Non-Polio Enterovirus C Replicate in Both Airway and Intestine Organotypic Cultures

Figures

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  Figure 1. Mutations acquired by CVA-13, CVA-20, and EV-C99 after two passages on Caco2 cells. For each mutation, the nucleotide position, the region of the genome, the nucleotide in the OM sample, the nucleotide in the P2 sample, and the amino acid substitution are indicated. Green indicates CVA-13, orange indicates CVA-20, and pink indicates EV-C99. CVA-13 (Coxsackievirus 13), CVA-20 (Coxsackievirus 20), EV-C99 (Enterovirus C 99), OM sample (original fecal sample), P2 (passage 2), aa (amino acid), 5′UTR (5′ untranslated region), VP3 (capsid protein 3), 2A (2A protease), VP4 (capsid protein 4), VP1 (capsid protein 1), 2C (2C protein), G (guanine), A (adenosine), C (cytosine), T (thymine).

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  Figure 2. Schematic representation of the experimental layout. Airway and intestinal tissues are isolated from both fetal and adult donors. Monolayers of each tissue and each donor are then generated. Infection is performed both apically and basolaterally and supernatant is collected at specified time points from both compartments. HAE (human airway epithelium), HIE (human intestinal epithelium).

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  Figure 3. Replication of CVA-13, CVA-20, and EV-C99 on fetal and adult-derived airway monolayers when apically infected. Viral load, plotted as Log 10, of CVA-13, CVA-20, and EV-C99 detected in supernatant samples via RT-qPCR and expressed as relative increase in RNA copies compared to time 0 h. (a) Replication kinetics in the apical compartment upon apical infection of adult-derived HAE. (b) Replication kinetics in the basolateral compartment upon apical infection of adult-derived HAE. (c) Replication kinetics in the apical compartment upon apical infection of fetal-derived HAE. (d) Replication kinetics in the basolateral compartment upon apical infection of fetal-derived HAE. The data represent mean ± SEM of two technical replicates of HAE inserts made from a pool of 14 donors (panel a) and of three biological replicates (panel b–d) with three technical replicates for each. The solid line indicates viral RNA in the apical compartment (apical collection) and the dotted line indicates viral RNA in the basolateral compartment (basolateral collection) of CVA-13 (blue), CVA-20 (orange), and EV-C99 (purple). * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

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  Figure 4. Immunofluorescence images of HAE infected with CVA-13, CVA-20, and EV-C99. Three-dimensional confocal images from an apical, basolateral, and side view at 8 h post-infection is shown for all viruses. (a) Ciliated cells are stained with β-tubulin (cyan), nuclei are stained with Hoechst 33342 (blue), cell boundaries are visualized by staining for Epcam (magenta), and viruses are stained for capsid protein VP1 (yellow). (b) Goblet cells are stained for Muc5AC (cyan), nuclei are stained with Hoechst 33342 (blue), cell boundaries are visualized with Epcam (magenta), and viruses are stained for capsid protein VP1 (yellow). (c) Basal cells are stained for p63 (light blue), nuclei are stained with Hoechst 33342 (blue), tight junctions are stained for zo-1 (magenta), and viruses are stained for capsid protein VP1 (yellow). The scale bar is 50 µm.

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  Figure 5. Replication of CVA-13, CVA-20, and EV-C99 on fetal and adult-derived intestinal monolayers when apically infected. Viral load, plotted as Log10, of CVA-13, CVA-20, and EV-C99 detected in supernatant samples via RT-qPCR and expressed as relative increase in RNA copies compared to time 0 h. (a) Replication kinetics in the apical compartment upon apical infection of adult-derived intestinal monolayer. (b) Replication kinetics in the basolateral compartment upon apical infection of adult-derived intestinal monolayer. (c) Replication kinetics in the apical compartment upon apical infection of fetal-derived intestinal monolayer. (d) Replication kinetics in the basolateral compartment upon apical infection of fetal-derived intestinal monolayer. The data represent mean ± SEM of three biological replicates with three technical replicates for each. The solid line indicates viral RNA in the apical compartment (apical collection) and the dotted line indicates viral RNA in the basolateral compartment (basolateral collection) of CVA-13 (blue), CVA-20 (orange), and EV-C99 (purple). * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

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  Figure 6. Immunofluorescence images of HIE infected with CVA-13, CVA-20, and EV-C99. Three-dimensional confocal images from an apical, basolateral, and side view at 8 h post-infection for all viruses. (a) Enterocytes are stained for villin (cyan), nuclei are stained with Hoechst 33342 (blue), cell boundaries are visualized by staining for Epcam (magenta), and viruses are stained for capsid protein VP1 (yellow). (b) Enteroendocrine cells are stained for ChgA (cyan), nuclei are stained with Hoechst 33342 (blue), tight junctions are stained for zo-1 (magenta), and viruses are stained for capsid protein VP1 (yellow). (c) Proliferating cells are stained for PCNA (cyan), nuclei are stained with Hoechst 33342 (grey), cell boundaries are visualized using Epcam (magenta), and viruses are stained for capsid protein VP1 (yellow). Arrows indicate some PCNA positive cells. The scale bar is 50 µm.