Rift Valley fever virus targets the maternal-foetal interface in ovine and human placentas

Abstract

Background: Rift Valley fever virus (RVFV) is an arbovirus of the order Bunyavirales that causes severe disease in ruminants and humans. Outbreaks in sheep herds are characterised by newborn fatalities and abortion storms. The association of RVFV infections with abortions of ovines and other ruminants is well recognized, whereas the pathology resulting in abortion has remained undescribed. Accumulating evidence suggests that RVFV is abortogenic in humans as well, warranting more research on the interaction of RVFV with the ruminant and human placenta.

Methodology/principal findings: Pregnant ewes were inoculated with a highly virulent strain of RVFV and necropsied at different days post infection. Tissues were collected and analysed by PCR, virus isolation, and immunohistochemistry. The results show that RVFV replicates efficiently in maternal placental epithelial cells before the virus infects foetal trophoblasts. Moreover, the virus was shown to bypass the maternal epithelial cell layer by directly targeting foetal trophoblasts in the haemophagous zone, a region of the ovine placenta where maternal blood is in direct contact with foetal cells. Abortion was associated with widespread necrosis of placental tissues accompanied with severe haemorrhages. Experiments with human placental explants revealed that the same virus strain replicates efficiently in both cyto- and syncytiotrophoblasts.

Conclusions/significance: This study demonstrates that RVFV targets the foetal-maternal interface in both ovine and human placentas. The virus was shown to cross the ovine placental barrier via two distinct routes, ultimately resulting in placental and foetal demise followed by abortion. Our finding that RVFV replicates efficiently in human trophoblasts underscores the risk of RVFV infection for human pregnancy.

Fig 1. Experimental design and primary outcome parameters ewes.

Ewes were inoculated intravenously with RVFV or mock inoculated at gestation day (gd) 55 (A) or day 78 (E) and euthanized at 4 (blue cross), 6 (green and purple crosses) or 7 (orange cross) days post inoculation (dpi). Purple numbers represent ewes that were mock-inoculated. Rectal temperatures (B, F), viremia by RT-qPCR (solid line, left y-axis), virus isolations (dotted line, right y-axis) (C, G) and the presence of RVFV in spleen and liver samples of the ewes (D, H) are depicted. Bars represent averages with SDs.

Fig 2. Detection of viral RNA and infectious virus in placentomes.

Viral RNA copies in placentomes as determined by RT-qPCR (black columns; left y-axis) and virus titres in placentomes as determined by virus isolation (grey columns; right y-axis). Results of experiment 1 (A) and 2 (B) represent means and SDs of 3 placentomes per foetus.

Fig 3. The ovine placenta at different stages of gestation.

(A) Schematic presentation of an ovine foetus, the cotyledons and their blood supply. Only the foetal parts of the placenta (cotyledons) are displayed, the uterus wall and maternal part of the placenta (caruncles) are not depicted. At the right, a cross section of a placentome is depicted, showing the maternal tissues in shades of pink, and the foetal villi in orange. Haemophagous zones at the base of the foetal villi are depicted in red. (B) A schematic overview of the cotyledon (center) with the different cell layers of the synepitheliochorial placenta at the left and the haemophagous zone at the right. In the synepitheliochorial placenta, the foetal blood is separated from maternal blood by several maternal and foetal cell layers. In the haemophagous zone maternal blood is in direct contact with the foetal trophoblasts. (C, D) HE staining of placentomes, the haemophagous zones and synepitheliochorial placenta at 1/3 gestation (C) and 1/2 gestation (D). Red interrupted lines indicate the boundaries between maternal and foetal tissues. F = foetal villus, M = maternal villus, MB = maternal blood. Notice the increase in the foetal/maternal villous interface in the synepitheliochorial placenta and the increase in size and erythrophagous activity of the trophoblasts of the haemophagous zone between 1/3 and 1/2 of the gestation period.

Fig 4. RVFV antigen in placentomes.

Immunohistochemical (IHC) detection of RVFV antigen in cross sections of the placentomes at 4 dpi (experiment 2) and 6 dpi (experiment 1). At 4 dpi (A) only small foci of antigen positive cells are visible throughout the placentome while at 6 dpi (F) almost the entire placentome stains positive for RVFV antigen. Higher magnification of the synepitheliochorial placenta (SP) at 4 dpi (B) shows strong labelling of the maternal epithelial cells with only an individual positively stained foetal trophoblast (black arrowhead). At 6 dpi both maternal and foetal cell layers are strongly stained (G). In the haemophagous zone (HZ) at 4 dpi (C) only small clusters of foetal trophoblasts stain positive for RVFV antigen while at 6 dpi (H) the entire foetal trophoblast lining of the haemophagous zone stains positive. Panels D, E, I and J represent cross sections of uninfected placentomes corresponding to B, C, G and H, respectively, showing absence of background IHC staining. Red interrupted lines indicate the boundaries between maternal and foetal tissues. F; foetal villus, M; maternal villus, MB; maternal blood. Bar = 5000 μm (A, F) or 100 μm (B-J).

Fig 5. Histopathology of the placentomes at imminent abortion.

HE staining of placentomes at 6 dpi. (A) Haemorrhages (black asterisk) and necrosis of maternal epithelial cells (arrowheads). Notice the relatively intact foetal epithelium. (B) Influx of neutrophils (red asterisk) and necrosis of maternal epithelium (arrowheads). F = foetal villus, M = maternal villus, Bar = 100 μm.

Fig 6. Detection of viral RNA and viral antigen in foetal organs.

Detection of RVFV RNA by RT-qPCR in of organ suspensions of foetal organs collected in experiment 1 (A) and experiment 2 (B). Bars represent averages with SDs. Staining of RVFV antigen in samples collected from liver (C), brain (D) umbilical cord (E) and leg muscle (F). Notice the strong staining of endothelial cells in the blood vessels within the various organs. Bar = 500 μm (D, E, F), 100 μm (C) or 20 μm (inset D).

Fig 7. RVFV in human placental explants.

Detection of viral RNA (A) and infectious virus (B) in human full term placental explants at different timepoints post infection. Viral RNA was detected by RT-qPCR and infectious virus by virus isolation. (C, D, E) Immunohistochemical staining of RVFV with mAb 4-D4, counterstained with haematoxylin. (C) Single villus with syncytiotrophoblasts (black arrowheads) staining negative for RVFV antigen with cytotrophoblasts (brown staining, red arrowhead) staining positive. (D) Single villus in which no cytotrophoblasts are present, with positive staining of syncytiotrophoblasts (black arrowheads). (E) Single villus showing absence of background IHC staining in non-infected control placental explant. Syncytiotrophoblasts are indicated with black arrowheads. (F) Immunohistochemical staining of epithelial cells in a non-infected placental villus with a mAb to cytokeratin showing both the syncytiotrophoblast layer (black arrowheads) and the cytotrophoblast layer (red arrowheads). Bar = 20 μm (C, D, E) or 50 μm (F).