We would like to thank Dr. Aaron Brault and Dr. Brandy Russell of the Centers for Disease Control and Prevention (CDC), USA for providing Zika viral strain PRVABC59. We thank Nicholas Ten of Yale University for copy-editing this manuscript. This work was supported by the Cedars-Sinai Medical Center Institutional Programmatic Research Award to V.A.

Hinweis: Eine schematische Darstellung der Arbeitsablauf ist in Abbildung 1 dargestellt. 1. Zellen <ol><li> Verwenden Sie Vero-Zellen für Zika Virusproduktion und Analyse von viralen Replikationszyklus. </li><li> Bereiten vollständige Wachstumsmedium enthaltend 10% fötales Rinderserum (FBS), 2 mM L-Glutamin, Penicillin (100 Einheiten / ml), Streptomycin (100 Einheiten / ml) und 10 mM HEPES. </li><li> Kultur Vero - Zellen mit dem angegebenen vollständige Wachstumsmedium bei 37 ° C mit 5% CO 2. </li></ol> 2. Zika Virusproduktion <ol><li> Ernten Sie die Vero-Zellen bei 80% Zelldichte unter Verwendung von 0,25% Trypsin in T-75-Kolben und die Zellen zu zählen. <ol><li> Aspirieren das Medium aus dem T-75 Kulturflasche und spülen Sie die Zellen mit 2 ml phosphatgepufferter Kochsalzlösung (PBS). </li><li> 2 ml 0,25% Trypsin und Inkubation Der Kolben wird bei 37 ° C für 5 min. </li><li> 8 ml des Serums Wachstumsmedium, das Trypsin zu inaktivieren. Pipettieren nach oben und unten zu suspend Zellen und übertragen Sie die Zellen in ein 15-ml-Tube. </li><li> Entfernen Sie 10 ul Zellen und mischen mit der gleichen Menge von 0,4% Trypanblau. Laden der vorbereiteten Mischung in die Zelle Zählkammer Dia- und erhalten Lebendzellanzahl eines automatisierten Zellzähler verwendet wird. </li></ol></li><li> Seed insgesamt 7 Millionen Zellen in einem 30 ml Volumen in einem T-160-Kolben. </li><li> Am nächsten Tag vorzubereiten eine geeignete Multiplizität der Infektion (0,01 bis 0,1 MOI) von Zika Virus Inoculum in einem 10 ml serumfreies Kulturmedium pro Flasche. </li><li> Entfernen Sie die verbrauchte Medien aus dem T-160-Kolben und fügen Sie dann den frisch viralen Inokulum hergestellt (10 ml). </li><li> Inkubieren der inokulierten Kolben in 37 ° C mit 5% CO 2 für 4-6 Std. Verbreiten Sie das Inokulum durch sanft die Kolben seitlich in jeder Stunde zu kippen. </li><li> Bei Beendigung der Inkubation ersetzt das Inokulum mit warmem Serum-ergänztem Wachstumsmedien (30 ml) für jeden Kolben. Dann weiter die Viruskultur für den nächsten 96 Stunden. </li><li> Stellen Sie sicher, das Fortschreiten der infection durch das Auftreten von viralen Plaques auf der Zellschicht Beobachtung eines Phasenkontrastmikroskop und nehmen Bilder (Abbildung 2) als bei 40 - facher und 200 - facher Vergrößerung erforderlich. </li></ol><img alt="Figur 2" src="/files/ftp_upload/54767/54767fig2.jpg" /> Abbildung 2:. Plaketten von Zika Virus auf einer Monoschicht von Vero - Zellen gebildet Hellfeldbilder von verschiedenen Vergrößerungen zeigen Zika viralen Plaques bei 48 hpi. Beachten Sie die Anwesenheit von gerundeten Zellfoci auf der Monoschicht. (Maßstabsbalken = 50 &amp; mgr; m) <a href="https://www.jove.com/files/ftp_upload/54767/54767fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www.jove.com/files/ftp_upload/54767/54767fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <ol start="8"><li> Erntezellkulturüberständen