Zika virus (ZIKV)-related neuropathology is an important global health concern. Several studies have shown that ZIKV can infect neural stem cells in the developing brain, but infection in the adult brain has not been examined. Two areas in the adult mouse brain contain neural stem cells: the subventricular zone of the anterior forebrain and the subgranular zone of the hippocampus. Here, using 6-week-old mice triply deficient in interferon regulatory factor (IRF) as a model, we show that blood-borne ZIKV administration can lead to pronounced evidence of ZIKV infection in these adult neural stem cells, leading to cell death and reduced proliferation. Our data therefore suggest that adult as well as fetal neural stem cells are vulnerable to ZIKV neuropathology. Thus, although ZIKV is considered a transient infection in adult humans without marked long-term effects, there may in fact be consequences of exposure in the adult brain.

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Tang et al., 2016 Tang H.

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et al. Zika Virus Infection during Pregnancy in Mice Causes Placental Damage and Fetal Demise. The radial-glial-derived cortical neural stem cells (NSCs) in the fetal brain appear to be especially impacted by ZIKV infection, either through greater susceptibility to the viral infection or virus-induced cytotoxicity. This same population is affected by inherited forms of microcephaly, suggesting that loss of these cells is responsible for the microcephaly after ZIKV infection. Indeed recent work demonstrated that ZIKV can infect human cortical NSCs and attenuate their growth and survival, when applied directly to either monolayer culture () or cerebral organoids or neurospheres (). Vertical transmission from ZIKV-infected murine dams to fetuses yielded virus in brain and histopathological evidence of cytotoxicity, supporting direct infection of NSCs. Effects of ZIKV on the placenta and secondary effects on brain may have also contributed ().

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Molnár Z. Neurogenic niches in the brain: help and hindrance of the barrier systems. In the adult brain, neurogenesis contracts after birth to just the anterior subventricular zone (SVZ) of the forebrain and the subgranular zone (SGZ) of the hippocampal dentate gyrus. These restricted niches contain progenitor cells that divide to produce neurons or glia, depending upon intrinsic and environmental cues. Neurogenic niches are characterized by a comparatively high vascular density and proximity to cerebrospinal fluid (CSF) (), allowing not just communication through signaling molecules but also proximity to circulating viruses.

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Figure 1 Systemic ZIKV Can Infect Neural Progenitor Cells in the Adult Brain Show full caption (A) Schematic of stem cell niches in adult mouse brain. Neural progenitor cells (NPCs) located in the subventricular zone (SVZ) in the anterior forebrain (red) adjacent to the cerebral ventricles contribute new neurons to the olfactory bulb. NPCs in the subgranular zone (SGZ) in the dorsal forebrain (red) contribute new neurons to the hippocampal dentate gyrus. LV, lateral ventricle; OB, olfactory bulb; Hipp, hippocampus. (B and C) Adult TKO mice were infected with ZIKV and then sacrificed, their brains were serially sectioned, and they were immunostained for ZIKV envelope protein (green). Evidence of ZIKV was found in the SVZ and SGZ, with less expression elsewhere in adult brain. High-power inset below with arrows (yellow) highlights immunoreactive cells. LV, lateral ventricle. Scale bar, 100 μm. (D) Marker expression during neural stem cell (NSC) differentiation from radial glia to mature neuron. (E–J) Confocal images and orthogonal projection of SVZ and SGZ regions co-stained for GFAP, Nestin, SOX2, and DCX with ZIKV envelop protein, evidencing ZIKV in NPCs and immature neuron populations. White circle outlines: infected cells. LV, lateral ventricle; SVZ, subventricular zone; DG, dentate gyrus. Scale bar, 10 μm. To examine the potential for virus infection in the brain, we screened serial coronal sections of whole brain from infected and mock-treated mice with the monoclonal 4G2 antibody that reacts with the flavivirus-specific family envelope protein. We observed dramatic immunoreactivity in proximity to the SVZ of the anterior forebrain, as well as the SGZ of the hippocampus ( Figures 1 A–1C), the two regions in mouse that maintain stem cell populations throughout adulthood, in infected (but not mock-infected) mice. In contrast there was less immunoreactivity in other regions of the brain under these conditions ( Figures S1 A–S1C), suggesting a particular tropism of the virus for proliferative regions of the brain. Quantification across major brain regions showed statistically significant selective vulnerability to these proliferative zones ( Figure S1 D).

