Zika Virus Targets Different Primary Human Placental Cells, Suggesting Two Routes for Vertical Transmission

Abstract

Zika virus (ZIKV) infection during pregnancy is linked to severe birth defects, but mother-to-fetus transmission routes are unknown. We infected different primary cell types from mid- and late-gestation placentas and explants from first-trimester chorionic villi with the prototype Ugandan and a recently isolated Nicaraguan ZIKV strain. ZIKV infects primary human placental cells and explants-cytotrophoblasts, endothelial cells, fibroblasts, and Hofbauer cells in chorionic villi and amniotic epithelial cells and trophoblast progenitors in amniochorionic membranes-that express Axl, Tyro3, and/or TIM1 viral entry cofactors. ZIKV produced NS3 and E proteins and generated higher viral titers in amniotic epithelial cells from mid-gestation compared to late-gestation placentas. Duramycin, a peptide that binds phosphatidylethanolamine in enveloped virions and precludes TIM1 binding, reduced ZIKV infection in placental cells and explants. Our results suggest that ZIKV spreads from basal and parietal decidua to chorionic villi and amniochorionic membranes and that targeting TIM1 could suppress infection at the uterine-placental interface.

Figure 1. Prototype Uganda and Nicaraguan 2016 ZIKV strains infect primary cell types from human placentas

Panel I: Primary human placental cells were infected with ZIKV MR766 and immunostained for E (A–E) and NS3 (F–J) proteins at 3 dpi. (A, B, F, G) Primary amniotic epithelial cells (AmEpC) at mid and late gestation were infected at MOI 0.1. (C, H) Trophoblast progenitor cells (TBPC) (P15), (D, I) Human placental fibroblasts (HPF), and (E, J) Human umbilical vein endothelial cells (HUVEC) (P5) were infected at MOI 1. Panel II: Primary AmEpC from mid and late gestation were infected with ZIKV Nica1–16 at MOI 0.001 and immunostained for E glycoprotein (A, B) and NS3 protein (C, D) at 3 dpi. Panel III: (A–C) Chorionic villus explants (8.4 wks) infected with ZIKV MR766 (1×10⁶ PFU) and immunostained for E and CK (A), NS3 and CK (B), and NS3 and Ki67 (C) in adjacent sections at 3 dpi. Left and right panels: higher magnification images. Arrowheads, cells expressing viral proteins; arrows, cells expressing Ki67. Images representative of n=4. Panel IV: Chorionic villus explants (8 wks) infected with ZIKV MR766 (1×10⁶ PFU) and immunostained for E and CK (A), NS3 and CK (B), and MHC class I HLA-G (differentiation antigen) and CK (C). Panel V: (A–F) Chorionic villus explant infected with Nica1–16 (5×10³ PFU) and immunostained for E and CK (A, D), NS3 and CK (B, E) and CD68 (Hofbauer cell/macrophage marker) and CK (C, F) in adjacent sections at 2 dpi. Villus core Hofbauer cells/Mϕ (circled) were positive for NS3 and E. Nuclei, blue (DAPI). VC, villus core. See also Figures S1 and S2 and Table S1.

Figure 2. Viral titers from ZIKV-infected AmEpC from mid-gestation are higher than those from late gestation

AmEpC isolated from mid- (21.2 and 22.6 wks) and late- (38.6 and 40.2 wks) gestation were infected with (A) MR766 at MOI 0.001, (B, D) Nica1–16 at MOI 0.001, and (C) Nica2–16 at MOI 0.009. Viral titers were determined by FFA at 3 dpi (A) and 5 and 7 dpi (B, C). (D) Viral titers at 5 and 7 dpi were quantified in ARPE-19 cells, stained with mAb 4G2 at 28 hpi. GA, gestational age. n=4 from 2 independent experiments, *p<0.05, **p<0.01, unpaired t-test. See also Table S1.

Figure 3. TIM1 is consistently expressed in primary placental cells, while Axl and Tyro3 expression varies according to cell type, differentiation state and individual donor

Expression of cofactors by immunoblotting in (A) differentiating CTB isolated from mid-gestation (n=4) and late-gestation placentas (n=1); (B) trophoblast cell lines JEG-3 and HTR8, (C) cells from 3 late-gestation amniotic membranes, including intact membranes, freshly isolated AmEpC before plating, and confluent P0 and P1 cells; (D) AmEpC (P1) from mid- and late-gestation placentas; (E) TBPC from early- and mid-gestation (P15) placentas; (F) HPF and HUVEC (P5). (G, H) Representative images of Axl and TIM1 staining in AmEpC from mid- (left) and late-gestation (right) placentas (n=6). See also Figure S2 and Table S3.