an der 96 Stunden Zeitpunkt und zentrifugieren der Überstand bei 300 xg für 10 min bei 4 ° C die Zelltrümmer zu entfernen. </li><li> Entfernen Sie vorsichtig den Überstand ohne störende pelle Schutt und Transfer to 15-ml-Röhrchen. Ultrazentrifugation bei 24.000 xg durchgeführt werden, um die Viruspartikel (optional) zu konzentrieren. </li><li> Lagern Sie die viralen Kulturüberständen in mehreren Aliquots bei -80 ° C. </li></ol> 3. Mess Titer Zika Virus durch Plaque-Assay <ol><li> Seed naive Vero - Zellen bei 1 × 10 5 Zellen pro Vertiefung in 2 ml Volumen einer 12-Well - Platte verwendet wird . </li><li> Am nächsten Tag vorbereiten serielle Verdünnungen von Virus-Kulturüberständen 10-fach von den T-160-Kolben gesammelt Serum-freien Medien. Entfernen Sie die verbrauchten Medien aus jeder Vertiefung und fügen 400 ul vorbereitet Inokulum auf Vero-Zellen in dreifacher Ausfertigung. </li><li> Inkubieren der inokulierten Kolben in 37 ° C mit 5% CO 2 für 4-6 Std. Verbreiten Sie das Inokulum durch sanft die Platte seitlich an jedem eine Stunde zu kippen. </li><li> Am Ende der Inkubation, ersetzen Sie das Inokulum mit Serum ergänzten Medium (2 ml pro Vertiefung). </li><li> Bei 48 Stunden nach der Impfung, zählen die Virusherde eine Phase contr mitast Mikroskop. Berechne die Virustiter als Plaque bildende Einheiten (PFU) pro ml. Siehe Abbildung 3 für Plaque - Test. </li><li> Fix und färben die Zellen in 4% Formaldehyd und 0,1% Kristallviolett-Lösung in 20% Ethanol für eine klare Darstellung von Plaques. </li></ol> 4. Zika virale Genom-Replikation Assay <ol><li> Seed naiver Vero - Zellen bei 1 × 10 5 Zellen pro Vertiefung in 2 ml Volumina einer 12-Well - Platte verwendet wird . Am nächsten Tag vorbereiten virale Inokula (MOI von 0,01 und 0,1; 400 &amp; mgr; l / Vertiefung) mit niedrigem und hohem Titer Serum freie Medien in dreifacher Ausfertigung mit. Für Mock-Infektion verwenden Serum freie Medien (400 &amp; mgr; l / Vertiefung) nur. </li><li> Wiederholen Sie die Schritte 3.2 bis 3.4. </li><li> Bei den 48 und 96 h Zeitpunkte, sammeln die Proben für die RNA und Immunzytochemie (ICC). <ol><li> Für die Ernte RNA-Proben, die Medien zu entfernen und dann 400 ul Lyselösung hinzufügen direkt in jede Vertiefung und sammeln die Lysaten. </li><li> Für ICC, entfernen Sie die Medien und tHenne fixieren die Zellen durch Zugabe von 1 ml Methanol in jede Vertiefung und die Platte für 30 Minuten bei 4 ° C inkubiert. </li><li> Aus dem Zelllysat, isolieren die Gesamt-RNA eine RNA-Extraktions-Kit gemäß den Anweisungen des Herstellers verwendet wird. Quantifizieren der RNA mit einem Spektralphotometer. </li></ol></li><li> Durchführen eines Zweischritt reverse Transkription quantitative PCR (RT-qPCR) das Genom ZIKV Gehalt von geernteten RNA zu bestimmen. <ol><li> Zur Umkehr der RNA transkribieren, zunächst eine 13 &amp; mgr; l-Reaktionsmischung aufgebaut, bestehend aus 5 ug isolierte RNA, 1 ul dNTPs (10 mM) und 1 ul zufälligen Hexamer (250 ng) in einer 0,2-ml-Röhrchen. Inkubieren der Röhrchen bei 65ºC für 5 min und dann für eine Minute auf Eis stellen. Anschließend fügen 1 ul reverse Transkriptase, 4 &amp; mgr; l Synthesepuffer 5fach Strang, 1 ul 0,1 M Dithiothreitol und 1 &amp; mgr; l RNase-Inhibitor zu dem