Figure 1
Figure 1. Characterization of HHV-8 acute infection in HUVECs. (A) Electron micrographs of HHV-8 particles produced and purified as described in “Materials and methods” (original magnification, × 50 000; Hitachi H-800 electron microscope; Hitachi, High Technology Europe, Krefeld, Germany). (B) Subconfluent HUVEC monolayers were mock infected (control) or infected with HHV-8 (HHV-8). Microscopic observation of HHV-8 infected HUVECs was performed at 48 hours after infection (a.i.) by a Nikon Eclipse TE2000-S microscope equipped for phase-contrast observation (10×/0.25 and 20×/0.45 NA Fluor objective lenses), using a DS camera head DS-5M and a DS camera control unit DS-L; Nikon). (C) Results of RT-PCR amplification of the indicated HHV-8 genes using RNA extracted from infected cells at different days after infection. β-Actin levels were determined in the same samples as control. Positive controls of amplification of the genes analyzed (C+) are shown. (D) The presence of the HHV-8 late K8.1 protein at 36 hours after infection was detected in HHV-8–infected HUVECs grown onto collagenated culture slides by immunofluorescence analysis using a Nikon Eclipse TE2000-S microscope equipped for fluorescence observation. Immunofluorescence micrograph (ii) and the corresponding bright-field image (i) are shown at original magnification × 4 (pictures were taken with a 4×/0.13 NA Fluor objective). All images were processed by Nero Photosnap Viewer 7 (Nero ACT, Karlsbad, Germany). Under the conditions described, approximately 20% of cells were positive for the K8.1 protein.

Characterization of HHV-8 acute infection in HUVECs. (A) Electron micrographs of HHV-8 particles produced and purified as described in “Materials and methods” (original magnification, × 50 000; Hitachi H-800 electron microscope; Hitachi, High Technology Europe, Krefeld, Germany). (B) Subconfluent HUVEC monolayers were mock infected (control) or infected with HHV-8 (HHV-8). Microscopic observation of HHV-8 infected HUVECs was performed at 48 hours after infection (a.i.) by a Nikon Eclipse TE2000-S microscope equipped for phase-contrast observation (10×/0.25 and 20×/0.45 NA Fluor objective lenses), using a DS camera head DS-5M and a DS camera control unit DS-L; Nikon). (C) Results of RT-PCR amplification of the indicated HHV-8 genes using RNA extracted from infected cells at different days after infection. β-Actin levels were determined in the same samples as control. Positive controls of amplification of the genes analyzed (C+) are shown. (D) The presence of the HHV-8 late K8.1 protein at 36 hours after infection was detected in HHV-8–infected HUVECs grown onto collagenated culture slides by immunofluorescence analysis using a Nikon Eclipse TE2000-S microscope equipped for fluorescence observation. Immunofluorescence micrograph (ii) and the corresponding bright-field image (i) are shown at original magnification × 4 (pictures were taken with a 4×/0.13 NA Fluor objective). All images were processed by Nero Photosnap Viewer 7 (Nero ACT, Karlsbad, Germany). Under the conditions described, approximately 20% of cells were positive for the K8.1 protein.

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