Postnatal Zika virus infection leads to morphological and cellular alterations within the neurogenic niche

Abstract

The Zika virus received significant attention in 2016, following a declaration by the World Health Organization of an epidemic in the Americas, in which infections were associated with microcephaly. Indeed, prenatal Zika virus infection is detrimental to fetal neural stem cells and can cause premature cell loss and neurodevelopmental abnormalities in newborn infants, collectively described as congenital Zika syndrome. Contrastingly, much less is known about how neonatal infection affects the development of the newborn nervous system. Here, we investigated the development of the dentate gyrus of wild-type mice following intracranial injection of the virus at birth (postnatal day 0). Through this approach, we found that Zika virus infection affected the development of neurogenic regions within the dentate gyrus and caused reactive gliosis, cell death and a decrease in cell proliferation. Such infection also altered volumetric features of the postnatal dentate gyrus. Thus, we found that Zika virus exposure to newborn mice is detrimental to the subgranular zone of the dentate gyrus. These observations offer insight into the cellular mechanisms that underlie the neurological features of congenital Zika syndrome in children.

Keywords: Brain development; Congenital Zika syndrome; Congenital malformation; Dentate gyrus; Hippocampus; Microcephaly; Neurogenesis; Subgranular zone; Zika virus.

Fig. 1.

Postnatal ZIKV infection leads to morphological alterations in the DG in a murine model. (A) Illustration of the postnatal infection model. Mice were injected intracerebroventricularly at postnatal day (P) 0 with either mock solution or 10⁴ plaque forming units (PFUs) of ZIKV-BR or ZIKV-AF, and samples were collected at P3 and P7. For the animals that had samples harvested at P14, the injection was milder, with only 30 PFU of ZIKV-BR injected. Image created with mindthegraph.com. (B) Weight gain (g) in the first 7 days following 1×10⁴ PFU intracranial injection of ZIKV-BR. n=20 mock-treated and 20 ZIKV-infected animals from six different litters. **P<0.005. (C) Weight gain (g) in the first 14 days post 30 PFU intracranial injection of ZIKV-BR. ****P<0.00005. (D) Viral load measured by plaque brain assay (in PFU/g). n=10 (3 mock-treated animals, 3 animals injected with 10⁴ PFU ZIKV-BR and analyzed at P3, and 4 animals injected with 10⁴ PFU ZIKV-BR and analyzed at P7). (E) Viral load measured by plaque hippocampal assay (in PFU/g). n=14 (5 mock-treated animals, 5 animals injected with 10⁴ PFU ZIKV-BR and analyzed at P7, and 4 animals injected with 30 PFU ZIKV-BR and analyzed at P14). (F,G) Representative images of the forebrain coronal hemisections of mice injected with mock solution or 10⁴ PFU of ZIKV-BR stained with Cresyl Violet at P7 for morphometric analysis. Blue lines show the limits of the forebrain area in the sections (external contours) and the pink lines show the dentate gyrus (DG). Scale bar: 500 μm. (H-K) Quantification of the external contour area and volume (H,J) and quantification of the DG area divided by the external contour area and volume (I,K). n=11 (5 mock-treated and 6 ZIKV-injected animals). *P<0.05; ****P<0.00001. (L-Q) Representative images of coronal sections of the DG at 3, 7 and 14 days post injection with mock solution or 10⁴ PFU of ZIKV-BR stained with DAPI. The white lines show the lengths of the DG blades. Scale bar: 100 μm. (R) Quantification of the lengths of the DG blades. n=30 (15 mock-treated and 15 ZIKV-injected animals). ns, not significant; ****P<0.00001. All data are presented as mean±s.e.m. Unpaired two-tailed Student's t-test was used for statistical analysis.

Fig. 2.

ZIKV infection alters apoptosis within the postnatal hippocampal DG. (A,B) Representative images of immunohistochemistry for activated caspase-3 (CAS3), a marker of apoptosis, in a DG coronal section of the mock and 10⁴ PFU ZIKV-BR groups at P3, counterstained with DAPI. (C) Quantification of CAS3⁺ cell density in the P3 DG. ns, not significant, P>0.05. n=8 (4 mock-treated and 4 ZIKV-injected animals). (D,E) Representative images of immunohistochemistry for CAS3 in a DG coronal section of the mock and 10⁴ PFU ZIKV-BR group at P7, counterstained with DAPI. (F) Quantification of CAS3⁺ cell density in the P7 DG. ****P<0.00001. n=18 (9 mock-treated and 9 ZIKV-injected animals). (G,H) Representative images of immunohistochemistry for CAS3 in a DG coronal section of the mock and 30 PFU ZIKV-BR groups at P14, counterstained with DAPI. (I) Quantification of CAS3⁺ cell density in the P14 DG. **P<0.001. n=6 (3 mock-treated and 3 ZIKV-injected animals). All data are presented as mean±s.e.m. Unpaired two-tailed Student's t-test was used for statistical analysis.

Fig. 3.

