Table 1.
2018 Samples Sequenced.
Fig 1.
Time-scaled phylogeny of Enterovirus D68.
A) A time-scaled phylogeny of VP1 segments, colored by region, is shown top-left. B) Key clusters (I, II, III), the largest from the 2018 season, are highlighted, colored by country. To display these clusters at a high resolution, the whole genome phylogenies are used. The 2014 and 2016 EV-D68 seasons are shown in orange and red boxes. C) A map shows the distribution of subclades by region from 2014–2018. Map from Mapbox and OpenStreetMap.
Fig 2.
Persistence and diversification of EV-D68 since 2014.
Multiple EV-D68 lineages persist from one biennial outbreak to another. (A) Zoomed-in view of the whole genome EV-D68 tree (for better time-resolution), showing 2018 subgroups in the B3 subclade. (B) Lineages which are ancestral to all of the samples in the 2014, 2016, and 2018 seasons diversify over time. The dotted black lines show the IQR of all samples taken during 2014, 2016, and 2018. (C) The change in number of lineages (as % of total lineages) per month for each season (left y-axis) and the mean number of samples per month (right y-axis).
Fig 3.
Age distribution of EV-D68 samples.
(A) The cumulative distribution over age separated by subclade; A2 being significantly more often detected in older persons. (B) The age of patients and subclade of their sample, for samples taken during 2014, 2016, and 2018 for which ‘age range 1’ (see section D of S1 Text) was available.
Table 2.
Linear model of subclade on age, showing the ages of patients in subclades A2 and A1 are significantly different from those in subclade B3. Subclades B2, B1, and C do not differ significantly from B3. Number of samples refers to the number of samples within a subclade for which exact age data was available.
Fig 4.
Molecular evolution of the EV-D68 capsid.
Panel A) Rendering of 5-fold symmetric arrangement of the crystal structure of the capsid protomer consisting of VP1, VP2, VP3 and VP4 [41], using different copies of the protomer to highlight different aspects of the capsid organization, evolution, and immunogenicity. The five subunits are labelled 1–5 (circled numbers). Subunits 1 and 4 are present in dark grey to complete the structure. Subunit 2 shows the surface exposed proteins in purple (VP1), sand (VP2), and green (VP3), subunit 3 displays the different putative epitopes [42] (note that the most variable parts of the BC- and DE-loops are not shown, as their structure is not resolved), and subunit 5 highlights variable positions in red, with the different proteins forming the surface indicated by pale colors. Panel B) The hypervariable BC- and DE-loops (partly missing from the structure in Panel A) in clusters and subclusters accumulated multiple changes since the root of the tree (the inferred root sequence patterns match those of the NY93 strain shown here). (BC-loop is AA positions 90–103; DE-loop is AA positions 140–148). Patterns for the C-terminus and a region of VP2 are shown in S2 Table. Panel C) Variable positions often fall into surface exposed parts of the protein (Fisher exact test, OR = 6, p < 10−16). Genes are highlighted by blue (VP4), orange (VP2), green (VP3), and purple (VP1). Grey boxes show, from left to right, the BC- and DE-loops, and C-terminus regions. Panels D&E) Phylogenetic VP1 trees are colored by the most common epitope patterns of the 6 and 8 most variable amino-acid positions in the BC- and DE-loops, respectively. Particularly note-worthy is the rapid turnover of BC-loop variants in the recent evolution of the A2 subclade. Patterns for the C-terminus region are shown in S6 Fig. See S8 Fig for a zoomed version of panels D & E with cumulative AA changes.