Global Distribution and Evolutionary History of Enterovirus D68, with Emphasis on the 2014 Outbreak in Ontario, Canada

FIGURE 1. Phylogenetic tree of EV-D68 detected globally and from Ontario, Canada. The phylogenetic tree based on the alignment of partial VP1 sequences (339 bp) of EV-D68 was inferred using the Neighbor-Joining method, evolutionary distances were calculated with the Maximum Composite Likelihood method (MCL), and 1,000 Bootstrap replicates were computed to estimate the accuracy of the phylogenetic inference using MEGA v6.06 (http://www.megasoftware.net). Ontario sequences from this study are identified with red filled circles. Sequences collected in 2014 from the USA, Italy, and Netherlands are marked with a blue square, green diamond, and light blue triangle, respectively. AFP sequences collected in 2013 obtained from GenBank are labeled with a green circle. Bootstrap values > 60% are shown next to the nodes. Branch lengths are drawn to the indicated scale, proportion of nucleotide substitutions per site. Some sub-clades are collapsed and labeled in bold to assist with visualization of other clades/sub-clades.

FIGURE 2. (A) Global distribution of EV-D68 as of December 2014. The location and corresponding year of specimen collection for EV-D68 VP1 sequences were obtained from the NCBI GenBank sequence database and color coded by country. EV-D68 clades and sub-clades documented in each country were assigned based on phylogenetic analysis of available partial (339 bp) VP1 nucleotide sequences as shown in Figure 1. Strains collected during 2014 are marked for Ontario (red), USA (blue), Netherlands (cyan), and Italy (green). The editable vector map of the world template was downloaded (http://www.presentationmagazine.com/world16maps-vector-editable-507.htm) and edited using PowerPoint and Adobe Photoshop. (B) The maximum clade credibility (MCC) tree of VP1 sequences of available strains including the ones from Ontario visualized by FigTree. Branches are color coded according to the world map in (A). Temporal x-axis scale represents the sampling dates. (C) Global distribution of clades, clusters and sub-clusters.

FIGURE 3. The phylogenetic relationships of the complete EV-D68 polyprotein gene (6567 nt) of Ontario strains (n = 2) to those available in NCBI’s GenBank sequence database (n = 36). Topology was constructed using the Neighbor-Joining analysis and the evolutionary distances were computed using the Maximum Composite Likelihood method available in MEGA v6.06. Bootstrap values (>70%) are shown as percentages derived from 1,000 samplings at the nodes of the tree. The scale bar represents the number of nucleotide substitutions per site. Strains from Ontario are marked with solid black circles.

FIGURE 4. (A) Schematic representation of EV-D68 genome. The black bars represent the location of nucleotide variation within strains of clade B2.b2. Height represents number of strains with specific nucleotide variation shown. (B) Nucleotide and amino acid identities between sub-clusters B2.b1 and B2.b2 (white area) and between the complete genomes of two strains from Ontario belonging to sub-clades B2.b1 and B2.b2 (gray area).