ZIKV infection affects cell proliferation within the hippocampal DG. (A,B) Representative images of immunohistochemistry for Ki67, a marker of cycling cells, in a DG coronal section of the mock and 10⁴ PFU ZIKV-BR groups at P3, counterstained with DAPI. (C) Quantification of Ki67⁺ cell density in the P3 DG. ns, not significant, P>0.05. n=8 (4 mock-treated and 4 ZIKV-injected animals). (D,E) Representative images of immunohistochemistry for Ki67 in a DG coronal section of the mock and 10⁴ PFU ZIKV-BR groups at P7, counterstained with DAPI. (F) Quantification of Ki67⁺ cell density in the P7 DG. *P<0.05. n=18 (9 mock-treated and 9 ZIKV-injected animals). (G,H) Representative images of immunohistochemistry for Ki67 in a DG coronal section of the mock and 30 PFU ZIKV-BR groups at P14, counterstained with DAPI. (I) Quantification of Ki67⁺ cell density in the P14 DG. *P<0.05. n=6 (3 mock-treated and 3 ZIKV-injected animals). All data are presented as mean±s.e.m. Unpaired two-tailed Student's t-test was used for statistical analysis.

Fig. 4.

ZIKV disrupts subgranular zone development and the positioning of SOX2⁺ cells. (A-L) Representative images of immunohistochemistry for SOX2⁺ cells, markers of neural progenitors, in hippocampi from mock-treated or ZIKV-BR-injected (10⁴ PFU for analysis at P3 or P7, 30 PFU for analysis at P14) animals at P3 (A-D), P7 (E-H) and P14 (I-L), counterstained with DAPI, reveal an abnormal formation of the subgranular zone. The white lines show the borders of the granule cell layer. (M) Quantification of SOX2⁺ cell density in the P3 DG. n=6 (3 mock-treated and 3 ZIKV-injected animals). (N) Quantification of SOX2⁺ cell density in the P7 DG. n=18 (9 mock-treated and 9 ZIKV-injected animals). All data are presented as mean±s.e.m. Unpaired two-tailed Student's t-test was used for statistical analysis. ns, not significant, P>0.05.

Fig. 5.

ZIKV infection leads to abnormal cellular features within the P7 DG. (A-D) Representative coronal sections of the DG from mock-treated and 10⁴ PFU ZIKV-BR-injected animals showing the distribution of SOX9⁺ cells. (E) Quantification of SOX9⁺ cell density. ns, non-significant, P>0.05. n=12 (6 mock-treated and 6 ZIKV-injected animals). (F-I) Representative coronal sections of the DG from mock-treated and 10⁴ PFU ZIKV-BR-injected animals showing the distribution of IBA1⁺ cells. (J) Quantification of IBA1⁺ cell density. *P<0.05. n=6 (6 mock-treated and 6 ZIKV-injected animals). (K-N) Representative coronal sections of the DG from mock-treated and 10⁴ PFU ZIKV-BR-injected animals showing the distribution of NEUN⁺ cells. (O) Quantification of NEUN⁺ cell density. **P<0.005. n=6 (3 mock-treated and 3 ZIKV-injected animals). (P-S) Representative coronal sections of the DG from mock-treated and 10⁴ PFU ZIKV-BR-injected animals showing the distribution of OLIG2⁺ cells. (T) Quantification of the OLIG2⁺ cell density. ns, not significant, P>0.05. n=12 (6 mock-treated and 6 ZIKV-injected animals). White lines mark the DG limits in the sections. All data are presented as mean±s.e.m. Unpaired two-tailed Student's t-test was used for statistical analysis.

Fig. 6.

ZIKV infection influences cellular composition within the hippocampal fissure region. (A,B) Representative images of immunohistochemistry for ZIKV non-structural protein 1 (NS1) and CAS3 on the coronal section of the hippocampus at P7 from mock-treated and 10⁴ PFU ZIKV-BR-injected animals. (C) Quantification of DAPI⁺ cell density in the hippocampal fissure. ****P<0.00005. n=36 (18 mock-treated and 18 ZIKV-injected animals). (D) Quantification of NEUN⁺, SOX9⁺, OLIG2⁺, IB4⁺ and IBA1⁺ cell density in the hippocampal fissure from mock-treated and 10⁴ PFU ZIKV-BR-injected animals. ns, not significant, P>0.05; *P<0.05; **P<0.005. n=6 (3 mock-treated and 3 ZIKV-injected animals). All data are presented as mean±s.e.m. Unpaired two-tailed Student's t-test was used for statistical analysis. (E-N) High-magnification representative images of the NEUN⁺, SOX9⁺, OLIG2⁺, IB4⁺ and IBA1⁺ staining in the cell clusters at the hippocampal fissure from P7 mock-treated and 10⁴ PFU ZIKV-BR-injected animals. Dashed squares (A,B) represent the regions of the hippocampal fissure that were analyzed.

Fig. 7.

Summary of the effects of neonatal (P0) ZIKV infection on postnatal DG development. Left: illustration of the typical DG, showing the distinct layers and characteristic shape of this structure. Right: representation of the ZIKV-infected DG. Besides being smaller, the DG has a vast disorganization of the subgranular zone and altered shape. Moreover, the cellular composition of the ZIKV-infected DG is altered. There is a reduction in the number of neurons and an increased number of microglial cells and astrocytes. At the hippocampal fissure level, the ZIKV-infected animals show ectopic microglial/neuronal clusters that could be a result of DG cell detachment. GL, granular layer; pRGC, progenitor radial glial cells. Image created with BioRender.com.