Table 1.
List of 2012 WNV isolates completely sequenced in this study.
Table 2.
Common nucleotide mutations present in the 2012 human WNV isolates, compared to the prototype strain WN-NY99 (AF196835).
Table 3.
Amino acid substitutions present in more than one of the 2012 human WNV isolates compared to the WN-NY99 (AF196835).
Fig 1.
Increasing evolutionary divergence of North American WNV strains over the time of collection.
Y-axis: Substitutions per site. X-axis: Years. Blue line: average divergence over sequence pairs within years; the numbers of base substitutions per site from averaging over all sequence pairs within each year are shown. Red line: divergence over sequence pairs between 1999 and other years; the numbers of base substitutions per site from averaging over all sequence pairs between 1999 and other years are shown. Green line: estimates of net evolutionary divergence between groups of sequences, 1999–2012: the numbers of base substitutions per site from estimation of net average between groups of sequences corresponding to each year are shown. Analyses were conducted in MEGA6 [24] using the Maximum Composite Likelihood model [46].
Fig 2.
Consensus maximum-likelihood tree of North American WNV ORFs, 1999–2012 (n = 870).
WNV genotypes are color-coded as NY99 (black), INTermediate (orange), WN02 (blue), SW/WN03 (purple) and cluster MW/WN06 (red). All WNV sequences derived from this study are labeled by black diamonds, and Nodes 1 to 6 containing these sequences are highlighted in green and shown in detail. Taxon names correspond to GenBank accession numbers and years of collection. Node-specific amino acid substitutions are shown for each node (see also S4 Table). For each node, states shown in red in the adjacent U.S. map are those from which strains have been isolated.
Table 4.
Selection pressure analysis of WNV strains collected in the U.S. in 2012.
Table 5.
Positively selected node-specific aa substitutions.
Fig 3.
Evolutionary fingerprint based on 1000 distribution samples (Datamonkey server www.datamonkey.org).
The plot depicts the estimate of the distribution of synonymous and non-synonymous rates inferred from alignment of WNV sequences (n = 77) from strains collected in the US in 2012. The ellipses reflect a Gaussian-approximated variance in each individual rate estimate, and colored pixels show the density of the posterior sample of the distribution for a given rate. The diagonal line represents the idealized neutral evolution scenario (ω = 1), points above the line correspond to positive selection (ω>1), and points below the line to negative selection (ω<1).
Fig 4.
Maximum clade credibility tree from Bayesian analysis of WNV strains from North America, 1999–2012 (n = 870).
A) WNV genotypes are color-coded in the branches of the tree as NY99 (black), WN02 (blue), SW/WN03 (purple) and cluster MW/WN06 (red). Nodes 1 to 6 containing WNV isolates from this study are highlighted in green and shown in detail. The mean time to the most recent common ancestor (tMRCA) is shown in each principal node. The 95% highest probability densities (95% HPD) for each node age are shown as blue bars. B) Bayesian coalescent inference of genetic diversity and population dynamics using the Bayesian Skyline plot. The X axis represents years of study and the Y axis, the relative genetic diversity product of the effective population size.