Reaktionsgemisch. Inkubiere das Rohr für weitere 5 min bei 25 ° C, gefolgt von 60 min bei 50 ° C und inactivaß die Reaktion durch Erhitzen auf 70 ° C für 15 min. </li><li> Führen Sie qPCR unter Verwendung von 1 &amp; mgr; l der resultierenden cDNA mit 12,5 ul 2x grünen Farbstoff Supermischung, die DNA-Polymerase und 1 ul 10 mM einzelnen Primer, die spezifisch für Zika Virus [Zika Virus pan-Genotyp-Primer (für: 5&#39;-AARTACACATACCARAACAAAGTGGT-3 &#39; Rev;: 5&#39;-TCCRCTCCCYCTYTGGTCTTG-3 &#39;), Zika Virus asiatischen Genotyp PRVABC59 Stamm-Primer (5&#39;-AAGTACACATACCAAAACAAAGTGGT-3&#39;; Rev: 5&#39;-TCCGCTCCCCCTTTGGTCTTG-3 &#39;)], oder Hepatitis-C-Virus (JFH RTQ F: 5 &#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3&#39;), oder zelluläre Housekeeping-Gen GAPDH (für: 5&#39;-CCACCTTTGACGCTGGG-3 &#39;; Rev: 5&#39;-CATACCAGGAAATGAGCTTGACA-3&#39;) in einem 25 ul Reaktionsvolumen. </li><li> Führen PCR unter Verwendung der Ausführungsbedingung 95 ° C für 15 sec und 60 ° C für 30 Sekunden (40 Zyklen) in einem Echtzeit-PCR-System. </li><li> Verwenden Sie GAPDH Expressionsniveau basierend auf cycle threshold (Ct) Wert der Z zu normalisierenika viralen Genoms Messung. Berechnen Sie die Delta - Ct (ACt) -Wert von Zika Genoms im Vergleich zu derjenigen von GAPDH und erhalten die 2 ACt Wert. Dann berechnen die Falte Änderung, indem das Verhältnis der normalisierten Zika Genom Inhalte zwischen infizierten und nicht infizierten Zellen zu den angegebenen Zeitpunkten nehmen. Siehe Abbildung 4 für ZIKV Genomreplikation Ergebnisse. </li></ol></li><li> Führen ICC das Methanol fixierten Zellen. <ol><li> Waschen der fixierten Zellen dreimal mit 1x PBS und Blockieren mit ICC Blockierungspuffer (3% Ziegenserum, 3% BSA, 0,1% Triton-X 100 in PBS). </li><li> Mit der Maus monoklonaler anti-dsRNA Antikörper J2 (1 ug / ml) in einer Verdünnung von 1: 100 in Blocking-Puffer und über Nacht bei 4 ° C inkubieren. </li><li> Waschen der Zellen mit 1x PBS und fügen sekundären Antikörper Ziege-anti-Maus-IgG-594 (1 ug / ml) in einer 1: 1000 Verdünnung in Blockierungspuffer und Inkubation für eine Stunde bei Raumtemperatur. </li><li> Wash-Zellen mit 1x PBS und Färbung für Kerne mit Hoechst-Farbstoff undbeachten Sie die Zellen , die ein Fluoreszenzmikroskop bei 100 - facher Vergrßerung (Abbildung 4) verwendet wird . </li></ol></li></ol> 5. Screening Zytokin-Bibliothek gegen eine Infektion Zika Virus <ol><li> Seed naive Vero - Zellen bei 1 × 10 5 Zellen pro Vertiefung einer 12-Well - Platte verwendet wird . Sobald die Zellen gebunden sind (6 Stunden nach der Aussaat), für jede Zytokin in den angegebenen Konzentrationen in biologischen Duplikaten (2 ml Volumen / Well). Enthalten Fahrzeug (PBS) allein Kontrolle. </li><li> Bei 12 Stunden nach der Behandlung, führen Zika Virusinfektion (MOI von 0,1 in 400 &amp; mgr; l pro Vertiefung). Fügen Negativkontrollen ohne Infektion (mock). </li><li> Bei 4 Stunden nach der Infektion, ersetzen Sie die viralen Inokulum mit Medien Zytokin behandelt (2 ml). Inkubieren der Zellen bei 37 ° C für weitere 44 Std. </li><li> Bei 48 Stunden nach der Infektion zählen virale Plaques ein Phasenkontrastmikroskop und erwerben repräsentative Bilder von infizierten Zellen bei 40 - facher Vergrößerung (Abbildung 5). </li></ol>

