FIG 1.

Generation of MDCK cells expressing X31 H3 HA. To establish X31 HA-expressing MDCK cell lines, the X31 HA mRNA gene was cloned into the pCAGGS vector and cotransfected with the pCB7 vector containing the hygromycin B gene. After transfection, hygromycin B-resistant clones were subcultured and screened by infection using WSN-sciIV virus complementation (A) and IFA (B). (A) WSN-sciIV virus complementation. Parental, WSN-expressing, and X31 HA-expressing MDCK cells were infected with WSN-sciIV at an MOI of 0.01. At 48 h postinfection, cell monolayers were imaged with a fluorescence microscope to observe GFP expression. Images are shown at ×10 magnification. Scale bar, 40 μm. (B) IFA. Parental, WSN-expressing, and X31 HA-expressing MDCK cells were fixed and stained with 2G9 (WSN) or X31 sera, followed by treatment with rabbit anti-mouse IgG FITC (green) and DAPI for nuclear staining (blue). The cell monolayers were imaged with a fluorescence microscope. Images are shown at ×40 magnification, pseudocolored and overlaid using Photoshop software. Scale bar, 10 μm.