. 2016 Jun 9;534(7606):267-71.

doi: 10.1038/nature18296. Epub 2016 May 11.

The Brazilian Zika virus strain causes birth defects in experimental models

Fernanda R Cugola¹, Isabella R Fernandes^{1

2}, Fabiele B Russo^{1

3}, Beatriz C Freitas², João L M Dias¹, Katia P Guimarães¹, Cecília Benazzato¹, Nathalia Almeida¹, Graciela C Pignatari^{1

3}, Sarah Romero², Carolina M Polonio⁴, Isabela Cunha⁴, Carla L Freitas⁴, Wesley N Brandão⁴, Cristiano Rossato⁴, David G Andrade⁴, Daniele de P Faria⁵, Alexandre T Garcez⁵, Carlos A Buchpigel⁵, Carla T Braconi⁶, Erica Mendes⁶, Amadou A Sall⁷, Paolo M de A Zanotto⁶, Jean Pierre S Peron⁴, Alysson R Muotri², Patricia C B Beltrão-Braga^{1

8}

Affiliations

V体育平台登录 - Affiliations

¹ University of São Paulo, Department of Surgery, Stem Cell Laboratory, São Paulo, São Paulo 05508-270, Brazil.
² University of California San Diego, School of Medicine, Department of Pediatrics/Rady Children's Hospital San Diego, Department of Cellular &Molecular Medicine, Stem Cell Program, La Jolla, California 92037-0695, USA.
³ Tismoo, The Biotech Company, São Paulo, São Paulo 01401-000, Brazil.
⁴ University of São Paulo, Department of Immunology, Neuroimmune Interactions Laboratory, São Paulo, São Paulo 05508-000, Brazil.
⁵ University of São Paulo, Department of Radiology and Oncology, USP School of Medicine, São Paulo, São Paulo 05403-010, Brazil.
⁶ University of São Paulo, Department of Microbiology, Institute of Microbiology Sciences, Laboratory of Molecular Evolution and Bioinformatics, São Paulo, São Paulo 05508-000, Brazil.
⁷ Institute Pasteur in Dakar, Dakar 220, Sénégal.
⁸ University of São Paulo, School of Arts, Sciences and Humanities, Department of Obstetrics, São Paulo, São Paulo 03828-000, Brazil.

PMID: 27279226
PMCID: PMC4902174 (VSports)
DOI: VSports app下载 - 10.1038/nature18296

The Brazilian Zika virus strain causes birth defects in experimental models

Fernanda R Cugola et al. Nature. 2016.

. 2016 Jun 9;534(7606):267-71.

doi: 10.1038/nature18296. Epub 2016 May 11.

Authors

Affiliations

¹ University of São Paulo, Department of Surgery, Stem Cell Laboratory, São Paulo, São Paulo 05508-270, Brazil.
² University of California San Diego, School of Medicine, Department of Pediatrics/Rady Children's Hospital San Diego, Department of Cellular &Molecular Medicine, Stem Cell Program, La Jolla, California 92037-0695, USA.
³ Tismoo, The Biotech Company, São Paulo, São Paulo 01401-000, Brazil.
⁴ University of São Paulo, Department of Immunology, Neuroimmune Interactions Laboratory, São Paulo, São Paulo 05508-000, Brazil.
⁵ University of São Paulo, Department of Radiology and Oncology, USP School of Medicine, São Paulo, São Paulo 05403-010, Brazil.
⁶ University of São Paulo, Department of Microbiology, Institute of Microbiology Sciences, Laboratory of Molecular Evolution and Bioinformatics, São Paulo, São Paulo 05508-000, Brazil.
⁷ Institute Pasteur in Dakar, Dakar 220, Sénégal.
⁸ University of São Paulo, School of Arts, Sciences and Humanities, Department of Obstetrics, São Paulo, São Paulo 03828-000, Brazil.

PMID: 27279226
PMCID: PMC4902174
DOI: 10.1038/nature18296

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 VSports手机版. Our model can be used to determine the efficiency of therapeutic approaches to counteracting the harmful impact of ZIKV(BR) in human neurodevelopment. .

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The authors declare no competing financial interests.

Figures

**Extended Data Figure 1. Impact of ZIKV^BR 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 ZIKV^BR 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 ZIKV^BR-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, ZIKV^BR 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 ZIKV^BR-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. Histopathological analysis of brains from ZIKV^BR-infected SJL pups**
Morphological aspect of hippocampus, thalamus, hypothalamus and cerebellum from brains of pups born from mothers infected with the ZIKV^BR. 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. 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 ZIKV^BR over time. b, Infection of NPCs with the ZIKV^BR and ZIKV^AF (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. 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. Human and chimp cerebral organoids infected with ZIKV**
a, Representative image of an entire cross-section of a cerebral human organoid infected with the ZIKV^BR (MOI = 0.1, 24 hours p.i.). Scale bar = 200 μm. b, Detail of the surface of a human organoid infected with the ZIKV^BR 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 ZIKV^BR 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 ZIKV^BR 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. ZIKV^BR 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, ZIKV^BR 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, ZIKV^BR-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, ZIKV^BR-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 ZIKV^BR-infected pups. h, Cell death gene expression signature in the brains of ZIKV^BR-infected pups (n = 2 mice per group; threshold = two-fold).

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 ZIKV^BR 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 ZIKV^BR 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 ZIKV^BR (MOI = 10) at 24 hours p.i. Scale bar = 25 μm. e, ZIKV^BR replication dynamics in NPCs (MOI = 10) (n = 2 technical replicates from RNA of two different donors). f, ZIKV^BR 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 ZIKV^BR (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. 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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Comment in

Viral Pathogenesis: Tracing the steps of Zika virus. (VSports手机版)
Hofer U. Hofer U. Nat Rev Microbiol. 2016 Jul;14(7):401. doi: 10.1038/nrmicro.2016.80. Epub 2016 May 23. Nat Rev Microbiol. 2016. PMID: 27211788 No abstract available.
Modeling Zika Virus Infection in Mice.
Rossi SL, Vasilakis N. Rossi SL, et al. Cell Stem Cell. 2016 Jul 7;19(1):4-6. doi: 10.1016/j.stem.2016.06.009. Cell Stem Cell. 2016. PMID: 27392219
Zika-related microcephaly in experimental models.
Peron JPS, Braga PCBB. Peron JPS, et al. Temperature (Austin). 2016 Aug 5;4(1):13-14. doi: 10.1080/23328940.2016.1208319. eCollection 2017. Temperature (Austin). 2016. PMID: 28417097 Free PMC article. No abstract available.

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1. Faria NR, et al. Zika virus in the Americas: Early epidemiological and genetic findings. Science. 2016 doi: 10.1126/science.aaf5036. - DOI (VSports) - PMC - PubMed
1. de Fatima Vasco Aragao M, et al. Clinical features and neuroimaging (CT and MRI) findings in presumed Zika virus related congenital infection and microcephaly: retrospective case series study. BMJ. 2016;353:i1901. doi: 10.1136/bmj.i1901. - "V体育安卓版" DOI - PMC - PubMed
1. Cao-Lormeau VM, et al. Guillain-Barre Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet. 2016 doi: 10.1016/S0140-6736(16)00562-6. - DOI - PMC - PubMed

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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

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