<ol>
	<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+-+Preliminary+Report.">Zika Virus Infection in Pregnant Women in Rio de Janeiro - Preliminary Report.</a> N Engl J Med. , 1-11 (2016).</li><li>Lucey, D. R., Gostin, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emerging+Zika+Pandemic:+Enhancing+Preparedness.">The Emerging Zika Pandemic: Enhancing Preparedness.</a> JAMA. 315 (9), 865-866 (2016).</li><li>Mlakar, 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=Zika+Virus+Associated+with+Microcephaly.">Zika Virus Associated with Microcephaly.</a> N Engl J Med. 374 (10), 951-958 (2016).</li><li>Schuler-Faccini, 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=Possible+Association+Between+Zika+Virus+Infection+and+Microcephaly.">Possible Association Between Zika Virus Infection and Microcephaly.</a> MMWR Morb Mortal Wkly Rep. 65 (3), 59-62 (2015).</li><li>Foy, B. D., 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=Probable+non-vector-borne+transmission+of+Zika+virus,+Colorado,+USA.">Probable non-vector-borne transmission of Zika virus, Colorado, USA.</a> Emerg Infect Dis. 17 (5), 880-882 (2011).</li><li>Kuno, G., Chang, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+sequencing+and+genomic+characterization+of+Bagaza,+Kedougou,+and+Zika+viruses.">Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses.</a> Arch Virol. 152 (4), 687-696 (2007).</li><li>Dick, G. W. <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.+II.+Pathogenicity+and+physical+properties.">Zika virus. II. Pathogenicity and physical properties.</a> Trans R Soc Trop Med Hyg. 46 (5), 521-534 (1952).</li><li>Dick, G. W., Kitchen, S. F., Haddow, A. J. <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.+I.+Isolations+and+serological+specificity.">Zika virus. I. Isolations and serological specificity.</a> Trans R Soc Trop Med Hyg. 46 (5), 509-520 (1952).</li><li>Oehler, E., 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+complicated+by+Guillain-Barre+syndrome--case+report,+French+Polynesia.">Zika virus infection complicated by Guillain-Barre syndrome--case report, French Polynesia.</a> Euro Surveill. 19 (9), 1-3 (2013).</li><li>Baronti, 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=Complete+coding+sequence+of+zika+virus+from+a+French+polynesia+outbreak+in+2013.">Complete coding sequence of zika virus from a French polynesia outbreak in 2013.</a> Genome Announc. 2 (3), e00500-e00514 (2014).</li><li>Lanciotti, R. S., 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=Genetic+and+serologic+properties+of+Zika+virus+associated+with+an+epidemic,+Yap+State,+Micronesia,+2007.">Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007.</a> Emerg Infect Dis. 14 (8), 1232-1239 (2008).</li><li>Lanciotti, R. S., 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=Phylogeny+of+Zika+virus+in+Western+Hemisphere,+2015+[Letter].">Phylogeny of Zika virus in Western Hemisphere, 2015 [Letter].</a> Emerg Infect Dis. 22 (5), (2016).</li><li>Musso, D., Nilles, E. J., Cao-Lormeau, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+spread+of+emerging+Zika+virus+in+the+Pacific+area.">Rapid spread of emerging Zika virus in the Pacific area.</a> Clin Microbiol Infect. 20 (10), O595-O596 (2014).</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. , 1-5 (2016).</li><li>Hamel, R., 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=Biology+of+Zika+Virus+Infection+in+Human+Skin+Cells.">Biology of Zika Virus Infection in Human Skin Cells.</a> J Virol. 89 (17), 8880-8896 (2015).</li><li>Faye, O., 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=Quantitative+real-time+PCR+detection+of+Zika+virus+and+evaluation+with+field-caught+mosquitoes.">Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes.</a> Virol J. 10, 311 (2013).</li><li>Chu, D., 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=Systematic+analysis+of+enhancer+and+critical+cis-acting+RNA+elements+in+the+protein-encoding+region+of+the+hepatitis+C+virus+genome.">Systematic analysis of enhancer and critical cis-acting RNA elements in the protein-encoding region of the hepatitis C virus genome.</a> J Virol. 87 (10), 5678-5696 (2013).</li><li>Hiratsuka, M., 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=Administration+of+interferon-alpha+during+pregnancy:+effects+on+fetus.">Administration of interferon-alpha during pregnancy: effects on fetus.</a> J Perinat Med. 28 (5), 372-376 (2000).</li><li>Ozaslan, E., 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=Interferon+therapy+for+acute+hepatitis+C+during+pregnancy.">Interferon therapy for acute hepatitis C during pregnancy.</a> Ann Pharmacother. 36 (11), 1715-1718 (2002).</li></ol>

