Avian influenza A (H5N1) viruses cause severe disease in humans 1,2 , 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 [3][4][5] . Laboratory experiments suggest that virusinduced cytokine dysregulation may contribute to disease severity [6][7][8][9] . 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 Tlymphocyte 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 COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. Influenza H5N1 viruses cause severe and often fatal disease in humans that is characterized by fulminant pneumonia and multi-organ failure 1,2 . High replication efficiency, broad tissue tropism and systemic replication seem to determine the pathogenicity of H5N1 viruses in animals [3][4][5] . To examine the relevance of these viral properties in the context of human disease, we carried out virological analyses in respiratory and non-respiratory specimens of 18 previously healthy individuals with influenza H5N1 who were admitted to referral hospitals in Ho Chi Minh City during the years 2004 and 2005, of whom 13 died. (Table 1). For comparison, we studied eight patients who were hospitalized during the same period with human influenza H3N2 or H1N1. These patients presented earlier in the course of illness (Table 1), which may be explained by their origin from Ho Chi Minh City or neighboring provinces, in contrast with H5N1 patients who were mostly from more distant provinces. Europe PMC Funders GroupDespite their presentation late in the course of illness, we were able to isolate virus from pharyngeal specimens of 12 of 16 H5N1-infected individuals (Table 2). Genetic characterization and phylogenetic analysis revealed that all viral strains were of the genotype Z, H5N1 sublineage of viruses prevalent in Vietnam, Cambodia and Thailand, as previously reported 10 . Pairwise comparison of all gene segments of viruses isolated from eight fatal and four surviving cases did not reveal unique amino acid changes in either group. No viruses contained Glu92 in the NS1 protein, which is associated with increased virulence of H5N1 viruses 6 , but all contained the recently reported PDZ-domain ligand ESEV 11 . An E627K substitution in the viral polymerase basic protein 2 (PB2), which is associated with adap...

In March and early April 2009, a new swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the United States. During the first few weeks of surveillance, the virus spread worldwide to 30 countries (as of May 11) by human-to-human transmission, causing the World Health Organization to raise its pandemic alert to level 5 of 6. This virus has the potential to develop into the first influenza pandemic of the twenty-first century. Here we use evolutionary analysis to estimate the timescale of the origins and the early development of the S-OIV epidemic. We show that it was derived from several viruses circulating in swine, and that the initial transmission to humans occurred several months before recognition of the outbreak. A phylogenetic estimate of the gaps in genetic surveillance indicates a long period of unsampled ancestry before the S-OIV outbreak, suggesting that the reassortment of swine lineages may have occurred years before emergence in humans, and that the multiple genetic ancestry of S-OIV is not indicative of an artificial origin. Furthermore, the unsampled history of the epidemic means that the nature and location of the genetically closest swine viruses reveal little about the immediate origin of the epidemic, despite the fact that we included a panel of closely related and previously unpublished swine influenza isolates. Our results highlight the need for systematic surveillance of influenza in swine, and provide evidence that the mixing of new genetic elements in swine can result in the emergence of viruses with pandemic potential in humans.

A highly pathogenic avian influenza virus, H5N1, caused disease outbreaks in poultry in China and seven other east Asian countries between late 2003 and early 2004; the same virus was fatal to humans in Thailand and Vietnam. Here we demonstrate a series of genetic reassortment events traceable to the precursor of the H5N1 viruses that caused the initial human outbreak in Hong Kong in 1997 (refs 2-4) and subsequent avian outbreaks in 2001 and 2002 (refs 5, 6). These events gave rise to a dominant H5N1 genotype (Z) in chickens and ducks that was responsible for the regional outbreak in 2003-04. Our findings indicate that domestic ducks in southern China had a central role in the generation and maintenance of this virus, and that wild birds may have contributed to the increasingly wide spread of the virus in Asia. Our results suggest that H5N1 viruses with pandemic potential have become endemic in the region and are not easily eradicable. These developments pose a threat to public and veterinary health in the region and potentially the world, and suggest that long-term control measures are required.