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. 2016 Aug:10:71-6.
doi: 10.1016/j.ebiom.2016.07.018. Epub 2016 Jul 16.

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex

Jean-Baptiste Brault  1 , Cécile Khou  2 , Justine Basset  2 , Laure Coquand  1 , Vincent Fraisier  1 , Marie-Pascale Frenkiel  2 , Bruno Goud  1 , Jean-Claude Manuguerra  2 , Nathalie Pardigon  2 , Alexandre D Baffet  3
Affiliations

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex

Jean-Baptiste Brault et al. EBioMedicine. 2016 Aug.

Abstract

The recent Zika outbreak in South America and French Polynesia was associated with an epidemic of microcephaly, a disease characterized by a reduced size of the cerebral cortex. Other members of the Flavivirus genus, including West Nile virus (WNV), can cause encephalitis but were not demonstrated to cause microcephaly. It remains unclear whether Zika virus (ZIKV) and other flaviviruses may infect different cell populations in the developing neocortex and lead to distinct developmental defects. Here, we describe an assay to infect mouse E15 embryonic brain slices with ZIKV, WNV and dengue virus serotype 4 (DENV-4). We show that this tissue is able to support viral replication of ZIKV and WNV, but not DENV-4. Cell fate analysis reveals a remarkable tropism of ZIKV infection for neural stem cells. Closely related WNV displays a very different tropism of infection, with a bias towards neurons. We further show that ZIKV infection, but not WNV infection, impairs cell cycle progression of neural stem cells. Both viruses inhibited apoptosis at early stages of infection. This work establishes a powerful comparative approach to identify ZIKV-specific alterations in the developing neocortex and reveals specific preferential infection of neural stem cells by ZIKV.

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Figures

Fig. 1
Fig. 1
Mouse cortical brain slices sustain ZIKV and WNV, but not DENV4 replication. a. Schematic representation of experimental approach. E15 mouse embryonic brains are sliced and cultured in modified cortical culture medium. Brain slices are then infected for 2 h with ZIKV, WNV and DENV-4 (6.105 FFU), all produced on C6/36 mosquito cells. Viruses are then washed out and brain slices cultivated for 24 to 48 h. b. Maximum likelihood phylogenetic tree inferred for sequences from NS5 genes of Flavivirus genus. The tick-borne encephalitis lineage was used as outgroup to root the tree. Viruses used in this study are underlined, ZIKV Pf13 strain is highlighted in red. c. Viral titration: supernatants were collected from previously infected brain slices and viral titers were quantified by focus forming assay (FFA) in Vero NK cells 24 and 48-hours post-infection. Error bars represent the standard deviations of results from three independent experiments (for each experiment, assays were performed in triplicate). d. Mouse embryonic brain slices infected at E15 with DENV-4, WNV or ZIKV, fixed after 24 h and immun-ostained with pan-flavivirus antibody directed against the E glycoprotein (4G2). VZ: ventricular zone; SVZ: sub-ventricular zone; IZ: intermediate zone; CP: cortical plate. Scale bar: 50 μm. e. High magnification image of a single ZIKV-infected cell in the VZ reveals clear radial glial morphology, with an apical process in contact with the ventricular surface and a basal process that extends all the way to the pial surface. Two viral factories can be observed in the perinuclear region. Scale bar: 5 μm. For each experiment, at least three infections were performed.
Fig. 2
Fig. 2
ZIKV, DENV-4 and WNV tropism of infection in the developing neocortex. a. Mouse embryonic brain slices infected with DENV-4, WNV or ZIKV and stained for RGP cell marker Pax6. Scale bars: 50 μm and 10 μm for insets. b. Mouse embryonic brain slices infected with DENV-4, WNV or ZIKV and stained for neuronal marker NeuN. Scale bars: 50 μm and 10 μm for insets. c. Quantification of the percentage of WNV- and ZIKV-infected cells positive for RGP cell marker Pax6 reveals strong preferential infection of these cells by ZIKV, but not by WNV. All infected cells within the first 50 μm of each brain slice were counted. d. Quantification of the percentage of WNV- and ZIKV-infected cells positive for neuronal marker NeuN reveals preferential infection of these cells by WNV, but not by ZIKV. e. Quantification of the percentage of Pax6 + and NeuN + cells in non-infected E15 brain slices, cultivated for 24 h. For each experiment, at least three infections were performed. Error bar indicate SD. ***p < 0.005 based on a Student's t-test.
Fig. 3
Fig. 3
ZIKV and WNV effect on cell cycle progression and apoptosis. a. Mouse embryonic brain slices infected with WNV, ZIKV or non-infected (NI) and stained for RGP cell marker Pax6 and mitotic marker phospho-Histone 3 (pH3). Yellow arrow indicates mitotic WNV-infected cell. Scale bar: 20 μm. b. Mouse embryonic brain slices infected with WNV, ZIKV or non-infected (NI) and stained for apoptotic marker cleaved caspase-3 (Cas3). Scale bars: 50 μm and 10 μm for insets. c. Quantification of the percentage of mitotic cells (pH3 +) out of total RGP cells (Pax6 +) reveals strong cell cycle progression defects in ZIKV-infected cells but not in WNV-infected cells. d. Quantification of the percentage of apoptotic cells (Cas3 +) out of total cells reveals decreased apoptosis in ZIKV (n = 3655 cells) and WNV-infected cells (n = 6644 cells), as compared to non-infected cells (n = 35,829 cells). For each experiment, at least three infections were performed. Error bar indicate SD. *p < 0.05; ***p < 0.005; ns, not significant, based on a Student's t-test.
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References

    1. Alsaleh K. The E glycoprotein plays an essential role in the high pathogenicity of European-Mediterranean IS98 strain of West Nile virus. Virology. 2016;492:53–65. - PubMed
    1. Baffet A.D. Cellular and subcellular imaging of motor protein-based behavior in embryonic rat brain. Methods Cell Biol. 2016;131:349–363. - PubMed
    1. Barkovich A.J. A developmental and genetic classification for malformations of cortical development: update 2012. Brain. 2012;135(Pt 5):1348–1369. - PMC - PubMed
    1. Calvet G. Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study. Lancet Infect. Dis. 2016 - PubMed
    1. Cao-Lormeau V.-M. Zika virus, French Polynesia, South Pacific, 2013. Emerg. Infect. Dis. 2014;20(6):1085–1086. - PMC - PubMed