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et al. Zika Virus Infection during Pregnancy in Mice Causes Placental Damage and Fetal Demise. Figure 2 ZIKV-Infected Adult NPCs Undergo Cell Death and Show Reduced Proliferation Show full caption (A and C) Representative images of SVZ (A) or SGZ (C) region from ZIKV-infected brains showing more CASP3+ cells (arrows) compared to regions in mock-infected brains. Scale bar, 100 μm. (B and D) Quantification of the number of CASP3+ cells relative to mock-infected brains. All data represent means ± SEM, n = 3 animals for each group. Student’s t test, ∗p < 0.05, ∗∗p < 0.01. (E) Adult TKO mice were infected with ZIKV or mock, injected with EdU after 6 days, then sacrificed after 2 hr. ZIKV-infected animals show reduced incorporation of EdU in both SVZ and SGZ, indicating reduced entry into S-phase. Scale bar, 100 μm. (F) Reduced Ki67 staining in ZIKV-infected mice in both SVZ and SGZ, indicating reduced cell proliferation. Scale bar, 100 μm. (G) Reduced phospho-histone H3 (pH3) staining in ZIKV-infected mice in both SVZ and SGZ, indicating reduced mitotic cells. Scale bar, 100 μm. (H–J) Stereological quantification of the number of EdU+, Ki67+, and pH3+ cells in each mouse was performed in every sixth section of the entire brains from three pairs of ZIKV and mock-infected animals. All data represent means ± SEM, n = 3 animals for each group. Student’s t test, ∗p < 0.05, ∗∗p < 0.01. ZIKV infection can lead to caspase-3 (CASP3) activation in both NPCs differentiated from human ESCs/IPSCs and embryonic mouse brain (). In order to determine whether systemic ZIKV infection can induce cell death in adult NPC populations, we stained for cleaved (i.e., activated) CASP3 in NPC niches. Mock-infected TKO mice showed scant evidence of CASP3+ cells. In contrast abundant CASP3+ cells were detected in ZIKV-infected brains within these neurogenic niches ( Figures 2 A–2D), and were also usually positive for Nestin. Similarly, the ZIKV staining colocalized with CASP3 in NPCs in the SVZ and SGZ ( Figures S2 I and S2J), suggesting that ZIKV infection can induce apoptotic cell death in adult NPCs in these regions.

We assessed the impact of systemic ZIKV infection on cell proliferation in niches in adult brain using the thymidine analog EdU and a series of cell-cycle markers. We performed EdU pulse-labeling in TKO mice 6 days after ZIKV or mock infection. Quantitative analysis at 2 hr after EdU injection showed that, in both neurogenic regions, although the brain size and volume of the SGZ and SVZ was not notably different ( Figure S2 H), ZIKV-infected mice had approximately a 4- to 5-fold reduction in the number of EdU+ cells per section, compared with mock ( Figures 2 E and 2H).

Consistent with the EdU-labeling results, there were many fewer cells positive for the proliferation marker Ki67 in the SVZ and SGZ, which was reduced by approximately 2- to 3-fold ( Figures 2 F and 2I). Similarly, there were many fewer cells positive for the mitotic marker phospho-Histone H3 in the SVZ and SGZ, reduced by approximately 2- to 4-fold ( Figures 2 G and 2J). Results were statistically significant and consistent across the three infected and three mock-infected animals. These results indicate that ZIKV infection leads to decreased NPC proliferation in the adult SGZ and SVZ.

Here we demonstrate that ZIKV exposure in adult mice shows infection of brain with a predilection for neurogenic niches and is associated with cellular apoptosis and reduction of cellular proliferation. Based upon the presence of the ZIKV antigen following exposure, we conclude that the virus was able to infect SVZ and SGZ niche cells to a much greater degree than non-neurogenic regions. This infection correlated with evidence of apoptosis and reduced numbers of cells evidencing DNA synthesis or proliferation. However, the relative contribution of these features, as well as the long-term effects, on the NPC niches remains unknown. Our results suggest that ZIKV infection can enter the adult brain and lead to neuropathology in mammals.

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Rhodes J.S. Neurogenesis, inflammation and behavior. The degree to which IFN-deficient mice model the extent and severity of flavivirus infection in humans is unknown. We recognize that healthy humans may be able to mount an effective antiviral response and prevent entry into the CNS, but it remains a possibility that some immunocompromised humans and even some apparently healthy humans may be susceptible in ways modeled by the TKO mice. It will be important to determine the extent of involvement of stem cell niches with less immunocompromised strains of mice. Brain inflammation in general, including IFN-α induction, can lead to reduction in adult neurogenesis (), and therefore the interaction between ZIKV infection and IFN signaling pathway and its impact on adult neurogenesis merit further investigation.

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et al. Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth. Neurotropic viruses can gain entry into the CNS and cause disease through different means (), but how ZIKV gains entry into the brain remains unknown. Current models include entry of the virus directly across the blood-brain barrier (BBB), across synapses from peripheral nerves, or through entry of infected microglia. Once across the BBB, the means of entry into NPCs may be through specific transmembrane receptors, such as the candidate AXL receptor (). The means by which infection leads to NPC death is also under active study. We hypothesize that the particular cell death in NPCs may be p53 mediated, in keeping with current models of human microcephaly ().