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

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

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

Cell lines and Known Receptors for Morbilliviruses.

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

<i>ISG15</i> expression during erythroid differentiation is independent of IFN signaling and partially dependent upon Epo signaling.

<p>(<b>A</b>) semi-quantitative RT-PCR analyses of <i>ISG15, Ube1L, UbcM8, Herc6, Irf7, Bcl-x_L</i> and <i>ß-Major-Globin</i> mRNA expression in WT or <i>IFNAR^-/-</i> differentiating primary erythroblasts. Bone marrow erythroblasts of each genotype were maintained in proliferation conditions (SCF, Epo and Dex) for one week and next induced to differentiate in response to Epo alone for three days. Cells were collected every 24 hours as indicated and RNA extracted. The experiment was normalized to <i>ß-Actin</i> mRNA expression. (<b>B</b>) Statistical analysis of the induction of the expression of <i>ß-Maj-Globin, Bcl-X_L</i>, <i>ISG15</i>, <i>Ube1L</i>, <i>UbcM8</i> and <i>Herc6</i> mRNA in differentiating WT and IFNAR^-/- erythroblasts. Quantification was performed as described in Mat & Met. Note the unchanged expression of a second housekeeping gene <i>HPRT</i>. au = arbitrary unit. (<b>C</b>) Whole cell protein extracts were prepared from WT or <i>IFNAR^-/-</i> erythroblasts maintained as in A and analyzed on a 10% acrylamide gel for <i>ISG15</i> expression using an anti-ISG15 antibody. Anti-ß-Actin and anti-GAPDH were used as loading controls. (<b>D</b>) Statistical analysis of the induction of ISG15 during erythroid differentiation as normalized to ß-Actin. Quantification was performed as described in Mat & Met. au = arbitrary unit. (<b>E and F</b>) A p53^-/- erythroid cell line expressing exogenous hBcl-X_L was switched from proliferation conditions (Epo, SCF, Dex) to differentiating medium in the presence or absence of Epo. Cells were collected every 24 hours as indicated and analyzed for their ability to (e) survive as measured by propidium iodide staining in flow cytometry analyses; (f) differentiate as shown by analysis of their morphology after benzidine/May-Grunwald staining. Note the significant induction of cell death in control cells maintained in absence of Epo; in contrast, hBCL-xL erythroblasts are strongly protected from apoptosis. (<b>G</b>) Cells were lyzed according to cell number and volume. ISG15 expression was analyzed on a 15% acrylamide gel using anti-ISG15 antibody, activation of the EpoR/STAT5 signaling pathway was monitored using anti-P-STAT5 antibody, differentiation was monitored using anti-Globin antibody and loading control was performed using anti-SAM68 antibody. (<b>H</b>) Mock and mscv-puro-STAT5^S710F transduced p53^-/- erythroid cell line maintained under proliferation conditions were lyzed and analyzed for ISG15 expression using anti-ISG15 antibody on a 10% acrylamide gel (Top panel). P-STAT5 was detected at a higher level in mscv-puro-STAT5^S710F transduced cells while only a modest increase in the total amount of STAT5 can be noted. Anti-β -Actin was used as a loading control.</p