OBJECTIVES:Human adenovirus type 7 (HAdV-7) has caused outbreaks of acute respiratory infections (ARIs) among children in multiple countries. This study aimed to investigate the clinical and genetic characteristics of HAdV-7 in hospitalized children with ARIs in China. METHODS:Respiratory samples were collected from hospitalized children with ARIs at 10 hospitals across Northern and Southern China between 2014 and 2025 for HAdV detection and genotyping. Clinical data of HAdV-7-positive patients were collected for clinical characteristics analysis. The whole genomes of isolated HAdV-7 strains were sequenced for bioinformatics analysis with those selected from GenBank. RESULTS:Among 250 HAdV-positive samples, 79 (31.60%) were HAdV-7, ranking second only to HAdV-114 (46.80%). Patients primarily presented with fever, cough, and sputum production. Some cases also complicated with gastrointestinal and neurological symptoms, skin rashes, and ocular diseases. Severe pneumonia occurred in 54.43% of patients, with one death. Whole genome sequence phylogenetic analysis revealed that HAdV-7 could be divided into two clusters. Cluster 1, including the prototype strain, had been rarely reported globally since the discovery of HAdV-7. Cluster 2 was the predominant cluster circulating worldwide, including China. Three novel amino acid substitutions were observed in the Loop 1 region of the Hexon protein, which might be associated with the severe disease phenotype and resulted in the emergence of a positive selection site. Recombination analysis identified a recombination event, which occurred as early as 1988. In vitro experiments demonstrated that HAdV-7 had comparable proliferation capacity with other genotypes of the HAdV-B species. CONCLUSIONS:HAdV-7 was an important pathogen of hospitalized children with ARIs in China, and associated with severe diseases and even death. Cluster 2 was the predominant epidemic cluster worldwide. Although the HAdV-7 genome was relatively conserved and stable, novel amino acid mutations have occurred.

During molecular surveillance of human adenoviruses (HAdVs) in children hospitalized with acute lower respiratory tract infections in Beijing, China, during 2014-2024, the most prevalent genotypes were HAdV-B114 (53.85%) and HAdV-B7 (27.18%). A novel recombinant genotype, HAdV-B117, was identified in 2 children <5 years of age with severe community-acquired pneumonia and serious complications. Genomic analysis revealed that HAdV-B117 arose from HAdV-B114 (P7H3F3) with the fiber gene from HAdV-B7. We observed amino acid substitutions and deletions in the pivotal regions of 3 major capsid proteins, and some were predicted to alter the protein structure. In vitro, the replication kinetics of HAdV-B117 were similar to those of HAdV-B3 and HAdV-B7. Clinical manifestations resembled severe pneumonia caused by HAdV-B3 or HAdV-B7. Both children recovered after treatment. The emergence of HAdV-B117 highlights the need for continuous genomic surveillance of HAdVs to detect novel recombinants with potential public health effects.

Influenza B virus (IBV) has circulated in the human population for a long time, yet the evolutionary mechanisms responsible for host adaptation remain poorly understood. Here we show that recent IBV strains exhibit an enhanced ability to evade the innate immune response and an increased replication efficiency compared with earlier strains. Our data indicate that the nonstructural protein 1 (NS1) of recent IBV strains interacts with TUFM and LC3B to induce mitophagy, leading to degradation of MAVS, suppression of interferon production and enhanced viral replication. In contrast, NS1 of earlier strains displays minimal ability to trigger mitophagy-mediated MAVS degradation. Sequence analyses show that, over the past two decades, IBV has acquired a phenylalanine (F)-to-leucine (L) substitution at residue 247 of NS1, altering its interaction with LC3B. A rescued recent IBV strain carrying the NS1-L247F mutation exhibits diminished NS1-LC3B binding, impaired mitophagy, and attenuated replication. Our study shows that adaptive evolution involving a single mutation in NS1 enables mitophagy-mediated innate immune evasion, contributing to IBV adaptation to the host.

