This study generated 260 new genomic sequences of influenza A(H1N1)pdm09 viruses collected in Yunnan province, China, between 2018 and 2023, providing new regional data for studying molecular characterization and evolution of A(H1N1)pdm09 within the global surveillance framework.
Abstract
Research on regional circulation and evolution of influenza A viruses before and after the COVID-19 pandemic is crucial for informing vaccine updates and antiviral drug development. This study generated 260 new genomic sequences of influenza A(H1N1)pdm09 viruses collected in Yunnan province, China, between 2018 and 2023. Comparative genomics analyses elucidated their evolutionary characteristics and dynamics. Epidemiological analysis identified key risk factors (sex, age, occupation) for influenza infection. Phylogenetic analyses revealed the sequence divergences between the vaccine strains and Yunnan circulating strains, especially in the 2020-2024 influenza seasons. The subclade reassortment events were extremely limited among these sequenced Yunnan strains, suggesting the reassortment may be not a major contributor for the circulation and evolution of influenza A(H1N1)pdm09 viruses in Yunnan during these influenza seasons. We detected the elevated evolutionary pressures acting on the specific gene segments, reflected in increased dN/dS ratios, particularly for envelope proteins. Furthermore, numerous amino acid substitutions (e.g., S185I/T) within HA antigenic epitopes and receptor binding sites were identified in most Yunnan strains, indicating potential roles of antigenic drift in modulating viral antigenicity and host adaptation. Notably, 17 amino acid substitutions in HA and NA (including HA: N156K) accumulated to higher frequencies during the 2022-2023 and 2023-2024 seasons. These changes likely represented the molecular signature of contemporary A(H1N1)pdm09 viruses in Yunnan. Collectively, this study explored the molecular evolutionary dynamics of A(H1N1)pdm09 viruses in Yunnan province during diverse influenza seasons, providing new regional data for studying molecular characterization and evolution of A(H1N1)pdm09 within the global surveillance framework.
Background Influenza is a major cause of acute respiratory infections worldwide. In tropical regions such as Sub‐Saharan Africa, influenza circulates year‐round with irregular peaks, yet genomic data guiding prevention strategies remain limited. This study characterized the genetic diversity and seasonal dynamics of influenza A(H1N1)pdm09 and A(H3N2) viruses circulating in Burkina Faso in 2024. Methods A cross‐sectional study was conducted from January to December 2024, including seven sentinel surveillance sites. Patients presenting with influenza‐like illness or severe acute respiratory illness were enrolled. Respiratory specimens were tested by real‐time RT‐PCR. Influenza‐positive samples with a cycle threshold ≤ 30 underwent whole‐genome sequencing using Oxford Nanopore and Illumina platforms. Phylogenetic analyses and clade assignment were performed using MEGA Version 12. Results Out of 2951 samples tested, 6.74% were positive for influenza viruses. Females had higher odds of influenza positivity than males (OR = 1.48; 95% CI: 1.11–1.98). A significantly higher risk of positivity was observed in the age groups of 5–15 years (OR = 1.67; 95% CI: 1.07–2.52; p = 0.02) and 25–50 years (OR = 2.54; 95% CI: 1.53–4.03; p < 0.001). Influenza A(H3N2) peaked in July, while A(H1N1)pdm09 peaked in October. Phylogenetic analysis of 43 genomes revealed co‐circulation of multiple clades within both subtypes. Conclusion Influenza A viruses circulating in Burkina Faso in 2024 showed substantial genetic diversity, underscoring the need for continuous genomic surveillance to inform vaccine strain selection and public health strategies in tropical Africa.
