In China, H9N2 subtype avian influenza outbreak is firstly reported in Guangdong province in 1992. Subsequently, the disease spreads into vast majority regions nationwide and has currently become endemic there. Over vicennial genetic evolution, the viral pathogenicity and transmissibility have showed an increasing trend as year goes by, posing serious threat to poultry industry. In addition, H9N2 has demonstrated significance to public health as it could not only directly infect mankind, but also donate partial or even whole cassette of internal genes to generate novel human-lethal reassortants like H5N1, H7N9, H10N8 and H5N6 viruses. In this review, we mainly focused on the epidemiological dynamics, biological characteristics, molecular phylogeny and vaccine strategy of H9N2 subtype avian influenza virus in China to present an overview of the situation of H9N2 in China.

Since the influenza pandemic occurred in 1918, people have recognized the perniciousness of this virus. It can cause mild to severe infections in animals and humans worldwide, with extremely high morbidity and mortality. Since the first day of human discovery of it, the “game” between the influenza virus and the host has never stopped. NS1 protein is the key protein of the influenza virus against host innate immunity. The interaction between viruses and organisms is a complex and dynamic process, in which they restrict each other, but retain their own advantages. In this review, we start by introducing the structure and biological characteristics of NS1, and then investigate the factors that affect pathogenicity of influenza which determined by NS1. In order to uncover the importance of NS1, we analyze the interaction of NS1 protein with interferon system in innate immunity and the molecular mechanism of host antagonism to NS1 protein, highlight the unique biological function of NS1 protein in cell cycle.

Influenza A virus is a negative-sense single-stranded RNA virus that belongs to Orthomyxoviridae family. Based on the antigenic characteristics of hemagglutinin (HA) and neuraminidase (NA) influenza viruses are classified into multiple subtypes. H9N2 belongs to the low pathogenic Avian Influenza Viruses (AIVs) and is one of the widely spread viruses in poultry, which can pose a threat to humans by directly infecting or providing internal genes for various zoonotic avian influenza strains. It has the potential to directly or indirectly participate in becoming an AIV that causes a human pandemic. When the virus enters a host, the innate immune system is activated first by pattern recognition receptors. The cytokines produced at the site of infection recruit innate immune cells and antigen-presenting cells and those cells subsequently transmit antigenic signals to adaptive immune cells (i.e. B cells and T cells), to trigger specific humoral and cellular immune responses. As a result, humoral and cellular immunity can clear virus and infected cells via antibody-mediated neutralization and cytotoxicity, respectively. Understanding how chicken immune systems respond to H9N2 is a top priority for effectively controlling the virus's spread and designing vaccines. In this review, we comprehensively discuss the role of the chicken immune system in defending against H9N2, and clarify the current limitations in understanding chicken immune responses to H9N2 virus, thereby providing potential directions for future research as research on the chicken respiratory mucosal immune system has been stagnant for more than 20 years especially on how the mucosal immune system in chicken responds to avian influenza.

Avian influenza virus (AIV) emerged and has continued to re-emerge, continuously posing great threats to animal and human health. The detection of hemagglutination inhibition (HI) or virus neutralization antibodies (NA) is essential for assessing immune protection against AIV. However, the HI/NA-independent immune protection is constantly observed in vaccines’ development against H7N9 subtype AIV and other subtypes in chickens and mammals, necessitating the analysis of the cellular immune response. Here, we established a multi-parameter flow cytometry to examine the innate and adaptive cellular immune responses in chickens after intranasal infection with low pathogenicity H7N9 AIV. This assay allowed us to comprehensively define chicken macrophages, dendritic cells, and their MHC-II expression, NK cells, γδ T cells, B cells, and distinct T cell subsets in steady state and during infection. We found that NK cells and KUL01⁺ cells significantly increased after H7N9 infection, especially in the lung, and the KUL01⁺ cells upregulated MHC-II and CD11c expression. Additionally, the percentages and numbers of γδ T cells and CD8 T cells significantly increased and exhibited an activated phenotype with significant upregulation of CD25 expression in the lung but not in the spleen and blood. Furthermore, B cells showed increased in the lung but decreased in the blood and spleen in terms of the percentages or/and numbers, suggesting these cells may be recruited from the periphery after H7N9 infection. Our study firstly disclosed that H7N9 infection induced local and systemic cellular immune responses in chickens, the natural host of AIV, and that the flow cytometric assay developed in this study is useful for analyzing the cellular immune responses to AIVs and other avian infectious diseases and defining the correlates of immune protection.

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.