In 2024, we identified and sequenced 52 avian influenza A (H9N2) virus strains in Laos. Using the established H9N2 genomic classification system, a novel HA gene clade of the A/chicken/Beijing/1/94-like (BJ/94-like) lineage, designated Clade 4.6.20, was identified. This new clade is phylogenetically distinct from the previously described clades, and the representative strains in this new Clade 4.6.20 presented a low cross reactivity to the antisera of other clades, suggesting antigenic drift of the viruses between the new Clade 4.6.20, and other clades in the dominant lineage of Clade 4.6. In addition, all the newly identified viruses in Clade 4.6.20 possessed HA-L226 and NP-N52 mutations, which are associated with human-type receptor binding and human MxA-related innate immunity escape, respectively. Our findings underscore the necessity of global surveillance network and cooperation to monitor the evolution of AIVs, update vaccine seed strains, and develop new vaccines with high effectiveness against H9N2 AIVs circulating globally, which threaten poultry and human health.

Abstract Clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) continue to circulate worldwide, posing zoonotic threats, especially with recent cattle outbreaks. The mechanisms by which these viruses adapt to mammalian hosts while maintaining a broad avian tropism remain poorly understood. Here, we demonstrate that two naturally occurring mutations (K222Q and S227R) in the hemagglutinin (HA) of a human-infecting H5N8 strain, first identified in 2020, enhance binding affinity for both α2-6-linked and Sialyl Lewis X (SLe X ) glycans, which may underlie the broad tissue binding and cross-species potential. Structural analyses reveal that these mutations expand receptor specificity for these glycans, which are abundant in the human respiratory tract and duck trachea, providing a possible molecular basis for cross-species transmission. Our findings suggest that clade 2.3.4.4b H5Ny viruses evolved dual receptor specificity as early as the 2020 Russian H5N8 strain, potentially contributing to sporadic human infections and widespread dissemination among birds and mammals.

Migratory birds play an important role in the spread of antimicrobial resistance (AMR); however, gaps in surveillance data from vital regions along migratory flyways across China limit the detection of emergent threats. Here, we assembled 340 metagenomes from 52 bird species covering 11 provincial administrative districts in China, presenting a specialized migratory microbial genome and gene catalog to archive the genomic and functional diversity of gut microbiomes in wild birds. This comprehensive migratory bird microbial genome and gene (MBGG) catalog includes 5823 metagenome-assembled genomes (MAGs), 13 072 plasmid sequences, and 44 974 viral genomes, which represent 1709 candidate species spanning 36 phyla. The catalog also contains over 20 million non-redundant protein-encoding genes, the use of which is confirmed by the mining of 15 678 secondary metabolite biosynthetic gene clusters, 1814 known antibiotic resistance genes, and 7219 virulence factors. The number of clinically critical ARGs identified in Grus japonensis was the highest, followed by Cygnus cygnus and Sibirionetta formosa, which indicated that these species are hotspot species of clinically critical AMR dissemination. Moreover, we mapped the profile of bacterial zoonotic/opportunistic pathogens carried by wild birds and evaluated their associations with publicly available genomes. Finally, the precise migratory movements for 10 bird species using a global positioning system tracking system help to assess the movement of microorganisms and AMR risk. Collectively, this valuable resource provides the basis for the integration and unification of global wild bird microbiomes, timely sharing, and assessing the uncertainty of migratory microbiomes in the future.

Reverse genetics, a cornerstone of virology, enables the de novo generation of viruses from cloned cDNA, allowing precise functional interrogation of viral biology. It is particularly vital for the Paramyxoviridae family, which includes major human and animal pathogens such as Nipah virus (NiV), peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV). Despite successful establishment for many paramyxoviruses, current reverse genetics systems face substantial challenges, including low rescue efficiency, system complexity, plasmid genetic instability, and difficulties in manipulation large genomic cDNA. In response, substantial improvements have been made across genomic templates, helper proteins, and host systems. Current advances include optimized promoter systems (T7, CMV), simplified plasmid backbones and engineered cell lines, whereas potential improvements mainly focus on rapid cDNA assembly techniques, high-capacity vectors and and AI-driven predictive design. With the development of AI-driven tools, the ability to complete partial viral genomes, automated codon optimization, enhanced viral rescue efficiency and streamline experimental workflows will be achieved in the future. Through continuous technological refinement and interdisciplinary integration, reverse genetics become more efficient and intelligent, significantly advancing both basic paramyxovirus research and the development of vaccines and therapeutics.