Figure 4. Axl is strongly expressed at the uterine-placental interface

Immunofluorescence staining of frozen sections from mid-gestation placentas for Axl (green) and cell type markers (red). (A) Axl and vimentin in cells of the basal decidua. (B) Axl and cytokeratin (mAb 7D3) in invasive CTB. (C) Axl colocalizes with CD68 (red), a marker of Hofbauer cells and macrophages (Mϕ). (D) Axl in CD31-positive vascular endothelial cells in a villus core. (E) Axl in villus core partially overlaps with vimentin, a marker of HPF. (F) Axl in the amniotic epithelium and cells of the mesenchymal portions of the amnion and chorion, where TBPC reside. Cytokeratin 7 (CK7) staining identifies CTB lining the surface of the chorionic mesenchyme. DAPI, blue. Images representative of n=4. See also Figure S2 and Table S3.

Figure 5. Axl and Tyro3 vary by site and gestational age, whereas TIM1 is consistently expressed

Axl, Tyro 3, and TIM1 expression in human placenta and fetal membranes was examined by immunohistochemical staining of fixed, paraffin-embedded placental biopsies from mid- and late-gestation (n=26). (A, D) Axl immunostaining in two mid-gestation amnions. (B, C, E, F) Mid-gestation AmEpC stain positively for Tyro 3 (B, E) and TIM1 (C, F). (G–O) Three zones of the maternal-placental interface from 3 placentas: amniotic epithelium on fetal side, bathed in amniotic fluid (G–I); chorionic villi of placenta with fetal blood vessels and villus core covered by STB in maternal blood (J–L); basal decidua containing invasive CTB from the placenta (M–O). (P–U) Interface of the amniochorion with parietal decidua from 3 placentas, including the amniotic epithelium (P–R) and the chorion, CTB layer and parietal decidua, containing invasive CTB (S–U). Images are representative of 26 donors (Table S2). Donor numbers are indicated in the lower right corner. See also Table S2.

Figure 6. Axl and TIM1 inhibitors block ZIKV infection differentially in primary placental cells

(A and B) Axl kinase inhibitor reduces infection of (i) MR766 and (ii) Nica2–16 (both MOI 0.014) in AmEpC (40.2 wks). (A) Representative immunofluorescence images stained for NS3 and (B) quantification of relative infection (%) (mean±SD) of 2 replicates of each condition from 2 independent experiments. **p<0.01, unpaired t-test. (C and D) TIM1 inhibitor (duramycin) potently reduces ZIKV MR766 (MOI 0.3) and Nica1–16 (MOI 0.001) infection of various placental cell types. (C) Representative immunofluorescence images of ZIKV MR766 infection in AmEpC (22.6 and 40.2 wks). NS3 in green. (D) (i) Relative infection of AmEpC with ZIKV MR766 (22.6 and 40.2 wks) and (ii) HPF, TBPC and HUVEC. n=2 for HPF and TBPC and n=1 for HUVEC. (iii) Relative infection of AmEpC with Nica1–16, n=3. (E) Virus titers from ZIKV MR766-infected AmEpC treated with duramycin at 3 dpi, quantified by FFA. n=2. D, E: **p<0.01, ***p<0.001, one-way ANOVA with Dunnett’s post-test.

Figure 7. A model of uterine-placental interface indicating ZIKV infection of numerous cell types and possible routes of transmission

(Left). Cross-section of the pregnant uterus and anchoring villus with CTB that remodel blood vessels that channel maternal blood to the placenta surface. The basic structural unit is the chorionic villus, composed of a stromal fibroblast core with fetal capillaries and Hofbauer cells/macrophages (Mϕ). During early development, villus CTB detach from the basement membrane and either fuse to form STB, which covers floating villi, or proliferate and aggregate into a column of extravillus CTB that differentiate and invade the basal decidua and uterine wall (invasive CTB). These anchoring villi establish physical connections between mother and fetus through attachment of CTB cell columns. (Right) The amniochorionic membrane circumscribes the fetal compartment extending from the margins of the placenta to surround the fetus, with AmEpC in direct contact with amniotic fluid. At 15–20 wks gestation, as the amniotic sac enlarges, the amniochorionic membranes fuse and contact the parietal decidua, and a subset of CTB, derived from TBPC in the chorion and invade the parietal decidua lining the uterine wall (invasive CTB). ZIKV infects primary placental cells that strongly express TIM1 (red). These include proliferating villus CTB in proximal cell columns and invasive CTB, Hofbauer cells, HPF and HUVEC in chorionic villi and TBPC and AmEpC in fetal membranes, defining a continuum of susceptible cells that represent two routes of ZIKV transmission from the basal decidua to chorionic villi and fetal circulation (Left), and parietal decidua to amniochorionic membranes, amniotic fluid and the fetus (Right). Modified from Pereira, et. al, 2005.