Research ArticleVIROLOGY

Rift Valley fever virus induces fetal demise in Sprague-Dawley rats through direct placental infection

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Science Advances  05 Dec 2018:
Vol. 4, no. 12, eaau9812
DOI: 10.1126/sciadv.aau9812
  • Fig. 1 Pregnant rats are more susceptible to death after RVFV infection, with virus homing to the liver and placenta.

    (A) Experimental design for E14 SD rats infected with RVFV. After delivery at E22, dams and pups were not disturbed until 5 days postdelivery (13 dpi). Euthanasia of surviving dams and pups occurred 18 dpi (10 days postdelivery). (B) Survival of RVFV-infected pregnant dams and nonpregnant SD rats (n = 3 to 6 per dose). The shaded area represents the 2- to 6-day clinical window when lethally infected pregnant rats were euthanized owing to severe disease. (C) vRNA (qRT-PCR; left) and infectious virus (VPA; right) in tissues from pregnant rats that succumbed (red squares; n = 17) between 2 and 6 dpi. (D) vRNA in tissue samples from pregnant rats that survived infection (blue circles; n = 11) and were euthanized 18 dpi. Placenta samples (open blue circles) were obtained at day of delivery (8 dpi). (E) Infectious virus measured by VPA in placental samples obtained from lethally infected (red squares) and surviving (blue circles) rats at the indicated dpi. CLN, cervical lymph node. Dotted horizontal lines represent the limits of detection (LOD) of the qRT-PCR (0.1 PFU/ml eq) and VPA (50 PFU). ND, not detected (below the LOD); PFU, plaque-forming unit; PFU/ml eq, PFU per milliliter equivalents; VPA, viral plaque assay; vRNA, viral RNA.

  • Fig. 2 RVFV causes pathology within the liver, uterus, and placenta of pregnant dams.

    H&E staining within the indicated tissues. (A) Images (20×) of liver, uterus, and placenta. (B) Images (60×) of placenta. Blue, white, yellow, orange, and green arrowheads highlight evidence of hemorrhaging, necrosis, vascular/perivascular congestion, calcification, and nucleated red blood cells, respectively.

  • Fig. 3 Infection of pregnant dams results in direct transmission of RVFV to the peritoneum and brain of pups.

    Pups delivered from (A) lethally infected and (B) surviving pregnant rats were tested for vRNA within the peritoneal cavity (left) and brain (right). In (A), the x axis represents the day the dams were euthanized owing to severe disease. In (B), the x axis represents the day after delivery, with day 10 representing surviving pups euthanized at the end of the study. For both graphs, red square data points indicate pup demise and blue circle data points indicate pup survival. Open data points are pup brain tissues; all closed data points are pup peritoneal cavity. (C) Photographic evidence of fetal resorption within the uterus of one of three dams that succumbed to RVFV infection. (D) Images (10×) of whole pups were examined for histological changes. H&E staining of a whole pup from a dam that succumbed to infection (right) or a corresponding uninfected control rat (left) euthanized at the same day of gestation. Blue, white, and black arrowheads highlight evidence of hemorrhaging, necrosis, and altered intestinal structure, respectively.

  • Fig. 4 Vertical transmission of RVFV in two dams resulted in stillborn pups with physical abnormalities and high viral titers.

    (A to D) Dam 1 was inoculated with 175 PFU and died giving birth at 8 dpi. Dam 1 delivered 13 stillborn pups, with 2 remaining in the uterus. (A) Pictures of individual pups are shown as numbered. Uterus with pups 13 and 14 within birth canal after demise. (B) vRNA within maternal tissue. (C) vRNA and (D) infectious virus within peritoneal cavity of indicated pups. The three pups not included in the graphs were used for other analyses. (E to H) Dam 2 was inoculated with 175 PFU and survived with no clinical signs of disease. Dam 2 delivered seven pups to full-term; five were dead and two were alive but subsequently consumed by the dam (data not available). (E) Pictures of individual pups as labeled. A normal pup from an uninfected dam is shown for comparison. (F) vRNA within maternal tissue. (G) vRNA and (H) infectious virus within peritoneal cavity of indicated pups. For all graphs, red square data points indicate either dam or pup demise. Blue circle data points indicate dam or pup survival.

  • Fig. 5 Evidence of variable survival and developmental outcome of pups resulting from vertical transmission.

    (A to E) Dam 3 was inoculated with 175 PFU and survived with no clinical signs of disease. Dam 3 delivered 10 pups to full-term; 6 were dead and 4 were alive. Two living pups were consumed by the dam within 2 days of birth (data not available). Two remaining pups survived to the end of the study (pups 7 and 8). (B) vRNA within maternal tissue. (C) vRNA and (D) infectious virus within peritoneal cavity of indicated pups. (E) Weight of surviving pups from dam 3 from days 5 to 10 neonate. The black line and the gray line represent growth curve calculated by linear regression modeling of pups 7 and 8, respectively. (F to J) Dam 4 was inoculated with 1300 PFU and survived with no clinical signs of disease. Dam 4 delivered nine stillborn pups. (F) Pictures of individual pups as labeled. (G) vRNA within maternal tissue. (H) vRNA and (I) infectious virus within peritoneal cavity of indicated pups. (J) Cage of dam 4 as it was found on day of pup delivery (E22; 8 dpi).

  • Fig. 6 RVFV replicates in human placental tissue, even the highly resistant syncytiotrophoblasts within placenta villi.

    Human chorionic villous tissue explants from donors 1 and 2 (A) were infected in vitro with RVFV at the indicated doses and then the supernatant was harvested at 0, 12, 24, 36, and 48 hours after infection for measurement of vRNA by qRT-PCR. (B) Immunofluorescent microscopy images of villi infected with 3 × 106 PFU RVFV for 48 hours. Uninfected control is shown on the left. DAPI (blue) stains DNA, J2 antibody (green) stains dsRNA of RVFV, cytokeratin 19 (red) stains epithelial cells, and actin (purple) stains all cells. (C) Fluorescent dsRNA signal was quantified from four human donors. Data from uninfected (n = 11) and infected (n = 16) villi are shown with symbols stratified by donor (closed circles, donor 1; open circles, donor 2; closed squares, donor 3; open squares, donor 4). **P < 0.01.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/12/eaau9812/DC1

    Fig. S1. RVFV is present in the ovary and uterus of nonpregnant female rats.

    Fig. S2. Detection of RVFV in the testes of male rats.

    Fig. S3. RVFV infection leads to fetal resorption.

    Fig. S4. Evidence of vertical transmission of RVFV in an RVFV-infected early-gestation (E5) pregnant dam, with high viral titers in the placenta.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. RVFV is present in the ovary and uterus of nonpregnant female rats.
    • Fig. S2. Detection of RVFV in the testes of male rats.
    • Fig. S3. RVFV infection leads to fetal resorption.
    • Fig. S4. Evidence of vertical transmission of RVFV in an RVFV-infected early-gestation (E5) pregnant dam, with high viral titers in the placenta.

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