Seasonal influenza causes a significant global disease burden every year. Currently licensed injectable influenza vaccines mainly elicit humoral immune responses with minimal mucosal immunity in the respiratory tract, limiting their ability to block viral infection and transmission. Messenger RNA (mRNA) vaccines formulated with lipid nanoparticles (LNPs) offer rapid design and flexibility and are highly immunogenic, yet share this limitation of mucosal immunity. Here, we developed influenza A and B mRNA vaccines (Am80-LNP/IFA and Am80-LNP/ IFB) using Am80-loaded LNP (Am80-LNP) encoding viral hemagglutinin (HA). Intramuscular immunization of mice with Am80-LNP/IFB (encoding influenza B/Victoria HA) at 1 and 10 & micro;g doses induced systemic HA-specific IgG responses comparable to those elicited by a commercial LNP formulation (SM102-LNP/IFB) and a licensed split vaccine. Notably, Am80-LNP/IFB markedly enhanced respiratory mucosal immunity, inducing 5.4-fold (1 & micro;g) and 11.8 -fold (10 & micro;g) higher virus-specific IgA in bronchoalveolar lavage fluid relative to SM102-LNP. In addition, Am80-LNP/IFB-10 increased lung CD4+ tissue-resident memory T (TRM) cells by 1.4-fold. Following lethal challenge of the influenza B virus, all mRNA-LNP/IFB vaccinated mice survived without weight loss, whereas the split-vaccinated mice experienced a transient decline to 88% of their initial body weight. Am80-LNP/IFB significantly reduced viral loads, rendering viral RNA undetectable in the lungs and nasal turbinates by 14 days post-infection (dpi) at a 10 & micro;g dose. In addition, the Am80-LNP/IFA vaccine (encoding A/ H1N1 HA) achieved complete viral clearance in nasal turbinates by 3 dpi, outperforming the split vaccine. These findings indicate that Am80-LNP enables injectable influenza mRNA vaccines to induce robust systemic immunity together with potent respiratory mucosal IgA and TRM responses, overcoming a major limitation of current inactivated vaccines.

Wild birds, as natural reservoirs of avian influenza viruses (AIVs), play a pivotal role in facilitating interregional virus dissemination during migration. To investigate the genetic diversity and transmission patterns of AIVs in the critical stopover ecosystems of the Tianshan Mountains’ northern piedmont—specifically along the East African-West Asian and Central Asian Flyways—we analyzed fecal samples from migratory birds at Moguhu Reservoir in 2018. Among 164 samples, 6 (3.66

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint synovial inflammation. Despite advancements in therapeutic strategies, the critical underlying mechanisms driving RA progression remain incompletely understood, and there is an ongoing need for novel therapeutic targets. We observed that high-level expression of extracellular cyclophilin A (eCypA) in patients with RA was associated with increased disease severity. Anti-CypA monoclonal antibody (mAb) treatment alleviated arthritis in both collagen-induced and collagen antibody-induced arthritis mouse models, outperforming anti-TNF-α mAb therapy. Single-cell RNA sequencing revealed that the anti-CypA mAb inhibited STAT3-mediated Th17 cell differentiation. Mechanistically, the binding of eCypA to the cell-surface receptor CD146 on CD4+ T cells promoted STAT3-mediated Th17 cell differentiation by increasing CD146 dimerization. Additionally, conditional CD146 knockout in CD4+ T cells suppressed Th17 cell differentiation and alleviated arthritis in a mouse model. In summary, our findings demonstrate that eCypA drives Th17 differentiation in RA by binding to CD146 and that blocking eCypA binding to CD146 is a promising strategy for RA treatment.

