Name: zika virus infection of cultured human fetal brain neural stem cells for immunocytochemical analysis
Uploaded: 2018-02-05T14:30:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis at JoVE.com

この作品は、ジョン s. ダン財団とヒトへの感染と免疫のテキサス大学医学部 (P.W.) の研究所からの資金によって支えられました。

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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>

著者の利害の衝突を宣言するあります。

文化、hNSCs を操作することは、さまざまな高スループット薬剤のスクリーニングの10、11,12,14、人間病気のモデリングから目的のために使用できる重要なツールを提供します 15,16,17。多くの質問がどのように人間の胎児...

ジカ (ZIKV) は、性的、または蚊ベクトルネッタイシマカとヒトスジシマカ蚊によって媒介するフラビです。ZIKV は、伝送の容易さのための深刻な公衆衛生の脅威として最近確認され、神経学的症状1を提携します。最も感染妊婦2,3に生まれた胎児の小頭症の開発は、神経学的な影響に関する。小頭症は胎児開発の間に、平均4以下の 2 標準偏差以下のまわりで、出生時、頭は一般的なサイズより小さい神経発達障害です。小さい頭囲ではさまざまな発達の遅れ、発作、ビジョンと難聴、摂食困難など併存疾患一般的が伴われます。
最近の研究は、動物モデルを使用か、誘導多能性幹細胞神経発達5,6,7,8ZIKV 感染症の影響を研究します。これらの研究は、ZIKV、別の種の使用の私達の知識に貢献している、または遺伝子組み換え細胞が時間がかかるかに ZIKV の効果を混同するかもしれない追加の変数を追加5,細胞の神経の開発6,7,8します。 ただし、hNSC 文化、このプロトコルで記述されている非付着 neurosphere 文化特にの難点は、文化は文化9を実施する使用される方法に非常に敏感です。メディア コンポーネント、または培養器の物理的な処理も変更は9セルから反応を引き出すのに十分です。これらの問題に対処するため、胎児の神経幹細胞に及ぼす ZIKV を機械論的に尋問するの in vitroひと胎児脳由来神経幹細胞 (hNSCs) 文化を開発しました。私たちのメソッドを使用して、hNSCs なく表現型変化10以上 80 通路維持されました。さらに、トリソミーなどの染色体の変化がいずれまたは最小11。この hNSC 文化は、非付着 neurosphere 文化として育ちます。神経幹細胞の利点の 1 つは球が二次元文化9に比べて生体内でニッチのより多くの反射は文化のユニークなニッチな環境を作成します。このプロトコルの別の利点は、複数の細胞のタイプから派生できる hNSC 文化 hNSC 生存と分化に関する特定の変数の影響を観察する研究者を有効にします。このプロトコルは、中枢神経系発達や障害に関する機構の質問に答えるために見ている個人に適用されます。次のプロトコルは、ZIKV に感染し、その後分化過程で感染の影響を観察する hNSCs を区別する hNSC 文化を展開する方法について説明します。様々 な種類の脳奇形11に貢献する ZIKV による障害の調査を進めるニューロンに hNSCs を区別するために長期的な使用のための hNSCs を格納するメソッドも含まれています。このプロトコルはまた感染や神経幹細胞の生存と分化の毒素など任意の環境の刺激の影響を理解しようとする研究者を対象と考えています。

<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

人間の胎児脳神経幹細胞は、人間発達神経生物学の様々 な刺激の影響を研究するユニークな非遺伝子組み換えモデル システムです。むしろ動物モデルまたは遺伝子組み換え多能性細胞を使用するよりも、ひと神経幹細胞は画面薬治療の効果を調べたり、個体差を調べるに効果的な培養システムを提供します。ここでは、様々 な神経細胞やアストロ サイト別プライミング手続きにそれらを区別するために、凍結、回収する、無血清のメディアと文化における人間の胎児脳神経幹細胞を展開に使用する方法について詳細なプロトコルを提供します。これらの細胞。さらに、我々 はジカ ウイルス感染症を勉強する人間の胎児脳神経幹細胞を使用する手順をについて説明します。

増殖期培養 hNSCs は、非付着性細胞 (図 1) として成長します。HNSC 通過直後多くの個々 のセルを集計して次の数日 (図 1 aと1 b) 形球を開始になります。健康的な球は、直径 1-2 mm 程度次の通路 (図 1) 9-10 日をする必要があります。2 mm よりも大きくなる球は暗い中心を開発して、培地で成長?...

