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

עבודה זו נתמכה על ידי בכספי קרן ג'ון דאן ס ו המכון זיהומים האנושי חסינות של הענף הרפואי אוניברסיטת טקסס (פ. וו).

<ol>
	<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>

המחברים יש שאין ניגודי אינטרסים להכריז.

היכולת תרבות ולטפל hNSCs מספק כלי קריטי שיכול לשמש למגוון רחב של מטרות של מידול מחלות אנושיות לתרופה תפוקה גבוהה הקרנת10,11,12,14, 15,16,17. שאלות רבות נותרו יש לטפל כמו מו?...

Zika וירוס (ZIKV) הוא flavivirus ששודרו או מינית, או על ידי יתוש וקטורים היתושים Aedes aegypti ו אדס מפוספס . ZIKV לאחרונה זוהה כאיום חמור לבריאות הציבור בשל הקלות של שידור, המזוהה עם סימפטומים נוירולוגיים1. ביותר לגבי תופעות נוירולוגיות היא התפתחות microcephaly ההתעסקות נולד האמהות נגועים בהריון2,3. Microcephaly היא הפרעה התפתחותיות איפה הראש הוא קטן יותר מהגודל טיפוסי במהלך התפתחות העובר, בלידה, עם ההיקף פחות מ- 2 סטיות תקן מתחת רשע4. היקף ראש קטן יותר מלווה בדרך כלל מגוון רחב של מחלות רקע כגון עיכובים התפתחותיים, התקפים, ראייה, אובדן שמיעה, האכלה קושי.
מחקרים שנעשו לאחרונה בשימוש בבעלי חיים או המושרה בתאי גזע pluripotent לחקור את ההשפעה של זיהום ZIKV neurodevelopment5,6,7,8. בעוד מחקרים אלה תרמו הידע שלנו על ZIKV, השימוש של מינים שונים או תאים מהונדסים עשויה לגזול זמן רב ו/או להוסיף משתנים נוספים אשר עשויים לבלבל את השפעת ZIKV על פיתוח עצבית תאים5, 6 , 7 , 8. עם זאת, הקושי עם תרבות hNSC, במיוחד את תרבות שאינם מחסידי neurosphere המתוארות פרוטוקול זה, היא כי התרבות היא רגישה מאוד שיטות השתמשו כדי לנהל את תרבות9. כל שינוי רכיבים בינונית, או אפילו את הטיפול הפיזי של כלי השיט תרבות, מספיקה כדי להפיק תגובה9תאים. כדי לטפל בבעיות אלה, פיתחנו תרבות אנושית עוברית המוח-derived עצבית תאי גזע (hNSCs) במבחנה mechanistically לחקור את השפעת ZIKV על תאי גזע עצביים בעובר. באמצעות השיטה שלנו, hNSCs היו נשמרים עבור מעל 80 קטעים ללא שינויים פנוטיפי לכאורה10. יתר על כן, שינויים כרומוזומלית כגון טריזומיה היו גם או מינימלי11. תרבות זו hNSC גדל כתרבות שאינו חסיד neurosphere. אחד היתרונות של neurospheres הוא הכדורים ליצור נישה סביבתית ייחודית בתרבות זו משקפת יותר ויוו נישות לעומת מימדי התרבויות9. יתרון נוסף של פרוטוקול זה הוא כי סוגי תאים מרובים הנגזרות התרבות hNSC, הפעלת חוקר לבחון את ההשפעה של משתנה נתון על הישרדות hNSC ובידול. פרוטוקול זה חל יחידים שמחפש תשובה מכניסטית שאלות לגבי התפתחות מערכת העצבים המרכזית או תפקוד לקוי. הפרוטוקול הבא מתאר כיצד להרחיב את תרבות hNSC להדביק עם ZIKV ולאחר מכן להבדיל את hNSCs כדי לבחון את ההשפעה של זיהום על תהליך התמיינות. זה כולל גם שיטות לאחסון hNSCs לשימוש ארוך טווח, וכדי להבדיל hNSCs לתוך סוגים שונים של נוירונים זה לאפשר את המשך החקירה של גירעונות ZIKV-induced תורם להפרת זכויות המוח11. אנו מאמינים שפרוטוקול זה הוא גם עניין חוקרים המבקשים להכיר את ההשפעה של כל גירוי סביבתיים כגון זיהום או רעלים על תאי גזע עצביים הישרדות ובידול.

