Este trabalho foi financiado por fundos do S. John Dunn Foundation e do Instituto para infecções humanas e imunidade da University of Texas Medical Branch (PW).

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	<li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> All countries and territories with active Zika virus transmisson. , (2016).</li><li>Brasil, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Infection+in+Pregnant+Women+in+Rio+de+Janeiro.">Zika Virus Infection in Pregnant Women in Rio de Janeiro.</a> N Engl J Med. 375 (24), 2321-2334 (2016).</li><li>Hills, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+Zika+Virus+Through+Sexual+Contact+with+Travelers+to+Areas+of+Ongoing+Transmission+-+Continental+United+States,+2016.">Transmission of Zika Virus Through Sexual Contact with Travelers to Areas of Ongoing Transmission - Continental United States, 2016.</a> MMWR Morb Mortal Wkly Rep. 65 (8), 215-216 (2016).</li><li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Facts about microcephaly. , (2016).</li><li>Garcez, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus+impairs+growth+in+human+neurospheres+and+brain+organoids.">Zika virus impairs growth in human neurospheres and brain organoids.</a> Science. 352 (6287), 816-818 (2016).</li><li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Disrupts+Neural+Progenitor+Development+and+Leads+to+Microcephaly+in+Mice.">Zika Virus Disrupts Neural Progenitor Development and Leads to Microcephaly in Mice.</a> Cell Stem Cell. 19 (5), 672 (2016).</li><li>Tang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Infects+Human+Cortical+Neural+Progenitors+and+Attenuates+Their+Growth.">Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth.</a> Cell Stem Cell. 18 (5), 587-590 (2016).</li><li>Wu, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertical+transmission+of+Zika+virus+targeting+the+radial+glial+cells+affects+cortex+development+of+offspring+mice.">Vertical transmission of Zika virus targeting the radial glial cells affects cortex development of offspring mice.</a> Cell Res. 26 (6), 645-654 (2016).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li><li>Wu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Region-specific+generation+of+cholinergic+neurons+from+fetal+human+neural+stem+cells+grafted+in+adult+rat.">Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat.</a> Nat Neurosci. 5 (12), 1271-1278 (2002).</li><li>McGrath, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Responses+of+Human+Fetal+Brain+Neural+Stem+Cells+to+Zika+Virus+Infection.">Differential Responses of Human Fetal Brain Neural Stem Cells to Zika Virus Infection.</a> Stem Cell Reports. 8 (3), 715-727 (2017).</li><li>Svendsen, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+rapid+and+long+term+growth+of+human+neural+precursor+cells.">A new method for the rapid and long term growth of human neural precursor cells.</a> J Neurosci Methods. 85 (2), 141-152 (1998).</li><li>Tarasenko, Y. I., Yu, Y., Jordan, P. M., Bottenstein, J., Wu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+growth+factors+on+proliferation+and+phenotypic+differentiation+of+human+fetal+neural+stem+cells.">Effect of growth factors on proliferation and phenotypic differentiation of human fetal neural stem cells.</a> J Neurosci Res. 78 (5), 625-636 (2004).</li><li>Barrows, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Screen+of+FDA-Approved+Drugs+for+Inhibitors+of+Zika+Virus+Infection.">A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection.</a> Cell Host Microbe. 20 (2), 259-270 (2016).</li><li>Jakel, R. J., Schneider, B. L., Svendsen, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+human+neural+stem+cells+to+model+neurological+disease.">Using human neural stem cells to model neurological disease.</a> Nat Rev Genet. 5 (2), 136-144 (2004).</li><li>Lopez-Garcia, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+stretch-induced+neurotrauma+model+for+medium-throughput+screening+in+vitro:+identification+of+rifampicin+as+a+neuroprotectant.">Development of a stretch-induced neurotrauma model for medium-throughput screening in vitro: identification of rifampicin as a neuroprotectant.</a> Br J Pharmacol. , (2016).</li><li>Mich, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+functionally+distinct+stem+cells+and+neurosphere-initiating+cells+in+adult+mouse+forebrain.">Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain.</a> Elife. 3, e02669 (2014).</li></ol>

Os autores têm sem conflitos de interesse para declarar.

