Research note: Molecular characteristics and genetic evolution of H1N1 avian influenza virus from wild birds in Shanghai, China

Abstract

The H1N1 influenza virus is a major pandemic and seasonal pathogen with a broad host range, posing a substantial threat to human health and underscoring the need for continuous surveillance. Wild birds, as natural reservoirs of avian influenza viruses (AIVs), carry H1N1 strains capable of reassorting with other influenza viruses, which can drive pandemic emergence. The global migration of wild birds facilitates the spread of these viruses, and their interactions with poultry increase the risk of cross-species transmission, further amplifying the public health threat. However, knowledge of H1N1 genetic diversity in wild birds remains limited. Database analysis shows 80% of avian-origin H1N1 isolates come from wild birds across over 40 countries, mainly in North America, Europe and Asia. This study characterized the molecular traits and genetic evolution of four H1N1 AIVs isolated from common teal and spot-billed ducks during 2019–2021. Phylogenetic and sequence analyses revealed these viruses cluster into distinct lineages, divergent from mammalian H1N1 strains, with complex genetic origins involving frequent recombination and high diversity. Frequent wild bird–poultry transmission elevates zoonotic risks. Our findings highlight wild birds’ critical role in H1N1 transmission and confirm their role as an H1N1 gene pool, emphasizing the need for sustained monitoring and research.

Introduction

The H1N1 influenza virus has been responsible for two major pandemics in history: the "Spanish" flu (H1N1) and the 2009 H1N1 pandemic, which resulted in tens of millions of deaths worldwide (Neumann et al., 2009). As a seasonal influenza virus, H1N1 causes approximately one billion infections annually, posing a significant threat to human health and imposing a heavy economic burden on societies.

The H1N1 influenza virus can infect not only humans but also various animals, such as swine, poultry, wild birds, horses, and marine mammals. It can bind to both sialic acid α−2,3-Gal and α−2,6-Gal receptors, thereby expanding its host tropism range (Koo et al., 2018; Matrosovich et al., 2000). Wild birds, as natural hosts of low-pathogenic avian influenza viruses (LPAIVs), play a crucial role in viral dissemination due to their long-distance migratory behavior (Ramey et al., 2015). Previous studies have shown that the H9N2 virus, carried by wild birds, can co-circulate with the H1N1 virus, potentially enhancing its adaptability to mammals through genetic recombination (Ge et al., 2018). The H1N1 virus isolated from wild birds has been shown to exhibit enhanced replication ability in both turkey and mouse models, suggesting that it may have potential pathogenicity to poultry and mammals (Petersen et al., 2018). Meanwhile, research reports indicate that the ongoing circulation of the H1N1 virus among wild birds may contribute genetic segments to highly pathogenic strains such as H5N1, thereby increasing the risk of recombination (Abente et al., 2017). Furthermore, H1N1 AIVs can establish long-term latent infections among poultry and wild birds. Monitoring areas where wild birds and domestic poultry interact has revealed that the H1N1 influenza virus co-circulates among these species, with evidence of close contact among these species, pigs, and humans, potentially increasing the risk of zoonotic transmission (Jessica et al., 2021).

Shanghai is a key stopover and wintering site on the East Asian–Australasian Flyway, hosting millions of migratory birds annually. AIV surveillance in wild birds has been ongoing here since 2006, with H5N6, H4N2, and H6N2 as the most frequently isolated subtypes (40% of total). However, long-term monitoring shows that H1N1 AIV prevalence is low, and knowledge of its genetic characteristics as well as its patterns of mutation and reassortment remains limited. In this study, wild bird samples collected in Shanghai during 2019–2021 were tested for AIVs, yielding four H1N1 isolates. Molecular and phylogenetic analyses were performed to clarify the epidemiological characteristics of H1N1 AIVs in wild bird populations. Overall, this study underscores the continued relevance and public health implications of H1N1 AIVs of wild bird origin.

