Origin of tested bat cell lines.

<p>Origin of tested bat cell lines.</p

Expression of human CD26/DPP4 confers MERS-CoV susceptibility to otherwise resistant bat cells.

<p>(A) Viral yields from MERS-CoV-resistant PESU-B5L, R05T, R06E, and Tb1Lu bat cells. Cells were transfected with a plasmid expressing human CD26/DPP4 or empty control plasmid and exposed 48 h later to MERS-CoV/EMC at an MOI of 3. Supernatants were harvested at 24 h after virus exposure for quantification of virus yields by plaque assay. (B) Same experiment: representative immunofluorescence assay (IFA) images of cells stained with anti-MERS-CoV spike protein antibody (green, top) or anti-human CD26/DPP4 antibody (red, bottom). (C) Merged IFA images demonstrate colocalization of MERS-CoV spike protein and CD26/DPP4. (D). Viral yields from MERS-CoV-susceptible bat cells transfected with a plasmid expressing human CD26/DPP4 or empty control plasmid using procedures identical to resistant cells in (A) except that cells were exposed to virus 24 h after transfection. Error bars indicate the standard deviation of duplicate samples.</p

Cell-surface expression of CD26/DPP4 on bat cells.

<p>Cells of different bat cell lines were analyzed by flow cytometry after staining with goat anti-human DPP4/CD26 antibody (red line) or control antibody (black lines).</p

Persistent MERS-CoV infection of bat cells induces downregulation of bat cell CD26/DPP4 expression.

<p>Bat cell lines susceptible to infection were infected with MERS-CoV/EMC (A) or MERS-CoV/Jor (B) at an MOI of 1. After 7 days, supernatants were harvested for virus yield analysis by plaque assay, and the cells were subcultured at a 1∶10 dilution in new flasks. Subsequently, the persistently infected cells were passaged at a 1∶10 dilution weekly. Error bars indicate the standard deviation of duplicate samples. (C and D) Same experiment: immunofluorescence assay (IFA) images of bat cells persistently infected with MERS-CoV/EMC (C) or MERS-CoV/Jor (D) at day +33 stained with anti-MERS-CoV spike protein antibody (green). (E) Same experiment: TEM images of bat cells persistently infected with MERS-CoV/EMC at day 56. (F) Flow cytometry data of CD26/DPP4 surface expression (red line: anti-human CD26-/DPP4 antibody; black line: control antibody) in persistently infected cells. (G) CD26/DPP4 expression in persistently infected EidNi/41.3 cells (day 63) as detected by western blot.</p

Six of ten tested bat cell lines are susceptible to MERS-CoV infection.

<p>(A and B) Ten different bat cell lines were exposed to MERS-CoV/EMC (A) or MERS-CoV/Jor (B) at an MOI of 1. Supernatants were harvested at days 0, 1, 3, and 5 after virus exposure, and virus yields were determined by plaque assay on Vero cells. Error bars indicate the standard deviation of triplicate samples. (C and D) Same experiment: immunofluorescence assay (IFA) images of bat cell lines exposed to MERS-CoV/EMC (C) or MERS-CoV/Jor. (D) 1 (D1) or 3 (D3) days after virus exposure and stained against MERS-CoV spike protein (green). (E) Same experiment: TEM images of bat cells infected with MERS-CoV/EMC at day 1 after virus exposure. Red arrows point at double-membrane vesicles (DMVs) typical of coronavirus infections.</p

<i>In vitro</i> detection of KRCV-1 RNA in infected cells using RNAscope^® <i>in situ</i> hybridization.

<p>(A) SHFV-infected MA-104 cells labeled with SHFV- (left) or KRCV-1-specific (right) probes. (B) KRCV-1-infected MARC-145 cells labeled with SHFV- (left) or KRCV-1-specific (right) probes. Positive results manifest as brown staining after amplification. All images were originally taken at 400X magnification.</p

Anti-human CD26/DPP4 antibody inhibits MERS-CoV infection of bat cells.

