclusivity/exclusivity, and adapted to compatibility with the Roche cobas5800/6800/8800 system as multiplex reaction. To evaluate the analytical performance of the new subtyping assay external quality assessment (EQA) samples were used as standards and quantified by digital-PCR. Limits of detection determined by 95% probit analysis were 754.0 digital copies (dcp)/ml for A(H1N1)pdm09, 148.0 dcp/ml for A(H3N2), 156 dcp/ml for A(H5) and 45.2 dcp/ml for the influenza A pan-target. Linearity was assessed for each subtyping target over at least four log-steps (r2: 0.9969–0.9998). The assay showed 100% agreement with EQA samples, eluates from the Friedrich Loeffler Institut (Germany) tested positive for different influenza A subtypes, and CE-IVD manual tests using 132 clinical samples. No false positives were detected in the exclusivity set. Our new subtyping assay is fully automated, easily scalable and can be used in surveillance and routine clinical settings enabling the detection of HPAIV infections in humans and may contribute to limit potential transmission chains at an early stage. Words: 250/250.

RESEARCH HIGHLIGHTS:Multiple independent HPAIV H5N1 euBB incursions occurred in Germany in 2023.HPAIV H5N1 euBB caused mass mortality in black-headed gulls and common terns.Outbreaks reduced gull and tern breeding populations by 16% and 6%, respectively.Rising H5-seroprevalence in terns may have contributed to the fade-out of genotype euBB.

Wild waterfowl and shorebirds are the primary reservoir of influenza A viruses in nature. The role of wild birds from other taxonomic groups remains insufficiently studied or is a subject of debate. This applies in particular to Passeriformes, the most diverse avian order, accounting for approximately 60% of the global bird population, where the role in circulation of influenza A viruses is underexplored. We used serological, virological, and PCR-based methods to survey avian influenza viruses in Passeriformes birds (65 species, 20 families) in Ukraine over a 20-year period, 2004-2025. Antibodies to influenza viruses were detected in serum and egg yolk of seven passerine species, with average seroprevalence 1.24% in sera and 8.94% in yolk samples. Seroprevalence varied across species, ranging from 1.96 to 27.2%. Virological screening resulted in the isolation of two viruses from Fieldfares (Turdus pilaris) of the subtypes H1N1 and H7N1. The overall infection rate based on virus isolation was 0.15%, while local infection rate in Fieldfares reached 11.1%. According to PCR results, 41 positive samples were detected, representing 3.61% of all tested birds (ranging from 1.42-9.1%), and by location ranged from 6.25-9.1%. Sequencing and phylogenetic analyses of H1N1 (Fieldfare), H7N1 (Fieldfare), H3N8 (Great Tit Parus major) influenza viruses confirmed them as Eurasian lineage low pathogenic avian influenza viruses and with close relatedness to viruses of the same subtypes circulating among wild waterfowl.

Highly pathogenic avian influenza of subtype H5N1 (HPAIV H5N1) reached Antarctica in late 2023/2024 breeding season, but the host ecology and movement processes shaping its incursion and spread remain poorly resolved. We investigated these mechanisms by integrating viral genomics, multi-season serology, and seabird movement data at King George Island (Fildes Peninsula, South Shetland Islands), a likely gateway between South America and the Antarctic Peninsula. Across three breeding seasons (2022/23–2024/25), we collected swabs and blood serum from key scavenging and predatory seabirds (Brown skua, South Polar skua, Southern Giant petrel) and paired these data with light-level geolocator tracks (10–16 full annual tracks per species) and breeding-season GPS tracks (10–20 individuals per species). Phylogenetic analyses including three newly generated HPAIV H5N1 genomes from infected Brown skuas placed these viruses within the South Georgia–Antarctic lineage, consistent with introduction via South Georgia followed by onward spread within the region. Longitudinal serology revealed a rapid increase in cumulative exposure in scavenging species, with antibodies against influenza A virus’ nucleoprotein and hemagglutinin subtype H5 rising to high levels by 2024/25 in Brown skuas (52.2%) and Southern Giant petrels (62.5%), but remaining markedly lower in South Polar skuas (12.5%), consistent with their more pelagic and less scavenging foraging behaviour. Tracking showed that migratory connectivity was greatest during the non-breeding period but contracted rapidly during colony return and early breeding. In contrast, network simulations parameterized by high-percentile breeding-season foraging distances indicated strong peninsula-wide connectivity and non-zero potential links toward South Georgia and other sub-Antarctic stepping-stones. Together, these results provide a mechanistic, host-based framework suggesting that HPAIV H5N1 likely reached Antarctica through the South Georgia pathway, but that its regional spread was shaped primarily by foraging ecology and breeding-season movement networks rather than by long-distance migration.