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

Abstract Emerging and re-emerging zoonotic viruses pose enormous challenges to public health worldwide. As an important livestock animal, pigs play a vital role in the evolution and spread of many zoonotic viruses. Hence, with the development of globalization and large-scale intensive farming, close human-pig contact increases the threat of zoonotic virus transmission. In this review, to facilitate disease prevention and control efforts, we summarized the prevalence and transmission characteristics of zoonotic viruses associated with pigs, such as influenza virus, coronavirus, and pseudorabies virus. Additionally, we emphasized novel detection techniques including rapid diagnostic tests, biosensor-based detection technology, high-throughput sequencing, and systematic viral epitope scanning. These techniques are instrumental in enabling cost-effective and convenient rapid detection procedures for broader implementation across diverse regions for effective surveillance of viral epidemics. To enhance virus surveillance capabilities and improve strategies for disease prevention in pigs, the improvement of our understanding of viral transmission modes combined with advancements in diagnostic technology is necessary.

Duck adenovirus 3 (DAdV-3) causes liver damage and bleeding, with morbidity rates ranging from 40 to 55% and mortality rates between 35 and 43%. Co-infection with other pathogens complicates disease control, significantly impacting the duck breeding industry. Currently, there have been no effective vaccines or treatments for DAdV-3. Therefore, rapid, specific, and sensitive detection methods are crucial for preventing and controlling this virus. Our study developed a lateral flow strip (LFS) detection method using recombinase polymerase amplification (RPA) and CRISPR/Cas12a. The RPA-CRISPR/Cas12a-LFS method, performed at 37°C, allowed for result visualization without sophisticated equipment. It targeted the DAdV-3 Fiber-2 gene and achieved a detection limit of 3.0 gene copies. Additionally, this method demonstrated high specificity, with no cross-reactivity to eight other avian viruses. The reaction time of RPA-CRISPR/Cas12a-LFS is only 45 min. Analysis of 95 waterfowl samples showed 98.95% consistency and agreement with quantitative polymerase chain reaction using the Fiber-2 RPA-CRISPR/Cas12a-LFS method. These findings highlighted the potential of this user-friendly, rapid, sensitive, and accurate detection method for on-site DAdV-3 detection.

ETHNOPHARMACOLOGICAL RELEVANCE:Lianhuaqingwen (LH), a traditional Chinese medicine, presents a broad-spectrum antiviral effect and has been widely used to treat influenza. Given the potential rise of drug-resistant influenza viruses, it is necessary to develop new antiviral drugs and explore combination therapies involving LH in tandem with existing antivirals such as Oseltamivir acid (Osel) or Baloxavir (Bal). These multidrug combinations could help effectively control the seasonal influenza epidemics and reduce the disease burden. AIM OF THE STUDY:This study aimed to evaluate the antiviral effects of LH, alone and in combination with Osel or Bal, against human seasonal influenza viruses in vitro and in vivo models. MATERIALS AND METHODS:The antiviral efficacy of LH alone and LH in combination with Osel/Bal against seasonal influenza A viruses (IAVs) (H1N1 and H3N2 subtypes) and influenza B viruses (IBVs) (BV- and BY-lineages) was assessed in vitro using MDCK cells. The median effective concentration (EC50) was determined, and the drug synergies were analyzed. Additionally, the antiviral activity of LH monotherapy and LH + Osel/Bal combination therapy were evaluated in vivo using an H1N1-infected BABL/c mouse model by monitoring changes in body weight, survival rate, lung viral titer, pathological damage, and inflammatory reaction. RESULTS:In vitro, LH alone and in combination with Osel/Bal exhibited antiviral activity against both IAVs and IBVs. The addition of LH to Osel/Bal improved the therapeutic efficacy compared to Osel/Bal alone. In vivo, LH monotherapy reduced body weight loss and increased the survival rates of H1N1-infected mice. LH in combination with Osel/Bal resulted in lower virus titers, more effective relief of pathological damage, and comparable low expression of inflammatory factors in the lungs of H1N1-infected mice compared to the use of Osel/Bal alone. Transcriptomic analysis of the lungs revealed that LH + Osel/Bal significantly increased the expression of genes associated with antiviral and anti-inflammatory effects. CONCLUSIONS:This study evaluated the antiviral effects of LH monotherapy and combination therapy with Osel/Bal against human seasonal influenza viruses in vitro and in vivo models. The results suggest that combining LH with Osel or Bal could enhance the antiviral efficiency for influenza viruses compared to the monotherapy using any of these three drugs.

