Research ArticleVIROLOGY

Human intestinal tract serves as an alternative infection route for Middle East respiratory syndrome coronavirus

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Science Advances  15 Nov 2017:
Vol. 3, no. 11, eaao4966
DOI: 10.1126/sciadv.aao4966
  • Fig. 1 Susceptibility of human primary intestinal cells, intestine explants to MERS-CoV, and identification of the replication intermediate in the stool specimen of a MERS patient.

    (A) Human primary intestinal cells were inoculated with MERS-CoV (left and middle) or mock-infected (right). At 24 hpi, cells were fixed and applied to immunofluorescence staining of MERS-CoV nucleocapsid protein (NP). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). The images show the representative results of one experiment. (B and C) Normal human small intestine explants were inoculated with MERS-CoV, as described in Materials and Methods. The data show representative results of the experiments independently performed twice. (B) The infected (left and middle) and mock-infected (right) explants were fixed at 20 hpi for immunofluorescence staining of MERS-CoV NP and the enterocyte marker CK19. Syncytia formation can be observed in the infected intestinal epithelium (middle). (C) At the indicated hpi, the explants (Tissue) and Matrigel together with culture medium (Matrigel) were harvested for the quantification of viral load. Data are means ± SD of viral loads in duplicated samples. (D) Chromatograph of the N gene sgRNA recovered from the stool specimen of a MERS patient show fusion of leader sequence, TRS, and N gene element.

  • Fig. 2 MERS-CoV infection and replication in human intestinal organoids.

    The differentiated intestinoids were inoculated with MERS-CoV (MOI ≈ 0.05) in duplicate and then re-embedded in Matrigel and maintained in culture medium. (A) At the indicated hpi, intestinoids (Intestinoid), cell-free Matrigel, and culture medium (Matrigel) were harvested for the quantification of viral load with RT-qPCR. Serially diluted MERS-CoV NP plasmids were used to generate a standard curve for the quantification. (B) The absolute viral loads in intestinoids were normalized with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA transcripts. (C) The dissolved Matrigel and culture medium were applied to viral titration with plaque assay. Data are means ± SD of one representative experiment independently repeated three times. (D and E) At 24 hpi, the infected and mock-infected intestinoids were fixed after disassociation and stained with the MERS-CoV NP antiserum and then applied to flow cytometry to evaluate the percentage of NP-positive cells. The histogram shows the results of one representative experiment. (E) Data are means and SD of three independent experiments. Student’s t test was used for data analysis. **P ≤ 0.01. (F) At 24 hpi, the infected (MERS-CoV) and mock-infected intestinoids, after fixation and immunofluorescence staining of MERS-CoV NP and CK19, were whole-mounted and imaged with a confocal microscope.

  • Fig. 3 MERS-CoV treatment with simulated gastrointestinal fluids.

    MERS-CoV solution [7.5 × 106 plaque-forming units (PFU), 500 μl] was mixed with 5 ml of FaSSGF, FeSSGF, FeSSIF, or DMEM and then incubated at 37°C for the indicated minutes. An aliquot (1 ml) of the virus/fluid mixture was sampled for virus titration in Vero-E6 cells in triplicate after neutralization with sodium hydroxide. The enterovirus EV71 and the human coronavirus hCoV-229E were treated with the same gastrointestinal fluids and titrated with TCID50 (median tissue culture infectious dose) assay in RD cells and plaque assay in Huh7 cells, respectively. Data are from a representative experiment independently performed three times.

  • Fig. 4 MERS-CoV replication and cell apoptosis in the polarized Caco-2 cells.