The authors have nothing to disclose.

Hier wird eine rationalisierte Protokoll zur Kultivierung Zika Virus in vitro dargestellt. Kritische Schritte, einschließlich Identifizierung optimale Endpunkte für Viruskultur erweitert, Titer zu messen und zu quantifizieren Genomreplikation bereitgestellt wurden. Zika-Virus ist ein menschlicher Krankheitserreger, so, beim Umgang mit infektiösen Erregern, die Biosicherheit Verfahren sind strikt befolgt werden. Ein Affennierenzelllinie, Vero, wurde für den Nachweis verschiedener virologische Tests verwendet...

Zika - Virus (ZIKV) ist eine wichtige menschliche Krankheitserreger im Zusammenhang mit Mikrozephalie und schlechte Schwangerschaft 1,4 Ergebnisse. ZIKV gehört zur Gruppe von medizinisch relevanten Flaviviren, die neurologische Defekte wie das Dengue-Fieber, West-Nil, und St. Louis-Enzephalitis-Viren verursachen können. Der Hauptmodus der viralen Übertragung durch den Moskito - Vektor Aedes aegypti, und zusätzlich hat die sexuelle Übertragung auch 5,6 berichtet. ZIKV hat ein großes globales Gesundheitsproblem geworden aufgrund der wachsenden geographischen Verteilung des Moskito-Vektor und seine starke Korrelation mit Geburtsfehler. ZIKV wurde isoliert zuerst 7,8 im Jahr 1947 von einem Sentinel - Rhesusaffen im Zika Wald, Uganda und der erste Mensch Fall wurde im Jahr 1952 berichtet. Personen, die mit ZIKV mit milden Symptomen vorhanden infiziert werden, wie Fieber, Hautausschlag, Kopfschmerzen, Konjunktivitis und Muskel / Gelenkschmerzen. Infizierte schwangere Frauen können ZIKV auf den sich entwickelnden Fötus 1 übertragen. ZIKV - Infektion hat sich auch Guillain-Barre - Syndrom in Verbindung gebracht worden, einer peripheren Nerven Auto-Immun - Erkrankung Demyelinisierung 9. Die Zika virale Genom besteht aus einer positiven Sinn, einsträngige RNA-Molekül, das etwa 10,8 Kilobasen lang ist. Die Struktur des Genoms ist als 5&#39;NCR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-2K-NS5-3&#39;NCR organisiert, wobei nicht-kodierenden Regionen (NCR) flankieren eine Protein-codierende Region 6. Ein einzelnes Polyprotein (3419 aa) übersetzt wird, dass ist Co- und post-translational in 10 kleinere Peptide gespalten. Sowohl die 5&#39;NCR und 3&#39;NCR RNA-Stem-Loop-Strukturen spielen eine entscheidende Rolle bei der Aufnahme des viralen Genoms Übersetzung und Replikation. Die strukturellen Komponenten des Genoms der Capsid, Membran besteht und Hüllproteine. Die nicht-strukturellen Proteine ​​sind entscheidend für die Genom-Replikation. Derzeit sind Zika Virusstämme gruppiert in drei Haupt Genotypen: West African, Ost - Afrika, Asien und 6,10-13. Es hat sich gezeigt , dass die East African Abstammung verbreiten nach Westafrika und Asien vorgeschlagen, wo es später weitere 12 entwickelt. Der asiatische Genotyp ist verantwortlich für die aktuellen Ausbrüche in Nord- und Südamerika. Zika Virus kann sowohl kultiviert werden in Mücke und Säugerzellen. Primäre Hautfibroblasten, unreife dendritische Zellen, kortikalen neuronalen Vorläuferzellen und Vero - Zellen sind empfänglich für eine virale Infektion Zika 10,14,15. Sowohl Typ I und Typ II - Interferonen wurden ZIKV Wachstum in Fibroblasten der Haut 15 gezeigt zu beschränken. Die Ziele dieser Studie sind eine stufenweise, detailliertes Protokoll für die Herstellung und das Testen des asiatischen Genotyp ZIKA Virusstamm PRVABC59 in einer Säugerzellkultursystem zur Verfügung zu stellen und den Nutzen dieser infektiösen Kultursystems als Medikamenten-Entwicklungsplattform zu demonstrieren. Diese Ressource hat das Potenzial, zu stark die Zika virale und neurologische Forschung profitieren weiter i erläuternts Mechanismen der viralen Pathogenese und Entwicklung von viralen Virulenz.

