Enhanced Enterovirus D68 Replication in Neuroblastoma Cells Is Associated with a Cell Culture-Adaptive Amino Acid Substitution in VP1

Figures

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  FIG 1. Growth curves of EV-D68 isolates in SK-N-SH and RD cells. Growth curve of clinical isolates of (sub)clades A, B1, B2, B3, and D1 and prototype EV-D68 strain Fermon in SK-N-SH cells (A) and RD cells (B) at an MOI of 0.01. Statistical analysis was performed using the one-way analysis of variance (ANOVA) compared to Fermon. Data are shown as mean ± SD from three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

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  FIG 2. Infection efficiency of different EV-D68 isolates in SK-N-SH and RD cells. Percentage of infected SK-N-SH cells (A) and RD cells (B) after infection with clinical isolates from A, B1, B2, B3, and D1 and prototype EV-D68 strain Fermon 8 h postinfection (MOI of 2). The infected cells were stained for VP1 antigen using immunofluorescence staining. Three high-power fields were captured per sample. Statistical analysis was performed using the one-way ANOVA in comparison with Fermon. Data are shown as mean ± SD from three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

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  FIG 3. Virus attachment of EV-D68 viruses to SK-N-SH and RD cells. Experiments were performed using flow cytometry. Attachment of clinical isolates from (sub)clades A, B1, B2, B3, and D1 and prototype strain Fermon to SK-N-SH cells (A) and RD cells (B). Heat-inactivated viruses were included as negative control. Statistical analysis was performed using the one-way ANOVA in comparison to Fermon. Data are shown as mean ± SD from three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

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  FIG 4. Virus attachment of clinical isolates B2/039, B2/947, and B3 after neuraminidase and heparinase III treatment. (A) Mean fluorescent intensity (MFI) levels of MAL [α(2,3)-linked sialic acid] and SNA [α(2,6)-linked sialic acid] with and without neuraminidase treatment. (B and C) Percentage of attachment of isolates B2/039, B2/947, and B3 with and without neuraminidase treatment of SK-N-SH cells (B) and RD cells (C). (D) MFI levels of heparan sulfate with and without heparinase III treatment. (E and F) Attachment of clinical isolates B2/039, B2/947, and B3 with and without heparinase III treatment on SK-N-SH cells (E) and RD cells (F). Statistical analysis was performed by paired t test. Data are shown as mean ± SD from three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

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  FIG 5. Attachment, internalization, and replication of clinical isolates B2/947, B3, and B2/039 in SK-N-SH and RD cells after heparinase III and NaClO3 treatment. SK-N-SH and RD cells were treated for 1 h with heparinase III, or cells were cultured for 5 days with NaClO3. After the treatment, cells were infected with clinical isolates at an MOI of 1. To remove the unbound viruses, cells were washed 3 times with PBS before cell lysis. Attached and intracellular viral RNA levels were determined by qPCR 1 h postinfection. Percentages of viral RNA levels were compared to values for untreated cells in SK-N-SH cells (A) and RD cells (B). Infectious virus particles of the supernatants from SK-N-SH cells (C) and RD cells (D) were determined by TCID50 10 h postinfection. Statistical analysis was performed using the one-way ANOVA. Data are shown as mean ± SD from three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.