Vertical Transmission of the Zika Virus Causes Neurological Disorders in Mouse Offspring

Abstract

The association between Zika virus (ZIKV) infection and congenital malformations such as microcephaly in infants is a public health emergency. Although various in vivo and in vitro models are used for ZIKV research, few animal models are available for resolving the effects of maternal ZIKV infection on neonatal development. Here, we established an immunocompetent mouse model via intrauterine inoculation. Our results confirmed that ZIKV, but not dengue virus, infection caused spontaneous abortions, brain malformations, ocular abnormalities, spinal cord defects and paralysis in mouse offspring. Aside from microcephaly and hippocampal dysplasia, eye abnormalities, including microphthalmia, thinner optic nerves, damaged retinae, and deficient visual projection, were also observed following ZIKV infection. Moreover, ZIKV-infected offspring showed a loss of alpha motor neurons in the spinal cord and cerebellar malformation, which may cause paralysis. ZIKV also impaired adult neurogenesis in neonatal mice. Due to its intact immunity, our rodent model can be used to systematically evaluate the impact of ZIKV on embryonic and neonatal development and to explore potential therapies.

Figure 1

Low birth rate, high mortality and severe brain pathology induced by intrauterine ZIKV inoculation. (a) Schematic depiction of intrauterine inoculation during pregnancy. Offspring mice were used in following studies. (b) Birth rate of neonatal mice. (c) ZIKV viral load in P14 tissues was measured by qRT-PCR. Bars indicate the mean. The gray dashed lines indicate the limit of detection. (d) Representative images of brains from P14 offspring. (e,f) Area (e) and perimeter (f) of the hemiencephalon of P14 mice. (g) Overview of ZIKV infection in 1 representative brain slice. CPu, caudate putamen (striatum); Po, posterior thalamic nuclear group; VPM, ventral posteromedial thalamic nucleus; VMH, ventromedial hypothalamic nucleus. (h) Percentage of ZIKV-infected cells in different brain regions. n = 5 samples from three independent experiments. (i) Representative images of the cortex stained for Cas3 (Caspase 3) and ZIKV. (j) Quantification of cortical thickness. (k) Percentage of Cas3-positive cells within the cortex. (j,k) n = 5 samples from three independent experiments. (l) Representative images of the hippocampus stained for Cas3 and ZIKV. (m) Percentage of ZIKV-infected cells in the DG, CA1, and CA3. n = 5 samples from three independent experiments. (n–p) Percentage of Cas3-positive cells within the DG(n), CA1(o), and CA3(p), respectively. n^Mock = 4, n^ZIKV = 5, n^DENV-2 = 4 samples from three independent experiments. Quantification data are presented as the mean ± SEM. One-way ANOVA with Bonferroni’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2

Visual defects caused by maternal ZIKV infection. (a) Representative images of P14 eyeballs. (b) Diameters of P14 eyeballs. (c) Representative images of P14 optic nerves. (d) Diameters of P14 optic nerves. (e) Representative images of the retina stained for Cas3 and ZIKV. White arrows, ZIKV-positive or Cas3-positive cells. (f) Percentage of ZIKV-infected cells within the INL and ONL. n = 10 samples from three independent experiments. (g) Percentage of Cas3-positive cells within the retina. n^Mock = 5, n^ZIKV = 9, n^DENV-2 = 5 samples from three independent experiments. (h–j) Retinal GCL and IPL thickness (h), INL thickness (i), and ONL thickness (j), respectively. n^Mock = 14, n^ZIKV = 13, n^DENV-2 = 11 samples from three independent experiments. (k) Quantification of retinal thickness. n^Mock = 14, n^ZIKV = 19, n^DENV-2 = 11 samples from three independent experiments. (l) AXL and ZIKV expression in the P7 retina. Quantification data are presented as the mean ± SEM. Student’s t-test (f) or one-way ANOVA with Bonferroni’s post hoc test, ***p < 0.001.

Figure 3

Few retinal ganglion cells and no starburst amacrine cells were left after maternal ZIKV infection. (a) P14 retinal Calretinin immunostaining. (b) Percentage of Calretinin-positive cells in the GCL. n^Mock = 7, n^ZIKV = 5, n^DENV-2 = 8 samples from three independent experiments. Data are presented as the mean ± SEM. One-way ANOVA with Bonferroni’s post hoc test, ***p < 0.001. (c) P14 retinal ChAT immunostaining. (d,e) Schematic depiction of a normal retina in mock- or DENV-2 infected mice (d) or a pathological retina in ZIKV-infected mice (e).

Figure 4

Impaired visual projection was induced by maternal ZIKV infection. (a) Schematic depiction of the anterograde tracing method used. (b) Brain atlases indicate the sites of brain slices in (c), (d), and (e), respectively. (c–e) Results of visual projection in the LGN (c), SC (d), and OX (e), respectively. (f) Mean gray value of visual projection fibers in the DLG, VLGM, SC, and OX. DLG and VLGM: n^Mock = 10, n^ZIKV = 14 samples; SC: n^Mock = 11, n^ZIKV = 10 samples; OX: n^Mock = 11, n^ZIKV = 9 samples from three independent experiments. Data are presented as the mean ± SEM. Student’s t-test, *p < 0.05, ***p < 0.001.

Figure 5

Severe movement defects were induced by maternal ZIKV infection. (a) Representative images of P14 offspring mice. The hind limbs of ZIKV-infected mice were paralyzed. (b) Quantification of the paralysis rate during P0-P28. (c) Representative images of lumbar spinal cords stained for Cas3 and ZIKV. White arrows, cavities. (d) Percentage of Cas3-positive cells within lumbar spinal cords. n^Mock = 4, n^ZIKV = 8, n^DENV-2 = 4 samples from three independent experiments. (e) Representative image of a P14 spinal cord to illustrate the different segments. (f) Nissl staining of P14 cervical, thoracic, and lumbar spinal cord. (g) Representative images of the cerebellum stained for Cas3 and ZIKV. Quantification data are presented as the mean ± SEM. One-way ANOVA with Bonferroni’s post hoc test, **p < 0.01, ***p < 0.001.

Figure 6

Impaired adult neurogenesis was induced by maternal ZIKV infection. (a) Immunofluorescence staining of Ki67 and ZIKV in the SVZ. (b) Immunofluorescence staining of Ki67 and ZIKV in the SGZ of the hippocampus. (c,d) Percentage of Ki67-positive cells in the SVZ (c) and SGZ (d), respectively. n^Mock = 9, n^ZIKV = 8 samples from three independent experiments. Data are presented as the mean ± SEM. Student’s t-test, *p < 0.05, ***p < 0.001.

Figure 7

Schematic summary of offspring pathological phenotypes caused by intrauterine ZIKV inoculation.