The ongoing circulation of highly pathogenic avian influenza (HPAI) A (H5N1) viruses, particularly clade 2.3.4.4b strains, poses a significant threat to animal and public health. Recent outbreaks in cattle highlight concerns about cross-species transmission and zoonotic spillover. Here, we found that the hemagglutinin (HA) protein from a cattle-infecting H5N1 virus has acquired slight binding to human-like α2-6-linked receptors while still exhibiting a strong preference for avian-like α2-3-linked sialic acid receptors. Immunohistochemical staining revealed HA binding to bovine pulmonary and mammary tissues, aligning with clinical observations. HA also binds effectively to human conjunctival, tracheal, and mammary tissues, indicating a risk for human transmission, notably in cases of conjunctivitis. High-resolution cryo-electron microscopy (cryo-EM) structures of this H5 HA in complex with either α2-3 or α2-6 receptors elucidate the molecular mechanisms underlying its receptor-binding properties. These findings provide critical insights into the tropism and transmission potential of this emerging pathogen.

Porcine epidemic diarrhea virus (PEDV) is a major coronavirus in swine, causing substantial economic losses in the industry. To deepen our understanding of the PEDV-host cell interactions, we performed whole-genome CRISPR/Cas9 screens on porcine IPEC-J2 and IPI-2I cell lines to identify key host factors essential for PEDV infection. Our study identified the Yip family 5 (YIPF5) protein as a critical host factor, where its knockout suppressed PEDV infection by specifically affecting the virus replication stage. YIPF5 interacts with viral non-structural protein (nsp) 3, 4, and 6, facilitating the formation of double-membrane vesicles (DMVs), essential for replication organelle biogenesis. The knockout of YIPF5 interferes with the interaction between nsp3 and nsp4, consequently impacting the formation of DMVs mediated by these proteins. These findings establish YIPF5 as a key host factor involved in DMV formation during PEDV infection, highlighting its potential as a therapeutic target. IMPORTANCE:Coronaviruses pose serious health threats to both humans and animals. Identifying host genes critical for porcine epidemic diarrhea virus (PEDV) infection can uncover new therapeutic targets and enhance our understanding of coronavirus pathogenesis. In this study, we conducted genome-scale CRISPR/Cas9 screens in two porcine cell lines (IPEC-J2 and IPI-2I) and identified YIPF5 as an essential host factor for PEDV replication. Our results demonstrate that YIPF5 plays a pivotal role in the formation of PEDV-induced double-membrane vesicles (DMVs), which are crucial for viral replication. These findings shed new light on the molecular mechanisms of PEDV and suggest YIPF5 as a therapeutic target.