C. Sawadogo, A. Cissé, N. Gouba et al.· Advances in Virology· 0 citations
Objective To elucidate the epidemiological distribution patterns of avian influenza virus (AIV) in the external environment of western Zhejiang from 2014 to 2025, analyze the molecular epidemiological characteristics of the H9N2 subtype, and assess its public health risks. Methods According to the Zhejiang Provincial Surveillance Program for Avian Influenza in Occupationally Exposed Populations and External Environments, real-time RT-PCR was used to detect AIV subtypes in environmental specimens. H9N2-positive samples with cycle threshold values <30 were inoculated into specific pathogen-free (SPF) embryonated chicken eggs for virus isolation, followed by whole-genome sequencing and bioinformatics analysis for phylogenetic and molecular characterization. Results A total of 7,762 specimens were tested from 2014 to 2025, with an overall positivity rate of 34.64% (2,689/7,762) for AIV. Significant differences in positivity rates were observed in seasons, regions, sampling sites, and specimen types (all p < 0.001). AIV activity peaked in winter and spring, with the highest rates detected in live poultry markets and chopping board swabs. The H9 was the predominant subtype, with co-circulation of multiple subtypes. All 48 H9N2 subtype isolates belonged to the G57 genotype, with the hemagglutinin (HA) and neuraminidase (NA) genes falling into the Y280-like branch, while the internal genes exhibited a mosaic pattern combining G1-like and F/98-like lineages. Molecular characterization analysis revealed multiple mammalian adaptive mutations, involving alterations in receptor-binding sites (T163N, H191N, T197D, T198V, Q234L, Q235M), antigenic epitopes (D280G, N285S), and glycosylation sites (218NRTF, 313NCSK). NA stalk deletion (62–64 aa), along with multiple mutations in the hemadsorption site (E/K368N, D369S/G, D401G/V, N402D, W403L/R, Q432H). Additionally, multiple key amino acid substitutions were also identified in the internal proteins. Conclusion The external environment in western Zhejiang exhibits a high prevalence of AIVs with pronounced spatiotemporal clustering. H9 was the dominant subtype and co-circulated with multiple subtypes, with live poultry markets and slaughterhouses identified as high-risk settings. The H9N2 subtype AIV has accumulated multiple mammalian adaptive mutations, and exhibits genetic linkages across eastern Chinese provinces. These findings collectively underscore the need for an integrated One Health surveillance and early-warning system to reduce the risk of human infections with avian influenza.
Shiwang Huang, B. Zhan, Min Wang et al.· Frontiers in Public Health· 0 citations
ABSTRACT Subtype H9N2 of the avian influenza virus (AIV) poses a growing threat to the poultry industry and public health. However, since 2014, there has been no systematic study of its genetic evolution, genotype, spatial dynamics, and pathogenicity in China. Therefore, we performed a large-scale sequence analysis of the genome of Chinese H9N2 viruses from 2014 to 2025 using public databases and laboratory isolates. We identified 52 different genotypes in 1,591 H9N2 viruses, including 4 previously recognized genotypes (G6, G57, G58, and G68) and 48 newly defined genotypes (G118–G163) in this study. G57 and G118 were the main epidemic genotypes in China from 2014 to 2025. Bayesian phylogeographic analysis showed that there were 12 obvious migration paths for the spread of H9N2 AIVs in China from 2017 to 2022. In particular, the South China region was the main transmission centre. H9N2 AIV continues to circulate in chickens and ducks in China and spreads to other hosts. The H9N2 AIVs with the G57 and G118 genotypes could effectively replicate in MDCK, CEF, A549, and HBE cells with titres of 0.97–8.5 lgTCID50/mL. The G57 and G118 genotypes H9N2 viruses preferentially bound to α-2,6-linked sialic acid glycopolymers (human receptors), and effectively replicate in multiple organs of mice and chickens and cause pathological changes in the lungs. Thus, it is necessary to strengthen the monitoring and prevention of H9N2 AIVs in China.
