Exosomes Facilitate Transmission of Enterovirus A71 From Human Intestinal Epithelial Cells

Figures

• 

  Figure 1.. Enterovirus A71 (EV-A71) actively replicates in differentiated C2BBe1 cells. (A) Flow cytometry results showing the percentage and mean fluorescence intensity of E-cadherin-, CDX-2-, and occludin-positive cells in undifferentiated and differentiated cells. (B) Transmission electron microscopy of undifferentiated and differentiated C2BBe1 cells to examine the presence of microvilli (scale bar for C2BBe1 cells, 1 μm; scale bar for differentiated C2BBe1 cells, 0.5 μm). (C) Differentiated C2BBe1 cells were infected with EV-A71 at a multiplicity of infection (MOI) of 10 for 12 hours. Immunofluorescence staining was performed to detect double-stranded ribonucleic acid (dsRNA). 4’,6-Diamidino-2-phenylindole (DAPI) staining was performed as internal control (scale bar, 50 μm/10 μm). (D) Flow cytometry results showing the percentage of EV-A71 3D-positive cells in differentiated cells infected with EV-A71. (E) Total protein was extracted from differentiated C2BBe1 cells infected with EV-A71 and analyzed by Western blotting with an antibody specific for EV-A71 3D protein. The expression of β-actin was used as an internal control. (F) Total RNA was isolated from differentiated C2BBe1 cells…

• 

  Figure 2.. Enterovirus A71 (EV-A71) viral particles exit differentiated enterocytes via a nonlytic pathway. (A) Differentiated and undifferentiated C2BBe1 cells were infected with EV-A71 at a multiplicity of infection (MOI) of 10, whereas RD cells were infected with EV-A71 at an MOI of 2. Phase contrast images of cells from mock- or EV-A71-infected groups were taken at 24 and 48 hours postinfection (h p.i.). (B) Cell viability of differentiated C2BBe1 cells, undifferentiated C2BBe1, and RD cells infected with EV-A71 were determined by MTT assays. (C) The viral titers of total lysates (supernatants + infected cells) and supernatants were quantified by plaque-forming assays. The data are presented as the means ± standard deviation (*, P < .05; **, P < .01; ***, P < .001).

• 

  Figure 3.. Enterovirus A71 (EV-A71) infection of differentiated C2BBe1 cells does not activate caspase 3 and is not affected by apoptosis inhibitors. (A) Differentiated and undifferentiated C2BBe1 were infected with EV-A71 at a multiplicity of infection (MOI) of 40. RD cells were infected with EV-A71 at an MOI of 0.2. Total protein was isolated from cells at different time points. Immunoblot assays were performed to detect the expression of procaspase 3, active caspase 3, and viral protein 3D. The expression of β-actin was used as an internal control. (B) Mock- and EV-A71-infected cells were fixed after 24 hours and allowed to react with mouse anti-EV-A71 3D and rabbit anti-active caspase 3 monoclonal antibodies (Abs), respectively. Phycoerythrin-conjugated antimouse immunoglobulin (Ig)G and fluorescein isothiocyanate-conjugated antirabbit IgG Abs were used for detection. (C) Differentiated C2BBe1 cells, undifferentiated C2BBe1, and RD cells were treated with Z-VAD-FMK (20 μM) or control medium and subsequently infected with EV-A71 at an MOI of 10 for undifferentiated and differentiated C2BBe1 cells and an MOI of 2 for RD cells. Total lysates (supernatants + infected cells) and supernatants…

