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

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

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

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

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

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

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

Genomic RNA (gRNA)-to-pfu ratio for RA27/3 and RV-Dz virions produced in HUVEC.

<p>Genomic RNA (gRNA)-to-pfu ratio for RA27/3 and RV-Dz virions produced in HUVEC.</p