Persistent Cytomegalovirus Infection in Amniotic Membranes of the Human Placenta

Abstract

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, including microcephaly, neurological deficits, hearing impairment, and vision loss. We previously reported that epithelial cells in amniotic membranes of placentas from newborns with intrauterine growth restriction and underlying congenital HCMV infection contain viral proteins in cytoplasmic vesicles. Herein, we immunostained amniotic membranes from 51 placentas from symptomatic and asymptomatic congenital infection with HCMV DNA in amniotic fluid and/or newborn saliva, intrauterine growth restriction, preterm deliveries, and controls. We consistently observed HCMV proteins in amniotic epithelial cells (AmEpCs) from infected placentas, sometimes with aberrant morphology. Primary AmEpCs isolated from mid-gestation placentas infected with pathogenic VR1814 proliferated and released infectious progeny for weeks, producing higher virus titers than late-gestation cells that varied by donor. In contrast to intact virion assembly compartments in differentiated retinal pigment epithelial cells, infected AmEpCs made dispersed multivesicular bodies. Primary AmEpCs and explants of amniochorionic membranes from mid-gestation placentas formed foci of infection, and interferon-β production was prolonged. Infected AmEpCs up-regulated anti-apoptotic proteins survivin and Bcl-x_L by mechanisms dependent and independent of the activated STAT3. Amniotic membranes naturally expressed both survivin and Bcl-x_L, indicating that fetal membranes could foster persistent viral infection. Our results suggest strengthening innate immune responses and reducing viral functions could suppress HCMV infection in the fetal compartment.

Figure 1

Immunohistochemical staining for human cytomegalovirus (HCMV)-infected cell proteins in amniotic membranes from placentas at delivery (additional information available in Table 1). Sample designations and sources are described in Materials and Methods. A–C: Uninfected controls. D and E: Uninfected amniotic membranes from preterm deliveries with intrauterine growth restriction (IUGR). F–I: Infected amniotic membranes from asymptomatic term deliveries and detection of HCMV DNA in newborn saliva. J–L: Infected amniotic membranes from diagnosed primary maternal HCMV infection. J and K: HCMV DNA in amniotic fluid. L: Congenital infection and stillbirth. M–R: HCMV-infected amniotic membranes from preterm deliveries. M: Diagnosed primary maternal infection with HCMV DNA in amniotic fluid. Scale bar = 50 μm. HIG, hyperimmune globulin.

Figure 2

A: Isolated amniotic epithelial cells (AmEpCs) were mock infected (control) or infected with VR1814 and fixed at 7 days postinfection (dpi). Immunostaining of stem cell markers (green) and viral proteins (red) in control (top panels) and infected cells (bottom panels). Nuclei were stained with DAPI (blue). Colocalization of green and blue signals (turquoise) and red and green signals (yellow). B: Immunoblotting of viral proteins. Lysates from mid-gestation (17 weeks) and late gestation (39 weeks) AmEpCs and ARPE-19 cells at indicated time points were immunoblotted with monoclonal antibodies to viral proteins and actin (loading control). CH160 was used to detect immediate early (IE) 1 and IE2. C: Numbers of IE1-positive cells stained with CH443 (IE1) and counterstained with DAPI were calculated at 2 and 10 dpi. At least 10 images (magnification ×200) from randomly selected areas were photographed using a Nikon Eclipse 50i microscope equipped with a SPOT 7.4 Slider camera controlled by SPOT 2.0 Advanced software (Diagnostic Instruments, Sterling, MI). Graph shows IE-positive cell numbers. D: Phase microscopy of HCMV-infected AmEpCs at the indicated times after infection. Numbers indicate titers of progeny virions in human foreskin fibroblasts. Foci of infected cells indicated by green dotted lines. E: Immunostaining for Ki-67 (green) and IE1 (CH443) (red) in uninfected control and VR1814-infected AmEpCs. Nuclei were stained with DAPI. Data are given as means ± SEM (C). Scale bars: 50 μm (A and E); 100 μm (D). Original magnifications: ×200 (A and E); ×100 (D). PFU, plaque-forming unit.