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:355px;">Sigma Life Science</td>
			<td style="width:119px;">D5796</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">HEPES</td>
			<td style="width:355px;">Life Technologies</td>
			<td style="width:119px;">15630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Glutamax</td>
			<td style="width:355px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">2.5% Trypsin, 10X [-] Phenol Red&#160;&#160;</td>
			<td style="width:355px;">Corning</td>
			<td style="width:119px;">25-054-C1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Stain 0.4%</td>
			<td style="width:355px;">Life Technologies</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Countess &#8211; Automated Cell Counter</td>
			<td style="width:355px;">ThermoFisher Scientific</td>
			<td style="width:119px;">C10227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Countess-cell counting&#160; chamber slides</td>
			<td style="width:355px;">ThermoFisher Scientific</td>
			<td style="width:119px;">C10283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Rneasy Mini Kit</td>
			<td style="width:355px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Nanodrop 2000</td>
			<td style="width:355px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-dsRNA antibody J2&#160;</td>
			<td>English &#38; Scientific Consulting Kft.</td>
			<td style="width:119px;">10010200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 594</td>
			<td style="width:355px;">Life Technologies</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SUPERSCRIPT III RT&#160;</td>
			<td style="width:355px;">Life Technologies</td>
			<td style="width:119px;">18080085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SYBR QPCR SUPERMIX W/ROX</td>
			<td style="width:355px;">Life Technologies</td>
			<td style="width:119px;">11744500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuantStudio12K Flex Real-Time PCR System</td>
			<td style="width:355px;">Thermo Fischer</td>
			<td style="width:119px;">4471088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">RNase-Free DNase</td>
			<td style="width:355px;">Promega</td>
			<td style="width:119px;">M6101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Vero Cell Line&#160;</td>
			<td style="width:355px;">ATCC</td>
			<td style="width:119px;">CCL-81</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">Zika viral strain PRVABC59</td>
			<td style="width:355px;">Centers for Disease Control and Prevention (CDC)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">IL-6</td>
			<td style="width:355px;">Peprotech</td>
			<td style="width:119px;">200-06&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">IL-1 alpha&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:355px;">Peprotech</td>
			<td style="width:119px;">200-01A&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">TNF-alpha</td>
			<td style="width:355px;">Peprotech</td>
			<td style="width:119px;">300-01A&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">Interferon alpha A&#160;&#160;</td>
			<td style="width:355px;">R &#38; D Systems</td>
			<td style="width:119px;">11100-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">Interferon beta</td>
			<td style="width:355px;">Peprotech</td>
			<td style="width:119px;">300-02BC</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">Interferon gamma</td>
			<td style="width:355px;">Peprotech</td>
			<td style="width:119px;">300-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Centrifuge 5415R</td>
			<td style="width:355px;">Eppendorf</td>
			<td style="width:119px;">5415R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Centrifuge 5810R</td>
			<td style="width:355px;">Eppendorf</td>
			<td style="width:119px;">5810R</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:33px;width:569px;">Nikon Eclipse Ti Immunofluorescence Microscope with Nikon Intenselight C-HGFI</td>
			<td style="width:355px;">Nikon</td>
			<td style="width:119px;">Visit Nikon for Request</td>
			<td></td>
		</tr>
	</tbody>
</table>

zika virus infectious cell culture system and the in vitro prophylactic effect of interferons

