Vorremmo ringraziare S. Werner per fornire i corvi utilizzati in questo studio e USDA, APHIS, WS, NWRC personale cura degli animali per la cura degli animali e monitoraggio. Menzione o l&#39;uso di un prodotto non implica l&#39;approvazione USDA. Finanziamento per questo studio è stato fornito da USDA, APHIS, servizi veterinari.

Il nostro protocollo è adattato da quello che abbiamo già pubblicato 7. Tutte le procedure che coinvolgono gli animali sono stati approvati dal Comitato Istituzionale cura degli animali ed uso del Dipartimento dell&#39;Agricoltura degli Stati Uniti (USDA), animale e vegetale servizio di ispezione sanitaria (APHIS), Wildlife Services (WS), National Wildlife Research Center (NWRC). 1. Crow Gavaging <ol><li> Stima tempo di passaggio di &#39;materiale cerebrale pseudo&#39; attraverso il tratto alimentare di corvi americani. <ol><li> Mescolare 5 ml di cotte e strapazzate uovo intero con il colorante blu e sonda gastrica 1 corvo con un 2 a gavage ago (Figura 1). </li><li> Controllare corvo ogni 30 minuti fino a quando / feci verde macchiato di blu non sono più escreti. </li></ol></li><li> Ottenere RML (ceppo Chandler)-infetti C57BL / 6 cervelli infetti e malati terminali mouse. </li><li> Generare un 20% di peso / volume normale e infetto omogeneizzato mouse cervello in un omogeneizzatore proiettile frullatore con 1xtampone fosfato salino (PBS), e perle di vetro, poi centrifugare a 3000 xg per 1 minuto per rimuovere le particelle più grandi. Rimuovere il surnatante e diluire con un volume uguale di PBS 1x per generare un 10% peso / volume in PBS sterile. Congelare a -80 ° C fino al momento dell&#39;uso. </li><li> 17 ore prima del gavage rimuovere il cibo, ma non l&#39;acqua, da penne d&#39;aria. </li><li> Casualmente destinare corvi ai gruppi di trattamento e per via orale sonda gastrica ogni corvo con 5 ml di normale o infetto omogeneizzato cervello di topo con un 2 in gavage ago. </li><li> Trasferimento corvi di gabbie individuali e di raccogliere tutta feci all&#39;interno di ogni gabbia per 4 ore dopo gavage (Figura 2). </li><li> Omogeneizzare feci pool per ogni corvo in un omogeneizzatore proiettile frullatore con perle di vetro fino ad ottenere una consistenza omogenea. * Feci gallina sono più liquidi e possono essere mescolati facilmente. </li><li> Per ogni corvo, diluire 500 ml di omogenato fecale in 9,5 ml di PBS 1x per un volume totale di 10 ml. </li><li> Centrifugare la homogena fecaleTE per 15 min a 1400 xg ed estrarre il surnatante. </li><li> Per minimizzare il rischio di una infezione microbica secondaria, trattare surnatante con 1 ml di 100 unità / ml di penicillina e 100 pg / ml streptomicina (Invitrogen, NY) per 100 ml di omogenato poi posto sotto luce UV a temperatura ambiente per 20 min per ridurre rischio di contaminazione virale e batterica. Dopo l&#39;esposizione ai raggi UV, i campioni Sonicare in un Mp sonicatore 3000 a creare 70 per 30 secondi per distruggere la membrana di eventuali microbi rimanenti. </li></ol> 2. Topo inoculazione <ol><li> Casualmente destinare topi per i seguenti gruppi di trattamento (Tabella 1): <ol><li> Gruppo 1 - topi trattamento positivo inoculati per via intraperitoneale (IP) di 