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The zoonotic H7N9 avian influenza virus emerged with the H9N2-origin internal gene cassette. Previous studies have reported that genetic reassortments with H9N2 were common in the first five human H7N9 epidemic waves. However, our latest work found that the circulating high pathogenicity H7N9 virus has established a dominant internal gene cassette and has decreased the frequency of reassortment with H9N2 since 2018. This dominant cassette of H7N9 was distinct from the cocirculating H9N2, although they shared a common ancestor. As a result, we suppose that this dominant cassette may benefit the viral population fitness and promote its continuous circulation in chickens., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Reassortment with the H9N2 virus gave rise to the zoonotic H7N9 avian influenza virus (AIV), which caused more than five outbreak waves in humans, with high mortality. The frequent exchange of genomic segments between H7N9 and H9N2 has been well-documented. However, the reassortment patterns have not been described and are not yet fully understood. Here, we used phylogenetic analyses to investigate the patterns of intersubtype and intrasubtype/intralineage reassortment across the eight viral segments. The H7N9 virus and its progeny frequently exchanged internal genes with the H9N2 virus but rarely with the other AIV subtypes. Before beginning the intrasubtype/intralineage reassortment analyses, five Yangtze River Delta (YRD A-E) and two Pearl River Delta (PRD A-B) clusters were divided according to the HA gene phylogeny. The seven reset segment genes were also nomenclatured consistently. As revealed by the tanglegram results, high intralineage reassortment rates were determined in waves 2-3 and 5. Additionally, the clusters of PB2 c05 and M c02 were the most dominant in wave 5, which could have contributed to the onset of the largest H7N9 outbreak in 2016-2017. Meanwhile, a portion of the YRD-C cluster (HP H7N9) inherited their PB2, PA, and M segments from the co-circulating YRD-E (LP H7N9) cluster during wave 5. Untanglegram results revealed that the reassortment rate between HA and NA was lower than HA with any of the other six segments. A multidimensional scaling plot revealed a robust genetic linkage between the PB2 and PA genes, indicating that they may share a co-evolutionary history. Furthermore, we observed relatively more robust positive selection pressure on HA, NA, M2, and NS1 proteins. Our findings demonstrate that frequent reassortment, particular reassorted patterns, and adaptive mutations shaped the H7N9 viral genetic diversity and evolution. Increased surveillance is required immediately to better understand the current state of the HP H7N9 AIV.

Highly pathogenic (HP) H7N9 avian influenza virus (AIV) emerged in China in 2016. HP H7N9 AIV caused at least 33 human infections and has been circulating in poultry farms continuously since wave 5. The genetic divergence, geographic patterns, and hemagglutinin adaptive and parallel molecular evolution of HP H7N9 AIV in China since 2017 are still unclear. Here, 10 new strains of HP H7N9 AIVs from October 2019 to April 2021 were sequenced. We found that HP H7N9 was primarily circulating in Northern China, particularly in the provinces surrounding the Bohai Sea (Liaoning, Hebei, and Shandong) since wave 6. Of note, HP H7N9 AIV phylogenies exhibit a geographical structure compatible with high levels of local transmission after unidirectional rapid geographical expansion towards the north of China in 2017. In addition, we showed that two major subclades were continually expanding with the viral population size undergoing a sharp increase after 2018 with an obvious seasonal tendency. Notably, the hemagglutinin gene showed signs of parallel evolution and positive selection. Our research sheds light on the current epidemiology, evolution, and diversity of HP H7N9 AIV that can help prevent and control the spreading of HP H7N9 AIV.

Avian influenza virus (AIV) H7N9 that emerged in 2013 in eastern China is a novel zoonotic agent mainly circulating in poultry without clinical signs but causing severe disease with high fatality in humans in more than 5 waves. Since the emergence of highly pathogenic (HP) H7N9 variants in 2016, it has induced heavy losses in the poultry industry leading to the implementation of an intensive nationwide vaccination program at the end of wave 5 (September 2017). To characterize the ongoing evolution of H7N9 AIV, we conducted analyses of H7N9 glycoprotein genes obtained from 2013 to 2019. Bayesian analyses revealed a decreasing population size of HP H7N9 variants post wave 5. Phylogenetic topologies revealed that two novel small subclades were formed and carried several fixed amino acid mutations that were along HA and NA phylogenetic trees since wave 5. Some of the mutations were located at antigenic sites or receptor binding sites. The antigenic analysis may reveal a significant antigenic drift evaluated by hemagglutinin inhibition (HI) assay and the antigenicity of H7N9 AIV might evolute in large leaps in wave 7. Molecular simulations found that the mutations (V135T, S145P, and L226Q) around the HA receptor pocket increased the affinity to α2,3-linked sialic acid (SIA) while decreased to α2,6-linked SIA. Altered affinity may suggest that HP H7N9 variations aggravate the pathogenicity to poultry but lessen the threat to public health. Selection analyses showed that the HP H7N9 AIV experienced an increasing selection pressure since wave 5, and the national implementation of vaccination might intensify the role of natural selection during the evolution waves 6 and 7. In summary, our data provide important insights about the genetic and antigenic diversity of circulating HP H7N9 viruses from 2017 to 2019. Enhanced surveillance is urgently warranted to understand the current situation of HP H7N9 AIV., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that they have no conflict of interest.

