H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P ​+ ​M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P ​+ ​M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.

Highly pathogenic avian influenza viruses (HPAIV), such as H5N1, H5N6, and H7N9, have been reported to frequently infect humans, but acute encephalitis caused by HPAIV in humans has been rarely reported. We report the first critical case of acute encephalitis with mild pneumonia caused by the H5N6 virus. On January 25 of 2022, a 6-year-old girl with severe neurological symptoms was admitted to our hospital and rapidly developed into seizures and coma. Brain imaging showed abnormalities. Electroencephalogram (EEG) presented abnormal slow waves. Cerebrospinal fluid (CSF) contained elevated protein (1.64 g/L) and white cells (546 × 10⁶/L). Laboratory investigations revealed abnormally elevated transaminases, lactate dehydrogenase, and cytokines in serum. A novel reassortant H5N6 virus was identified from the patient’s serum, CSF, and tracheal aspirate specimens. Phylogenic analysis indicated that this virus was a novel reassortant avian-origin influenza A (H5N6) virus that belonged to clade 2.3.4.4b. This patient was diagnosed with acute encephalitis and discharged from the hospital accompanied by a language barrier. An epidemiological investigation confirmed that wild waterfowls were the direct source of infection in this case. Our study highlights the urgent need to pay attention to acute encephalitis caused by HPAIV.

Influenza A virus (IAV) infection initiates a complex interplay of cell death modalities, including apoptosis, necroptosis, pyroptosis, and their integration, known as PANoptosis, which significantly impacts host immune responses and tissue integrity. These pathways are intricately regulated by viral proteins and host factors, contributing to both viral clearance and pathogenesis-related tissue damage. This review comprehensively explores the molecular mechanisms underlying these cell death processes in influenza infection. We highlight the roles of key regulatory proteins, such as ZBP1 (Z-DNA binding protein 1) and RIPK3 (receptor-interacting protein kinase 3), in orchestrating these responses, emphasizing the dual roles of cell death in both antiviral defense and tissue injury. Furthermore, we discuss emerging therapeutic strategies targeting these pathways, aiming to enhance antiviral efficacy while minimizing collateral tissue damage. Future research should focus on targeted approaches to modulate cell death mechanisms, aiming to reduce tissue damage and improve clinical outcomes for patients with severe influenza.

H9N2 avian influenza viruses (AIVs) pose an increasing threat to the poultry industry worldwide and have pandemic potential. Vaccination has been principal prevention strategy to control H9N2 in China since 1998, but vaccine effectiveness is persistently challenged by the emergence of the genetic and/or antigenic variants. Here, we analysed the genetic and antigenic characteristics of H9N2 viruses in China, including 70 HA sequences of H9N2 isolates from poultry, 7358 from online databases during 2010-2020, and 15 from the early reference strains. Bayesian analyses based on hemagglutinin (HA) gene revealed that a new designated clade16 emerged in April 2012, and was prevalent and co-circulated with clade 15 since 2013 in China. Clade 16 viruses exhibited decreased cross-reactivity with those from clade 15. Antigenic Cartography analyses showed represent strains were classified into three antigenic groups named as Group1, Group2 and Group3, and most of the strains in Group 3 (15/17, 88.2%) were from Clade 16 while most of the strains in Group2 (26/29, 89.7%) were from Clade 15. The mean distance between Group 3 and Group 2 was 4.079 (95%CI 3.605-4.554), revealing that major switches to antigenic properties were observed over the emergence of clade 16. Genetic analysis indicated that 11 coevolving amino acid substitutions primarily at antigenic sites were associated with the antigenic differences between clade 15 and clade 16. These data highlight complexities of the genetic evolution and provide a framework for the genetic basis and antigenic characterization of emerging clade 16 of H9N2 subtype avian influenza virus.