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. Influenza A virus (IAV) can infect diverse host species, from wild and domestic birds to mammalian species, and the pathogenesis of IAV is complex due to its remarkable genetic variability. The genome of IAV contains eight RNA segments that encode at least 17 viral proteins, including 8 initially identified proteins (PB2, PB1, PA, HA, NP, NA, M1, and NS1), 2 splicing variants of the M and NS genes (M2 and NS2) (1-3), and the recently identified proteins PB1-N40 (4), PB1-F2 (5), PA-X (6), M42 (7), NS3 (8), PA-N155, and PA-N182 (9). PB1-N40 is an N-terminally truncated version of the PB1 protein that lacks the transcriptase function but can still interact with other polymerase complex subunits and regulate virus replication in a specific genetic background (4). PB1-F2, encoded by an alternative open reading frame (ORF) of PB1, has multiple functions, including the induction of apoptosis (10), aggravation of inflammation (11,12), and secondary bacterial infection (13). PA-X is a frameshift product of the ribosome and acts to decrease the virulence of the 1918 H1N1 virus in mice (6). PA-N155 and PA-N182 are N-terminally truncated forms of PA, and the mutant viruses lacking these two proteins exhibit attenuated in vitro replication and pathogenicity in mice relative to the wild-type (wt) virus (9). M42 is the M2 isoform with an alternative ectodomain that can functionally replace M2 and support efficient viral replication (7). Selman et al. have identified NS3 as the isoform of NS1 and speculated that the codon providing NS3 expression could be associated with host adaptation and the overcoming of the species barrier (8)....

PA-X is a novel protein encoded by PA mRNA and is found to decrease the pathogenicity of pandemic 1918 H1N1 virus in mice. However, the importance of PA-X proteins in current epidemiologically important influenza A virus strains is not known. In this study, we report on the pathogenicity and pathological effects of PA-X deficient 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza H5N1 viruses. We found that loss of PA-X expression in pH1N1 and H5N1 viruses increased viral replication and apoptosis in A549 cells and increased virulence and host inflammatory response in mice. In addition, PA-X deficient pH1N1 and H5N1 viruses up-regulated PA mRNA and protein synthesis and increased viral polymerase activity. Loss of PA-X was also accompanied by accelerated nuclear accumulation of PA protein and reduced suppression of PA on non-viral protein expression. Our study highlights the effects of PA-X on the moderation of viral pathogenesis and pathogenicity.

Influenza poses a severe threat to human health in the world. However, developing a universal anti-viral strategy has remained challenging due to the presence of diverse subtypes as well as its high mutation rate, resulting in antigenic shift and drift. Here we developed an antiviral strategy using iron oxide nanozymes (IONzymes) to target the lipid envelope of the influenza virus.Methods: We evaluated the antiviral activities of our IONzymes using a hemagglutination assay, together with a 50% tissue culture infectious doses (TCID50) method. Lipid peroxidation of the viral envelope was analyzed using a maleic dialdehyde (MDA) assay and transmission electron microscopy (TEM). The neighboring viral proteins were detected by western blotting.Results: We show that IONzymes induce envelope lipid peroxidation and destroy the integrity of neighboring proteins, including hemagglutinin, neuraminidase, and matrix protein 1, causing the inactivation of influenza A viruses (IAVs). Furthermore, we show that our IONzymes possess a broad-spectrum antiviral activity on 12 subtypes of IAVs (H1~H12). Lastly, we demonstrate that applying IONzymes to a facemask improves the ability of virus protection against 3 important subtypes that pose a threat to human, including H1N1, H5N1, and H7N9 subtype.Conclusion: Together, our results clearly demonstrate that IONzymes can catalyze lipid peroxidation of the viral lipid envelope to inactivate enveloped viruses and provide protection from viral transmission and infection.

The PA-X protein, arising from ribosomal frameshift during PA translation, was recently discovered in influenza A virus (IAV). The C-terminal domain ‘X’ of PA-X proteins in IAVs can be classified as full-length (61 aa) or truncated (41 aa). In the main, avian influenza viruses express full-length PA-X proteins, whilst 2009 pandemic H1N1 (pH1N1) influenza viruses harbour truncated PA proteins. The truncated form lacks aa 232–252 of the full-length PA-X protein. The significance of PA-X length in virus function remains unclear. To address this issue, we constructed a set of contemporary influenza viruses (pH1N1, avian H5N1 and H9N2) with full and truncated PA-X by reverse genetics to compare their replication and host pathogenicity. All full-length PA-X viruses in human A549 cells conferred 10- to 100-fold increase in viral replication and 5–8 % increase in apoptosis relative to corresponding truncated PA-X viruses. Full-length PA-X viruses were more virulent and caused more severe inflammatory responses in mice. Furthermore, aa 233–252 at the C terminus of PA-X strongly suppressed co-transfected gene expression by ∼50 %, suggesting that these terminal 20 aa could play a role in enhancing viral replication and contribute to virulence.

Since May 2014, highly pathogenic avian influenza H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals, are not known. We characterized the virus receptor-binding affinity, pathogenicity, and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired a binding affinity for human-like SA␣2,6Gal-linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less-severe pathogenicity than the parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans. O n 7 May 2014, the Chinese National Health and Family Planning Commission (NHFPC) announced the first human case of avian H5N6 influenza virus infection (1). Subsequently, three more human infections with H5N6 virus cases were reported in the winter of 2014-2015 (2, 3). Between 30 December 2015 and 2 January 2016, the NHFPC notified the World Health Organization of two additional human cases of avian H5N6 virus infection. All six human infections were presented as acute respiratory distress syndrome (ARDS) of which three were fatal. Five cases had a common history of contact with or exposure to poultry or livebird markets before disease onset (1-3), suggesting zoonotic transmission. Sequence analyses of the human H5N6 isolates indicated that the virus was derived from clade 2.3.4.4 avian H5N6 viruses that are circulating in poultry in China (1, 2, 4). Avian H5N6 influenza virus was first isolated from mallards in North America in 1975 (5). In China, H5N6 virus first emerged in 2010 and has since been extensively circulating in both domestic and wild birds (6-9). Recent surveillance data from the Ministry of Agriculture of China indicate that H5N6 viruses have become enzootic in domestic poultry. Unlike the worldwide distribution H5N2 and H5N8 viruses (10-12), prevailing H5N6 viruses appear to be largely confined to China and Laos (13). We recently characterized the novel H5N6 viruses in poultry (14); however, their zoonotic capability and characteristics are poorly understood. In the present study, we examined the emergent H5N6 virus for its genetic characteristics, receptor binding properties, pathogenicity, and transmissibility in mice and ferrets. MATERIALS AND METHODS Ethical