Questo lavoro è stato supportato anche da finanziamenti NIAID (R01AI074951, U54AI057168) e la Penn frontiere Genome Institute di SC; dal NIH T32HG000046 di TSM, dal NIH T32GM07229 e T32AI007324 di LRS.

Parte 1: RNAi in 384 piastre <ol><li> Standardizzazione dei reagenti utilizzati per lo screening è essenziale. Testiamo singoli lotti di media Schneider, siero fetale bovino, e reagenti colorazione. Poi abbiamo aliquota e utilizzare lo stesso lotto per l&#39;intero schermo. </li><li> Per ogni test, si consiglia di testare diverse linee cellulari differenti per determinare quale è più riconducibile al biologico lettura. Usiamo tipicamente la S2-DRSC linea. </li><li> Crescere le cellule di Drosophila a 25 ° C in mezzi di comunicazione completa di Schneider. <ol class="lalpha"><li> Schneider mezzi di comunicazione </li><li> 10% di calore FBS inattivato </li><li> 1X L-glutammina </li><li> 1X Pen / strep antibiotici </li></ol></li><li> Cellule di Drosophila sono semi-aderenti e sono diversi passaggi senza tripsinizzazione ogni quattro giorni pipettando le cellule fuori i palloni e diluendo le cellule 1:10 in un pallone fresca. Le cellule devono essere in fase di log per esperimenti. </li><li> Piastra-to-plate e giorno per giorno variabilità sono ridotti al minimo attraverso l&#39;utilizzo di gestione automatica dei liquidi per tutte le aggiunte liquido multi-pozzetti. Usiamo un WellMate (Matrix). </li><li> Preparare tubi WellMate. <ol class="lalpha"><li> Spray hood e WellMate con il 70% di etanolo. </li><li> Rimuovere il tubo dalla confezione e spruzzare con il 70% di etanolo. </li><li> Inserire cassetta tubi di erogazione posizione sul WellMate. </li><li> Tubi primo con 25 ml di etanolo al 70%. </li><li> Sterilizzare tubi in etanolo per 15 minuti, allora primo con un altro 25 ml di etanolo. </li><li> Sciacquare tubi di priming con 50 ml di PBS sterile. </li></ol></li><li> Staccare le cellule dal pallone e contare. Celle a pellet in modo da avere 25% in più del necessario (300xg, 5 min). Il numero di cellule seminate in ogni pozzetto deve essere ottimizzata in base alla lunghezza del saggio. Quando si esegue l&#39;analisi automatizzata delle immagini, un monostrato unico senza aggregazione cellulare è l&#39;ideale per la segmentazione delle immagini. </li><li> Risospendere le cellule in pellet nel siero senza mezzi di comunicazione di Schneider a 1.7x10 6 cellule / ml. </li><li> Eseguiamo lo schermo in 384 piastre pre-aliquotati con una libreria disponibile in commercio del dsRNA ad una concentrazione di 250ng/well. Diversi pozzi di ogni piatto vengono lasciati vuoti per i controlli, che vengono aggiunti manualmente. Noi usiamo dsRNA a luciferasi come controllo negativo. Noi usiamo dsRNA contro Thread (DIAP) come controllo per la robusta RNAi. Abbattere di questo anti-apoptotica risultati fattore di morte cellulare drammatico ed è un modo rapido visivo per valutare la qualità del abbattere. Infine, usiamo un dsRNA virale contro il giornalista, in questo caso i beta-galacosidase, come controllo positivo per confermare che siamo in grado di inibire l&#39;infezione virale con il nostro test di lettura. Se spotting dsRNA nel piatto al momento della semina delle cellule, aliquota 1 dsRNA microlitri in un angolo del pozzo per consentire la facile visualizzazione, quindi si centrifuga dsRNA verso il fondo del pozzo (300xg, 1 min) prima di aggiungere le cellule. </li><li> Usa WellMate per aggiungere 10 microlitri cellule preparate in precedenza per ciascun pozzetto. </li><li> Spin cellule su piastre da centrifugazione 300xg, 1 min. </li><li> Incubare a 25 ° C incubatore per 45 minuti. </li><li> Aggiungere 20 l completa di Schneider media e centrifuga 300xg, 1 min. </li><li> Piastre posto in umidificata contenitori Tupperware foderato con impregnato d&#39;acqua tovaglioli di carta. Il volume di un 384 è ben piccolo: così, l&#39;evaporazione può avere grandi effetti sulla biologia, che a sua volta può portare a cambiamenti artefatta nel read-out dei pozzi di bordo. Per ovviare a questo, piastre devono essere mantenuti sotto umidità elevata. </li><li> Incubare per tre giorni per consentire knockdown robusto. Non c&#39;è bisogno di cambiare i mezzi di comunicazione o di aprire i contenitori umidificata in questo periodo. </li></ol> Parte 2: Infezione con virus vaccinico <ol><li> Sterilizzare multicanale aspiratore in Quatricide per 15 minuti, poi sciacquare in etanolo al 70%. </li><li> Preparare WellMate come descritto sopra. </li><li> Preparare Schneider mezzi di comunicazione con il 2% FBS relative alle infezioni da diluire multimediale completo 1:5 in siero media liberi. </li><li> Filtro di riserva virus greggio attraverso siringa 0,8 um filtro per rimuovere detriti cellulari o utilizzare virus purificato. </li><li> Diluire virus vaccinico nel 2% dei media siero Schneider ad essere molteplicità di infezione (MOI) di 2. </li><li> Rimuovere PBS da tubi WellMate, e il primo con virus diluito fino a quando il tubo è libero di bolle d&#39;aria. </li><li> Usa sterilizzati multi-canale collettore di aspirazione e aspiratore per rimuovere delicatamente i media dalle tavole di dsRNA cellule trattate con Drosophila. </li><li> Dispensare 30 microlitri per virus e sulle piastre con il WellMate. </li><li> Centrifugare piastre 1 minuto a 300xg. </li><li> Sterilizzare tubi WellMate. Il primo con 25 ml di 10% Bleach, attendere 10 minuti, e il primo con un altro candeggina 25 ml al 10%. </li><li> Tubi primo con 50 mL di acqua seguita da 50 mL di etanolo al 70%. Ritorna tubi al suo confezionamento. </li><li> Ritorna piatti per umidificare Tupperware. Incubare a 25 ° C per 48 ore. </li></ol> Parte 3: Lastre di colorazione <ol><li> Fissare le piastre in 15 microlitri di formaldeide al 4% / PBS per 10 minuti. Liquido viene rimosso utilizzando un multi-canale collettore di aspirazione e vuoto ad ogni passo. Una volta che le piastre sono stati risolti, attrezzature sterili e reagenti non sono più necessari. </li><li> Rimuovere fix e utilizzare WellMate aggiungere 30 microlitri PBST (PBS + 0,1% Triton-X). Incubare 10 minuti e ripetere. WellMate operare come descritto sopra, ad eccezione di tubi non ha bisogno di essere sterilizzato. </li><li> Per rilevare l&#39;infezione