1 ml di omogenato fecale da corvi oralmente gavaged con 5ml infettato cervelli del mouse. </li><li> Gruppo 2 - Negativo topi inoculati trattamento IP con 1 ml di omogenato fecale da corvi oralmente gavaged con 5ml normali cervelli del mouse. </li><li> Diluire infected e normale omogeneizzato cervello di topo per i gruppi 3 e 4 a 1:100 w / v in PBS 1x. </li><li> Gruppo 3 - topi di controllo positivi inoculato IP con 1 ml di omogenato infetto topo-cervello. </li><li> Gruppo 4 - Negativo topi di controllo inoculati IP con 1 ml di omogenato normale mouse del cervello. </li></ol></li></ol><table border="1"><tbody><tr><td></td><td> Gruppo di trattamento </td><td> Numero di animali </td></tr><tr><td> Gruppo 1 </td><td> Scrapie + Crow Feci </td><td> 100 </td></tr><tr><td> Gruppo 2 </td><td> Scrapie - Crow Feci </td><td> 25 </td></tr><tr><td> Gruppo 3 </td><td> Cervello Scrapie + mouse </td><td> 10 </td></tr><tr><td> Gruppo 4 </td><td> Scrapie - cervello di topo </td><td> 5 </td></tr></tbody></table> . Tabella 1 Stato Scrapie di inoculo (+ positivo, negativo -) e il numero di animali utilizzati 7. <ol start="2"><li> Intraperitoneale inoculare il mouse: <ol><li> Scruff mouse dorsale collo di pelliccia con il pollice e l&#39;indice e delicatamente ruotare per esporre lato ventrale. </li><li> Elevare estremità posteriore del mouse in modo testa è leggermente inferiore. </li><li> Inserire 25 ago G 1 centimetro attraverso la pelle, 1 centimetro laterale della linea mediana, e di 1-2 cm anteriormente alla sacroiliaca usando una siringa-ago di bloccaggio. </li><li> Iniettare 1 ml trapiantare lentamente nel topo cavità del corpo. </li></ol></li></ol> 3. Monitoraggio mouse <ol><li> Controllo topi ogni giorno fino esprimono segni clinici di topo scrapie. I segni clinici possono includere: cifosi, atassia, coda rigida, la mancanza di governare, deperimento, e letargia. </li><li> Punteggio topi per ognuno dei 6 segni clinici quando segni visibili sono evidenti, in cui 0 = nessuna visibile, 1 = moderato, e 2 = grave. </li><li> Eutanasia topi quando punteggi totali giornalieri per ogni segno raggiungere ≥ 8 per 1 giorno, ≥ 6 ininterrottamente per 3 giorni, oa 365 giorni dopo l&#39;inoculazione (dpi). </li><li> Cervelli raccolto subito dopo euthanasia e conservare a -70 ° C. </li><li> Per confermare una diagnosi di scrapie, digerire campioni di cervello con 3 ml di un 50 mg / ml di soluzione di proteinasi-K (PK) diluito come segue: 3.1. microlitri PK, 12.5 ml di EDTA 500 mM, pH 8, 109.39 ml di PBS 1x per 30 min a 45 ° C, poi inattivare il PK aggiungendo 8 microlitri tampone di caricamento e incubando i campioni a 95 ° C per 5 min. Campioni caricare su un 12% SDS-PAGE gel, elettroforesi e trasferimento su membrana di PVDF e Immobilon blocco con 5% di latte scremato in 0,2% di Tween 20 in PBS per 1 ora a temperatura ambiente. Poi sonda con Bar224 anti-PrP anticorpo monoclonale coniugato con perossidasi di rafano, diluita in Superblocco, per 1 ora a temperatura ambiente. Risciacquare membrana per 1 ora con PBS-0.2% Tween 20. Per visualizzare, incubare Western Blot 5 minuti con il substrato chemiluminescente e immagine su un sistema di documentazione gel G-box. </li></ol>

