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.

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.

H9N2 influenza viruses have been circulating worldwide in multiple avian species, and regularly infect pigs and humans. Recently, a novel protein, PA-X, produced from the PA gene by ribosomal frameshifting, was demonstrated to be an antivirulence factor in pandemic 2009 H1N1, highly pathogenic avian H5N1 and 1918 H1N1 viruses. However, a similar role of PA-X in the prevalent H9N2 avian influenza viruses has not been established. In this study, we compared the virulence and cytopathogenicity of H9N2 WT virus and H9N2 PA-X-deficient virus. Loss of PA-X in H9N2 virus reduced apoptosis and had a marginal effect on progeny virus output in human pulmonary adenocarcinoma (A549) cells. Without PA-X, PA was less able to suppress co-expressed GFP in human embryonic kidney 293T cells. Furthermore, absence of PA-X in H9N2 virus attenuated viral pathogenicity in mice, which showed no mortality, reduced progeny virus production, mild-to-normal lung histopathology, and dampened proinflammatory cytokine and chemokine response. Therefore, unlike previously reported H1N1 and H5N1 viruses, we show that PA-X protein in H9N2 virus is a pro-virulence factor in facilitating viral pathogenicity and that the pro-or antivirulence role of PA-X in influenza viruses is virus strain-dependent.

BackgroundThe influenza viruses circulating in animals sporadically transmit to humans and pose pandemic threats. Animal models to evaluate the potential public health risk potential of these viruses are needed.Methodology/Principal FindingsWe investigated the guinea pig as a mammalian model for the study of the replication and transmission characteristics of selected swine H1N1, H1N2, H3N2 and avian H9N2 influenza viruses, compared to those of pandemic (H1N1) 2009 and seasonal human H1N1, H3N2 influenza viruses. The swine and avian influenza viruses investigated were restricted to the respiratory system of guinea pigs and shed at high titers in nasal tracts without prior adaptation, similar to human strains. None of the swine and avian influenza viruses showed transmissibility among guinea pigs; in contrast, pandemic (H1N1) 2009 virus transmitted from infected guinea pigs to all animals and seasonal human influenza viruses could also horizontally transmit in guinea pigs. The analysis of the receptor distribution in the guinea pig respiratory tissues by lectin histochemistry indicated that both SAα2,3-Gal and SAα2,6-Gal receptors widely presented in the nasal tract and the trachea, while SAα2,3-Gal receptor was the main receptor in the lung.Conclusions/SignificanceWe propose that the guinea pig could serve as a useful mammalian model to evaluate the potential public health threat of swine and avian influenza viruses.

BackgroundH9N2 influenza A viruses have undergone extensive reassortments in different host species, and could lead to the epidemics or pandemics with the potential emergence of novel viruses.MethodsTo understand the genetic and pathogenic features of early and current circulating H9N2 viruses, 15 representative H9N2 viruses isolated from diseased chickens in northern China between 1998 and 2010 were characterized and compared with all Chinese H9N2 viruses available in the NCBI database. Then, the representative viruses of different genotypes were selected to study the pathogenicity in mice with the aim to investigate the adaptation and the potential pathogenicity of the novel H9N2 reassortants to mammals.ResultsOur results demonstrated that most of the 15 isolates were reassortants and generated four novel genotypes (B62-B65), which incorporated the gene segments from Eurasian H9N2 lineage, North American H9N2 branch, and H5N1 viruses. It was noteworthy that the newly identified genotype B65 has been prevalent in China since 2007, and more importantly, different H9N2 influenza viruses displayed a diverse pathogenicity to mice. The isolates of the 2008-2010 epidemic (genotypes B55 and B65) were lowly infectious, while two representative viruses of genotypes B0 and G2 isolated from the late 1990s were highly pathogenic to mice. In addition, Ck/SD/LY-1/08 (genotype 63, containing H5N1-like NP and PA genes) was able to replicate well in mouse lungs with high virus titers but caused mild clinical signs.ConclusionSeveral lines of evidence indicated that the H9N2 influenza viruses constantly change their genetics and pathogenicity. Thus, the genetic evolution of H9N2 viruses and their pathogenicity to mammals should be closely monitored to prevent the emergence of novel pandemic viruses.