    (A and B) The polarized Caco-2 cells were inoculated with MERS-CoV in duplicate from the apical or basolateral side of the monolayer with an MOI of 0.1. At the indicated hpi, cell-free media were harvested from the upper and bottom chambers for viral load quantification. The experiments were independently performed twice. A representative experiment is presented. (A) Viral gene copy number in the medium collected from the upper chamber (Apical release) and bottom chamber (Basolateral release) after apical inoculation (Apical inoc) and basolateral inoculation (Basolateral inoc). (B) Viral titer in the media harvested at 24 hpi detected by plaque assay. No plaque was detected in the media at 2 hpi. (C and D) The polarized Caco-2 cells were inoculated from the apical or basolateral side with MERS-CoV at an MOI of 2 or mock-inoculated. (C) At 24 hpi, cells were fixed after dissociation and applied to flow cytometry to detect the expression of MERS-CoV NP and activated caspase-3. The left and middle panels are the histograms of one representative experiment. The right panel presents means and SD of three independent experiments. *P ≤ 0.05. (D) At 24 hpi, the polarized Caco-2 cells seeded on polycarbonate membranes were fixed and applied to immunofluorescence staining of MERS-CoV NP (red) and imaged en face.

  • Fig. 5 MERS-CoV enteric infection in hDPP4 transgenic mice.

    hDPP4 mice were inoculated with MERS-CoV via direct intragastric gavage or intragastrically injected as described in Materials and Methods. (A) (a to c) Representative histopathology in small intestines at days 1, 3, and 5 after inoculation, respectively. (d and e) Small intestines of pantoprazole-pretreated mice at day 5 after inoculation. (f) Small intestine of a mock-infected mouse. H&E staining, magnification ×200. (B) Identification of MERS-CoV–infected cells in the intestines of the inoculated hDPP4 mice with immunostaining of NP and cell type markers. (a to c) Virus-positive enterocytes in mice at day 1 after inoculation. The infected enterocytes (c) form syncytia. (d to f) Virus-infected cells in mouse intestines at days 5 and 8 after inoculation, respectively. Arrows show NP-positive cells in the lamina propria, including the NP/CD68 double-positive macrophage (f). The arrowhead (in d) indicates virus-positive cells in Peyer’s patch. (g) Virus-positive cells in the colon of an inoculated mouse. (h) Costaining of NP/CD3 in the mock-infected mouse. (C) Three intragastrically injected mice of the indicated groups were sacrificed at the indicated day after inoculation; intestine homogenates were applied for the quantification of viral load by RT-qPCR. The gray and black bars represent the viral loads of the mice pretreated with pantoprazole and mock-treated with PBS, respectively. Data are means and SD of three mice.

  • Fig. 6 Inflammation and MERS-CoV infection in the lung tissues of hDPP4 mice.

    (A) Lung tissues in the mice without pantoprazole pretreatment (a) versus those in pantoprazole pretreatment (b and c) at day 5 after intragastric MERS-CoV inoculation. (d) Lung pathology in an inoculated mouse at day 8 after intragastric inoculation without pantoprazole pretreatment. (f) Lung tissue of a mock-infected mouse. H&E staining, magnification ×200. (e) Immunostaining shows virus-positive cells (green) in the lung tissue of a mouse at day 8 after intragastric inoculation. (B) Lung tissues were harvested from three intragastrically injected mice at the indicated day after inoculation and homogenized for the quantification of viral load by RT-qPCR. The gray and black bars represent the viral loads of the mice pretreated with pantoprazole and mock-treated with PBS, respectively. Data are means and SD of three mice.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/11/eaao4966/DC1

    fig. S1. MERS-CoV replication in human primary intestinal epithelial cells.

    fig. S2. Expression of human DPP4 on the surface of epithelial cells in intestinoids.

    fig. S3. Formation of intact epithelial barrier in the polarized Caco-2 cells after Transwell culture.

    fig. S4. The virus-positive cells in small intestine of an intragastrically inoculated mouse and those in the lung tissue of an intranasally inoculated mouse.

    fig. S5. Identification of live viruses in the lung and brain homogenates and increased viral load in the brain tissues of intragastrically injected mice.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. MERS-CoV replication in human primary intestinal epithelial cells.
    • fig. S2. Expression of human DPP4 on the surface of epithelial cells in intestinoids.
    • fig. S3. Formation of intact epithelial barrier in the polarized Caco-2 cells after Transwell culture.
    • fig. S4. The virus-positive cells in small intestine of an intragastrically inoculated mouse and those in the lung tissue of an intranasally inoculated mouse.
    • fig. S5. Identification of live viruses in the lung and brain homogenates and increased viral load in the brain tissues of intragastrically injected mice.

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