Materials and methods

Sample collection

Between 2019 and 2021, AIV surveillance was carried out in the Jiuduansha (31°06′–31°14′ N, 121°46′–122°15′ E) and Nanhui Dongtan (30°51′–31°06′ N, 121°50′–121°51′ E) wetlands in Shanghai, China. A total of 3,338 oropharyngeal and cloacal swabs were collected from apparently healthy wild birds. The samples were stored in viral transport medium (2 mL), transported to the laboratory at 4 °C within 24 hours, and stored at −80 °C until analysis.

Virus detection and sequencing

Viral RNA was extracted using the MagMAX™ Pathogen RNA/DNA Kit (Applied Biosystems, USA) with a MagMAX-96 Express Instrument, following the manufacturer’s protocol. Influenza A virus (IAV) was detected by real-time reverse transcription polymerase chain reaction (RT-qPCR) with matrix gene primers and a probe (WHO, 2009). Viral RNA from positive samples was reverse transcribed to generate cDNA using the Uni12 primer (5′-AGC AAA AGC AGG-3′) and the PrimScript™ II 1st Strand cDNA Synthesis Kit (Takara, Japan). The eight viral gene segments were amplified with universal primers (Hoffmann et al., 2001).

Sequence and phylogenetic analysis

As of May 1, 2025, all available H1N1 influenza virus sequences were downloaded from the Global Initiative on Sharing Avian Influenza Data (GISAID) EpiFlu database (GISAID Initiative) to perform host, temporal, and geographical distribution analyses. We aligned and compiled our sequences using DNAMAN 7.0 software, and retrieved related reference sequences from GenBank (National Center for Biotechnology Information). All sequences were aligned using MEGA X, and low-quality sequences were filtered out to retain high-quality datasets for phylogenetic analysis. Phylogenetic trees were constructed using the neighbor-joining method in MEGA X, with the Kimura 2-parameter model and supported by 1,000 bootstrap replicates. For genotyping, duplicate sequences (sharing identical sampling year, host species, and location within a clade) were removed, retaining one representative sequence per group. Gene groups (lineages) were defined based on a sequence identity threshold of 95%. Accordingly, phylogenetic clades were delineated as distinct lineages or sublineages.

Results and discussion

Prevalence of H1N1 viruses in global

The H1N1 influenza virus is characterized by rapid mutation and high transmissibility. It re-emerges annually with varying intensity and has been responsible for multiple global pandemics. To better characterize its epidemiological features, we downloaded all available H1N1 data (as of May 1, 2025) from the GISAID EpiFlu database (GISAID Initiative), resulting in a total of 166,945 isolate records. Based on host origin, the isolates were categorized as follows: 90.02% were of human origin, 9.63% were of animal origin, and the remainder were from environmental, laboratory, or unknown sources (Fig. 1a). Among animal-origin isolates, mammalian strains accounted for 93.51% and avian strains 6.49% (Fig. 1b); swine-derived isolates made up 99.43% of mammalian strains (Fig. 1c). Among avian isolates, 80.84% came from wild birds and the remainder from ducks, chickens, and other avian species (Fig. 1d).

Fig. 1.

Spatial and temporal distribution of global H1N1 isolates. (a)-(d) Proportion chart of H1N1 isolates classified by different hosts. (e) The global distribution of H1N1 isolates in human and avian species. Map generated using ArcGIS version 10.2. Size of each circle represents the number of isolates and the legend circle represents 300 isolates. The shades of orange represent the number of H1N1 isolates from human in different countries. Red, blue, green, and yellow colors represent the number of H1N1 isolates from wild duck, duck, chicken, and other avian, respectively. (f) Number of H1N1 isolates from wild birds in the world from 1976 to 2024.

Global distribution and host analysis of these isolates showed that H1N1 viruses have been detected in avian samples from over 40 countries, with the majority reported from North America, Europe, and three Asian nations (Korea, Japan, and China) (Fig. 1e). The database contained 1,075 avian H1N1 isolates, over 80% of which were derived from wild birds. Annual wild bird-derived H1N1 isolates have risen substantially since 2002, a trend likely driven by expanded global AIV surveillance in wild bird populations (Fig. 1f). Given the strong research focus on highly pathogenic H5/H7 viruses and prevalent strains like H9N2 (Abente et al., 2017; Ramey et al., 2015), knowledge of H1N1 transmission dynamics in wild birds remains limited. Therefore, sustained surveillance and research focused on H1N1 AIVs in wild birds are essential for the early detection and preparedness against potential influenza pandemics.