Zika-Virus (ZIKV) ist eine aufstrebende Erreger, die fetale Entwicklungsstörungen wie Mikrozephalie verbunden ist, Augenfehler und Wachstumsstörungen. ZIKV ist ein RNA - Virus der Familie Flaviviridae. ZIKV wird hauptsächlich durch Stechmücken übertragen, kann aber auch als auch sexuellen Kontakt durch mütterliche fetalen vertikale Übertragung verteilt werden. Bis heute gibt es keine zuverlässige Behandlung oder Impfung Optionen zur Verfügung, die mit dem Virus infiziert zu schützen. Die Entwicklung einer reproduzierbaren, effektive Zika Virus infektiös Zellkultursystem ist von entscheidender Bedeutung für die molekularen Mechanismen der ZIKV Replikation sowie Drogen-und Impfstoff-Entwicklung zu studieren. In dieser Hinsicht ist ein Protokoll einer Säugerzelle basierenden in vitro Zika Viruskultursystem für die virale Produktion und Wachstumsanalyse beschreibt , wird hier berichtet. Einzelheiten zur Bildung von Plaques durch Zika Virus auf einer Zellmonoschicht und Plaque-Assay für Virustiter Messung dargestellt. Viralen Genomreplikation kineticsund doppelsträngigen RNA-Genom replicatory Zwischenprodukte werden bestimmt. Diese Kultur Plattform zum Screenen gegen eine Bibliothek von einer kleinen Gruppe von Cytokinen, was zu der Identifizierung von Interferon-α (IFN-α), IFN-β und IFN-γ als potente Inhibitoren von Zika virales Wachstum genutzt wurde. Zusammengefasst ein in vitro - Infektions Zika Viruskultursystem und verschiedene virologische Tests sind in dieser Studie gezeigt, die das Potenzial hat, die Forschungsgemeinschaft bei der Aufklärung die Mechanismen der viralen Pathogenese und die Entwicklung der viralen Virulenz stark profitieren. Antivirale IFN-alpha kann weiter als Prophylaktikum, Post-Expositions-Prophylaxe zu bewerten und Behandlungsoption für Zika Virusinfektionen in Hochrisikogruppen, einschließlich der infizierten schwangeren Frauen.

Ein Zika - Virusstamm (PRVABC59; GenBank - Zugangsnummer KU501215) des asiatischen Genotyp wurde in dieser Studie 12 genutzt. Vero - Zellen bei 80% Konfluenz wurden zur Untersuchung der de novo Zika Virusinfektion verwendet. Für die virale Produktion und anschließende virologischen Charakterisierung, eine frühe Passage (P3) Zika Virus wurde verwendet. Die viralen Plaques wurden am zweiten Tag der Infektion beobachtet. Zika viralen Nachkommen von der anfänglich inf...

Watch this Scientific Journal Video about Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons at JoVE.com

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.

Zika Virus Infectious Zellkultursystem und die<em&gt; In Vitro</em&gt; Prophylaktische Wirkung von Interferonen

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

zika-virus-infectious-cell-culture-system-vitro-prophylactic-effect

Research

JoVE Journal

Immunology and Infection

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

20.7K Aufrufe.  Cedars-Sinai Medical Center. 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.

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

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Nachweis von infektiösen Viren aus Field-gesammelt Moskitos von Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Forschung

Immunologie und Infektion

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualizing Dengue Virus durch Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Eine primäre Neuron Culture System zur Erforschung des Herpes Simplex Virus Latenz und Reaktivierung

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In-vitro-Analyse Myd88-vermittelte zelluläre Immunantwort gegen das West-Nil-Virus-Infektion Mutantenstamm

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophilen-Isolierung und Analyse zur Bestimmung ihrer Rolle in der Lymphom-Zellempfindlichkeit therapeutische Mittel

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Rettung und Charakterisierung des rekombinanten Virus aus einer neuen Welt Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika-Virus spezifische diagnostische Epitop Entdeckung

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Schnelle, sichere und einfache manuelle am Krankenbett Nukleinsäure-Extraktion für den Nachweis des Virus in Vollblut-Proben

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

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.

Bewertung der Zika-Virus-spezifischen T-Zell-Reaktionen in Organe der Immunabwehr von infizierten Ifnar1- / - Mäusen

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

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Ein Ex-Vivo Huhn Primärkultur Bursitis-Zelle Modell zur Bursitis Infektionskrankheit Virus Pathogenese

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...