A statistical phylogeography of influenza A H5N1

Abstract

The geographic diffusion of highly pathogenic influenza A H5N1 has largely been traced from the perspective of the virus's victims. Birds of a variety of avian orders have been sampled across localities, and their infection has been identified by a general genetic test. Another approach tracks the migration from the perspective of the virus alone, by way of a phylogeography of H5N1 genetic sequences. Although several phylogenies in the literature have labeled H5N1 clades by geographic region, none has analytically inferred the history of the virus's migration. With a statistical phylogeography of 192 hemagglutinin and neuraminidase isolates, we show that the Chinese province of Guangdong is the source of multiple H5N1 strains spreading at both regional and international scales. In contrast, Indochina appears to be a regional sink, at the same time demonstrating bidirectional dispersal among localities within the region. An evolutionary trace of HA(1) across the phylogeography suggests a mechanism by which H5N1 is able to infect repeated cycles of host species across localities, regardless of the host species first infected in each locale. The trace also hypothesizes amino acid replacements that preceded the first recorded outbreak of pathogenic H5N1 in Hong Kong, 1997.

Fig. 1.

H5N1 migration events through hemagglutinin DELTRAN parsimony. (A) DELTRAN parsimony phylogeny for locality character on a parsimony gene tree for 192 H5N1 hemagglutinin (HA) sequences sampled across 20 Eurasian localities. Branches are colored by locality. Changes in color represent migration events. (B) Typical randomized Monte Carlo trial (no. 9732) for DELTRAN parsimony phylogeny for locality. The tree based on the real sample localities supports 66 migration events without consideration of the outgroup. The Monte Carlo trial supports 147 migration events without the outgroup. The trees were colorized in Mega 3.1 (10).

Fig. 2.

Map of H5N1 migration events DELTRAN parsimony: hemagglutinin, neuraminidase, and concatenation. Migration events in DELTRAN parsimony phylogenies of 192 H5N1 nucleotide sequences sampled across 20 Eurasian localities 1996–2005 (3 ≤ n ≤ 29 isolates per locality). Orange vectors are statistically significant (α = 0.05) under an upper-tail Monte Carlo test of 10,000 trials and a sparse false discovery rate (sFDR) correction: hemagglutinin (HA) (a), neuraminidase (NA) (b), and their concatenation (HANA) (c). Nonsignificant vectors are color-coded by Monte Carlo P value: the brighter the yellow, the greater the support. Quintiles are defined by breaks in ranked P values of > 0.01, except within the final quintile. The maps are based on satellite pictures made available in Google Earth (http://earth.google.com).

Fig. 3.

Evolutionary trace for HA₁ across three H5N1 migration events: Guangdong to Hong Kong 1997, Guangdong to Thailand 2003, and Guangdong to Qinghai 2005. Each of the three migration events are supported by large bootstrap values in the original gene tree. The three migration events included the same change at residue position 263 (H5 numbering) in antigenic site E. Guangdong to Hong Kong and Guangdong to Thailand also shared a change at position 156, a site for possible N-linked glycosylation (7). The A156T replacement apparent here is associated with increased adaptation to and virulence in poultry (8). Of the 19 amino acid replacements depicted in HA₁ across the three migration events depicted in the figure, all except four occurred elsewhere in the tree. All residue changes in the Guangdong-to-Thailand migration were identified as part of the natural variation apparent across the 192 sequences included in the migration analysis. In contrast, only changes at two sites (140, 262) appeared a part of the natural variation for the Guangdong-to-Qinghai migration, emblematic of the differentiation of the QH-like sublineage that has since spread west into Europe and Africa (15). Most of the changes in the globular head represented here appear to arise from escape mutations in antigenic sites near receptor binding site residues (2). One change, at site 129 in the Guangdong-to-Thailand migration, occurred in the receptor binding domain itself. Previous work (e.g., ref. 8) defined H5N1 amino acid replacements by directly comparing field isolates. The trace instead infers replacements through H5N1's evolutionary history, including some of those leading to the first H5N1 outbreak in Hong Kong in 1997. The histidine at site 138 for the Guangdong-to-Hong Kong migration appears a historic artifact now abandoned. The asparagine at site 94 evolves from aspartic acid and is not ancestral, contrary to analyses of 1997 isolates alone.