Prevalence of H1N1 viruses in wild birds in Shanghai

Between 2019–2021, 3,339 oropharyngeal and cloacal swab samples were collected from healthy wild birds in Shanghai, China; RT-qPCR testing yielded 538 AIV-positive samples (16.11%). We characterized the HA and NA genes of most positive samples, with this study focusing primarily on four isolated H1N1 strains: one from a common teal, designated as A/common teal/Shanghai/NH110923/2019 (H1N1) (abbreviated as NH110923), and three from spot-billed ducks: A/Spot-billed duck/Shanghai/JDS110424/2020 (H1N1) (JDS110424), A/Spot-billed duck/Shanghai/JDS110425/2020 (H1N1) (JDS110425), and A/Spot-billed duck/Shanghai/JDS110431/2020 (H1N1) (JDS110431). Whole-genome sequencing was successfully completed for all isolates, with the exception of the PB1 gene sequence of JDS110424, which failed to be sequenced, likely due to sample degradation. Sequencing results were deposited in GenBank under accession numbers MT747873–MT747880, OR569039–OR569045, OR574150–OR574157, and OR569029–OR569036.

Molecular characterization

Molecular characterization revealed that the HA cleavage site amino acid motif of all four H1N1 isolates was PSIQSR↓GLF, indicating low-pathogenic avian influenza virus (LPAIV) properties. Although wild bird-transmitted influenza viruses are typically low-pathogenic (Fouchier and Guan, 2013), wild birds can also carry highly pathogenic avian influenza viruses (HPAIV) that may disperse during long-distance migrations (Abente et al., 2017; Ramey et al., 2015). The HA protein substitution 226Q –linked to heightened disease severity in the 2009 H1N1 pandemic (Koo et al., 2018) –was detected at the human receptor-binding site of these isolates. No other mutations associated with mammalian adaptation or enhanced virulence were identified.

Sequence analysis

In this study, the three 2020 isolates (JDS110424, JDS110425, JDS110431) were nearly identical, with 99.5%–100% nucleotide identity across their seven genes. By contrast, their Polymerase Acidic (PA) genes showed 95.8%–99.9% nucleotide identity. The 2019 isolate (NH110923) shared relatively low nucleotide identity (93.8%–99.0%) with the three 2020 isolates across all eight genomic segments.

BLAST analysis showed that the HA, NA, M, and Nucleoprotein (NP) genes of the three 2020 isolates were closely related to those of A/Mallard (Anas platyrhynchos)/South Korea/KNU2021-5/2021 (H1N1). Their PB2 and PB1 genes clustered with A/duck/Kaohsiung/20WB0201-46/2020 (H3N8) and A/Wild Duck/South Korea/KNU2020-110/2020 (H9N2), respectively. Meanwhile, their PA and NS genes were highly homologous to A/Common Teal (Anas crecca)/South Korea/KNU2021-22/2021 (H8N4) and A/duck/Taipei/18WB0137-6-10-E1/2018 (H7N1), respectively. For isolate NH110923 (2019), PB2, PB1, and NS genes shared the highest nucleotide identity with A/wild duck/South Korea/KNU2018-26/2020 (H1N1); HA and NA genes were closely related to A/duck/Taipei/19WB0254-19-E1/2019 (H1N1). PA and NP genes showed the highest homology to A/Bean Goose (Anser fabalis)/South Korea/KNU2021-50/2021 (H5N8) and A/duck/Taipei/19WB0280-21/2019 (H4N1), respectively, and its M gene clustered with A/duck/Mongolia/652/2019 (H4N6).

Phylogenetic analyses

Analyzing the evolutionary dynamics of H1N1 across hosts is critical for clarifying its epidemic patterns in different species. Based on collected H1N1 gene sequences, we performed phylogenetic analysis and constructed evolutionary trees categorized by host type (wild birds, other avian species, humans, swine, other mammals). The HA and NA genes of the four isolates were closely related to those from wild ducks and other avian species in Europe and Asia, but distantly related to human, swine, and other mammalian isolates (Fig. 2a, 2b). Consistent with previous research showing that wild bird H1N1 viruses are highly similar to swine and domestic poultry strains (Jessica et al., 2021), we observed that a wild bird isolate (A/Blue-winged teal/Mexico/HA UIFMVZ322/2016) clustered with prevalent swine strains (Fig. 2a), indicating potential genetic segment recombination.

