CD26/DPP4 cell-surface expression in bat cells correlates with bat cell susceptibility to Middle East respiratory syndrome coronavirus (MERS-CoV) infection and evolution of persistent infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently isolated betacoronavirus identified as the etiologic agent of a frequently fatal disease in Western Asia, Middle East respiratory syndrome. Attempts to identify the natural reservoirs of MERS-CoV have focused in part on dromedaries. Bats are also suspected to be reservoirs based on frequent detection of other betacoronaviruses in these mammals. For this study, ten distinct cell lines derived from bats of divergent species were exposed to MERS-CoV. Plaque assays, immunofluorescence assays, and transmission electron microscopy confirmed that six bat cell lines can be productively infected. We found that the susceptibility or resistance of these bat cell lines directly correlates with the presence or absence of cell surface-expressed CD26/DPP4, the functional human receptor for MERS-CoV. Human anti-CD26/DPP4 antibodies inhibited infection of susceptible bat cells in a dose-dependent manner. Overexpression of human CD26/DPP4 receptor conferred MERS-CoV susceptibility to resistant bat cell lines. Finally, sequential passage of MERS-CoV in permissive bat cells established persistent infection with concomitant downregulation of CD26/DPP4 surface expression. Together, these results imply that bats indeed could be among the MERS-CoV host spectrum, and that cellular restriction of MERS-CoV is determined by CD26/DPP4 expression rather than by downstream restriction factors

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

Origin of tested bat cell lines.

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

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

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

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