A capacidade de cultura e manipular hNSCs fornece uma ferramenta crítica que pode ser usada para uma variedade de finalidades da modelagem de doenças humanas para10,11,12,14, de despistagem de drogas de alto rendimento 15,16,17. Muitas perguntas continuaram a ser abordadas como cérebro f...

Vírus Zika (ZIKV) é um flavivirus transmitido sexualmente ou por mosquitos vetores Aedes aegypti e Aedes albopictus mosquitos. ZIKV foi recentemente identificada como uma ameaça grave de saúde pública devido à sua facilidade de transmissão e sintomas neurológicos1de afiliados. Um dos mais relativas aos efeitos neurológicos é o desenvolvimento de microcefalia em fetos nascidos de mães grávidas infectadas2,3. A microcefalia é um distúrbio neurológico, onde a cabeça é menor do que o tamanho típico durante o desenvolvimento fetal e ao nascimento, com a circunferência menor que 2 desvios-padrão abaixo da média4. A menor circunferência da cabeça é geralmente acompanhada por uma variedade de comorbidades tais como atrasos no desenvolvimento, convulsões, visão e perda de audição e dificuldade de alimentação.
Estudos recentes têm utilizado modelos animais ou induzida por células-tronco pluripotentes para estudar o efeito da infecção ZIKV em neurodesenvolvimento5,6,7,8. Enquanto estes estudos têm contribuído para o nosso conhecimento de ZIKV, o uso de diferentes espécies ou células geneticamente modificadas podem ser demorado e/ou adicionar variáveis adicionais que podem confundir o efeito da ZIKV no desenvolvimento neural células5, 6 , 7 , 8. no entanto, a dificuldade com a cultura do hNSC, particularmente a cultura de não-aderente neurosphere descrita neste protocolo, é que a cultura é muito sensível aos métodos utilizados para realizar a cultura9. Qualquer alteração em componentes médios, ou mesmo a manipulação física do navio cultura, é suficiente para provocar uma reação de9células. Para solucionar esses problemas, nós desenvolvemos uma cultura humana fetal derivado do cérebro neural células-tronco (hNSCs) em vitro para interrogar mecanicamente o efeito da ZIKV em células-tronco neurais fetais. Usando nosso método, hNSCs foram mantidos por mais de 80 passagens sem alterações fenotípicas aparente10. Além disso, alterações cromossômicas, como trissomia eram um none ou mínimo11. Esta cultura hNSC cresce como uma cultura de não-aderente neurosphere. Uma vantagem do neurospheres é que as esferas criam um nicho ambiental na cultura que é mais reflexiva de nichos na vivo em comparação com as culturas bidimensional9. Outra vantagem deste protocolo é que vários tipos de células podem ser derivados da cultura hNSC, permitindo que um investigador observar o impacto de uma determinada variável na sobrevivência do hNSC e diferenciação. Este protocolo é aplicável a indivíduos que olham para responder perguntas mecanicistas sobre o desenvolvimento do sistema nervoso central ou disfunção. O protocolo seguinte descreve como expandir uma cultura hNSC para infectar com ZIKV e posteriormente, diferenciar o hNSCs para observar o impacto da infecção sobre o processo de diferenciação. Ele também inclui métodos para armazenar hNSCs para o uso a longo prazo e hNSCs se diferenciar em vários tipos de neurônios que permitam a investigação dos défices induzida por ZIKV contribuindo para de malformação do cérebro11. Acreditamos que este protocolo também é de interesse para os investigadores procuram entender o impacto de qualquer estímulo ambiental, tais como infecção ou toxinas na sobrevivência de células-tronco neurais e diferenciação.