<ol>
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Nessun conflitto di interessi dichiarati.

Dimostriamo una procedura per documentare il passaggio del materiale PrP Res attraverso il sistema digestivo di corvi. Abbiamo usato metodi convenzionali per determinare se i corvi hanno la possibilità di traslocare il materiale PrP Res per aree geografiche indenni. Altri hanno valutato la resistenza di PrP Res ai ruminanti 19-21 e roditori 22,23 fluidi digestivi, entrambi i quali non è riuscito a eliminarlo. La futura applicazione di queste tecniche dovrebbe essere applicato ad altri carnivori 24 in quanto potrebbero anche potenzialmente incontrare materiale PrP Res in carogne e trasportare questo materiale attraverso il paesaggio favorendo la diffusione delle malattie da prioni. Abbiamo aggiunto colorante alimentare blu al nostro uova strapazzate &#39;pseudo cervello&#39; materiale, fornendo un modo semplice per valutare il tempo approssimativo passaggio del cervello omogeneizzato mouse attraverso il tubo digerente di corvi. Consigliamo altri considerano questa tecnica per primo insPECT colore feci esistente di animali di studio e quindi utilizzare coloranti alimentari che è più contrastanti. Ciò consentirà per una facile identificazione del colore cibo materiale colorato che ha attraversato gli animali dello studio. Abbiamo scelto di utilizzare coloranti alimentari per stimare il tempo di passaggio, ma altri sono stati utilizzati pigmenti fluorescenti 25, ossido di ferro 26, marcatori colorati di plastica 27, e scaglie metalliche colorate (cioè scintillio) 28. Minimizzare la minaccia di una infezione microbica secondaria da feci d&#39;aria e correttamente inoculo topi sono punti di partenza fondamentali per gli studi mouse prioni che richiedono periodi di incubazione lunghi. Se uno di queste considerazioni sono violati, la mortalità precoce mouse è suscettibile di provocare. Un altro possibile strumento di indagine è la proteina di serie misfolding amplificazione ciclica o sPMCA per valutare campioni di feci d&#39;aria nel corso del tempo per stabilire quanto tempo dopo corvi sonda gastrica passano materiale infetto, conla necessità di un saggio biologico mouse. I recenti progressi nella LCMP analisi delle feci di Pulford, et al. Indicano che l&#39;amplificazione dei livelli di prioni minuti può essere rilevata nelle feci di 29 mammiferi, suggerendo LCMP potrebbe anche essere uno strumento utile per valutare le feci aviaria. Il biotest sui topi e l&#39;approccio sPMCA potrebbero essere utilizzati per valutare l&#39;infettività residua nelle feci di corvi gavaged con materiale CWD-infettati. Una linea di topi transgenici cervidized sarebbe necessario e intracerebrale contro inoculazione intraperitoneale darebbe risultati più rapidi. Spazzini aviaria, come corvi, avvoltoi, aquile, potrebbero svolgere un ruolo nella diffusione di malattie TSE, cioè CWD in Nord America. Queste specie possono consumare tessuti CWD-positive dalle carcasse malate o interiora (nel caso di cervidi cacciatori-ucciso) e traslocare materiale infettivo nelle loro feci di zone di libero CWD o popolazioni di cervidi. Come la pratica della somministrazione di grano cervidi, in natura o in cattivitàimpostazioni, attira corvi che può defecare sulla fonte di cibo e di essere inavvertitamente consumati dai cervidi, è quindi una pratica ad alto rischio (Vercauteren osservazione personale). Inoltre, anche se le probabilità di animali privi di CWD incontrando materiale PrP Res attraverso le feci casuale deposizione può essere bassa, le aree in cui si concentrano corvi e quindi le loro feci, come qui di seguito i siti dormitori comunali, potrebbero diventare delle aree ad alto rischio di trasmissione di malattie dal prioni sono così persistenti nell&#39;ambiente 30,31.

Encefalopatie spongiformi trasmissibili (TSE) sono fatali disturbi infettivi neurodegenerative che colpiscono la fauna selvatica, animali domestici, e gli esseri umani. L&#39;agente infettivo delle malattie TSE sembra essere isoforme mal ripiegate o patogeni (PrP Res) di proteine ​​prioniche 1. Malattie TSE animale includono sindrome del dimagrimento cronico (CWD) in cervo mulo (Odocoileus hemionus), cervi dalla coda bianca (Odocoileus virginianus), alce (Cervus elaphus), e alce (Alces alces); scrapie in ovini e caprini; encefalopatia spongiforme bovina ( BSE) nel bestiame domestico; encefalopatia visone in visone d&#39;allevamento; encefalopatia spongiforme felina nei gatti; esotico encefalopatia spongiforme ungulati in zoo esotico rumina della famiglia dei bovidi, e l&#39;encefalopatia spongiforme nei primati non umani 8. Il singolo malattia TSE umana, malattia di Creutzfeldt-Jakob variante è rara e pensato per essere acquisita da consumare PrP Res-Contamincibo ated 9. Allo stesso modo, la BSE può infettare l&#39;uomo se manzo contaminato è consumato 10. Di tutte le malattie TSE, scrapie e CWD sono gli unici due con epidemie autosufficienti e fonti per l&#39;infezione si presume essere da animale ad animale 2,3,11 così come da fonti ambientali 4-6. La ricerca suggerisce che la maggior parte delle malattie EST richiedono notevoli tempi di incubazione lunghi di esposizione eventi naturali di PrP Res alla manifestazione dei segni clinici 2-4,6,8 e delle specie apparenti ostacoli minimizzano, ma non eliminano il rischio di trasmissione interspecie 12-14 . Identificare i meccanismi per la diffusione del prione infettivo (PrP Res) materiale è estremamente importante per rispondere alle domande su come le malattie EST muovono attraverso il paesaggio. Indagini sperimentali hanno suggerito che gli insetti 15,16, pollame e suini 17, e corvi americani (Corvus brachyrhynchos) 7,18 sono vettori passivi o dispersori di materiale PrP Res. Passaggio di materiale PrP Res attraverso il sistema digestivo di corvi è stato recentemente documentato, dimostrando il ruolo che possono svolgere in dispersione delle malattie TSE 7. Questi risultati rendono plausibile che i corvi, uno spazzino, potrebbero incontrare, consumano, e trasportano materiale infetto tramite le feci deposizione, in aree indenni. Le procedure dimostriamo qui sono stati usati per documentare il passaggio del materiale PrP Res attraverso il sistema di digestione di corvi e notevolmente facilitare l&#39;applicazione di questi metodi per altri scavenger e modelli carnivoro specie-specifiche in ricerche future correlate. In questo studio sono stati utilizzati i metodi convenzionali per indagare su un mezzo non convenzionali di traffico di materiale PrP Res, che potrebbe contribuire alla diffusione e gravità complessiva della materia PrP Res.