<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

תאי גזע עצביים במוח העובר האנושי מהווים מערכת מודל ייחודי שאינו-מהונדסים לחקור את ההשפעה של גירויים שונים על האדם הנוירו-ביולוגיה התפתחותית. מעדיף מאשר להשתמש במודל חיה או תאים pluripotent המושרה מהונדסים, תאי גזע עצביים אנושי לספק מערכת יעילה במבחנה כדי לבחון את ההשפעה של טיפולים, תרופות מסך, או לבחון הבדלים אינדיבידואליים. כאן, אנו מספקים את הפרוטוקולים מפורט עבור שיטות המשמשות להרחבתן של תאי גזע עצביים במוח העובר האנושי בתרבות עם מדיה ללא סרום, כדי להבדיל אותם לתוך תת עצביים שונים, האסטרוציטים באמצעות הליכים שונים לקרקע, כדי להקפיא ולשחזר תאים אלה. יתר על כן, אנו מתארים הליך של שימוש בתאי גזע עצביים במוח העובר האדם ללמוד Zika בנגיף.

HNSCs בתרבית בשלב המקדימות שלהם יגדל ככל שאינם מחסידי neurospheres (איור 1). מיד לאחר המעבר hNSC, יהיו הרבה תאים בודדים, אשר צבירה, להתחיל הספירות טופס בבימים הקרובים (איור 1A ו 1B). הספירות בריא צריך להיות כ 1-2 מ מ בקוטר של 9-10 ימים בעקבות מעבר (<stron...

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

מאמר זה מפרט את שיטות המשמשות כדי להרחיב את תאי גזע עצביים במוח העובר האנושי תרבות, כמו גם כיצד להבדיל אותם לתוך תת עצביים שונים, האסטרוציטים, תוך שימת דגש על השימוש בתאי גזע עצביים ללמוד Zika בנגיף.

זיהום בנגיף Zika של תאי גזע עצביים במוח העובר האנושי בתרבית לניתוח 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 ...

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

Research

JoVE Journal

Developmental Biology

6.5K Views.  University of Texas Medical Branch. מאמר זה מפרט את שיטות המשמשות כדי להרחיב את תאי גזע עצביים במוח העובר האנושי תרבות, כמו גם כיצד להבדיל אותם לתוך תת עצביים שונים, האסטרוציטים, תוך שימת דגש על השימוש בתאי גזע עצביים ללמוד Zika בנגיף.

Video: 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.

בידוד וכמוני Immunocytochemical אפיון של תאים ראשוניים myogenic וFibroblasts משרירי שלד אדם

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

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.

מערכת תרבית תאי זיהומיות וירוס זיקה ואת<em&gt; במבחנה</em&gt; אפקט מניעתי של אינטרפרונים

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

Immunology and Infection

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.

הערכה של תגובות T-cell Zika וירוס ספציפיים באיברים Immunoprivileged של עכברים נגועים Ifnar1- / -

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.

דגמי Zika בנגיף של ה בפיתוח המוח האנושי במבחנה באמצעות תאי גזע נגזר Organoids מוחי

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

Neuroscience

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.

יצירת מודלים העכבר עבור וירוס Zika-induced הפרעות נוירולוגיות באמצעות הזרקת גרם אסטרטגיות: עובריים Neonatal, למבוגרים

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.

בידוד וקוונפיקציה של וירוס Zika ממספר איברים בעכבר

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.

פיתוח מודל חוץ גופי של זיהום בנגיף זיקה באורגנואידים מוחיים

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.

הקמת מודל של ניוון עצבי הנגרם על ידי נגיף זיקה בעכבר בוגר

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.

התמיינות תאי גזע עצביים אנושיים לאבות עצביים ואסטרוציטים

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

זיהום בנגיף Zika של תאי גזע עצביים במוח העובר האנושי בתרבית לניתוח Immunocytochemical

Summary

Explore More Videos

Chapters in this video