Fig. 2.

Phylogeny of HA and NA genes of H1N1 isolates. In the phylogenetic tree, HA genes (c, n = 479) and NA genes (d, n = 435) sequences were respectively classified into different gene groups. The size of the pink circle indicated neighbour-joining bootstrap values, values less than 75% were not shown. Four H1N1 isolates characterized in this study are indicated by red five stars. The region and host information of major clades are labeled in different colors next to the phylogenetic tree.

To further characterize the limited avian-origin H1N1 strains, we divided sequences into distinct lineages and sublineages (Fig. 2c, 2d). In brief, avian H1N1 HA and NA genes fall into three lineages: Eurasian, North American, and South American. The Eurasian HA lineage was subdivided into six host- and geography-defined sublineages (Europe-Asia-1/2/3, Europe, Asia-1/2; Fig. 2a); the North American HA lineage contained two sublineages (North America-1/2). Most Europe-Asia-1 and North America-1 strains were from domestic poultry, while other sublineages were mainly from wild birds (with occasional detection in poultry). The South American and North America-2 lineages were geographically restricted, with strains isolated only from wild birds and poultry in South America and North America, respectively. Europe-Asia-2/3 were hybrid Eurasian branches, indicating ongoing genetic exchange and evolution; the four isolates in this study clustered within Europe-Asia-3. Asia-1/2 strains were almost exclusive to Asia, and Europe lineage strains dominated in Europe.

Similar to HA gene patterns, the NA gene's North America-1 and Worldwide lineages were primarily from poultry, while others were mostly from wild birds (Fig. 2d). Both North America-2 and South American NA lineages showed strong geographical restriction, mirroring their HA counterparts. Europe-Asia NA strains were widespread in Eurasia, whereas Europe and Asia lineages dominated their respective regions. Genotyping revealed a complex Eurasian H1N1 evolutionary history. Our isolates clustered within Eurasian/Asian sublineages, indicating this region is a major H1N1 AIV endemic area. Recombination analysis showed divergent phylogenetic positions between the 2020 and 2019 isolates (data not shown). Wild bird isolates spread between Asia and Europe, continuously exchanging genes with poultry viruses–a process that may facilitate cross-species transmission (Fouchier and Guan, 2013). Given extensive wild bird migration, these isolates likely represent novel viruses from complex recombination events. Their ongoing evolution and spread highlight the critical need for sustained wild bird H1N1 surveillance.

In conclusion, this study characterized the genetic and biological features of H1N1 AIVs. Phylogenetic analysis divided these viruses into distinct lineages, with our isolates clustering within the Eurasian lineage–clearly divergent from human, swine, and other mammalian influenza viruses. The high similarity of the four isolates to AIVs along the East Asia–Australasia migration route, and their highest homology with known avian and poultry strains, suggests complex genetic origins and distinct diversity. This pattern is consistent with the high recombination tendency of wild bird AIVs. Therefore, sustained surveillance and research on wild bird H1N1 AIVs are critical for understanding pandemic emergence, evolution, and cross-species transmission.

Data availability statement

The epidemiological data of H1N1 influenza viruses used were downloaded from GISAID (GISAID Initiative). We gratefully acknowledge the authors and laboratories for sharing the AIVs sequences in the GISAID and NCBI databases. The AIV sequences generated in this study have been deposited in the NCBI GenBank (National Center for Biotechnology Information) with accession numbers MT747873-MT747880, OR569039-OR569045, OR574150-OR574157, and OR569029-OR569036, respectively.

CRediT authorship contribution statement

Ling Tang: Writing – original draft, Software, Investigation, Formal analysis, Data curation. Rui Wang: Writing – review & editing, Validation, Supervision, Resources. Guimei He: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.

Disclosures

The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.