vaccinia usiamo un β-galattosidasi giornalista guidato da un inizio / fine promotore vaccinia. </li><li> Rimuovere le cellule di liquido e incubare in blocco (PBST con il 2% BSA) per 10 minuti. </li><li> Diluire l&#39;anticorpo primario in blocco, rimuovere il liquido e aggiungere 15 microlitri di anticorpi ad ogni pozzetto usando un pipettatore multicanale ripetizione (Matrix). </li><li> Spin down piastre (300xg, 1 min), coprire con un adesivo trasparente, e incubare a 4 ° C durante la notte. </li><li> Rimuovere l&#39;anticorpo primario, e usare il WellMate per lavare i pozzi in PBST 3 volte, a 10 minuti ciascuno. </li><li> Diluire anticorpo secondario fluorescente (FITC) e nucleari macchia in blocco, e aggiungere 15 microlitri di pozzi con un pipettatore multicanale ripetere. </li><li> Incubare le piastre 1 ora a temperatura ambiente, al riparo dalla luce. </li><li> Lavare i pozzetti in PBST 3 volte, a 10 minuti ciascuno utilizzando la WellMate. </li><li> Guarnizione piastra con adesivo trasparente, coprire e conservare a 4 ° C fino a tre settimane. </li></ol> Parte 4: Imaging and Image Analysis <ol><li> Utilizzare un microscopio automatico a 20X di ingrandimento alle piastre immagine. </li><li> Catturare le immagini dei nuclei totale (DAPI) e le cellule infettate (CIC). Ottimizzare il tempo di esposizione per ogni canale singolarmente al fine di massimizzare la gamma dinamica. </li><li> Ottimizzare la funzione di messa a fuoco automatica prendendo una pila Z di immagini, 1um distanziate, che attraversano i piani sopra e sotto il piano previsto di messa a fuoco. Una volta che il piano focale ottimale si trova, regolare le impostazioni di messa a fuoco automatica conseguenza. </li><li> Immagine l&#39;intera piastra, catturando almeno tre immagini al bene sia nel canale Hoechst (DAPI) e il canale di virus (CIC). Scegliere i siti da diverse regioni diverse all&#39;interno di un bene al fine di catturare immagini che rappresentano il bene nel suo complesso. </li><li> Usa MetaXpress software per scrivere un diario di segmentare l&#39;immagine e l&#39;utilizzo dei moduli Nuclei Conte per calcolare il numero totale di cellule positive per la colorazione nucleare (cellule totali) e infezioni (CIC) per ogni piatto. </li><li> Calcolare l&#39;infezione per cento (100 celle * FITC positivi / totale) e log trasformare. </li><li> Per identificare i candidati che usiamo un punteggio Z robusta basata sulla gamma media e interquartile di ogni piatto. Questo metodo è insensibile ai valori anomali e dati non simmetrico, e fornisce quindi una maggiore potenza a identificare anche colpi deboli o moderati 10. Per ogni piatto, la mediana viene sottratto dai dati di log-trasformati. Poi i valori risultanti sono divisi di 0,74 volte il range interquartile (la differenza tra i valori del percentile 75 ° e il 25 ° percentile). Il fattore 0,74 viene utilizzato per approssimare una distribuzione normale standard. </li><li> Il punteggio robusto Z è una misura della distanza in deviazioni standard dalla media del piatto. Ad esempio, un robusto Z-score di 2 indica l&#39;infezione% del pozzo è ~ 2 deviazioni standard dalla mediana piatto o p &lt;0,05. </li><li> Analizzare il numero di cellule per identificare i candidati citotossici. Calcolare il punteggio Z robusto dei conteggi nucleare. I candidati con un robusto Z &lt;-2 in schermi doppio sono da considerarsi citotossici. </li><li> Pozzi che hanno un punteggio robusto Z per cento delle infezioni&gt; 2 o &lt;-2 nelle schermate duplicato (p &lt;0,0025) senza citotossicità sono considerati potenziali candidati dalla schermata principale. </li><li> Candidati positivi sono convalidati utilizzando reagenti indipendente. dsRNAs sono generati contro i geni di interesse da un&#39;altra regione del mRNA e testato come sopra. </li><li> Questi geni per il quale due dsRNAs indipendenti hanno lo stesso fenotipo può considerare per ulteriori studi. </li></ol> Parte 5: immagini rappresentative e interpretazione dei tassi di infezione <img src="/files/ftp_upload/2137/2137fig1.jpg" alt="Figura 1" /> Pozzi Figura 1. Infette non mostrano alcuna colorazione per le proteine ​​del virus vaccinico, mentre un rappresentante e contiene le cellule infettate colorazione positiva per vaccinia-espresso beta-galattosidasi (βgal) di proteine, come misurato da microscopia di immunofluorescenza.Virus = verde, nuclei = blu. <img src="/files/ftp_upload/2137/2137fig2.jpg" alt="Figura 2" /> Figura 2. Automatizzato software di analisi dell&#39;immagine quantifica l&#39;infezione in ogni immagine utilizzando i parametri impostati dall&#39;utente. In un&#39;immagine rappresentativa, il numero totale dei nuclei delle cellule e il numero di cellule che esprimono l&#39;antigene virale sono conteggiati in base alle dimensioni e l&#39;intensità della colorazione al di sopra della colorazione di fondo locale. <img src="/files/ftp_upload/2137/2137fig3.jpg" alt="Figura 3" /> Figura 3. Knockdown di filo (DIAP) serve come controllo positivo per la robusta riduzione RNAi di geni bersaglio. Knockdown di questo anti-apoptotica fattore porta alla morte cellulare drammatico. Nuclei = blu. <img src="/files/ftp_upload/2137/2137fig4.jpg" alt="Figura 4" /> Knockdown Figura 4. Luciferasi della funge da controllo negativo per l&#39;effetto del doppio filamento di RNA su trattamento delle infezioni. L&#39;esaurimento delle luciferasi non ha alcun effetto su infezioni rispetto alle cellule non trattate. Virus = verde, nuclei = blu. <img src="/files/ftp_upload/2137/2137fig5.jpg" alt="Figura 5" /> Figura 5. Knockdown di proteine ​​βgal serve come controllo positivo per una riduzione di infezione, dato che il test utilizza livelli di proteina βgal come una lettura di infezione. Esaurimento dei risultati βgal una diminuzione della percentuale di cellule infette. Virus = verde, nuclei = blu. <img src="/files/ftp_upload/2137/2137fig6.jpg" alt="Figura 6" /> Figura 6. Knockdown di cellulari fattore Rab5 si traduce in una diminuzione della percentuale di cellule infette. Dal momento che Rab5 è noto per partecipare a endocitosi, questo fattore contribuisce probabile ingresso del virus vaccinico. Ciò rappresenta un outlier esempio dallo schermo. Virus = verde, nuclei = blu.