Genotype S H9N2 avian influenza virus, which has been predominant in China since 2010, contributed its entire internal gene cassette to the genesis of novel reassortant influenza viruses, including H5Nx, H7N9 and H10N8 viruses that pose great threat to poultry and humans. A key feature of the genotype S H9N2 virus is the substitution of G1-like M and PB2 genes for the earlier F/98-like M and PB2 of genotype H virus. However, how this gene substitution has influenced viral adaptability of emerging influenza viruses in mammals remains unclear. We report here that reassortant H5Nx and H7N9 viruses with the genotype S internal gene cassette displayed enhanced replication and virulence over those with genotype H internal gene cassette in cell cultures as well as in the mouse models. We showed that the G1-like PB2 gene was associated with increased polymerase activity and improved nuclear accumulation compared with the F/98-like counterpart, while the G1-like M gene facilitated effective translocation of RNP to cytoplasm. Our findings suggest that the genotype S H9N2 internal gene cassette, which possesses G1-like M and PB2 genes, is superior to that of genotype H, in optimizing viral fitness, and thus have implications for assessing the potential risk of these gene introductions to generate emerging influenza viruses., (© 2019 Blackwell Verlag GmbH.)

Polymerase acidic (PA) protein is a multifunctional regulator of influenza A virus (IAV) replication and pathogenesis. In a previous study, we reported that nucleolin (NCL) is a novel PA-interacting host protein. In this study, we further explored the role of NCL during highly pathogenic H5N1 avian influenza virus infection. We found that depletion of endogenous NCL in mammalian cells by siRNA targeting during H5N1 infection resulted in significantly increased viral polymerase activity, elevated viral mRNA, cRNA and vRNA synthesis, accelerated viral replication, and enhanced apoptosis and necrosis. Moreover, siRNA silencing of NCL significantly exacerbated the inflammatory response, resulting in increased secretion of IL-6, TNF-α, TNF-β, CCL-4, CCL-8, IFN-α, IFN-β and IFN-γ. Conversely, overexpression of NCL significantly decreased IAV replication. Collectively, these data show that NCL acts as a novel potential antiviral factor during H5N1 infection. Further studies exploring the antiviral mechanisms of NCL may accelerate the development of new anti-influenza drugs.

Unlabelled: PA-X is a newly discovered protein that decreases the virulence of the 1918 H1N1 virus in a mouse model. However, the role of PA-X in the pathogenesis of highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype in avian species is totally unknown. By generating two PA-X-deficient viruses and evaluating their virulence in different animal models, we show here that PA-X diminishes the virulence of the HPAIV H5N1 strain A/Chicken/Jiangsu/k0402/2010 (CK10) in mice, chickens, and ducks. Expression of PA-X dampens polymerase activity and virus replication both in vitro and in vivo. Using microarray analysis, we found that PA-X blunts the global host response in chicken lungs, markedly downregulating genes associated with the inflammatory and cell death responses. Correspondingly, a decreased cytokine response was recapitulated in multiple organs of chickens and ducks infected with the wild-type virus relative to those infected with the PA-X-deficient virus. In addition, the PA-X protein exhibits antiapoptotic activity in chicken and duck embryo fibroblasts. Thus, our results demonstrated that PA-X acts as a negative virulence regulator and decreases virulence by inhibiting viral replication and the host innate immune response. Therefore, we here define the role of PA-X in the pathogenicity of H5N1 HPAIV, furthering our understanding of the intricate pathogenesis of influenza A virus., Importance: Influenza A virus (IAV) continues to pose a huge threat to global public health. Eight gene segments of the IAV genome encode as many as 17 proteins, including 8 main viral proteins and 9 accessory proteins. The presence of these accessory proteins may further complicate the pathogenesis of IAV. PA-X is a newly identified protein in segment 3 that acts to decrease the virulence of the 1918 H1N1 virus in mice by modulating host gene expression. Our study extends these functions of PA-X to H5N1 HPAIV. We demonstrated that loss of PA-X expression increases the virulence and replication of an H5N1 virus in mice and avian species and alters the host innate immune and cell death responses. Our report is the first to delineate the role of the novel PA-X protein in the pathogenesis of H5N1 viruses in avian species and promotes our understanding of H5N1 HPAIV., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)