H3N2 influenza A viruses [A(H3N2)] circulate as seasonal influenza in humans worldwide, resulting in a huge disease burden. Adaptation study of A(H3N2) in mice could provide a basis for preclinical evaluation of antivirals and vaccines targeting A(H3N2) and identify the genetic markers responsible for the viral adaptation, replication, and pathogenesis. Lung-to-lung passaging of wild-type (WT) A(H3N2) strain was performed in C57BL/6J mice. Amino acid (AA) mutations occurred during the passaging and temporal dynamics of these mutations were identified using the next-generation sequencing. We determined the polymerase activity of the ribonucleoprotein (RNP) complex containing mutation genes and compared the pathogenicity between the mouse-adapted (MA) and A(H3N2)-WT strains based on body weight change, survival rate, lung index, lung viral load, and lung pathology of the infected mice. The A(H3N2)-MA strain was obtained after seventeen lung-to-lung passages in mice. 14 AA mutations in the PB2, PB1, PA, HA, NP, and M1 genes were identified in the A(H3N2)-MA strain compared to the A(H3N2)-WT strain. In addition, the polymerase activity of the RNP complex containing mutation genes was increased, and the pathogenicity of the MA virus is significantly higher than that of the WT strain. One A(H3N2)-MA strain has been developed that can infect and kill mice. The MA strain showed stronger replication ability and pathogenicity than the A(H3N2)-WT strain. This A(H3N2)-MA model provides a valuable basis for evaluating the effects of drugs and vaccines and for studying pathogenesis.

Introduction:Respiratory pathogens pose a complex challenge for public health systems. In the winter of 2023, multiple respiratory pathogens showed staggered epidemic waves. Additionally, co-infections involving various pathogens were observed, resulting in significant disease burdens. Understanding the epidemiological dynamics of these pathogens is essential for supporting public health systems in the prevention and control of respiratory infectious diseases. Methods:Respiratory samples were collected from patients in Beijing presenting with influenza-like symptoms to detect 27 respiratory pathogens using multiplex qPCR. Results:Four distinct epidemic waves were identified. The first wave was a pre-winter outbreak of Mycoplasma pneumoniae (M. pneumoniae). This was then followed by successive waves of influenza A and B viruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibited a resurgence by the end of February 2024. Age-dependent susceptibility varied, with SARS-CoV-2 and influenza A/B peaking in the 30-40-year age group. Conversely, adenovirus, rhinovirus, M. pneumoniae, Moraxella catarrhalis (M. catarrhalis), and Haemophilus influenzae (H. influenzae) were more common in adolescents and the elderly. Furthermore, 18.8% of cases were identified as co-infections with more than two pathogens. H. influenzae was found to frequently co-infect with viral and bacterial pathogens. Conclusions:Respiratory pathogens exhibited different prevalence trends during the first influenza season following the COVID-19 pandemic. Influenza viruses showed a higher peak incidence and delayed seasonality. Moreover, the co-circulation of viral and bacterial infections increased the complexity of respiratory infections. Interestingly, staggered epidemic waves between SARS-CoV-2 and influenza A/B viruses were observed. Consequently, SARS-CoV-2 may become a seasonal virus, causing epidemics alongside influenza viruses. However, further research is needed to elucidate its epidemiological patterns. The co-circulation of these epidemic viruses and other respiratory pathogens underscores the need for enhanced diagnostic and intervention strategies, including vaccination campaigns.

Seasonal influenza activity significantly decreased in China during the coronavirus disease 2019 (COVID-19) pandemic, yet the H3N2 virus led to three epidemic waves. Understanding the characteristics of H3N2 epidemic viruses is essential for recognizing influenza during COVID-19 and for updating vaccines. In this study, we analyzed 579 respiratory samples from patients exhibiting influenza-like symptoms, collected in 2019–2022, leading to the successful sequencing of 36 complete H3N2 genomes. Genomic analysis indicated that the epidemic strains from these periods belonged to different hemagglutinin (HA) clades and exhibited phylogenetic divergence from the concurrently used vaccine strains. Significant antigenic differences were identified through cross-hemagglutination inhibition (HI) and cross-microneutralization (MN) assays. Furthermore, pathogenicity studies showed that representative strains replicated in Madin-Darby canine kidney‌ (MDCK) cells, with varying abilities, and all replicated more effectively at 37 °C compared to 33 °C. These strains also replicated well in the respiratory tracts of mice and guinea pigs. The findings indicate a mismatch between circulating H3N2 viruses and recommended vaccine strains, highlighting the need for improved international cooperation and epidemiological surveillance of influenza viruses post-COVID-19. Optimizing effective vaccine strain update strategy and developing a universal influenza vaccine are crucial for future preparedness.