<ol>
	<li>Ramadan, N., Flockhart, I., Booker, M., Perrimon, N., Mathey-Prevot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+high-throughput+RNAi+screens+in+cultured+Drosophila+cells.">Design and implementation of high-throughput RNAi screens in cultured Drosophila cells.</a> Nat Protoc. 2, 2245-2264 (2007).</li><li>Chotkowski, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus+infection+of+Drosophila+melanogaster+induces+a+protective+RNAi+response.">West Nile virus infection of Drosophila melanogaster induces a protective RNAi response.</a> Virology. 377, 197-206 (2008).</li><li>Blondel, D., Petitjean, A. M., Dezelee, S., Wyers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+in+Drosophila+melanogaster+cells:+regulation+of+viral+transcription+and+replication.">Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication.</a> J Virol. 62, 277-284 (1988).</li><li>Sessions, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+insect+and+human+dengue+virus+host+factors.">Discovery of insect and human dengue virus host factors.</a> Nature. 458, 1047-1050 (2009).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+RNAi+screening+in+Drosophila+S2+cells:+what+have+we+learned+about+host-pathogen+interactions?.">Genomic RNAi screening in Drosophila S2 cells: what have we learned about host-pathogen interactions?.</a> Curr Opin Microbiol. 11, 262-270 (2008).</li><li>Hao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+RNAi+screen+identifies+host+genes+important+for+influenza+virus+replication.">Drosophila RNAi screen identifies host genes important for influenza virus replication.</a> Nature. 454, 890-893 (2008).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+activity+coupled+with+fatty+acid+biosynthesis+is+required+for+viral+replication.">COPI activity coupled with fatty acid biosynthesis is required for viral replication.</a> PLoS Pathog. 2, e102-e102 (2006).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+reveals+a+specific+sensitivity+of+IRES-containing+RNA+viruses+to+host+translation+inhibition.">Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition.</a> Genes Dev. 19, 445-452 (2005).</li><li>Moser, T. S., Jones, R. G., Thompson, C. B., Coyne, C. B., Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Kinome+RNAi+Screen+Identified+AMPK+as+Promoting+Poxvirus+Entry+through+the+Control+of+Actin+Dynamics.">A Kinome RNAi Screen Identified AMPK as Promoting Poxvirus Entry through the Control of Actin Dynamics.</a> PLoS Pathog. 6, e1000954-e1000954 (2010).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4, 67-73 (1999).</li><li>Echeverri, C. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+in+cultured+cells:+a+user's+guide.">High-throughput RNAi screening in cultured cells: a user's guide.</a> Nat Rev Genet. 7, 373-384 (2006).</li></ol>

