Fig. 1.

PDCoV infection induces apoptosis in vitro. (A) CPE and DNA fragmentation in PDCoV-infected cells. ST cells were mock-infected or infected with PDCoV. At the indicated time points after infection, PDCoV-specific CPE were photographed using an inverted microscope at a magnification of 200× (top panels). For DNA fragmentation assay, DNA was extracted from mock- or PDCoV-infected cells and nucleosomal DNA fragmentation of cells was analyzed by agarose gel electrophoresis (bottom panel). As a positive control (PC), cells were treated with staurosporine for 24 h to induce apoptosis. Lane M represents a 1-kb ladder as a DNA molecular-weight size marker. (B) TUNEL labeling of PDCoV-infected cells. Mock-infected control and PDCoV-infected cells fixed at 24 hpi were labeled with TUNEL (green) and sequentially stained with an anti-PDCoV-N antibody (red). Cells were counterstained with DAPI, and photomicrographs of TUNEL labeling and N protein staining in virus-infected cells were obtained using a confocal microscope at 400× magnification. In a merged image, all TUNEL-positive cells were localized within the nuclei of the corresponding PDCoV-infected cells. (C) Cell death analysis by flow cytometry with dual Annexin V-PI cell labeling. PDCoV-infected cells collected at different time points were subjected to dual Annexin V and PI labeling and analyzed by FACS. Lower left quadrants represent intact cells (Annexin V negative/PI negative); Lower right quadrants represent early apoptotic cells (Annexin V positive/PI negative); Upper right quadrants indicate late apoptotic and/or necrotic cells (Annexin V positive/PI negative); and Upper left quadrants indicate necrotic cells (Annexin V negative/PI positive). The figure is representative of three independent experiments. The graph on the right represents the percentage of each quadrant and the non-significant percentages of Annexin V-positive and PI-negative cells were excluded.