<table><tbody><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>F12</td><td>Gibco</td><td>11765-054</td><td></td></tr><tr><td>Glucose (10%)</td><td>Sigma</td><td>G8644</td><td></td></tr><tr><td>HEPES (1M)</td><td>Corning/cellgro</td><td>25-060-c1</td><td></td></tr><tr><td>Pen Strep (100x)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I4011</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Transferrin</td><td>Sigma</td><td>T2036</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma</td><td>S5261</td><td></td></tr><tr><td>Heparin Sigma</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>basic fibroblast growth factor</td><td>R&amp;amp;D system</td><td>233-FB</td><td></td></tr><tr><td>epidermal growth factor</td><td>R&amp;amp;D system</td><td>236-EG</td><td></td></tr><tr><td>leukemia inhibitory factor</td><td>R&amp;amp;D system</td><td>7734-LF</td><td></td></tr><tr><td>Laminin</td><td>Invitrogen</td><td>23017-015</td><td></td></tr><tr><td>2.5% trypsin</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>Trypsin inhibitor</td><td>Sigma</td><td>T6522</td><td></td></tr><tr><td>Poly-D-Lysine hydrobromide(PDL)</td><td>Sigma</td><td>P6407-5MG</td><td></td></tr><tr><td>B-27 supplement</td><td>Gibco/Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650-100ml</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline</td><td>Corning/cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A4378-25G</td><td></td></tr><tr><td>Normal goat serum</td><td>Jackson ImmunoResearch Lab</td><td>005-000-121</td><td></td></tr><tr><td>Triton X-100</td><td>FisherBiotech</td><td>BP151-500</td><td></td></tr><tr><td>Nestin antibody</td><td>BD Transduction Laboratories</td><td>611659</td><td>1:200 dilution</td></tr><tr><td>Class III beta-tubulin antibody (TuJ1)</td><td>Covnce</td><td>MMS-435p</td><td>1:2000 dulution</td></tr><tr><td>GFAP antibody</td><td>Millipore</td><td>AB5804</td><td>1:1000 dilution</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>D1306</td><td>1:2000 dilution</td></tr><tr><td>ZIKV antibody</td><td>World Reference Collection for Emerging Viruses and Arboviruses at the University of Texas Medical Branch</td><td></td><td>1:2000 dilution</td></tr><tr><td>Fluoromount G</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Forma</td><td>Model# 3110</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo fisher Scientific</td><td>75004221</td><td></td></tr><tr><td>Biological safety cabinet</td><td>Forma scientific Claas II A/B3</td><td>Model# 1284</td><td></td></tr><tr><td>Freezer (-80 &amp;deg;C)</td><td>Forma scientific,Inc</td><td>model# 8516</td><td></td></tr><tr><td>Microscope(phase contrast image)</td><td>Hp 2230 workstation</td><td>Product#NOE25us#ABA</td><td></td></tr><tr><td>Microscope (epifluorescent image)</td><td>Nikon Eclipse 80i</td><td></td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon TE2000-E microscope with C1si confocal system</td><td></td><td></td></tr></tbody></table>

zika virus infection of cultured human fetal brain neural stem cells for immunocytochemical analysis

Células-tronco neurais cérebro fetal humano são um sistema único modelo não-geneticamente modificados para estudar o impacto de vários estímulos na neurobiologia do desenvolvimento humano. Prefiro a usar um modelo animal ou células pluripotentes induzidas geneticamente modificados, células-tronco neurais humanas fornecer um sistema eficaz em vitro para examinar os efeitos dos tratamentos, drogas de tela, ou examinar as diferenças individuais. Aqui, nós fornecemos os protocolos detalhados para os métodos utilizados para expandir as células-tronco neurais cérebro fetal humano na cultura com media serum-free, para diferenciá-los em vários subtipos neuronais e astrócitos através de procedimentos diferentes de escorva, para congelar e recuperar Estas células. Além disso, descrevemos um procedimento de usando células-tronco neurais cérebro fetal humano para estudar a infecção pelo vírus Zika.

HNSCs cultivadas em fase proliferativa crescerá como neurospheres não-aderente (Figura 1). Imediatamente após a passagem do hNSC, haverá muitas células individuais, que irão agregar e começar a esferas de forma nos próximos dias (figura 1A e 1B). As esferas saudáveis devem ser aproximadamente 1-2 mm de diâmetro 9-10 dias após uma passagem (Figura 1). Esferas que crescem ma...