High pathogenicity avian influenza (HPAI) H5 viruses of the Goose/Guangdong lineage continue to diversify through recurrent reassortment, sustaining an evolving threat to animal health and transboundary disease control in Europe. While multiple incursions of HPAI H5 clade 2.3.4.4b viruses have been documented, the regional and ecological contexts that facilitate reassortment during ongoing circulation remain insufficiently characterized. In this study, we combined nationwide avian influenza surveillance in Germany with whole-genome sequencing and Bayesian phylogeographic reconstruction to investigate virus evolution during the 2024-2025 season. Phylogenetic analyses identified multiple genetically distinct HPAI H5 clade 2.3.4.4b genotypes, including several reassortants that emerged during the observation period. The inferred evolutionary histories indicate repeated virus introductions into Germany, followed by local diversification and onward dissemination across Europe. Spatio-temporal reconstruction consistently highlighted coastal regions along the North Sea and Baltic Sea as major interfaces of viral exchange, in line with their role as convergence zones of migratory bird flyways. Rather than representing a primary source of viral lineages, phylogeographic analyses identified Germany as prominent node within the European HPAI network, consistent with a central role in virus movement and mixing. This pattern was observed across multiple genotypes, supporting a generalized role of this region in HPAI evolution. Together, these findings provide a continental-scale perspective on HPAI virus dynamics and emphasize the value of integrated surveillance approaches combining wild bird monitoring and whole-genome sequencing. From an epidemiological standpoint, expanding surveillance frameworks to include low pathogenic avian influenza viruses is likely to improve early detection of reassortment events and enhance preparedness for the emergence of novel HPAI variants.

Avian influenza viruses undergo frequent genetic reassortment, which can coincide with phenotypic changes in transmission, pathogenicity, and host species niche. Since 2020, clade 2.3.4.4b H5 high pathogenicity avian influenza viruses (HPAIVs) have driven a global panzootic, causing mass mortality in wild birds, poultry, and, for the first time, repeated spillover infections in a variety of mam-malian species. This resurgence of H5 HPAIV has coincided with a dramatic increase in the number of circulating reassortant strains; however, the scale, impact and drivers of these reassortants remain unknown. Here, we combined statistical and phylodynamic modelling to reconstruct the global evolutionary dynamics of H5Nx viruses across four epizootic seasons (2020-2024). We identified 209 genetically distinct reassortants, stratified into three transmission categories based on their phylogenetic and epidemiological profiles. Accounting for sampling depth and HPAIV incidence, we estimated that reassortants emerged most frequently in Asia, but ‘major’ reassortants associated with increased host range, inter-seasonal persistence, and long-range dissemination, more frequently emerged from Europe. Altogether, reassortant emergence followed an episodic pattern in which most reassortants were transient, but 3% seeded large clusters of secondary reassortants soon after their own emergence. Statistical modelling revealed that reassortant success was strongly shaped by ecological factors, including circulation in specific wild bird orders and the ability to infect a wider range of host niches. Reassortant dispersal was linked to poultry trade intensity, particularly in North America. Collectively, our findings reveal reassortment dynamics in H5 HPAIVs and identify key virological and ecological drivers underpinning the emergence and global spread of successful reassortants. These insights support the importance of enhanced surveillance to track evolution of H5 HPAIV and identify traits relevant for consideration in pandemic risk assessment. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, BB/V011286/1, BB/X006204/1, BB/X006166/1, BB/Y007271/1, BB/Y007298/1 Biotechnology and Biological Sciences Research Council - Institute Strategic Grants, BBS/E/RL/230002C, BBS/E/RL/230002D Medical Research Council, MR/Y03368X/1 National Natural Science Foundation of China, https://ror.org/01h0zpd94, 32061123001, 32425053, 32200416 National Key Research and Development Program of China, 2023YFC2307500 European Union, 727922, 874850, 101094685, 101084171, 874735 Fonds National de la Recherche Scientifique, F.4515.22 Fonds voor Wetenschappelijk Onderzoek — Vlaanderen, G098321N