Nessun conflitto di interessi dichiarati.

Genoma di screening RNAi in Drosophila fornisce un metodo robusto ed efficiente per l&#39;esame della componente cellulare di infezione virale. Un certo numero di virus di infettare le cellule di mammifero Drosophila, che può essere utilizzato per identificare i componenti della risposta immunitaria ospite intrinseca, e fattori proteici ospitante è richiesta per la replicazione virale, che possono rappresentare nuovi bersagli terapeutici. Prima di eseguire uno schermo, il dosaggio deve essere attentamente ottimizzati per il tipo cellulare, numero di cellulare, e il livello di infezione, e deve essere ben controllato, sia positivi che negativi virali e obiettivi cellulari 11. E &#39;importante garantire una sufficiente separazione tra controlli positivo e negativo prima dello screening per massimizzare la gamma dinamica del test. Una volta che lo schermo è stato effettuato, i candidati possono essere suddivisi in categorie funzionali per determinare quali meccanismi ospitare contribuiscono alle infezioni. Per i virus sui mammiferi, come vaccino, il contributo di omologhi mammiferi da RNAi dovrebbero essere determinati come parte di analisi secondaria. Presi insieme, questi metodi di screening robusti ci permetterà di ottenere informazioni sui meccanismi complessi da cui i virus interagiscono con i loro cellule ospiti.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell culture</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Schneider&#x2019;s media</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11720</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX 100X</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>35050</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin (pen/strep)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>F6178</td>
<td>&nbsp;</td>
<tr>
<td>Screening</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Genome-wide dsRNA library</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>384 well plates, black with clear bottom</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3712</td>
<td>&nbsp;</td>
<tr>
<td>Aluminum seals</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>6569</td>
<td>&nbsp;</td>
<tr>
<td>Clear seals</td>
<td>&nbsp;</td>
<td>Denville</td>
<td>B1212-4</td>
<td>&nbsp;</td>
<tr>
<td>Well Mate</td>
<td>&nbsp;</td>
<td>Matrix</td>
<td>201-10001</td>
<td>&nbsp;</td>
<tr>
<td>24 pin manifold</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>3-000-101</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B2261</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescently-labeled secondary antibodies</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ImageXpressMicro automated microscope</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MetaXpress image analysis software</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

rnai screening for host factors involved in vaccinia virus infection using drosophila cells