<table border="0" cellpadding="0" cellspacing="0" width="545">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RML Chandler strain mouse-adapted scrapie</td>
			<td style="width:112px;">Rocky Mountain Laboratories</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RC57BL/6 mice</td>
			<td style="width:112px;">Hilltop Lab Animals</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">American crows</td>
			<td style="width:112px;">wild captured</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Phosphate buffered Saline</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">70011-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Sonicator</td>
			<td>Misonix</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Proteinase-K solution</td>
			<td style="width:112px;">Roche</td>
			<td align="right" style="width:137px;">3115887001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Loading buffer</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0007 and 0009</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Bis-tris SDS PAGE 12% gel</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0342</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Immobilon PVDF membrane</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">1SEQ00010</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Tween 20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:137px;">P2287</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bullet blender homogenizer</td>
			<td style="width:112px;">Braintree Scientific</td>
			<td style="width:137px;">BBX24B</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">2.3 mm Zirconia/silica beads</td>
			<td style="width:112px;">BioSpec Products</td>
			<td style="width:137px;">11079125Z</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bar224 anti-PrP monoclonal antibody</td>
			<td style="width:112px;">Cayman Chemical</td>
			<td align="right" style="width:137px;">10009035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Superblock</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td align="right" style="width:137px;">37517</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">chemiluminescent substrate</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">WBKLS0500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">G-box gel documentation system</td>
			<td style="width:112px;">Syngene</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

procedures for identifying infectious prions after passage through the digestive system of an avian species

Prionica infettiva (PrP Res) materiale è probabile che la causa della fatale, neurodegenerative l&#39;encefalopatia spongiforme trasmissibile (TSE) malattie 1. Trasmissione di malattie TSE, quali sindrome del dimagrimento cronico (CWD), si presume essere da animale ad animale 2,3 così come da fonti ambientali 4-6. Spazzini e carnivori hanno un potenziale di traslocare il materiale PrP Res attraverso il consumo e l&#39;escrezione di carogne CWD contaminati. Studi recenti hanno documentato il passaggio di materiale PrP Res attraverso il sistema digestivo di corvi americani (Corvus brachyrhynchos), un nordamericano tesoro comune 7. Descriviamo procedure utilizzate per documentare il passaggio del materiale PrP Res attraverso corvi americani. Crows sono stati gavaged con RML-ceppo di topi-adattato scrapie e le loro feci sono stati raccolti 4 ore dopo sonda gastrica. Feci Crow sono stati poi riuniti e iniettato per via intraperitoneale inTopi C57BL / 6. I topi sono stati monitorati giornalmente finché non espressi segni clinici di topo scrapie e sono stati successivamente sottoposti a eutanasia. Topi asintomatici sono stati monitorati fino a 365 giorni dopo l&#39;inoculazione. Western blot è stata condotta per confermare lo stato di malattia. I risultati hanno rivelato che i prioni rimangono contagiosi dopo aver viaggiato attraverso il sistema digestivo di corvi e sono presenti nelle feci, causando la malattia nei topi di prova.

