Evolutionary and Functional Diversity of the 5′ Untranslated Region of Enterovirus D68: Increased Activity of the Internal Ribosome Entry Site of Viral Strains during the 2010s

Figures

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  Figure 1. Genetic diversity of the 5′ UTR of EV-D68 (A) Average pairwise genetic diversity of the whole genome of EV-D68 among the 374 strains collected between 1962 and 2016, calculated using a sliding window of 100 nucleotides with a step size of 10 nucleotides. Schematic figure for the structure of whole genome is shown at the top. (B) Phylogenetic trees of the viral strains, constructed using the maximum likelihood method for the VP1 gene (left) and the 5′ UTR (right). Branches are colored by lineages identified by the phylogenetic tree for the VP1 gene. Bootstrap values >60% at major branches are shown.

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  Figure 2. RNA secondary structure of the 5′ UTR of EV-D68. Predicted RNA secondary structure of the 5′ UTR of EV-D68 (Fermon/1962) is shown. Locations of mutations in the consensus sequence of viral strains in each phylogenetic lineage are indicated by dots except variable region. The Green, blue, and red indicate mutation sites in lineages 1, 2, and 3, respectively. The same figure in high resolution is available in Figure S1.

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  Figure 3. IRES activities of the 5′ UTR of EV-D68 in various cell lines. (A) IRES activities of the 5′ UTR of Fermon/1962 and recently circulating strains of EV-D68 as determined in various cell lines. The activities were measured by luciferase activity and normalized to Fermon/1962. N.C. indicates a negative control, which was a construct lacking the 5′ UTR of EV-D68. (B) IRES activities of 5′ UTR of chimeric sequences of Fermon/1962 and lineage 2 strain (Ph397/2013) in A172 cells. The RNA sequence in each stem-loop (I–VI) or variable region (VR) was exchanged between the two strains. The activities were measured by luciferase activity and normalized to the backbone sequence. Data represent the mean ± standard deviation of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

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  Figure 4. Growth kinetics of Fermon/1962 and Ph397/2013 (lineage 2) in A172 cells. A172 cells were infected with a virus at a MOI of 0.01. Supernatant and cellular lysates were collected at 12, 24, and 48 h after infection. Viral RNA levels in cellular lysates (A) and supernatants (B) were measured by quantitative RT-PCR and shown as differences in Cq values, compared with data at 12 h. Data for cellular lysates at 48 h were unavailable because of cellular death, due to the cytopathic effect. Progeny viruses in the supernatants were titrated using the 50% tissue culture infective dose (TCID50) method in RD cells. (C) Data represent the mean ± standard deviation of three independent experiments. n.s., not significant.