Human Dendritic Cells Transmit Enterovirus A71 via Heparan Sulfates to Target Cells Independent of Viral Replication

Figures

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  FIG 1. Human dendritic cells transmit EV-A71 C1 to target cells. (A to D) DCs were exposed to EV-A71 C1 (MOI 0.5) for 2 days, and after extensive washing cocultured with RD99 cells. DCs were treated with Ribavirin (RBV; 10 μM) to control for a replicating infection in DCs during transmission. After coculture, supernatant and cells were collected at different time points and analyzed by RT-qPCR and flow cytometry, respectively. (A to C) Flow cytometry analyses of DC transmission to RD999 is shown for one representative donor (A) and combined data for different donors on infection of CD1a-negative RD99 cells by flow cytometry (B, N = 4) and virus production in coculture (C, N = 4). The symbols represent independent donors mean ± s.d. (flow cytometry) or mean ± SEM (RT-qPCR) of duplicates. (D) Images show the morphological changes of RD99 cells indicating the cytopathic effects 48 h after incubation with a representative DC donor.

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  FIG 2. Human dendritic cells transmit EV-A71 C5 to target cells. (A to D) Transmission experiments with EV-A71 C5 are performed as described in Fig. 1. (A) Populations plots of one representative donor are shown to indicate the percentages of gated cells, (B, N = 4) followed by the combined percentage of EV-A71 VP1 positive RD99 cell (CD1a-negative population) as detected by flow cytometry and (C, N = 4) the total amount of viral copies as detected by RT-qPCR. The symbols represent independent donors mean ± s.d. (flow cytometry) or mean ± SEM (RT-qPCR) of duplicates. (D) Images show the morphological changes of RD99 cells indicating the cytopathic effects 48h after incubation with a representative DC donor.

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  FIG 3. Human dendritic cells transmit EV-A71 C1 to target cells. (A to D) DCs were exposed to EV-A71 C1 or EV-A71 C5 (MOI 0.5) for 2 days in the absence or presence of replication inhibitor ribavirin (10 μM). Next, DCs were washed extensively and cocultured with RD99 cells. DCs from Fig. 1 and Fig. 2 were collected and analyzed by flow cytometry, respectively, at the moment of coculture (48 h) or 1 day (72 h) after transmission. (A) Populations plots of one representative DC donor are shown to indicate the percentages of gated cells followed by the combined percentage of (B, N = 4) EV-A71 C1 and (C, N = 3) EV-A71 C5 in the DC population (CD1a-positive population) as determined by flow cytometry. The symbols represent independent donors mean ± s.d. of duplicates.

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  FIG 4. Heparan sulfates are involved in EV-A71 C1 transmission by DCs. (A to D) Transmission experiments with EV-A71 C1 are performed as described in Fig. 1. DCs were preincubated with either anti-DC-SIGN antibodies or soluble heparin (5 mg/mL). After coculture, supernatant and cells were collected at different time points, and analyzed by RT-qPCR and flow cytometry, respectively (A) Populations plots of one representative DC donor are shown to indicate the percentages of gated cells, (B, N = 4) followed by the combined percentage of EV-A71 C1 positive cells RD99 cells (CD1a-negative population) as determined by flow cytometry, (C, N = 4) and the total amount of viral copies as detected by real-time PCR. The symbols represent independent donors mean ± s.d. (flow cytometry) or mean ± SEM (RT-qPCR) of duplicates. (D) Images are included to show the morphological changes of RD99 cells 48 h after incubation with a representative DC donor.

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  FIG 5. Heparan sulfates are involved in EV-A71 C5 transmission by DCs. (A to D) Transmission experiments with EV-A71 C5 are performed as described in Fig. 4. (A) Populations plots of one representative DC donor are shown to indicate the percentages of gated cells, (B, N = 4) followed by the combined percentage of EV-A71 C5 positive cells RD99 cells (CD1a-negative population) as determined by flow cytometry, (C, N = 4) and the total amount of viral copies as detected by real-time PCR. The symbols represent independent donors mean ± s.d. (flow cytometry) or mean ± SEM (RT-qPCR) of duplicates. (D) Images are included to show the morphological changes of RD99 cells 48h after incubation with a representative DC donor.