Article Navigation Wild-type Measles Virus Infection Upregulates Poliovirus Receptor-Related 4 and Causes Apoptosis in Brain Endothelial Cells by Induction of Tumor Necrosis Factor-Related Apoptosis-lnducing Ligand

Small numbers of brain endothelial cells (BECs) are infected in children with neurologic complications of measles virus (MV) infection. This may provide a mechanism for virus entry into the central nervous system, but the mechanisms are unclear. Both in vitro culture systems and animal models are required to elucidate events in the endothelium. We compared the ability of wild-type (WT), vaccine, and rodent-adapted MV strains to infect, replicate, and induce apoptosis in human and murine brain endothelial cells (HBECs and MBECs, respectively). Mice also were infected intracerebrally. All MV stains productively infected HBECs and induced the MV receptor PVRL4. Efficient WT MV production also occurred in MBECs. Extensive monolayer destruction associated with activated caspase 3 staining was observed in HBECs and MBECs, most markedly with WT MV. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not Fas ligand, was induced by MV infection. Treatment of MBECs with supernatants from MV-infected MBEC cultures with an anti-TRAIL antibody blocked caspase 3 expression and monolayer destruction. TRAIL was also expressed in the endothelium and other cell types in infected murine brains. This is the first demonstration that infection of low numbers of BECs with WT MV allows efficient virus production, induction of TRAIL, and subsequent widespread apoptosis

wtPDV infects Vero cells.

<p>Vero and VDS cells were infected at an MOI of 0.1. (A) CPE observed in Vero and VDS cultures infected with wtPDV/NL88n, wt PDV/USA2006, wtCDV and wtMV at 2 dpi by phase contrast microscopy (Magnification X100). Foci of rounded cells are indicated by arrows. (B) Cells were infected with wtPDV/NL88n, wtCDV and wtMV, fixed, permeabilised and stained with SSPE serum and rabbit anti-human FITC; nuclei were stained with propidium iodide. Images were taken using a Nikon Eclipse TE2000-U UV microscope (x400). (C) Vero cells and VDS cells were infected with wtPDV/NL88n, wtPDVUSA2006, wtMV and wtCDV for up to 5 days. Titres were determined by TCID₅₀ in VDS cells. The results are representative of two independent experiments.</p

Anti-β1 integrin treatment of Vero cells increases infection of wtPDV and Edmonston MV.

<p>(A) Vero cells were examined for β1 integrin expression by staining cells with anti-β1 integrin antibody or mouse isotype control followed by fixation and staining with rabbit anti-mouse FITC. Nuclei were stained with DAPI. Immunofluorescent images were taken using a Nikon Eclipse TE2000-U UV microscope (x100). (B) and (C) Vero cells were incubated with anti-β1 integrin (blocking) antibody or with control mouse isotype, prior to infection (MOI 0.1) for 2 days (Onderstepoort CDV and Edmonston MV) or 5 days (wtPDV/USA2006). (B) Cells were fixed before incubating with SSPE serum followed by staining with rabbit anti-human FITC and analysed by flow cytometry. (C) Virus was harvested and the titre determined by TCID₅₀/ml in VDS cells. The results are representative of two independent experiments.</p

Sodium chlorate and heparinase treatment decreases cell fusion.

<p>Vero cells were untreated or treated with 30 mM or 60 mM sodium chlorate for 2 days prior to infection (MOI 0.1) with Schwarz-GFP MV or wtPDV/USA2006. (A) CPE and GFP expression by Schwarz-GFP MV was examined at 2 days by dual phase contrast-UV microscopy (top panel). Cultures of wtPDV were fixed and permeablised at 5 dpi before staining with SSPE antibody and rabbit anti-human FITC (bottom panel). Nuclei were stained with DAPI. (B) Vero cells were treated with 10 U/ml of heparinase for 90 min and infected at an MOI of 0.1 with Schwarz GFP MV or wtPDV/NL88n. CPE was examined at 2 days by dual phase contrast-UV microscopy (top panel) and wtPDV CPE by phase contrast microscopy (bottom panel). Images were taken using a Nikon Eclipse TE2000-U UV microscope (X100).</p

wtPDV infection and binding is increased in CHO-proHB-EGF cells.

<p>CHO-empty and CHO-proHB-EGF cells were (A) examined for pro-HB-EGF expression by staining with goat anti-HB-EGF antibody or control goat serum followed by fixation and staining with rabbit anti-goat FITC. (B) Inoculated with wtPDV/USA2006, wtPDV/NL88n, wtCDV or wtMV (MOI 0.1) for 2 days. Cells were viewed by phase contrast microscopy (1st panel) or fixed before staining with SSPE serum and rabbit anti-human FITC (all other panels). Images were taken using a Nikon Eclipse TE2000-U UV microscope (X400). (C) Monolayers were inoculated with wtPDV/USA2006, wtCDV or wtMV (MOI 10) at 4°C for 2 hr. After washing, Sybr green qRT-PCR was carried out and the copy number of virus RNA determined from a standard curve.</p

Cell lines and Known Receptors for Morbilliviruses.

<p>Cell lines and Known Receptors for Morbilliviruses.</p

Increased titres of wtPDV are obtained in CHO cells expressing either SLAM or PVRL4.

<p>Cells were infected (MOI of 0.1) with wtPDV/USA2006 (all CHO cell lines) and wtCDV (CHO, CHO-DSLAM and CHO-PVRL4). Virus was harvested at 1 to 5 dpi from wtPDV and wtCDV infected cultures and the titre determined by TCID50 in VDS cells. The results are representative of two independent experiments.</p

Virus Origin and Isolation.

<p>Virus Origin and Isolation.</p

Infection of B95a cells with wtPDV is partially inhibited by anti-SLAM antibody.

<p>B95a cells were incubated with anti-SLAM monoclonal antibody or mouse isotype control prior to infection at an MOI of 0.1 with (A) Edmonston MV, Schwarz GFP MV, wtCDV and wtPDV/NL88n at MOI of 0.1 for 2 days followed by fixation and staining (with the exception of Schwarz-GFP MV) with SSPE serum followed by rabbit anti-human FITC and analysed by flow cytometry. (B) Virus was harvested from cells infected with wtMV, wtCDV and wtPDV/NL88n and determination of titres by TCID₅₀ in VDS cells. The results are representative of two independent experiments.</p

Omar et al data tables

Data files for graphical figures with SD