Enterovirus Replication and Dissemination Are Differentially Controlled by Type I and III Interferons in the Gastrointestinal Tract

Figures

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  FIG 1. Human FcRn is necessary and sufficient for echovirus infection of murine-derived primary enteroids. (A) Murine enteroids were generated from Lgr5+ crypts isolated from the small intestines of five 10-day-old neonatal C57BL/6J (WT) mice. Confocal microscopy images of enteroids immunostained with cytokeratin-19 in green and actin in red (left) or mucin-2 in green and actin in red (right) ~10 days after culturing are shown. (B) WT (white) or hFcRnTg32 (red) enteroids were generated from small intestine tissue from 10-day-old neonatal mice and infected with 106 PFU of neutral red-incorporated E5. Viral titers (log10 PFU per milliliter) were assessed in the cell culture supernatants at the indicated time points. (C) Heatmap of reads per kilobase per million (RPKM) values of the type I IFNs Ifna4 and Ifnb1 and the type III IFNs Ifnl2 and Ifnl3 from bulk RNASeq of uninfected (mock) or E5-infected hFcRnTg32 enteroids at 24 h postinfection. In the key at the right, purple indicates higher reads, and gray denotes no reads detected. At the bottom are viral fragments per kilobase per million (FPKM) values from the samples shown at the top. Yellow indicates high viral RNA reads, and gray…

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  FIG 2. Expression of human FcRn is not sufficient for echovirus infection by the enteral route in vivo. (A) Table of the six genotypes used in this study. Shown is the expression of mouse or human FcRn, IFNAR, and IFNLR among these genotypes. (B) Survival of the indicated genotype of mice inoculated with 106 PFU of E5 by oral gavage for 7 days postinoculation. The log rank test was used to analyze the statistical differences of the survival rates. (C to H) At 3 dpi, animals were sacrificed, and viral titers in the stomach (C), duodenum (D), jejunum (E), ileum (F), large intestine (G), and stool (H) were determined by TCID50 assays. In all panels, titers are shown as log10 TCID50 per milliliter, with the limit of detection indicated by a dotted line. Data are shown as means ± standard deviations, with individual animals shown as each data point. Data are shown with significance determined by a Kruskal-Wallis test with Dunn’s test for multiple comparisons (*, P < 0.05; **, P < 0.005; ***, P < 0.0005).

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  FIG 3. Type I IFNs control echovirus dissemination from the GI tract. Seven-day-old pups were orally inoculated with 106 PFU of E5, and at 3 dpi, animals were sacrificed for viral titration and histology. (A to D) Viral titers in the blood (A), liver (B), pancreas (C), and brain (D). In all panels, titers are shown as log10 TCID50 per milliliter, with the limit of detection indicated by a dotted line. Data are shown as means ± standard deviations, with individual animals shown as each data point. (E) Hybridization chain reaction (HCR) RNA fluorescence in situ hybridization (RNA-FISH) from liver sections of hFcRnTg32, hFcRnTg32-IFNAR−/−, or hFcRnTg32-IFNLR−/− neonatal mice at 3 dpi using probes against the E5 genome (green) and albumin (red). DAPI-stained nuclei are shown in blue. Bars, 100 μm. In panels A to D, data are shown with significance determined by a Kruskal-Wallis test with Dunn’s test for multiple comparisons (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001).

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  FIG 4. Type III IFNs restrict persistent echovirus infection in the GI epithelium. Seven-day-old neonatal mice were orally inoculated with 106 PFU of E5, and at 7 dpi, animals were sacrificed for viral titration and tissue collection. (A to F), Viral titers in the stomach (A), duodenum (B), jejunum (C), ileum (D), large intestine (E), and stool (F). In all panels, titers are shown as log10 TCID50 per milliliter, with the limit of detection indicated by a dotted line. Data are shown as means ± standard deviations, with individual animals shown as each data point. Significance was determined using a Kruskal-Wallis test with Dunn’s test for multiple comparisons (*, P < 0.05; **, P < 0.005). (G) At 3 dpi, animals were sacrificed, and the entire GI tract was removed and Swiss rolled, followed by histological sectioning. HCR of hFcRnTg32-IFNAR−/− or hFcRnTg32-IFNLR−/− pups at 3 dpi was performed using probes against the E5 genome (green) and DAPI (blue). Bars, 100 μm. Zoomed-in views of specific regions of the hFcRnTg32-IFNLR−/− images are shown on the right. (H) Quantification of three independent tile scans using confocal microscopy of each region of the small intestine based on the number…

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  FIG 5. In vivo replication of echoviruses is specific for enterocytes. (A) Hybridization chain reaction (HCR) RNA-FISH of uninfected small intestine sections using specific probes against Alpi, Muc2, or Chga (in green), as indicated at the bottom. DAPI-stained nuclei are shown in blue. A no-probe-containing control is shown on the left. In all panels, the white box is shown zoomed in (magnification, approximately ×6) at the right using the probes indicated in red. Red arrows in the Alpi section denote goblet cells based on morphology that were not positive for Alpi, as expected. (B to D) Seven-day-old hFcRnTg32-IFNLR−/− neonatal mice were orally inoculated with 106 PFU of E5; at 3 dpi, animals were sacrificed; and the entire small intestine was removed and Swiss rolled for subsequent histological sectioning. Shown are representative images of ileum tissue using probes for E5 (green in all panels) and either Alpi (B), Muc2 (C), or Chga (D) (red in all panels). DAPI-stained nuclei are shown in blue. In all panels, white boxes denote zoomed-in areas shown on the right, which include black-and-white images, as indicated. Bars, 50 μm. (E) Quantification of confocal images was performed using…