Zifeng Pang, Peiting Zhong, Cuishan Mai et al.· Emerging Microbes and Infect...· 0 citations
The H3 subtype avian influenza virus (AIV) poses a substantial global public health threat due to its high host adaptability and ongoing evolution. The recent emergence of novel H3N8 and H3N3 AIVs associated with cross-species transmission underscores the urgent need for enhanced epidemiological surveillance. In this study, we conducted surveillance and characterization of H3 AIVs based on a total of 737 poultry samples collected across 21 Chinese provinces from November 2022 to December 2023. Of these, 69 (9.4%) tested positive for H3 AIV by RT-qPCR, and one H3N8 isolate and ten H3N3 isolates were obtained for whole-genome characterization. We performed whole-genome sequencing, phylogenetic analysis, reassortment inference, and evaluation of key amino acid substitutions, alongside antigenic characterization using hemagglutination inhibition (HI) assays and an in vivo mouse challenge experiment. The H3N8 isolate was identified as a triple-reassortant virus possessing the Eurasian avian H3 gene, the North American avian N8 gene, and H9N2-derived internal genes. The H3N3 isolates represented reassortant viruses that had acquired the HA gene from the novel H3N8 AIV lineage, the NA gene from H10N3 AIV, and internal genes from H9N2 AIV. All isolates exhibited HA cleavage sites characteristic of low pathogenic avian influenza viruses. Additionally, several amino acid substitutions previously associated with enhanced mammalian adaptation were identified, including L89V and I292V in PB2 and H436Y in PB1. In a BALB/c mouse challenge experiment, the representative H3N8 virus established infection without prior adaptation and replicated predominantly in the upper respiratory tract, with detectable viral RNA in respiratory tissues and limited extrapulmonary dissemination. Antigenic analysis revealed no cross-reactivity between the novel H3 AIVs and H5, H7, or H9 AIVs as measured by HI. Based on molecular and phylogenetic characterization, antigenic assessment, and preliminary mammalian infection data, our findings provide evidence suggesting a potential public health risk. We recommend intensified surveillance of H3 AIVs in poultry and accelerated vaccine development to curb viral spread and improve public health preparedness.
Xue Wang, Hao Shi, Peidong Li et al.· The Veterinary Journal· 0 citations
Respiratory viral infections, particularly Influenza A and SARS‐CoV‐2, remain major public health concerns in endemic settings such as Pakistan, where high influenza‐like illness burden and limited genomic data underscore the need for surveillance to guide vaccine strategies. This study assessed respiratory virus circulation and genetic diversity among ILI cases at a secondary‐care hospital in Islamabad during the 2025–2026 winter season. Among 170 qRT‐PCR‐tested cases, Influenza A was detected in 24.1%, SARS‐CoV‐2 in 1.17%, and Influenza B was not detected. Influenza A positivity was slightly higher in females (25.0%), with highest burden in individuals aged 11–20 years. Whole‐genome sequencing of 21 Influenza A(H3N2) isolates revealed co‐circulation of subclade K (52.4%), J.2.2 (28.6%), and J.2 (9.5%). Phylogenetic analysis showed subclade K clustering with recent isolates from North America, Oceania, Europe, and WHO‐recommended 2026–2027 Northern Hemisphere H3N2 vaccine strains, whereas J.2 and J.2.2 aligned with earlier vaccine‐like strains. NA analysis showed predominance of clade B.4.2.2 associated with K‐lineage and relatedness to recent North American strains. Subclade K exhibited conserved HA substitutions (K2N, S144N, N158D, I160K, Q173R, T328A) and HA2 S49N with limited variation. No antiviral resistance mutations were detected. Overall, findings demonstrate predominance and genetic stability of H3N2 subclade K and support continuous genomic surveillance for vaccine strain selection and influenza preparedness.
Aamir Javed, Jamal Hayat, S. A. Haider et al.· Journal of Medical Virology· 0 citations
The H9N2 subtype of avian influenza A virus has been endemic in poultry populations across Asia and the Middle East, posing ongoing economic and public health concerns. Since their initial detection in Pakistan in the 1990s, H9N2 viruses have caused repeated outbreaks in commercial poultry, leading to substantial economic losses and raising concerns about zoonotic transmission. To characterize the recent genetic evolution and zoonotic potential of circulating strains, five H9N2 isolates were obtained from poultry in Pakistan between January and March 2023. Phylogenetic analysis of the haemagglutinin gene revealed that all five isolates belong to the G5.3.2, formerly known as B2, sub-lineage of the G1 Eurasian lineage, in accordance with the revised global classification system for H9 viruses. Comparative genomic analysis confirmed that all eight gene segments were closely related to previously reported Pakistani strains, with no evidence of recent reassortment, suggesting localized persistence and ongoing genetic drift. Notably, several mammalian-adaptive mutations were identified in internal gene segments of the isolates, suggesting a potential risk of cross-species transmission. In addition, all five isolates exhibited dual receptor-binding characteristics, recognizing both α2,3-linked (avian-type) and α2,6-linked (human-type) sialic acid glycans, with a stronger affinity for α2,6-linked glycans. These findings underscore the need for continuous surveillance and risk assessment of H9N2 viruses circulating in Pakistan.
Kai-Yun Tan, Nousheen Arshad, Salman L. Butt et al.· Avian Pathology· 0 citations