Figure 5. RNA replication of CSFV occurs on the membranes of autophagosome-like vesicles. (A) PK-15 cells infected with CSFV (MOI = 1) for 48 h. The cells were fixed and processed for indirect immunofluorescence using antibodies against the CD63 and CSFV proteins (E2 or NS5A), and the targeted protein staining was detected with secondary antibodies conjugated to FITC and TRITC as described in Materials and Methods. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, E2 and NS5A staining is shown in green (a and e), CD63 staining is shown in red (b and f), and the signals of colocalization are shown in yellow in the merged images (c and g). The higher magnification images are shown in (d and h) from the white-square frame-enclosed region in (c and g). Scale bar: 10 μm. One of 3 independent experiments is shown. (B) 3D4/2 cells were infected and analyzed as in (A). Scale bar: 10 μm. One of 3 independent experiments is shown. (C) PK-15 (a and c) and 3D4/2 (b and d) cells were infected with CSFV at an MOI of 1 for 48 h. Subsequently, IEM analysis was performed using specific monoclonal antibodies against the CSFV NS5A and E2 proteins, and the targeted proteins were detected with a secondary antibody conjugated to 12-nm colloidal gold particles. The immunogold labeling showing the localization of CSFV proteins on the membranes of autophagosome-like vesicles is indicated by black arrows. Scale bar: 500 nm. One of 3 independent experiments is shown.