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, 534 (7606), 267-71

The Brazilian Zika Virus Strain Causes Birth Defects in Experimental Models

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The Brazilian Zika Virus Strain Causes Birth Defects in Experimental Models

Fernanda R Cugola et al. Nature.

Abstract

Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (family Flaviviridae) and was first described in 1947 in Uganda following blood analyses of sentinel Rhesus monkeys. Until the twentieth century, the African and Asian lineages of the virus did not cause meaningful infections in humans. However, in 2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy epidemic on the Yap Island in Micronesia. Patients experienced fever, skin rash, arthralgia and conjunctivitis. From 2013 to 2015, the Asian lineage of the virus caused further massive outbreaks in New Caledonia and French Polynesia. In 2013, ZIKV reached Brazil, later spreading to other countries in South and Central America. In Brazil, the virus has been linked to congenital malformations, including microcephaly and other severe neurological diseases, such as Guillain-Barré syndrome. Despite clinical evidence, direct experimental proof showing that the Brazilian ZIKV (ZIKV(BR)) strain causes birth defects remains absent. Here we demonstrate that ZIKV(BR) infects fetuses, causing intrauterine growth restriction, including signs of microcephaly, in mice. Moreover, the virus infects human cortical progenitor cells, leading to an increase in cell death. We also report that the infection of human brain organoids results in a reduction of proliferative zones and disrupted cortical layers. These results indicate that ZIKV(BR) crosses the placenta and causes microcephaly by targeting cortical progenitor cells, inducing cell death by apoptosis and autophagy, and impairing neurodevelopment. Our data reinforce the growing body of evidence linking the ZIKV(BR) outbreak to the alarming number of cases of congenital brain malformations. Our model can be used to determine the efficiency of therapeutic approaches to counteracting the harmful impact of ZIKV(BR) in human neurodevelopment.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. Impact of ZIKVBR infection in the C57BL/6 and SJL mice
a, Scheme for infecting mice and the follow up analyses. Pregnant females at approximately day 10–13 of gestation were challenged with 4 x 1010 PFUs of ZIKVBR via an intra-venous route. Their pups were analyzed immediately after birth for signs of malformation. b, A representative pup from mock-infected and the ZIKVBR-infected C57BL/6 mice. Scale bar = 1 cm. c, C57BL/6 pups born with no gross morphological changes or size differences compared to mock controls (n = 21 pups from three separate litters, error bars, s.e.m, t-test). Scale bar = 1 cm. d, e, CT analysis confirmed lack of anatomical alterations (n = 21 pups from three separate litters, error bars, s.e.m, t-test). f, ZIKVBR RNA was not detected in the brains of six C57BL/6 pups. g, Cell death pathway signature revealed by qPCR gene expression in the brains of the ZIKVBR-infected SJL pups (n = 2 technical replicates of pooled RNA from two pups each group; threshold = two-fold). h, Heatmap representation of misregulated genes in “g”.
Extended Data Figure 2
Extended Data Figure 2. Histopathological analysis of brains from ZIKVBR-infected SJL pups
Morphological aspect of hippocampus, thalamus, hypothalamus and cerebellum from brains of pups born from mothers infected with the ZIKVBR. Arrowheads indicate intranuclear vacuoles and “empty” nuclei aspect with chromatin margination observed in thalamus and hypothalamus. Scale bar from left to right = 100 μm, 100 μm, 50 μm and 10 μm.
Extended Data Figure 3
Extended Data Figure 3. Impact of ZIKV infection in human NPCs and neurons