• 

  Figure 4.. Enterovirus A71 (EV-A71) increases the release of exosomes from differentiated C2BBe1 cells and human intestinal organoids. (A) Differentiated C2BBe1 cells were infected with EV-A71 at a multiplicity of infection of 10 for 24 hours. Exosomes purified from mock- and EV-A71-infected samples were subjected from total protein isolation. Immunoblotting analysis was performed using antibodies specific for CD63, calnexin, TSG101, and EV-A71 3D. Total protein extracted from supernatants and cells collected from infected samples were used as control. (B) Flow cytometric results showing the percentage of CD63-positive vesicles released from mock- and EV-A71-infected differentiated C2BBe1 cells. (C) FluoroCet Exosome quantitation kit was performed to quantify the exosomes extracted from differentiated C2BBe1 cells that were mock and EV-A71 infected. (D) Cryoelectron microscopy images of the exosomes purified from the differentiated C2BBe1 cells infected with EV-A71. (E) Intestinal organoids were infected with EV-A71 for 48 hours. Immunofluorescence staining was performed using antibodies specific for double-stranded ribonucleic acid (dsRNA). (scale bar, 50 μm/10 μm). (F) Supernatants and…

• 

  Figure 5.. Exosomes secreted from differentiated C2BBe1 cells and human intestinal organoids contain enterovirus A71 (EV-A71) components. (A) Differentiated C2BBe1 and intestinal organoids were infected with EV-A71 at a multiplicity of infection of 10 for differentiated C2BBe1 cells and with 2 × 104 plaque-forming units virus per organoid. The supernatants were collected for exosome isolation, and immunoblotting analysis was performed using antibodies specific for EV-A71 VP0 protein, calnexin, CD63, and TSG101. (B) Quantitative reverse-transcription polymerase chain reaction was performed to detect the expression levels of EV-A71 5’UTR ribonucleic acid (RNA) in exosomes harvested from differentiated C2BBe1 cells and intestinal organoids that were mock and EV-A71 infected. (C) Transmission electron microscope images of the exosomes purified from differentiated C2BBe1 cells infected with EV-A71 are shown (i and ii). The viral stock solution was applied onto an electron microscope grid to observe the viral particles (iii). Arrow indicates the presence of a viral particle (ii). Scale bar, 50 nm. h p.i., hours postinfection; vRNA, viral RNA segments.

• 

  Figure 6.. Exosomes are able to transmit enterovirus A71 (EV-A71) components and establish active replication in other cells. (A) Exosomes collected from mock- and EV-A71-infected intestinal organoids were collected and labeled with DiD (uninfected exosomes, exosomes collected from mock-infected intestinal organoids; infected exosomes, exosomes collected from EV-A71 infected intestinal organoids). The DiD-labeled exosomes were then added to RD cells, and immunofluorescence staining was performed to detect the expression of viral protein at 6 hours posttreatment. The EV-A71 virus stock was prepared using the freeze-thaw method and was used a positive control (scale bar, 20 μm). (B) Exosomes isolated from differentiated C2BBe1 cells and intestinal organoids infected with EV-A71 were used to infect RD cells, and the viral titers were quantified at different time points by plaque-forming assays. DAPI, 4’,6-diamidino-2-phenylindole; h p.i., hours postinfection; PFU, plaque-forming units.

• 

  Figure 7.. Exosome inhibitors suppress replication of enterovirus A71 (EV-A71) in differentiated C2BBe1 cells and improve survival rates of infected animals. (A) Differentiated C2BBe1 cells were treated with GW4869 (1 and 3 μM) or ketotifen (5 and 10 μM) and then infected with EV-A71 at a multiplicity of infection (MOI) of 10. Total cell lysates (supernatants and infected cells) were collected at 24 hours postinfection (h p.i.), and viral titers were quantified. (B) The plaque assay was also performed to quantify the released virus particles in the culture supernatants. (C) RD cells were treated with GW4869 (1 and 3 μM) or ketotifen (5 and 10 μM) and then infected with EV-A71 at an MOI of 2, and total cell lysates were collected at 24 h p.i. Plaque assays were performed to quantify the viral titers. (D) The released virus particles in the culture supernatants of RD cells were quantified with plaque-performing assay. (E) Transgenic mice expressing hSCARB-2 (hSCARB2-TG) mice were intragastrically administered 50 μL phosphate-buffered saline ([PBS] n = 9) or GW4869 (3 mM) (n = 10) 2 hours before EV-A71 challenge (2 × 106 plaque-performing units [PFU]) via the oral route. The PBS or GW4869 were…