Watch this Scientific Journal Video about Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis at JoVE.com

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Este artigo detalha os métodos que são usados para expandir as células-tronco neurais cérebro fetal humano na cultura, bem como diferenciá-los em vários subtipos neuronais e astrócitos, com ênfase na utilização de células-tronco neurais para estudar a infecção pelo vírus Zika.

Infecção por vírus Zika do cérebro Fetal humano culta células-tronco neurais para análise de Immunocytochemical

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

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Research

JoVE Journal

Developmental Biology

6.4K visualizações.  University of Texas Medical Branch. Este artigo detalha os métodos que são usados para expandir as células-tronco neurais cérebro fetal humano na cultura, bem como diferenciá-los em vários subtipos neuronais e astrócitos, com ênfase na utilização de células-tronco neurais para estudar a infecção pelo vírus Zika.

Vídeo: Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

Isolamento e Quantitative Immunocytochemical caracterização de células Miogênica primários e fibroblastos de músculo-esquelético humano

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Biologia do Desenvolvimento

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Virus Zika sistema de cultura celular Infecciosas e do<em&gt; In Vitro</em&gt; Efeito profilático de interferões

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Imunologia e Infecção

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Avaliação das respostas de células T Zika vírus específicos em órgãos Immunoprivileged de infectados Ifnar1- / - ratos

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

Modelagem de infecção de vírus Zika do desenvolvimento cérebro humano In Vitro usando células-tronco derivadas de Organoids Cerebral

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Neurociência

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

Estabelecer modelos de Mouse para doenças neurológicas induzida por vírus Zika usando estratégias de injeção Intracerebral: embrionárias, Neonatal e adulto

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

Isolamento e quantificação do vírus Zika de múltiplos órgãos em um mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Developing an In Vitro Model of Zika Virus Infection in Cerebral Organoids

Source: Salick, M. R., et al. <a href="https://app.jove.com/v/56404">Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids.</a> J. Vis. Exp. (2017).
This video demonstrates the establishment of an in vitro model of Zika virus infection in a developing brain. Human cerebral organoids containing rosettes of stem cells, neural progenitor cells (NPCs), and neurons are infected with the Zika virus. The virus infects NPCs and triggers apoptosis, disrupting organoid development.

Desenvolvimento de um modelo in vitro de infecção pelo vírus Zika em organoides cerebrais

1. Creating Cerebral Organoids from iPSC / hESC 2D Culture (Day 0)
Note: This protocol assumes that stem cell (SC) maintenance is conducted in xeno-free ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Infectious Diseases

Zika Virus Infection of Fetal Neural Stem Cells

Source: McGrath, E. L., et al., <a href="https://app.jove.com/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018)
This video demonstrates a procedure to infect human fetal neural stem cells with Zika virus. The infected cells can be used for further studies.

Source: McGrath, E. L., et al., Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis. J. Vis. Exp. (2018)
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Establishing a Model of Zika Virus-Induced Neurodegeneration in an Adult Mouse

Source: Herrlinger, S. A. et al. <a href="https://app.jove.com/v/56486">Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult.</a> J. Vis. Exp. (2018)
The video demonstrates intracerebral injection of Zika virus suspension in an adult mouse to induce neurodegeneration. An anesthetized mouse is secured in a stereotaxic frame, and a hole is drilled into the exposed skull for injection. A suspension of the Zika virus is injected into the brain tissue. The viral infection causes inflammation, leading to neurodegeneration.

Estabelecendo um modelo de neurodegeneração induzida pelo vírus Zika em um camundongo adulto

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Adult ...

Differentiating Human Neural Stem Cells into Neural and Astrocyte Progenitors

Source: McGrath, E. L., et al. <a href="https://app.jove.com/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018).
This video demonstrates the method to differentiate human neural stem cells from neurospheres into neural and astrocyte progenitors using enzymatic treatment and culturing in a growth factor-rich medium, which eventually leads to maturation into specialized progenitor cells.

Diferenciando células-tronco neurais humanas em progenitores neurais e astrócitos

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Stock medium preparation and stem ...

Infecção por vírus Zika do cérebro Fetal humano culta células-tronco neurais para análise de Immunocytochemical

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