a, Scheme of the in vitro experiments using hPSCs. The cells were differentiated into NPCs, neurons, neurospheres and cerebral organoids to test the impact of ZIKVBR over time. b, Infection of NPCs with the ZIKVBR and ZIKVAF (MOI = 1) at 24 and 96 hours p.i. Scale bar = 25 μm. c, Aspects of iPSC-derived human neurons after ZIKV infection (MOI = 1) at 24 and 96 hours p.i. Scale bar = 200 μm (Bright field) and scale bar = 25 μm (immunofluorescence). d, Viral replication dynamics in human NPCs over time (MOI = 1) (n = 2 technical replicates from two different donors; error bars, s.e.m). e, Viral replication dynamics in human neurons over time (MOI = 1) (n = 2 technical replicates from two different donors; error bars, s.e.m). f, Dynamics of NPCs toxicity over time after ZIKV infection (MOI = 1), indicating no significant differences among the different viruses (n = 2 technical replicates from two different donors; error bars, s.e.m). g, h, Viral replication dynamics of ZIKV in human neurons over time at MOI = 10 and MOI = 1, respectively (n = 2 technical replicates from two different donors; error bars, s.e.m; one-way ANOVA).
Extended Data Figure 4
Extended Data Figure 4. Impact of ZIKV infection in human neurospheres
a, Representative bright-field images of ZIKV infection (MOI = 1) at 24 and 96 hours p.i. Scale bar = 400 μm. b, Alterations in neurospheres diameter over time (MOI = 1) (n = 22 neurospheres from two different donors for each time-point in each condition; unpaired t-test, ****P < 0.0001). c, ZIKV replication dynamics in neurospheres (MOI = 1) (n = 3 technical replicates from two different donors). d, Representative bright-field images of ZIKV infection (MOI = 1) at 96 hours p.i. Scale bar = 400 μm and 1000 μm (Mock). The dotted circle describes the neurospheres borders indicating the immunostained regions in e and f. e, Immunostaining of neurospheres infected with ZIKV at 96 hours p.i. at MOI = 1 (left panel) or MOI = 10 (right panel), revealing a qualitative reduction of proliferative cell migration from Mushashi (Mus)-positive cells. Scale bar = 50 μm.
Extended Data Figure 5
Extended Data Figure 5. Human and chimp cerebral organoids infected with ZIKV
a, Representative image of an entire cross-section of a cerebral human organoid infected with the ZIKVBR (MOI = 0.1, 24 hours p.i.). Scale bar = 200 μm. b, Detail of the surface of a human organoid infected with the ZIKVBR at 24 hours p.i. (MOI = 0.1). Marginal zone (MZ) and cortical plate (CP) delineated by doted white lines. Scale bar = 200 μm. c, Detail of the surface of a human organoid infected with the ZIKVBR at 96 hours p.i. (MOI = 0.1). Notice the significant tissue damage and reduction in the CP related to 24 hours p.i. Scale bar = 200 μm. d, A representative characterization of CP and ventricular zone (VZ) in human organoid infected with the ZIKVBR at 24 and 96 hours p.i. (MOI = 0.1). Scale bar = 50 μm. e, Reduction in the cortical thickness measured by the extension of TBR1-positive layer of cells in human organoids at 96 hours p.i. (MOI = 0.1). (n = 3 replicates from three human cell lines; error bars, s.e.m; t-test, *P = 0.0203). f, Reduction in the cortical thickness measured by the extension of CTIP2-positive cells layer in human organoids at 96 hours p.i. (MOI = 0.1). (n = 3 replicates from three human cell lines; error bars, s.e.m; t-test, ***P = 0.001). g, Nuclear size (diameter) of cleaved caspase-3 positive apoptotic cells in human organoids at 96 hours p.i. (MOI = 0.1). (n = 10 organoids/slides from three human cell lines; error bars, s.e.m; t-test, ***P = 0.0004). Scale bar = 50 μm. h, Percentage of TUNEL-positive cells in relation to controls (doted line) at 24 and 96 hours p.i. (MOI = 0.1). (n = 10 organoids/slides from three human cell lines; error bars, s.e.m; ANOVA, **P = 0.0042). i, Percentage of TBR1-positive cells in non-primate organoids (Chimp) in relation to controls (doted line) at 24 and 96 hours p.i. (MOI = 0.1) (n = 3 organoids from two donors, error bars, s.e.m; ANOVA). j, Percentage of CTIP2-positive cells in non-primate organoids (Chimp) in relation to controls (doted line) at 24 and 96 hours p.i. (MOI = 0.1) (n = 3 organoids from two donors; error bars, s.e.m; ANOVA). k, Viral replication dynamics in chimpanzee organoids over time (MOI = 0.1) (n = 3 replicates from two donors; error bars, s.e.m; ANOVA).
Figure 1
Figure 1. ZIKVBR infection in SJL mice
