Polarized Entry of Human Parechoviruses in the Airway Epithelium

Figures

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  Figure 1. HPeV1 and HPeV3 replication kinetics in nasal HAE cell cultures. Relative increase in HPeV 50% tissue culture infectious dose (TCID50) titers in the apical (A) and basolateral (B) HAE samples, and relative increase in the HPeV RNA copies determined by RT-qPCR analysis in the apical (C) and basolateral (D) samples following either apical (dotted line) or basolateral inoculation (solid line) of the HAE cultures. Data represents the mean and standard error of mean for four technical and four biological replicates. *p-values <4e-4. Dpi; days post infection.

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  Figure 2. Human parechoviruses target p63 positive basal cells in the airway epithelium. Confocal image stacks of HPeV1- and HPeV3-infected HAE cultures triple-stained by (A) ciliated cell marker β-tubulin (red), basal cell marker p63 (purple) and HPeV antibody (green) or (B) secretory cell marker mucin 5B (red), basal cell marker p63 (purple) and HPeV antibody (green). Nuclei stained by DAPI (blue). Scale bars, 20 μm.

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  Figure 3. Effect of αV ITG and HS blocking on HPeV replication. Relative increase in HPeV1 (A) and HPeV3 (B) 50% tissue culture infectious dose (TCID50) titers in apical samples following function blocking ITGαV mAb pretreatment (15 μg/ml or 30 μg/ml) of the HAE cultures or heparin pretreatment (5 mg/ml) of the virus inoculum. Mean and standard error of mean for three biological replicates is shown. Basolateral sampling data not shown.

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  Figure 4. Overview of the significantly differentially expressed genes (DEGs) induced by HPeV infection at day 3 post infection (p < 0.05, gene expression fold change >1.2 log2). (A) RT-qPCR analysis of HPeV replication in the HAE apical compartment at the time of microarray sample preparation. (B) Venn diagram showing the total number, overlap and differences in the DEGs induced by HPeV1 and HPeV3. (C) Number of up- and downregulated genes among the four subsets of DEGs; true common, common, HPeV1-specific and HPeV3-specific. (D) Heatmap of the true common DEGs induced similarly by both HPeV1 and HPeV3. (E) Heatmap of the common DEGs induced differently by HPeV1 and HPeV3. (F) Heatmap of the DEGs induced exclusively upon HPeV3 infection. Gene expression fold change scale in the heatmaps is normalized by row values.

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  Figure 5. Top 15 most significantly over-represented biological processes among the gene ontologies of the (A) true common, (B) common and (C) HPeV3-specific up- and downregulated DEG subsets. Number of DEGs associated with each biological process shown along y-axis and -log10 p-values on the x-axis.