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

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

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

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

Virus Origin and Isolation.

<p>Virus Origin and Isolation.</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

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

Cell lines and Known Receptors for Morbilliviruses.

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

Increased infection of wtPDV infection in CHO cells expressing either SLAM or PVRL4.

<p>(A) CHO-CD46, CHO-MSLAM and CHO-PVRL4 cells were stained with their respective receptor antibodies or mouse isotype control, fixed and stained with rabbit anti-mouse FITC. CHO cells were stained in the same manner with anti-CD46 antibody (B) CHO, CHO-CD46, and CHO-MSLAM cells were infected with wtPDV/NL88n and wtCDVUSA2006 (C) CHO, CHO-DSLAM, CHO-PVRL4 and VDS cells with wtPDV/USA2006, wtCDV and wtMV at MOI of 0.1 for 2 days. Cells were, permeabilised, fixed and stained with SSPE serum followed by rabbit anti-human FITC. Nuclei were either stained with propidium iodide and images take in a Leica TCS/NT confocal microscope or stained with DAPI and images taken using a Nikon Eclipse TE2000-U UV microscope (x400).</p