Usutu Virus Infects Human Placental Explants and Induces Congenital Defects in Mice

Abstract

Usutu virus (USUV) is a neurotropic mosquito-borne flavivirus that has dispersed quickly in Europe these past years. This arbovirus mainly follows an enzootic cycle involving mosquitoes and birds, but can also infect other mammals, causing notably sporadic cases in humans. Although it is mainly asymptomatic or responsible for mild clinical symptoms, USUV has been associated with neurological disorders, such as encephalitis and meningoencephalitis, highlighting the potential health threat of this virus. Among the different transmission routes described for other flaviviruses, the capacity for some of them to be transmitted vertically has been demonstrated, notably for Zika virus or West Nile virus, which are closely related to USUV. To evaluate the ability of USUV to replicate in the placenta and gain access to the fetus, we combined the use of several trophoblast model cell lines, ex vivo human placental explant cultures from first and third trimester of pregnancy, and in vivo USUV-infected pregnant mice. Our data demonstrate that human placental cells and tissues are permissive to USUV replication, and suggest that viral transmission can occur in mice during gestation. Hence, our observations suggest that USUV could be efficiently transmitted by the vertical route.

Keywords: Usutu virus; arbovirus; flavivirus; placenta; vertical transmission.

Figure 1

Kinetics of USUV infection in placental cell lines and effect on cell growth and viability. JAR cells or HIPECs have been infected by USUV at MOI 3 at different times before proceeding to analyses. (a) Indirect immunofluorescence realized against USUV Env antigen (green: Env; blue: DAPI) at 16 or 48 h post-infection (hpi). Images are representative of at least three independent experiments, and three independent fields each time. Scale bar = 50 μm. (b) Quantification of the percentage of infected cells determined by flow cytometry, via intracellular staining of Env antigen. (c) Quantification of cell growth between JAR cells and HIPECs upon USUV infection, by counting viable cell number at different time points. (d) Cell mortality was evaluated by trypan blue staining for JAR cells and HIPECs at different times upon USUV infection. In (b–d), symbols represent mean ± SEM for three independent experiments. In each experiment, a two-way ANOVA statistical test was performed and indicated a significant difference between cell lines (*, p < 0.05).

Figure 2

JAR cells and HIPEC allow USUV productive replication cycle with different efficiencies. HIPECs (left column) or JAR cells (right column) have been infected by USUV at MOI 1 or 10 during indicated times (hpi: hours post-infection) before proceeding to analyses. (a) Quantification of the proportion of infected cells determined by flow cytometry, via intracellular staining of Env antigen. Symbols represent the mean ± SEM for three independent experiments for HIPECs and four independent experiments for JAR cells. (b) Quantification of viral RNA extracted from infected cells, upon normalization by actin mRNA and by the level of viral RNA at 6 hpi for MOI 1. Histograms represent mean ± SEM for three independent experiments. (c) Quantification of viral RNA released in cell supernatant. Symbols represent the mean ± SEM for five independent experiments. (d) Quantification of infectious particles released in cell supernatant. Histograms represent mean ± SEM for five independent experiments. In (a–d), a two-way ANOVA statistical test was performed to evaluate a statistical difference between cell lines and MOI (ns, non-significant; *, p < 0.05; **, p < 0.005).

Figure 3

Characterization of innate immune responses of HIPEC and JAR cells upon USUV infection. (a) And (c) volcano plot representing differences in normalized mean mRNA expression in JAR cells (a) or HIPECs (c) 16 h upon USUV infection compared to non-infected cells, from three independent experiments. mRNAs exhibiting significant differences upon infection are represented by colored circles (Student’s t-test p-value ≤ 0.05 and log2 ratio ≥ 2 or ≤ −2). (b–d) Detail of the under- and over-expressed mRNAs upon USUV infection in JAR cells (b) or HIPECs (d).

Figure 4

Human first- and third-trimester placentas are permissive for USUV replication. (a) Pipeline of placental explant infection and histoculture. (b) Left columns: cross sections of placental alkaline phosphatase immunohistochemistry and hematoxylin staining of placental villi from histoculture at day 15, observed by bright field microscope. Image representative from at least three independent experiments. Scale bar = 100 μm. Right columns: fluorescence-based TUNEL assay done on cross section of placental villi from histoculture at day 15. Image representative from three independent experiments (NI: non-infected; USUV UV: infection by UV-inactivated USUV). (c) Heat-map representing the level of viral RNA expression (normalized by actin) in placental tissues upon infection by USUV or by UV-inactivated virus (USUV UV), for six independent experiments (NI: non-infected). The ΔCT values are represented by a double gradient color map (blue: high ΔCT = no amplification of viral RNA; red: low ΔCT = amplification of viral RNA; and crossed gray: no data). A one-way ANOVA statistical test was performed to evaluate a statistical difference between conditions, followed by Tukey’s multiple comparison test (ns, non-significant; *, p < 0.05; ***, p < 0.0005; ****, p < 0.0001). (d) Anti-USUV Env immunohistochemistry done on term placental villi from histoculture at day 15. Blue staining corresponds to hematoxylin coloration of the nucleus and brown staining correspond to the USUV Env detection. Scale bar = 50 μm (NI: non-infected). (e) Quantification of viral RNAs (red circles: first-trimester placenta; blue squares: term placenta; and left Y-axis) and infectious particles (blue triangles, term placenta, and right Y-axis) released in supernatant by placenta histocultures at different times upon USUV infection (dpi: days post-infection). Symbols represent the mean ± SEM for three to six independent experiments. (f) Results of anti Env immunofluorescence realized upon reinfection of Vero cells incubated with supernatant of placental histocultures, either from first-trimester (left panels) or term (right panels) placentas, done with supernatants collected at day 15 after infection by USUV or UV inactivated USUV (ND: not determined). Blue: DAPI, green: USUV Env antigen.

Figure 5

USUV can achieve congenital infection of immunocompetent mice and causes occasional fetal demise. C57BL/6 pregnant mice were inoculated with USUV by subcutaneous (footpad) route with 104 TCID50 in 50 µL of PBS. (a) Brain and blood of suckling mice were collected after the birth for the brain and 2 weeks later for blood. Viral burden was measured by RT-qPCR assay and indicated by TCID50 equivalent per g or per ml. (b) Fetal demise in USUV-infected mice after cesarean section in the second third of gestation. Spleen (c), placenta (d), and mammary glands (e) of infected pregnant mice were collected and viral burden was measured by RT-qPCR assay. Organs were harvested at E6 and E12 stages of gestation for spleen and placenta, and 2 weeks after delivery for mammary glands.