Comparison of tissue tropism and host response to enteric and respiratory enteroviruses

Figures

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  Fig 1. EV-D68 and EV-D94 tissue tropism (A-C) and IFN-λ induction (D-F). Tissues were inoculated with equivalent multiplicity of infection (MOI) of EV-D94 and EV-D68 and replication was assessed by RT-qPCR. Respiratory (A) and intestinal (B) tissues were inoculated apically and washed 3 times at 3 hours post-infection (hpi). Residual virus was quantified after the washes. Apical samples were then collected at the indicated time points for viral RNA quantification. Neural tissues (C) were incubated with viral suspension and viral RNAs extracted from tissue lysate after inoculation (2hpi) and 3 days later were compared. IFN-λ mRNA levels (D-F) were measured in tissue lysates collected 3dpi and fold change relative to non-infected tissues was calculated with the ΔΔCt method. Significance between viruses is shown with black stars. In D, E and F, significance relative to mock-infected is shown with grey stars.

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  Fig 2. Differential replication of EV-D94 and EV-D68 in respiratory tissues. A) Viral replication at 33°C and 37°C. Viral replication was quantified as in Fig 1A and 1B) 3D projections of confocal images of infected respiratory tissues at 1dpi. Nuclei were stained with DAPI (blue), viral RNA with J2 antibody (green) and ciliated cells with anti-beta IV tubulin antibody (red). Images were acquired in the same area at different z-stacks and 3D projections (with apical tissue side on top) were generated with Imaris. The corresponding 2D images are available in S2 and S3 Figs with co-staining of virus and ciliated or basal cells respectively. C) Binding assay. Tissues were infected 1h 4°C, washed extensively and viral loads were quantified in tissue lysates by RT-qPCR. D) Binding assay in tissues pretreated or not with sialidase. Tissues were washed 3 times with PBS and incubated for 1h at 37°C with sialidase or with control buffer. After extensive washing, binding assay was run as in C. Treated and untreated tissues were processed in parallel for sialic acid staining (S4 Fig).

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  Fig 3. Single step growth curve assay of EV-D68 and EV-D94 grown at 33°C. Viral loads were quantified in tissue lysates by RT-qPCR at the indicated time points (A). IFN-λ (B), IFN-β (C) and IFN-α (D) mRNAs were quantified from the same tissue lysates as in A) and fold changes were calculated relative to the average induction observed for the two viruses at the time of infection (0hpi) with the ΔΔCT method. In A) Statistical analysis was performed comparing at each time point the replication between the two viruses while for B to D, significance was calculated at each time point, relative to IFN induction by the same virus at 0hpi.

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  Fig 4. Comparison of the transcriptomic profile of non-infected respiratory and intestinal tissues. A) Gene expression in RNA reads per million (rpm) of mock-infected respiratory and intestinal tissues. A threshold of 1rpm determines expressed genes (horizontal/vertical lines). 2-fold changes determine “tissue-up” genes. Expressed genes were divided in 5 groups: orange (intestinal specific transcripts), brown (transcripts highly expressed in intestinal tissues), turquoise (transcripts equally expressed in both tissues), pink (transcripts highly expressed in respiratory tissues), blue (respiratory specific transcripts). Unexpressed genes in both tissues (‹1 rpm) are represented in green. B) Functions associated to each group of genes in A). Functions were defined by the top enriched uniport keywords associated to each group of genes. Circle sizes represent p-value. The list of genes for all categories is provided in the GEO database with accession number GSE184488.

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  Fig 5. Significantly enriched pathways after infection by EV-D94 or EV-D68 in respiratory and intestinal tissues. A). Genes with average log2 fold change increase/decrease of 0.5 versus the mock were considered for pathway analysis. Pathway with p-value < 0.00001 were classified as enriched. Yellow bars represent upregulated pathways while purple bars represent downregulated ones. Bar size illustrates p-value. 1* Cell-Cell communication; 2* Cellular responses to external stimuli; 3* Developmental biology; 4* DNA repair; 5* Extracellular matrix organization; 6* Gene expression (Transcription); 7* Hemostasis; 8* Metabolism; 9* Organelle biogenesis and maintenance; 10* Signal transduction. Functional annotations were obtained from reactome.org. B) Heatmap of selected immune genes. The FC values for each transcript are represented in S7 Fig. Images depicting intestinal and respiratory tissue sections were obtained from Mattek and from [25]. Another representation of these data is available in S6 Fig and at [http://genebrowser.unige.ch/enterovirus/].