Figure 3

A–C: Immunostaining of human cytomegalovirus (HCMV) gB (green) and pp28 (red) (A), gB (green) and pp65 (red) (B), and gB (green) and CD63 (red) (C) in VR1814-infected amniotic epithelial cells (AmEpCs) and ARPE-19 cells at indicated time points. Blue dotted lines delineate sites of colocalization. Arrowhead and pink circle also indicate colocalization. D: Immunostaining of caveolin 1 (green) and gB (red) in VR1814-infected AmEpCs. E: Infected AmEpCs from early and late gestation released progeny virions. Conditioned media from VR1814-infected AmEpCs from different gestational ages were collected every other day and titered in human foreskin fibroblasts (HFFs). F: Progeny virions from both early gestation (17 weeks, left y axis) and late gestation (32 weeks, right y axis) AmEpCs retained tropism for epithelial cells, as indicated by titration on AmEpCs (23.5 weeks' gestation). G: VR1814 inoculum was pre-incubated with medium alone or serial dilutions of anti-gB monoclonal antibody (Mab; TRL345), anti-pentamer MAb (TRL310, 1F11), hyperimmune globulin (HIG; Cytotect), or negative control antibody (Synagis) and then added to AmEpCs (22.5 weeks' gestation). Percentages of infected cells were determined by counting immediate early 1 (CH443)-positive cells and normalized to counts from the no antibody control. Data points represent means of two independent experiments each with three replicates (G). Scale bar = 50 μm (A–D). Original magnification, ×200 (A–D). dpi, days postinfection; GA, gestational age; PFU, plaque-forming unit.

Figure 4

A: Immunostaining of interferon regulatory factor (IRF) 7 in mock-infected control (Cont.) and VR1814, TB40/E or Δ131A mutant virus-infected cells at indicated time points. Nuclei were stained with DAPI. B: Human cytomegalovirus (HCMV)-infected cells activate IRF7. Cell lysates from mock-infected control and VR1814-infected (inf.) amniotic epithelial cells (AmEpCs) at indicated time points were immunoblotted with antibodies to phospho-IRF7 (pIRF7), IRF7, and actin (loading control). Results are representative of at least four independent experiments. C: Levels of interferon (IFN)-β in conditioned media from mock-infected control and TB40/E or Δ131A-infected AmEpCs were quantified by enzyme-linked immunosorbent assay (ELISA). D: Levels of IFN-β in conditioned media from mock-infected control and VR1814-infected (inf.) AmEpCs from three donors were quantified by ELISA. E–J: Up-regulation of IFN-β in an explant of amniochorionic membrane (19.3 weeks' gestation) infected ex vivo with VR1814. Immunostaining of CK19 (green; E), IFN-β (green; F), and immediate early (IE) 1 (red; E and F) at 5 days postinfection (dpi). The dashed box in F indicates the area shown at high magnification in H–J. G: No specific staining was observed using isotype control antibodies. Scale bar = 20 μm (A and H–J); 50 μm (E–G). Original magnifications: ×200 (A and E–G); ×400 (H–J).

Figure 5

A: IL-6 levels in conditioned media from mock-infected control (cont.) and VR1814-infected amniotic epithelial cells (AmEpCs) quantified by enzyme-linked immunosorbent assay. Similar results were obtained with four cell preparations. B: Human cytomegalovirus (HCMV) infection activates STAT3 and up-regulates anti-apoptotic proteins. Cell lysates from mock-infected control and VR1814-infected AmEpCs or ARPE-19 cells at indicated times were immunoblotted with antibodies to phospho-STAT3 (pSTAT3), STAT3, Bcl-2, Bcl-x_L, survivin, and actin (loading control). Results are representative of at least three independent experiments. C and D: Immunostaining of survivin (B), Bcl-x_L (C), and immediate early (IE) 1 (CH443) in mock-infected control and VR1814-infected cells. Nuclei were stained with DAPI. Blue dashes delineate foci of infection. E: Effects of STAT3 inhibitors on Bcl-x_L and survivin expression. Mock-infected control and VR1814-infected AmEpCs were cultured with medium alone (no treat), STAT3 inhibitors S31-201 (100 μmol/L), WP6001 (5 μmol/L), or STA21 (50 μmol/L), or the vehicle dimethyl sulfoxide (DMSO). Cell lysates at 3 days postinfection (dpi) were immunoblotted with antibodies to Bcl-x_L, survivin, and actin (loading control). Results are representative of at least four independent experiments. F: Cell lysates from mock-infected control and VR1814-infected AmEpCs from different gestational ages were immunoblotted with antibodies to Bcl-2, Bcl-x_L, survivin, and actin (loading control). Scale bar = 50 μm (C and D). Original magnification, ×200 (C and D).

Figure 6

Representative immunohistochemical staining for survivin and Bcl-x_L in amniotic membranes of placentas from uninfected control, preterm delivery, congenital human cytomegalovirus (HCMV) infection, and congenital infection with preterm delivery. Thirty-four membranes (13 expressing HCMV proteins) were examined for survivin expression, and 17 membranes (8 expressing HCMV proteins) were examined for Bcl-x_L expression. Sample designations and sources are described in Materials and Methods. A: Section of SF21 adjacent to the section in Q stained with isotype control for rabbit monoclonal antibody to anti-survivin. B: Section of SF21 adjacent to the section in R stained with control antisera for rabbit polyclonal anti–Bcl-x_L antibody. Survivin and Bcl-x_L immunostaining of amniotic membranes: control (uninfected; C–F); control with preterm delivery (G and H); congenital HCMV infection (I–L); and congenital HCMV infection with preterm delivery (M–R).