<p>RoNi/7.1 or Huh-7 cells (control) were treated with increasing concentrations (0, 1.25, 2.5, 5, 10, and 20 µg/ml) of anti-human CD26/DPP4 antibody or control antibody and then exposed to MERS-CoV/EMC at an MOI of 1. (A) After 24 h, viral yields in supernatants were determined by plaque assay. (B) Cellular infection was determined by immunofluorescence assay (IFA) with an anti-MERS-CoV spike protein antibody (green). (B left) The percentage of infected cells was analyzed by high content imaging. (B right) Representative IFA images. Error bars indicate the standard deviation of triplicate samples.</p

<i>In vitro</i> detection of SHFV RNA in infected cells using RNAscope^® <i>in situ</i> hybridization.

<p>(top): uninfected control HeLa cell slides. (top left) Result RNAscope^® in situ hybridization with a negative-control target probe targeting the bacterial <i>dapB</i> gene. (top right) Result with a positive-control target probe targeting the human <i>POLR2A</i> gene. (center and bottom) Uninfected- or SHFV-infected MA-104 cells treated with unspecific (<i>dapB</i>) or SHFV-specific target probes. Positive results manifest as brown staining after amplification (top right, bottom left). All images were originally taken at 400X magnification.</p

<i>In situ</i> detection of SHFV RNA from tissue sections from an SHFV-infected rhesus monkey using RNAscope^® <i>in situ</i> hybridization.

<p>(A) Liver sections from an uninfected or SHFV-infected rhesus monkey labeled with unspecific (<i>dapB</i>) or SHFV-specific target probes. Top: all images were originally taken at 200X magnification. Bottom: all images were originally taken at 400X magnification. Positive results manifest as brown staining. (B) Detection of SHFV RNA in brain, spleen, and additional liver sections of the same animal (original magnification 400X). Positive results manifest as brown staining after amplification. (C) Quantification of SHFV RNA-positive foci in brain, liver, and spleen sections by counting; four fields were counted per tissue section of 200X-magnified images (<i>p</i> value calculated by multiple t-test analysis with GraphPad Prism 6 software).</p

Alphavirus infection causes actin rearrangements into actin foci that are Rac1- and Arp3-dependent and that co-localize with Rac1, PIP5K1-α, and E2.

<p>(<b>A</b>) Representative confocal images of mock-, VEEV-, CHIKV-, or RVFV-infected HeLa cells (MOIs = 0.5, 5, or 3, respectively). Cells were fixed and stained with virus-specific antibodies (VEEV and CHIKV E2, RVFV nucleoprotein; shown in green) and phalloidin (red) 18 h (VEEV) or 24 h (CHIKV, RVFV) after infection. Nucleus staining is shown in blue. Representative actin foci are indicated by asterisks. (<b>B</b>) High-content quantitative image-based analysis was used to measure infection rates of VEEV, CHIKV, and RVFV (left panel), and the number of actin foci per cell (number of actin foci/total cell number, right panel). Analysis is based on single Z sections. ***, <i>p</i> < 0.0001, Student's <i>t</i> test (between the sample and mock). (<b>C</b>) VEEV-infected HeLa cells (MOI = 0.5) were fixed in formalin at the indicated time points, stained, and analyzed as in (<b>B</b>). (<b>B</b>–<b>C</b>) Values represent the mean ± SD, n ≥12. (<b>D</b>) Representative confocal images of VEEV-infected HeLa cells (MOI = 0.5) pretreated with the Rac1 inhibitor EHT1864 or Arp3 inhibitor CK548. Cells were fixed 18 h after virus addition and stained with VEEV E2-specific antibody (green), phalloidin (red), and a nuclear stain (blue). (<b>E</b>) High-content quantitative image-based analysis was used to measure infection rates of VEEV and the number of actin foci per cell. (<b>F</b>) Confocal images of VEEV-infected HeLa cells (MOI = 5). Co-localization of hemagglutinin (HA)-tagged PIP5K1-α (top panel) or Rac1 (bottom panel) (blue), actin (red), and VEEV E2 (green), at a single z section is shown (left panel). Insets: zoom on actin filaments indicated by white arrows. Single channel intensities were measured along lines crossing different actin clusters (right panel). VEEV was added to HeLa cells that were reverse-transfected with a plasmid encoding HA-tagged PIP5K1-α or tetracycline-induced T-Rex HeLa cells that expressed Rac1 fused to eGFP. Cells were fixed 20 h later, permeabilized, and stained with VEEV E2-specific antibody, phalloidin, and an antibody against HA.</p