Genetic and phenotypic characterization of recently discovered enterovirus D type 111

Figures

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  Fig 1. Comparative analysis of the four EV-D111 full-length genomes. A similarity plot was drawn by comparing the genomic sequence of the strain BAN-06-150 with those of the four other EV-D111 strains sequences in this study.

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  Fig 2. Predicted secondary structure of the cis-acting replicating element of EV-D111. The triplet known to be directly involved in the uridylation of the protein 3B is in red.

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  Fig 3. Multiple alignment of genomes of EV-Ds and echovirus 7 in the region of the recently discoverd short open reading frame. The alignment starts at nucleotide position 591 of the E-7 strain Wallace genome. The uORF start codon is highlighted in green; the in-frame stop codons are highlighted in red. The 11 nt-long sequence perfectly conserved at the beginning of the uORF is shaded in grey.

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  Fig 4. Schematic representation of the genetic data available for the known EV-D111 strains. Solid bars cover the genomic regions whose sequences are available. The asterisk indicates the short region used to draw the phylogram featured as S1 Fig.

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  Fig 5. Phylogenetic trees based on different genomic regions. The organization of the enterovirus genome is schematized in the middle of the picture; the genomic regions used to draw the phylogenetic trees are shaded. In the trees, the EV-D111 labels are colour-coded according the colours used in Fig 4.

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  Fig 6. Comparative analysis of EV-D94 and EV-D111 full-length genomes. A similarity plot was drawn by comparing the genomic sequence of the strain EV-D94 19–04 with those of other EV-Ds.

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  Fig 7. Acid-sensitivity assay. EV-D68 strain Fermon, EV-D70 strain J670/71 and EV-D111 strain OUP-05-059 suspensions were incubated at neutral or acid pH before being titrated on RD cells. The error bars indicate the 95% confidence intervals calculated on triplicates.

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  Fig 8. Susceptibility of different cell lines to EV-D111 strain OUP-05-059. Serial 10-fold dilutions of a virus suspension were used to infect cells in 96-well plates (10 wells per dilution for each cell line). Cell monolayers were checked microscopically 6 days post-infection to detect cytopathic effects. For each dilution, the coloured squares indicate the number of wells in which cytopathic effects were detected.

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  Fig 9. Protection of cells by antibody D171 against virus-induced cytopathic effect. Cells seeded in 96-well plates were pre-incubated with or without antibody D171. Plates were than used to titrate CV-B3 strain Nancy, PV-1 strain Sabin and EV-D111 strain OUP-05-059 suspensions. The error bars indicate the 95% confidence intervals calculated on triplicates.

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  Fig 10. Detection of virus replication in L and C6/C6 cells. PV-1 strain Sabin, EV-D70 strain J670/71 and EV-D111 strain OUP-05-059 suspensions were used to inoculate L cell monolayers; C6/36 monolayers were inoculated with EV-D70 and EV-D111 only. RNA was extracted from clarified supernatant 1 hour post-inoculation and 7 days post-inoculation and EV RNA was detected through real-time RT-PCR. Virus replication leads to an increase of the viral RNA concentration during the course of time and to a drop of cycle threshold (Ct) values. Therefore, negative values on x-axis reveal virus replication.

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  Fig 11. Replication kinetics assay in L cells. PV-3 strain Sabin and EV-D111 strain OUP-05-059 suspensions were used to inoculate L cell monolayers (1,000 TCID50 and 100 TCID50 per well, respectively). After three freeze-thaw cycles, well contents were harvested and clarified, and viruses were titrated on RD cells. Errors bars indicate the 95% confidence interval calculated on 4 wells. Titres lower than 101.8 TCID50.mL-1 cannot be measured.