In autumn 2025, an unprecedented mass mortality event was observed among the western migrating subpopulation of Eurasian cranes ( Grus grus ) in Germany. Systemic infection with highly pathogenic avian influenza virus H5N1, clade 2.3.4.4b, genotype DI.2.1, was identified as the cause of acute death. The gregarious behavior of cranes at feeding and resting sites likely has contributed to the rapid and massive dissemination of viruses within the crane population. ### Competing Interest Statement The authors have declared no competing interest. European Union, 101084171

Highly pathogenic avian influenza viruses (HPAIV) of the H5 goose/Guangdong (gs/GD) lineage have repeatedly emerged in Germany since 2006. Rooted in the respective gs/GD lineages, HPAIV in Germany have genetically diversified into a plethora of clades and subclades and evolved into an assortment of sub- and genotypes. This technical note summarizes the genotype differentiation procedure, lists the successfully assigned genotypes and supplies corresponding reference sequences.

In Europe, highly pathogenic avian influenza (HPAI) virus circulates in avian wildlife, undergoing frequent reassortment, sporadic introductions in domestic birds, and spillover to mammals. An H5N1 clade 2.3.4.4b reassortant, EA-2023-DG, affecting wild and domestic birds was detected in western Europe in November 2023. Six of its RNA segments came from the EA-2021-AB genotype, but the polymerase basic 2 and polymerase acidic segments originated from low pathogenicity avian influenza viruses. Discrete phylogeographic analyses of concatenated genomes and single polymerase basic 2 and polymerase acidic segments suggested reassortment in summer 2023 near the southwestern Baltic Sea. Subsequent continuous phylogeographic analysis of all concatenated EA-2023-DG genomes highlighted circulation in northwestern Europe until June 2024 and long-distance dispersal toward France, Norway, England, Slovakia, Switzerland, and Austria. Those results illustrate the value of phylodynamic approaches to investigate emergence of novel avian influenza virus variants, trace their subsequent dispersal history, and provide vital clues for informing outbreak prevention and intervention policies.

High-pathogenicity avian influenza viruses (HPAIVs) of the goose/Guangdong lineage are enzootically circulating in wild bird populations worldwide. This increases the risk of entry into poultry production and spill-over to mammalian species, including humans. Better understanding of the ecological and epizootiological networks of these viruses is essential to optimize mitigation measures. Based on full genome sequences of 26 HPAIV samples from Iceland, which were collected between spring and autumn 2022, as well as 1 sample from the 2023 summer period, we show that 3 different genotypes of HPAIV H5N1 clade 2.3.4.4b were circulating within the wild bird population in Iceland in 2022. Furthermore, in 2023 we observed a novel introduction of HPAIV H5N5 of the same clade to Iceland. The data support the role of Iceland as an utmost northwestern distribution area in Europe that might act also as a potential bridging point for intercontinental spread of HPAIV across the North Atlantic.

In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b H5N1 infections in dairy cows were first reported from Texas, USA. Rapid dissemination to more than 190 farms in 13 states followed. Here, we provide results of two independent clade 2.3.4.4b experimental infection studies evaluating (i) oronasal susceptibility and transmission in calves to a US H5N1 bovine isolate genotype B3.13 (H5N1 B3.13) and (ii) susceptibility of lactating cows following direct mammary gland inoculation of either H5N1 B3.13 or a current EU H5N1 wild bird isolate genotype euDG (H5N1 euDG). Inoculation of the calves resulted in moderate nasal replication and shedding with no severe clinical signs or transmission to sentinel calves. In dairy cows, infection resulted in no nasal shedding, but severe acute mammary gland infection with necrotizing mastitis and high fever was observed for both H5N1 genotypes/strains. Milk production was rapidly and drastically reduced and the physical condition of the cows was severely compromised. Virus titers in milk rapidly peaked at 108 TCID50/mL, but systemic infection did not ensue. Notably, adaptive mutation PB2 E627K emerged after intramammary replication of H5N1 euDG. Our data suggest that in addition to H5N1 B3.13, other HPAIV H5N1 strains have the potential to replicate in the udder of cows and that milk and milking procedures, rather than respiratory spread, are likely the primary routes of H5N1 transmission between cattle.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

Several subtypes and many different genotypes of highly pathogenic avian influenza viruses of subtype H5 clade 2.3.4.4b have repeatedly caused outbreaks in Germany. Four new highly pathogenic avian influenza genotypes emerged in November 2023 after reassortment with low pathogenicity precursors, replacing genotype BB, which had dominated in Europe since 2022.