Le procedure utilizzate dimostrano che il sistema digestivo dei corvi non elimina PrP Res infettività 4 ore dopo la sonda gastrica di omogenato cerebrale scrapie 7. Tutti i venti corvi che sono stati gavaged con materiale PrP Res successivamente trasmessi materiale PrP Res tramite le feci di topi. Topi malati sono stati identificati dalla manifestazione di segni topo-scrapie clinici e conferma malattia è stata completata da analisi Western Blot. Investigation del tempo di ritenzione del materiale ingerito da corvi rivelato tempo passaggio nel tratto alimentari per un corvo gavaged essere 4 ore, in base alla presenza di colorante nelle feci (Figura 1). Tutti gli feci sono stati raccolti con pipette monouso e raggruppati per ogni linea (Figura 2). Tossicità acuta da feci d&#39;aria non trattati si è verificato quando greggio surnatante fecale è stato iniettato in topi IP di test pilota. Trattando inoculare fecale con penicillina e Streptomycin, luce UV, e sonicazione abbiamo alleviato il problema. Tutti i topi inoculati con uno scrapie-positivo cervello di topo (9/9) o feci dal corvi scrapie inoculati (66 * / 84) hanno sviluppato segni clinici e risultati positivi mediante analisi Western blot (Figura 3), che illustra che la scrapie facilmente passare attraverso l&#39; sistema digestivo dei corvi e malattia causata. * Diciotto i topi sono morti entro 3 giorni di inoculazione, probabilmente a causa di tossicità. Tutti tranne uno (1/23) topi inoculati scrapie-negativi sono risultati negativi mediante analisi Western Blot. Noi ipotizziamo che questo mouse è stato inavvertitamente inoculato con scrapie-positivo d&#39;aria feci invece di feci da un corvo scrapie-negativo. <img alt="Figura 1" fo:content-width="6in" src="/files/ftp_upload/50853/50853fig1.jpg" width="500px" /> Figura 1. Crow trattenuto manualmente e oralmente gavaged con 5 ml di uovo intero mescolato con colorante blu (A) e Blue / feci verde macchiato di 4 ore dopo gavage (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50853/50853fig1highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . <img alt="Figura 2" fo:content-width="6in" src="/files/ftp_upload/50853/50853fig2.jpg" width="500px" /> Figura 2. Feci Crow collezione con pipetta prima di omogeneizzazione. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50853/50853fig2highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . <img alt="Figura 3" fo:content-width="6in" src="/files/ftp_upload/50853/50853fig3.jpg" width="500px" /> Figura 3. . Rappresentante SDS-PAGE Western Blot di cervello di topo da biotest NBH-normale omogenato di cervello di topo, scrapie-negativo, gabbia TX 4 - cervello di un topo inoculato con le feci di un corvo scrapie-inoculato. Con (+) e without (-). PK (proteinasi K) digestione <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50853/50853fig3highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> .

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Spazzini hanno un potenziale di traslocare infettive trasmissibili prioni dell&#39;encefalopatia spongiforme nelle loro feci in aree indenni. Abbiamo metodi di dettaglio utilizzati per determinare se i prioni scrapie mouse adattati rimangono contagiosi dopo il passaggio se il tratto digestivo di corvi americani (Corvus brachyrhynchos), un consumatore comune di animali morti.

Procedure per l&#39;identificazione Infettive prioni dopo il passaggio attraverso l&#39;apparato digerente di una specie aviaria

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

procedures-for-identifying-infectious-prions-after-passage-through

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.0K Views.  USDA.  Spazzini hanno un potenziale di traslocare infettive trasmissibili prioni dell'encefalopatia spongiforme nelle loro feci in aree indenni. Abbiamo metodi di dettaglio utilizzati per determinare se i prioni scrapie mouse adattati rimangono contagiosi dopo il passaggio se il tratto digestivo di corvi americani (Corvus brachyrhynchos), un consumatore comune di animali morti. 

Video: Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Identificare i geni disregolazione indotta da sarcoma di Kaposi associato Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Ricerca

Immunologia e infezioni

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Sviluppo di un marcatore negativo selezionabile per Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

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.

Visualizzazione virus Dengue attraverso Alexa Fluor Etichettatura

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

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Viral Determinazione Concentrazione Attraverso placca saggi: Sistemi overlay per mezzo di tradizionali e nuovi

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

Misure di sicurezza e procedure operative in un (A) BSL-4 di laboratorio: 1. livello di biosicurezza 4 Suit Laboratorio Suite entrata e di uscita Procedure

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

Misure di sicurezza e procedure operative in un (A) laboratorio BSL-4: 2. Pratiche generali

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

Misure di sicurezza e procedure operative in un (A) BSL-4 di laboratorio: 4. procedure di imaging medico

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

Misure di sicurezza e procedure operative in un (A) BSL-4 di laboratorio: 3. aerobiologia

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

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

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

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

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

Zika Virus System infettiva coltura cellulare e la<em&gt; In Vitro</em&gt; Effetto profilattico di interferoni

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

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolando i linfociti del sistema immunitario intestinale piccolo per Mouse

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...