Fig 1.
Study design configuration for wt BVDV and ddBVDV pregnant bovine trial.
DPG: days post gestation: DPC: days post challenge infection.
Table 1.
Summary of heifer gestation and infection dates.
Fig 2.
Extracted bovine uteruses and schematic anatomy of bovine placenta.
The photographs in the upper part show uteruses extracted from the heifers during necropsy. The dark red structures visible on the surface of the organs shown on the upper left photograph represent the cotyledons as indicated by an arrow. Another arrow points at a caruncle. The anatomy of the bovine placenta is illustrated in the schemes below. The drawing on the left shows the uterus with the fetus whereas the scheme on the right represents a magnified image of the placentome (marked by a box in the left scheme) illustrating its location and how the structures are in contact represented by the fetal (red) and maternal (blue) epithelium, from which the cell lines used in our analyses were derived (F3 and BCEC, respectively).
Fig 3.
Detection of viral RNA in the tissues taken from fetuses at the indicated time points.
Orange triangles and blue dots stand for the animals infected with wt BVDV or ddBVDV, respectively. The positioning of the triangles and dots reflects the amount of viral RNA detected in copy number per mg total RNA.
Fig 4.
Detection of viral RNA in fetal tissues via RNAscope.
The pictures represent the results of RNAscope in situ hybridization of the indicated tissues. The labelling lists from left to right the animal number, the day of slaughter, the virus used for infection and the tissue from which the sample was taken. Red color represents positive staining for BVDV genomic RNA.
Fig 5.
Detection and quantification of BVDV genome in tissues of the heifers.
Orange triangles and blue dots indicate the animals infected with wt BVDV or ddBVDV, respectively. The positioning of the triangles and dots reflects the amount of viral RNA detected in copy number per mg total RNA.
Fig 6.
Detection via RNAscope of genomic RNA of ddBVDV in the palatine tonsil of animal 751 and of wt BVDV in Peyers Patches of animal 741, respectively (both from necropsy at 6 DPI).
The labeling lists from left to right the animal number, the day of slaughter, the virus used for infection and the tissue from which the sample was taken. Red color represents positive staining.
Fig 7.
Detection of viral RNA in placenta samples.
Orange triangles and blue dots stand for the animals infected with wt BVDV or ddBVDV, respectively. The positioning of the triangles and dots reflects the amount of viral RNA detected (in copies per mg total isolated RNA).
Table 2.
Summary of the results obtained for the placenta samples from the wt virus infected animals via qRT-PCR and in situ hybridization.
Fig 8.
Detection via RNAscope of BVDV genomic RNA in placenta tissue.
The pictures represent the results of RNAscope in situ hybridization of the specified placenta samples. The labeling lists from left to right the animal number, the day of slaughter, the virus used for infection and the tissue from which the sample was taken. Red color represents positive staining. In the right picture in the middle row inscriptions and arrows highlight the structural organization of the placenta.
Fig 9.
Transcriptional changes in the trophoblast cell line F3 and the caruncle epithelial cell line BCEC-1 upon infection with wt BVDV and ddBVDV.
(A) Vulcano plot showing the statistical significance of differentialy regulated genes of the trophoblast cell line F3 infected either with ddBVDV or wt BVDV. Genes in the rectangle are shown in C. The number of genes downregulated or upregulated by more than twofold are given in the graph (left and right bottom, respectively) (B) Vulcano plot showing the statistical significance of differentialy regulated genes of the caruncle epithelial cell line BCEC-1 infected either with ddBVDV or wt BVDV. Genes in the rectangle are shown in E. Meaning of numbers given in the graph are same as in (A) (C) The relative expression of the 20 genes with the highest “fold change” in F3 cells are shown. (D) The relative expression of the 20 genes with the highest “fold change” in BCEC-1 cells are shown. Genes showing up as differentially expressed in both F3 and BCEC-1 cells are highlighted in yellow (E) Comparison of gene expression levels in non-infected BCEC-1 and F3 cells. Expression levels for the two cell lines are given as log 2 in columns 3 and 4, respectively, whereas column 2 shows the ratio of the levels found in BCEC-1 and F3. Genes found among the 20 highest regulated genes in both cell lines are highlighted in yellow, whereas blue highlights genes found only in F3 cells (C) and green labels those found only in BCEC-1 (D). (F) The interactome of the genes listed in (E) (G, H) Gene enrichment analyses of upregulated genes (≥ 3 fold) of F3 (G) and BCEC-1 (H) using DAVID software.
Fig 10.
Growth curves of wt BVDV and ddBVDV mutant on bovine placenta cell lines.
Cells of the lines indicated on top of the diagrams were infected with a MOI of 0.001 with either of the two viruses. Freeze/thaw extracts of infected cultures were prepared at the indicated time points p.i. and viral titers were determined as described [53,79] with detection of infected cells via indirect immunofluorescence using monoclonal antibody code 4 (mAb 8.12.7 [87]) and FITC anti mouse as secondary antibody.
Table 3.
Real-time quantitative PCR primers.