a, SJL pups born with IUGR. Scale bar = 1 cm. b, Total body weight, crowm-rump and skull measurements in pups born from infected animals (n = 6 pups, comprising 3 mice from 2 separate litters; error bars, s.e.m; t-test, **P < 0.01). c, ZIKVBR RNA detected in SJL pup tissues (n = 6 pups, comprising 3 mice from 2 separate litters; error bars, s.e.m; t-test). d, Histopathological aspect of the cortical organization (brackets) in infected brains, including intranuclear vacuoles, and “empty” nuclei aspect with chromatin margination in neurons (arrows). Scale bar = 100 μm (left panels), 50 μm (middle panels) and 10 μm (right panels). e, ZIKVBR-infected brains displayed a reduced cortical layer thickness (n = 6 pups, comprising 3 mice from 2 separate litters; error bars, s.e.m; t-test, ***P < 0.001). Infected brains have fewer cells/layer (n = 6 pups, comprising 3 mice from 2 separate litters; error bars, s.e.m; t-test, **P < 0.1). f, ZIKVBR-infected cortical neurons have pronounced nuclei (diameter) (cortical n = 31; deep cortical n = 21 and medulla n = 41 nuclei; error bars, s.e.m; two-way ANOVA, ****P < 0.001). g, Ocular malformations (arrow) in the ZIKVBR-infected pups. h, Cell death gene expression signature in the brains of ZIKVBR-infected pups (n = 2 mice per group; threshold = two-fold).
Figure 2
Figure 2. ZIKV infection in vitro
a, Relative expression of TAM receptors (n = 2 technical replicates from two pooled different donors, error bars, s.e.m; t-test; ***P < 0,01). b, Expression of TAM receptors in NPCs after ZIKVBR infection (MOI = 10) at 48 hours p.i. (n = 2 technical replicates from two pooled different donors, error bars, s.e.m). c, TEM detection of ZIKVBR viral particles 24 hours p.i. at MOI = 10 (red arrowheads) inside NPCs (top panels) and neurons (bottom panels). Viral factories (yellow arrowhead). Immature viral particles (white arrowheads). Scale bars = 0.5 μm/40.000× (top left); 200nm/80.000× (top right); 0.2 μm/50.000× (bottom left); 50 nm/3000.000× (bottom right). d, Immunofluorescence revealed susceptibility to infection in NPC and neurons with the ZIKVBR (MOI = 10) at 24 hours p.i. Scale bar = 25 μm. e, ZIKVBR replication dynamics in NPCs (MOI = 10) (n = 2 technical replicates from RNA of two different donors). f, ZIKVBR replication dynamics in neurons (MOI = 10) (n = 2 technical replicates from RNA of two different donors). g, NPC death measured by FAC with two different cell gating sizes (P1 and P2). Apoptosis (left panel), necrosis (middle panel) and late apoptosis (right panel) (MOI = 10), 48 hours p.i. (n = 2 technical replicates from two different donors; error bars, s.e.m; two-way ANOVA, *P < 0.5). h, Representative images of human neurospheres infected with ZIKVBR (MOI = 10; 96 hours p.i.). Scale bar = 200 μm. i, Alterations in neurosphere diameter over time (MOI = 10) (n = 25 neurospheres from two different donors for each time point; unpaired t-test, ****P < 0.0001). j, ZIKV replication dynamics in neurospheres (MOI = 10) (n = 2 technical replicates from two different donors).
Figure 3
Figure 3. Cortical alterations in human brain organoids infected with ZIKV
a, Representative image of a human cerebral organoid showing the marginal zone (MZ), cortical plate (CP) and ventricular zone (VZ), delineated by doted white lines. Scale bar = 200 μm. b, Representative images of the CP stained for CTIP2, TBR1, MAP2 or TUJ1 (neurons). Scale bar = 50 μm. c, Representative images of the proliferative regions in the VZ stained for Ki67, PAX6, and cleaved caspase-3 (Cas3). Scale bar = 50 μm. d, Percentage of TBR1-positive cells in relation to mock-infected controls (doted line) (MOI = 0.1) (n = 3 replicates from three human cell lines; error bars, s.e.m; ANOVA, **P = 0.0025). e, Percentage of CTIP2-positive cells (MOI = 0.1) (n = 3 replicates from three human cell lines; ****P< 0.001 and *P = 0.0430). f, Percentage of PAX6-positive cells (MOI = 0.1) (n = 3 replicates from three human cell lines; error bars, s.e.m; ANOVA, *P = 0.0221; ). g, Percentage of Ki67-positive cells (MOI = 0.1) (n = 3 replicates from three human cell lines; error bars, s.e.m; ANOVA, ****P< 0.001). h, Percentage of Sox2-positive cells (MOI = 0.1) (n = 3 replicates from three human cell lines; error bars, s.e.m; ANOVA, ****P = 0.003). i, Percentage of cleaved-caspase3-positive cells (Cas3) (MOI = 0.1) (n = 3 replicates from three human cell lines; error bars, s.e.m; ANOVA, ****P = 0.002).

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