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

この記事は、ひと胎児脳神経幹細胞文化だけでなく、様々 な神経細胞やアストロ サイト、ジカ ウイルス感染症を研究する神経幹細胞の使用に重点を置いてにそれらを区別する方法を展開に使用する方法を詳しく説明します。

培養ひと胎児脳神経幹細胞の免疫細胞化学的解析のジカ ウイルス感染症

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

zika-virus-infection-cultured-human-fetal-brain-neural-stem-cells-for

Research

JoVE Journal

Developmental Biology

6.4K ビュー.  University of Texas Medical Branch. この記事は、ひと胎児脳神経幹細胞文化だけでなく、様々 な神経細胞やアストロ サイト、ジカ ウイルス感染症を研究する神経幹細胞の使用に重点を置いてにそれらを区別する方法を展開に使用する方法を詳しく説明します。

ビデオ: 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.

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

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

発生生物学

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.

ジカウイルス感染細胞培養系と<em&gt;インビトロ</emインターフェロンの&gt;予防効果

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

免疫・感染学

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.

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

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.

脳オルガノイドを派生開発人間の脳の培養幹細胞を使用してジカ ウイルス感染症をモデル化

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

神経科学

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.

脳内注入戦略を使用してジカ ウイルス性神経疾患のマウスモデルの確立: 胎生期、新生児、およびアダルト

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.

マウスの複数の臓器からのジカウイルスの単離と定量

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.

脳オルガノイドにおけるジカウイルス感染のin vitroモデルの開発

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

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)
...

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.

成体マウスにおけるジカウイルス誘発性神経変性のモデル確立

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.

ヒト神経幹細胞の神経前駆細胞とアストロサイト前駆細胞への分化

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

培養ひと胎児脳神経幹細胞の免疫細胞化学的解析のジカウイルス感染症

要約

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この動画の章

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

ジカウイルス感染細胞培養系と

ジカウイルス特異 Immunoprivileged 臓器感染 Ifnar1^-/-マウスの T 細胞応答の評価

脳オルガノイドを派生開発人間の脳の培養幹細胞を使用してジカウイルス感染症をモデル化

脳内注入戦略を使用してジカウイルス性神経疾患のマウスモデルの確立: 胎生期、新生児、およびアダルト

マウスの複数の臓器からのジカウイルスの単離と定量

脳オルガノイドにおけるジカウイルス感染のin vitroモデルの開発

Zika Virus Infection of Fetal Neural Stem Cells

成体マウスにおけるジカウイルス誘発性神経変性のモデル確立

ヒト神経幹細胞の神経前駆細胞とアストロサイト前駆細胞への分化

培養ひと胎児脳神経幹細胞の免疫細胞化学的解析のジカ ウイルス感染症

要約

さらに動画を探す

この動画の章

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

ジカウイルス感染細胞培養系と

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

脳オルガノイドを派生開発人間の脳の培養幹細胞を使用してジカ ウイルス感染症をモデル化

脳内注入戦略を使用してジカ ウイルス性神経疾患のマウスモデルの確立: 胎生期、新生児、およびアダルト

マウスの複数の臓器からのジカウイルスの単離と定量

脳オルガノイドにおけるジカウイルス感染のin vitroモデルの開発

Zika Virus Infection of Fetal Neural Stem Cells

成体マウスにおけるジカウイルス誘発性神経変性のモデル確立

ヒト神経幹細胞の神経前駆細胞とアストロサイト前駆細胞への分化

培養ひと胎児脳神経幹細胞の免疫細胞化学的解析のジカウイルス感染症

ジカウイルス特異 Immunoprivileged 臓器感染 Ifnar1^-/-マウスの T 細胞応答の評価

脳オルガノイドを派生開発人間の脳の培養幹細胞を使用してジカウイルス感染症をモデル化

脳内注入戦略を使用してジカウイルス性神経疾患のマウスモデルの確立: 胎生期、新生児、およびアダルト