Research ArticleEMERGING INFECTIONS

Zika virus–related neurotropic flaviviruses infect human placental explants and cause fetal demise in mice

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Science Translational Medicine  31 Jan 2018:
Vol. 10, Issue 426, eaao7090
DOI: 10.1126/scitranslmed.aao7090
  • Fig. 1 ZIKV-related flaviviruses cause fetal demise in mice that can be prevented by mAb treatment.

    Wild-type pregnant dams were inoculated subcutaneously on embryonic day 6.5 (E6.5) by footpad injection with 102 focus-forming units (FFU) of West Nile virus (WNV) or 103 FFU of Powassan virus (POWV), chikungunya virus (CHIKV), Mayaro virus (MAYV), or phosphate-buffered saline (PBS) (mock), and tissues were harvested on either E13.5 or E18.5, as indicated. (A to C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of viral RNA (vRNA) burden on E13.5 in the placenta (A), fetal head (B), and maternal spleen (C). (D) Fetal survival as assessed on E18.5. (E) Photograph of littermate mock- and WNV-infected fetuses at E18.5. (F) Interferon (IFN)–stimulated gene (ISG) mRNA expression as measured by qRT-PCR from placenta and fetal head on E13.5 after E6.5 inoculation with WNV, POWV, CHIKV, or MAYV. (G to H) Pregnant mice were inoculated subcutaneously with WNV (102 FFU) via footpad injection on E6.5 and then treated on E8.5 with a single intraperitoneal injection of 300 μg of anti-WNV monoclonal antibody (mAb) (humanized E16) or isotype control mAb (anti-CHIKV human mAb 4N12) at E8.5. Fetal heads and placentas were harvested on E13.5 for viral titer measurement by qRT-PCR (G) or on E18.5 to assess fetal survival (H). Antibody treatment experiments are pooled from three independent experiments with one to two pregnant female dams per experiment. vRNA burden and ISG expression data represent the means ± SEM for at least n = 6 tissues per group from four or five infected dams in at least two independent experiments. Each data point represents a biological replicate. In (A) to (C) and (F), data were analyzed by one-way analysis of variance (ANOVA). In (G), data were analyzed by Mann-Whitney test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). The n for survival data is indicated above each bar. Survival data were analyzed by χ2 test (****P < 0.0001).

  • Fig. 2 Placental histology on E18.5 after infection with WNV and CHIKV.

    Wild-type pregnant mice were inoculated with 102 FFU of WNV or 103 FFU of CHIKV or PBS (mock) via footpad injection on E6.5, and placentas were harvested on E18.5 for histological analysis. (A to C) Hematoxylin and eosin (H&E) staining of placentas was subsequently performed on mock-infected (A), CHIKV-infected (B), and WNV-infected (C) fetuses. Top row, lower magnification views. Bottom two rows, higher magnification views of the decidua (D; middle row) and labyrinth (L; bottom row). SpT indicates spongiotrophoblasts within the junctional zone (JZ). (D) Placental diameter of mock-, CHIKV-, and WNV-infected placentas. Data are representative of two independent experiments with n = 3 tissue samples for histological analysis and n = 7 fetuses from two or three infected dams for placental diameter measurements. Scale bars, 200 μm (top row) and 50 μm (bottom two rows). Data in (D) were analyzed by ANOVA test (**P < 0.01 and ***P < 0.001).

  • Fig. 3 Fetal brain histology on E18.5 after infection with WNV and CHIKV.

    Wild-type pregnant mice were inoculated with 102 FFU of WNV or 103 FFU of CHIKV or PBS (mock) via footpad injection on E6.5, and fetuses were harvested on E18.5 for histological analysis. (A to D) H&E staining was performed on mock-infected (A), CHIKV-infected (B), and WNV-infected (C and D) fetuses. Red arrows indicate necrosis of the brain in a severe case of WNV fetal disease. Data are representative of two independent experiments with n = 3 fetuses each. Scale bars, 200 μm (top rows) and 100 μm (bottom rows).

  • Fig. 4 WNV tissue tropism in the mouse placenta and fetus.

    Wild-type pregnant mice were inoculated with 102 FFU of WNV or PBS (mock) via footpad injection on E6.5, and fetuses were harvested on E13.5 for analysis by RNA in situ hybridization (ISH). (A and B) Low- and high-magnification images of RNA ISH staining of mock- and WNV-infected placenta on E13.5. D, decidua; JZ, junctional zone; L, labyrinth; UC, umbilical cord. Scale bars, 1 mm (A) and 100 μm (B). (C to G) Low- and high-magnification images of RNA ISH staining of mock- and WNV-infected fetuses on E13.5. WNV RNA ISH staining in severely infected fetuses (C, right, and D to G), including the forebrain (D), hindbrain (E), spinal cord (F), and myocardium (G). Data are representative of two independent experiments with n = 3 placentas or fetuses each. Scale bars, 1 mm (C) and 100 μm (D to G).

  • Fig. 5 Viral growth curves in human placental explants.

    (A to F) Human placenta villi, decidua, and fetal membrane explants from three donors (21 to 23 weeks of gestation) were inoculated with 5 × 106 FFU of ZIKV (Zika virus), WNV, POWV (A to C), CHIKV, or MAYV (D to F), incubated at 37°C for 2.5 hours, washed, and then cultured in DMEM/F12. Small aliquots of cell explant supernatants were collected at 3, 12, 24, 36, and 48 hours and analyzed by focus-forming assay. Data represent the means ± SEM from three or four organoid explants per experiment from three separate donors under each condition.

  • Fig. 6 Immunofluorescence staining of virus-infected human placental explants.

    Confocal micrographs of human placental tissues (chorionic villi, decidua, and fetal membrane) from four donors (16 to 23 weeks of gestation) that were inoculated with the indicated viruses [mock (PBS), ZIKV, WNV, POWV, CHIKV, and MAYV] for 48 hours. Immunofluorescence microscopy was performed for vRNA [using anti-dsRNA (double-stranded RNA) antibody; green] and tissue counterstained for cytokeratin-19 (Cyt-19; a marker of epithelial-derived cells, including trophoblasts) or actin (in red, as indicated below each panel). DAPI (4′,6-diamidino-2-phenylindole)–stained nuclei are shown in blue. White boxes denote zoomed regions shown at bottom right. Images are representative of four explants per experiment from four separate donors under each condition. Scale bar, 50 μm.

Supplementary Materials

  • Supplementary Material for:

    Zika virus–related neurotropic flaviviruses infect human placental explants and cause fetal demise in mice

    Derek J. Platt, Amber M. Smith, Nitin Arora, Michael S. Diamond, Carolyn B. Coyne, Jonathan J. Miner*

    *Corresponding author. Email: miner{at}wustl.edu

    Published 31 January 2018, Sci. Transl. Med. 10, eaao7090 (2018)
    DOI: 10.1126/scitranslmed.aao7090

    This PDF file includes:

    • Fig. S1. Survival of pregnant dams after infection at E6.5 and vRNA burden in pregnant dams after inoculation on E9.5.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primary data.

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