Patogeni virali rappresentano un pericolo significativo per la salute pubblica, non solo può causare epidemie virus naturale della malattia umana, ma il loro uso potenziale nella bioterrorismo è anche una preoccupazione. Una migliore comprensione dei fattori cellulari che l&#39;infezione impatto avrebbe facilitato lo sviluppo di terapie molto bisogno. I recenti progressi nella interferenza dell&#39;RNA (RNAi), la tecnologia accoppiata con il sequenziamento completo del genoma di organismi diversi ha portato alla ottimizzazione del genoma, a base di cellule perdita di funzione di schermi. Cellule di Drosophila sono particolarmente suscettibili di genoma scala schermi a causa della facilità ed efficienza di RNAi in questo sistema 1. Soprattutto, una grande varietà di virus possono infettare le cellule di Drosophila, compreso un certo numero di virus dei mammiferi di interesse medico e agricolo 2,3,4. Precedenti schermi RNAi in Drosophila hanno identificato fattori dell&#39;ospite che sono richiesti per varie fasi dell&#39;infezione da virus, compresa l&#39;iscrizione, la traduzione e la replicazione dell&#39;RNA 5. Inoltre, molti dei fattori cellulari necessari per la replicazione virale nelle cellule in coltura Drosophila sono anche limitante nelle cellule umane infettate con questi virus 4,6,7,8, 9. Pertanto, l&#39;identificazione di fattori dell&#39;ospite cooptati durante l&#39;infezione virale presenta nuovi obiettivi per la terapia antivirale. Qui vi presentiamo un protocollo generalizzata per un high-throughput schermo RNAi per identificare fattori cellulari coinvolti nella infezione virale, utilizzando vaccinia virus come esempio.

Watch this Scientific Journal Video about RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells at JoVE.com

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Fattori dell&#39;ospite romanzo coinvolti nella infezione virale può essere identificato tramite cell-based genome-wide perdita della funzione di screening RNAi. A<em&gt; Drosophila</emCellulari&gt; modello di coltura è particolarmente suscettibile a questo approccio a causa della facilità ed efficienza di RNAi. Qui mostriamo questa tecnica utilizzando<em&gt; Vaccinia</emVirus&gt; come esempio.

Lo screening RNAi per fattori di accoglienza che partecipano Vaccinia Infezione da virus utilizzando Drosophila Celle

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

rnai-screening-for-host-factors-involved-vaccinia-virus-infection

Research

JoVE Journal

Immunology and Infection

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

9.4K Views.  University of Pennsylvania. Fattori dell'ospite romanzo coinvolti nella infezione virale può essere identificato tramite cell-based genome-wide perdita della funzione di screening RNAi. A Drosophila modello di coltura è particolarmente suscettibile a questo approccio a causa della facilità ed efficienza di RNAi. Qui mostriamo questa tecnica utilizzando Vaccinia come esempio.

Video: RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Dissezione Host-virus Interazione in replica litico di un Herpesvirus modello

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Ricerca

Immunologia e infezioni

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS-Mediated Forward Genetica screening per la identificazione di geni coinvolti nella resistenza Nonhost


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Metodologia per la generazione efficiente di proteine del virus vaccinia taggate fluorescentmente

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

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.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

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 ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un approccio semplice ed efficiente per costruire Mutant vaccinia vettori di virus

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Saggi Microscopia-based per high-throughput screening dei fattori di accoglienza che partecipano<em&gt; Brucella</em&gt; L&#39;infezione di cellule HeLa

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

Saggi per caratterizzare nuovi endoteliale regolatori coinvolti nella risposta infiammatoria di screening

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

Infezioni da arbovirus come strumenti di Screening per l'identificazione di fattori virali immunomodulatori e Host antivirale

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

La formazione immagine interazione cellula nella Mucosa trachea durante l'infezione da Virus influenzale usando la microscopia Intravital due-fotone

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Istituzione dell'infezione virale e analisi dell'interazione ospite-Virus in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...