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...

For microbial pathogens, phylogeographic differentiation seems to be relatively common. However, the neutral population structure of Salmonella enterica serovar Typhi reflects the continued existence of ubiquitous haplotypes over millennia. In contrast, clinical use of fluoroquinolones has yielded at least 15 independent gyrA mutations within a decade and stimulated clonal expansion of haplotype H58 in Asia and Africa. Yet, antibiotic-sensitive strains and haplotypes other than H58 still persist despite selection for antibiotic resistance. Neutral evolution in Typhi appears to reflect the asymptomatic carrier state, and adaptive evolution depends on the rapid transmission of phenotypic changes through acute infections.Many bacterial taxa can be subdivided into multiple, discrete clonal groupings (clonal complexes, or ecotypes) that have diverged and differentiated as a result of clonal replacement, selective sweeps, periodic selection, and/or population bottlenecks (1). Geographic isolation and clonal replacement can also result in phylogeographic differences between bacterial pathogens from different parts of the world (2), even within young, genetically monomorphic pathogens (3) (supporting online material text) such as Mycobacterium tuberculosis (4) and Yersinia pestis (5). Typhi is a genetically monomorphic (6), human-restricted bacterial pathogen that causes 21 million cases of typhoid fever and 200,000 deaths per year, predominantly in southern Asia, Africa, and South America (7). Typhi also enters a carrier state in rare individuals [such as Mortimer's example of "Mr. N the milker" (8)], who can shed high levels of these bacteria for decades in the absence of clinical symptoms. Genome sequences are available from strains CT18 (9) and Ty2 (10), but † To whom correspondence should be addressed. E-mail: achtman@mpiib-berlin. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts the global diversity, population genetic structure, and evolutionary history of Typhi were poorly understood. It has been speculated that Typhi evolved in Indonesia, which is the exclusive source of isolates with the z66 flagellar antigen (11).We investigated the evolutionary history and population genetic structure of Typhi by mutation discovery (12) We anticipated that housekeeping genes would exhibit diminished levels of nucleotide diversity, π, as a result of purifying selection, and that pathogenicity genes would exhibit elevated levels as a result of diversifying selection. However, π did not differ significantly with gene category (P > 0.05, analysis of variance) (table S1). Purifying selection should result in Ka/Ks (the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site) values that are less than 1.0 and diversifying selection should result in ratios higher than 1.0. A trend in this direction was observed (table S1), but it was not particularly strong. We therefore concluded that these 88 BiPs largely reflect the lack of strong selection a...