Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.

/pdf/fatal-outcome-of-human-influenza-a-h5n1-is-associated-with-2li6tz2o9n.pdf

Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia

The outbreak of highly pathogenic avian influenza of the H5N1 subtype in Asia, which has subsequently spread to Russia, the Middle East, Europe, and Africa, has put increased focus on the role of wild birds in the persistence of influenza viruses. The ecology, epidemiology, genetics, and evolution of pathogens cannot be fully understood without taking into account the ecology of their hosts. Here, we review our current knowledge on global patterns of influenza virus infections in wild birds, discuss these patterns in the context of host ecology and in particular birds' behavior, and identify some important gaps in our current knowledge.

/pdf/global-patterns-of-influenza-a-virus-in-wild-birds-45p3uenzex.pdf

Global patterns of influenza A virus in wild birds

The cytokine storm has captured the attention of the public and the scientific community alike, and while the general notion of an excessive or uncontrolled release of proinflammatory cytokines is well known, the concept of a cytokine storm and the biological consequences of cytokine overproduction are not clearly defined. Cytokine storms are associated with a wide variety of infectious and noninfectious diseases. The term was popularized largely in the context of avian H5N1 influenza virus infection, bringing the term into popular media. In this review, we focus on the cytokine storm in the context of virus infection, and we highlight how high-throughput genomic methods are revealing the importance of the kinetics of cytokine gene expression and the remarkable degree of redundancy and overlap in cytokine signaling. We also address evidence for and against the role of the cytokine storm in the pathology of clinical and infectious disease and discuss why it has been so difficult to use knowledge of the cytokine storm and immunomodulatory therapies to improve the clinical outcomes for patients with severe acute infections.

Into the Eye of the Cytokine Storm

Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions. The emergence of new strains will continue to pose challenges to public health and the scientific communities. A prime example is the recent emergence of swine-origin H1N1 viruses that have transmitted to and spread among humans, resulting in outbreaks internationally. Efforts to control these outbreaks and real-time monitoring of the evolution of this virus should provide us with invaluable information to direct infectious disease control programmes and to improve understanding of the factors that determine viral pathogenicity and/or transmissibility.

/pdf/emergence-and-pandemic-potential-of-swine-origin-h1n1-3dy3x98p24.pdf

Emergence and pandemic potential of swine-origin H1N1 influenza virus.

Conservation of gene order in vertebrates is evident after hundreds of millions of years of divergence, but comparisons of the Arabidopsis thaliana sequence to partial gene orders of other angiosperms (flowering plants) sharing common ancestry approximately 170-235 million years ago yield conflicting results. This difference may be largely due to the propensity of angiosperms to undergo chromosomal duplication ('polyploidization') and subsequent gene loss ('diploidization'); these evolutionary mechanisms have profound consequences for comparative biology. Here we integrate a phylogenetic approach (relating chromosomal duplications to the tree of life) with a genomic approach (mitigating information lost to diploidization) to show that a genome-wide duplication post-dates the divergence of Arabidopsis from most dicots. We also show that an inferred ancestral gene order for Arabidopsis reveals more synteny with other dicots (exemplified by cotton), and that additional, more ancient duplication events affect more distant taxonomic comparisons. By using partial sequence data for many diverse taxa to better relate the evolutionary history of completely sequenced genomes to the tree of life, we foster comparative approaches to the study of genome organization, consequences of polyploidy, and the molecular basis of quantitative traits.

Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events

The H5N1 influenza viruses transmitted to humans in 1997 were highly virulent, but the mechanism of their virulence in humans is largely unknown. Here we show that lethal H5N1 influenza viruses, unlike other human, avian and swine influenza viruses, are resistant to the antiviral effects of interferons and tumor necrosis factor alpha. The nonstructural (NS) gene of H5N1 viruses is associated with this resistance. Pigs infected with recombinant human H1N1 influenza virus that carried the H5N1 NS gene experienced significantly greater and more prolonged viremia, fever and weight loss than did pigs infected with wild-type human H1N1 influenza virus. These effects required the presence of glutamic acid at position 92 of the NS1 molecule. These findings may explain the mechanism of the high virulence of H5N1 influenza viruses in humans.

Lethal H5N1 influenza viruses escape host anti-viral cytokine responses.

Previous studies have associated influenza virus-induced expression of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), with influenza pathogenesis in the human respiratory tract and have suggested that alpha and beta interferons are the first cytokines recruited to counteract such infection. However, we report here that TNF-α has powerful anti-influenza virus activity. When infected with influenza virus, cultured porcine lung epithelial cells expressed TNF-α in a dose-dependent manner. Expression of TNF-α was induced only by replicating virus. TNF-α showed strong antiviral activity against avian, swine, and human influenza viruses, and the antiviral effect of TNF-α was greater than that of gamma or alpha interferon. These findings suggest that TNF-α serves as the first line of defense against influenza virus infection in the natural host.

Tumor Necrosis Factor Alpha Exerts Powerful Anti-Influenza Virus Effects in Lung Epithelial Cells

In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8+ T cells from inbred chickens (B2/B2) infected with an H9N2 influenza virus to naive inbred chickens (B2/B2) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8+ T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses.

Cross-Reactive, Cell-Mediated Immunity and Protection of Chickens from Lethal H5N1 Influenza Virus Infection in Hong Kong Poultry Markets

Alveolar macrophages constitutively reside in the respiratory tracts of pigs and humans. An in vivo role of alveolar macrophages in defending against influenza viruses in mice infected with a reassorted influenza virus, 1918 HA/NA:Tx/91, was reported, but there has been no report on an in vivo role of alveolar macrophages in a natural host such as a pig using currently circulating human influenza virus. Here we show that in vivo depletion of alveolar macrophages in pigs by dichloromethylene diphosphonate (MDPCL2) treatment results in 40% mortality when pigs are infected with currently circulating human H1N1 influenza viruses, while none of the infected control pigs died. All infected pigs depleted of alveolar macrophages suffered from more severe respiratory signs than infected control pigs. Induction of tumor necrosis factor alpha in the infected pigs depleted of alveolar macrophages was significantly lower than that in the lungs of infected control pigs, and the induction of interleukin-10, an immunosuppressive cytokine, significantly increased in the lungs of infected pigs depleted of alveolar macrophages compared to infected control pigs. When we measured antibody titers and CD8 + T lymphocytes expressing gamma interferon (IFN-γ), lower antibody titers and a lower percentage of CD8 + T lymphocytes expressing IFN-γ were detectable in MDPCL2-treated infected pigs than in phosphate-buffered saline- and liposome-treated and infected pigs. Taken together, our findings suggest that alveolar macrophages are essential for controlling H1N1 influenza viruses in pigs.

Sang Heui Seo

Papers

Lethal H5N1 influenza viruses escape host anti-viral cytokine responses.

Tumor Necrosis Factor Alpha Exerts Powerful Anti-Influenza Virus Effects in Lung Epithelial Cells

Cross-Reactive, Cell-Mediated Immunity and Protection of Chickens from Lethal H5N1 Influenza Virus Infection in Hong Kong Poultry Markets

Alveolar Macrophages Are Indispensable for Controlling Influenza Viruses in Lungs of Pigs

The NS1 gene of H5N1 influenza viruses circumvents the host anti-viral cytokine responses.