Western blot analysis of purified vaccine and wtRV virions and viral proteins in the infected HUVEC.

<p>Vero and HUVEC were infected with RA27/3 and RV-Dz at MOI = 5. Virions were pelleted from the culture media by ultracentrifugation and resuspended in an SDS-sample buffer. The cell monolayers were washed with PBS and proteins were then extracted with RIPA buffer. Proteins were separated by a 4–12% NuPage gel, either nonreducing (E1, C, β-actin) or reducing (E2), and then the blots were probed with rubella E1, E2 and C-specific MAb to identify RV structural proteins. The blots were also probed with β-actin MAb to demonstrate equal protein loading for the analysis of the cell lysates and show purity of the pelleted virions. Representative results of two independent experiments are shown.</p

Effects of RV infection on cell proliferation and mitosis.

<p>(<b>A</b>) Growth curves of mock-infected and RV-infected HUVECs. HUVEC were mock infected or infected with RV-Dz at MOI=10 and then counted daily. The data are results of 2 independent experiments each performed in duplicate. (<b>B</b>) Mitosis in infected HUVEC. At 2 dpi the mock infected and infected HUVEC (RV-Dz, MOI=10) were immunostained by IFA with capsid MAb and DAPI to quantitate infected cells and mitotic figures. Mitotic indexes (MI) were calculated as % cells with mitotic figures in two duplicate wells. Note RV-antigen positive mitotic cell in red circle. (<b>C</b>) Cell cycle analysis of infected HUVEC. Serum-starved HUVECs were mock-infected or infected with RV-Dz at MOI=10. Histograms of cell cycle analysis at 1 dpi show DNA content of propidium iodide-stained cells by flow cytometry and % of cells in each phase of the cell cycle. The representative results of two independent experiments are shown.</p

Rubella particles and replication complexes in RA27/3 infected HUVEC and Vero cells.

<p>Representative images of rubella virions (the red arrows) and replication complexes (the white arrow) in cytopathic vacuoles (CPV) observed by transmission electron microscopy in HUVEC and Vero cells infected with RV-Dz at MOI = 50 at 24 hpi. Inserts represent enlarged images of the replication complex and virions that are marked with the arrows. Scale bars correspond to 500 nm.</p

Quantitation of positive-strand RV RNA by <i>in situ</i> hybridization.

<p>(A) Representative images from at least 3 independent experiments showing the results of the RNA-FISH for positive-strand RV RNA in HUVECs mock infected or infected with RA27/3 or RV-Dz at 6 dpi. Outlines of cell boundaries were created in the AxioVision software (Zeiss, Germany) by using PPIB dots as reference points. Note the presence of cells with high hybridization signal in RA27/3 infected HUVECs. (B) Distribution of dots (plus-strand genomic RNA) per cell in infected HUVECs with countable signal (up to 150 dots/cell). The dots were counted in 7–10 randomly selected microscopic fields (total 120 cells). Combined data from two separate experiments are presented in a box-and-whisker plot such that the edges of the boxes represent the 25th and 75th percentiles, the horizontal line in the box is the median, and the whiskers show the range of values. Significance was determined using ANOVA, followed by post hoc analysis using the Tukey test for multiple comparisons (**, P < 0.01; ***, P < 0.001).</p

Expression of viral structural proteins in infected HUVEC.

<div><p>(<b>A</b>) Kinetics of viral protein synthesis. HUVEC were mock infected (M) or infected with RV-Dz at an MOI=5. Proteins were separated by 4-12% NuPage gel, either nonreducing (E1, C, β-actin) or reducing (E2), and then the blots were probed with rubella E1, E2 and C-specific MAb and β-actin MAb.</p> <p>(<b>B</b>) Spatial distribution of E1, E2 and C proteins in infected cells. HUVECs were infected with RV-Dz at an MOI=5 on chamber slides and processed for indirect immunofluorescence at 2 dpi using E1, E2 and capsid-specific MAb. Nuclei were counterstained with DAPI.</p></div

Comparative analysis of vaccine and wtRV persistence in HUVEC.

<p>(A) Growth curves of RA27/3 and RV-Dz at different MOIs. (B) Percentage of E1-positive cells in the infected monolayers at different times post-infection. Representative results of at least two independent experiments are shown. (C) Phase contrast images and results of immunofluorescence assay (E1 staining) of HUVEC infected with RA27/3, RV-Dz or mock-infected at different times post-infection. Nuclei were counterstained with DAPI.</p

Secretion of infectious rubella virions into growth media.

<p>Vero and HUVEC were infected with RA27/3 and RV-Dz at MOI = 5. Media and cells were collected at 2 dpi and titered on Vero cells. Note that secretion of WT and vaccine viruses from HUVEC were equally effective.</p

Productive infection of HUVEC with low passage wtRV.

<p>(<b>A</b>–<b>B</b>) Kinetics of RV replication in HUVEC, Vero and A549 cells. Cells were infected with RV-Dz at an MOI of 0.05 or 5. Cell culture supernatants (A) or cell lysates (B) were titered in duplicate on Vero cells. Data are presented as a mean value +/- standard deviation of two independent experiments each performed in duplicate. The data were analyzed by two-way ANOVA with the Bonferroni posttests for correcting for multiple comparisons (*, P<0.05; **, P<0.01; ***, P<0.001). (<b>C</b>) Quantitation of intracellular rubella genomic RNA. HUVEC, Vero and A549 cultures were infected with RV-Dz at an MOI of 5. Genomic RNA was quantitated by RT-qPCR. GAPDH mRNA was used for normalization in the comparative threshold cycle method. Data are presented relative to the genomic RNA amount at 4 hpi. The results represent the mean of at least two independent experiments each done in duplicate. (<b>D</b>) Phase contrast pictures of cells at 5 dpi either mock infected or RV-Dz infected at MOI=5. Note cytopathic effect of wtRV in A549. (<b>E</b>) Representative images of rubella virions observed by TEM in HUVEC infected with RV-Dz at MOI=50 at 24 hpi. (<b>F</b>) Representative images of rubella virions and (<b>G</b>) replicative complexes observed by TEMin Vero cells infected with RV-Dz at MOI=50 at 24 hpi. Inserts represent enlarged images from the replicative complex and virions that are marked with the red arrows. Bars, 100 nm.</p

Quantitation of negative-strand RV RNA by <i>in situ</i> hybridization.

<p>(A) The representative images from at least 3 independent experiments showing the results of the RNA-FISH for negative-strand RV RNA and influenza NP-negative strand RNA (negative control) in HUVECs infected with RA27/3 and RV-Dz at 2 dpi. Outlines of cell boundaries were created in the AxioVision software by using PPIB dots as reference points. (B) Distribution of the dots (negative-strand genomic RNA) per cell in infected HUVEC. The dots were counted in 7–10 randomly selected microscopic fields (total 120 cells). The combined data from two separate experiments are presented in a box-and-whisker plot such that the edges of the boxes represent the 25th and 75th percentiles, the horizontal line in the box is the median, and the whiskers show the range of values. Significance was determined using ANOVA, followed by post hoc analysis using the Tukey test for multiple comparisons (**, P < 0.01; ***, P < 0.001).</p

Detection of RV dsRNA in infected cells by immunofluorescence.

<p>RV infected and mock-infected HUVECs (A) or Vero cells (B) were fixed with methanol and examined for presence of dsRNA by indirect immunofluorescence using dsRNA-specific antibody. Nuclei were counterstained with DAPI.</p