Gli autori desiderano ringraziare alcuni membri del laboratorio per i loro preziosi suggerimenti per lo sviluppo di agroinfiltration di assemblaggio e test in vitro. Questo lavoro è stato finanziato da una borsa di studio presso la University of California. Questo studio è stato sostenuto in parte da un finanziamento della National Science Foundation (cbet-1144237).

1. Materiale vegetale <ol><li> Nicotiana benthamiana da utilizzare nel saggio incapsidamento dovrebbe essere a 4 fase foglie (piante settimane circa 3-4 anni). </li></ol> 2. La consegna e l&#39;espressione di componenti funzionali del genoma virale di piantare cellule agroinfiltration <ol><li> Giorno 1: Il ceppo di Agrobacterium (es. EH 105 o GV 3101) ospitante il pCass BMV-1 RNA, RNA BMV 2 e 3 1,2 BMV RNA sono coltivate su piastre di agar LB integrato con 50 mg / ml di kanamicina (seleziona per il vettore pCASS) e 100mg/ml di rifampicina (seleziona per l&#39;Agrobacterium). L&#39;incubazione viene effettuata a 28 ° C per due giorni. </li><li> Giorno 3: Seminare singola colonia dalla piastra di LB agar in 2 ml di brodo LB contenente 50 mg / ml di kanamicina e 100 mg / ml di rifampicina per 1 giorno a 28 ° C in agitatore orbitale impostata a 250 RPM. </li><li> 4 ° giorno: Inoculare 50 ml del supplemento brodo LBed a 50 mg / ml di kanamicina e 100 mg / ml di rifampicina, 10 mM MES pH 5,6 100 mM e acetosiringone con un ml di coltura in 250 ml di beuta per 16 ore a 28 ° C su agitatore orbitale a 250 rpm . </li><li> Giorno 5: Trasferire la cultura (600 OD, quando ha raggiunto 1,0) ad un tubo di Oak Ridge o sterile provetta Falcon e centrifugare per 10 minuti a 5000 rpm a 4 ° C. </li><li> Sciogliere il pellet in 10 ml di 10 mM MgCl 2 e centrifugare per 10 minuti a 5000 giri a 4 ° C, Ripetere questa fase una volta per assicurare la rimozione completa del antibiotico. </li><li> Sospendere il precipitato in 10 ml di tampone contenente 10 mM MgCl 2 e 10 mM MES (pH 5,6). </li><li> Misurare la OD 600 per ogni coltura di BMV 1 RNA, RNA 2 e 3 RNA e regolare a 0,1 OD 600 utilizzando 10 mM MgCl 2 e 10 mM MES (pH 5,6). </li><li> Miscelare 1 ml ciascuno tutti e tre 0.1 OD 600 sospensioni cultura. </li><li> Aggiungere 100 mM di acetosiringone, mix gentmente e mantenere la miscela indisturbato a temperatura ambiente per 3 ore. </li><li> Disegnare la sospensione cultura in 1 ml. siringa senza ago. </li><li> Infiltrati la sospensione di sopra della cultura nel lato abassiale di 2-3 ben espanse foglie di N. benthamiana (5 foglie di scena, le piante 3-4 settimane di età) premendo delicatamente la siringa alla metà della superficie della foglia abassiale. </li><li> Ripetere questa procedura per infiltrazioni altre foglie. </li><li> Mantenere le piante infiltrati nella casa verde con 24 ° C. </li><li> Harvest infiltrato foglie 3 o 4 giorni dopo l&#39;infiltrazione (dpi). </li></ol> 3. Purificazione di virioni BMV <ol><li> Raccogliere N. benthamiana lascia agroinfiltrated con una miscela contenente tutti e tre di tipo selvatico agrotransformants BMV. </li><li> Macinare foglie in un mortaio e pestello sterile con un volume uguale (w / v) di estrazione tampone BMV (0,5 M NaAc, 0,08 M ​​MgAc, pH 4,5 e volume 1/100 di β-mercaptoetanolo, che deve essere aggiunto appena prima dell&#39;uso) 3. Per massimizzare la resa virus, si raccomanda di aggiungere sabbia lavata acido (~ 0.5-1 g) che facilita la distruzione cellulare semplice macinazione ed efficiente. </li><li> Filtrare l&#39;estratto di foglie con 2-3 strati di panno di mussola e raccogliere la raffica. </li><li> Ancora macinare la porzione di trattenuta sul panno di mussola con un volume uguale di tampone di estrazione BMV al peso iniziale di materiale fogliare. </li><li> Ripetere la procedura di filtrazione con un panno di mussola. Questa procedura deve essere condotta a 4 ° C. </li><li> Trasferire la soluzione filtrato in un tubo da centrifuga e aggiungere uguale volume di pre-vortice e refrigerati cloroformio (o shake) per 5 minuti alla temperatura ambiente fino al colore della sospensione diventa verde chiaro. </li><li> Centrifugare emulsionato per 15 minuti a 12.000 RPM a 4 ° C. </li><li> Raccogliere la fase acquosa e mescolare per 30 minuti su agitatore magnetico per rimuovere ogni residuo di cloroformio. </li><li> Trasferire la sospensione in una provetta sterile ultracentrifuga. </li><li&gt; Underlay 1 ml. 10% di saccarosio (preparato in tampone di estrazione virus) come un cuscino 3. </li><li> Centrifugare a 30.000 rpm per 3 ore in una ultracentrifuga Beckman a 4 ° C. </li><li> Eliminare il supernatante e sospendere il pellet nel volume desiderato di tampone sospensione BMV (per preparare tampone diluita sospensione BMV tampone di estrazione a 1/10 con acqua distillata sterile). </li><li> La preparazione virione parzialmente purificato dal passo precedente viene sottoposto a centrifugazione in gradiente di 10-40% di densità di saccarosio per 150 min a 28000 rpm a 4 ° C. </li><li> Raccogliere il gruppo virus manualmente o da un frazionatore. La band virus apparirà blu sotto illuminazione a luce bianca. </li><li> Diluire la soluzione di saccarosio contenente campione di virus almeno il 50% con tampone virus sospensione. </li><li> Centrifugare diluita virus contenente una soluzione di saccarosio per 3 ore a 30.000 rpm in una ultracentrifuga Beckman a 4 ° C. </li><li> Infine sospendere il altamente purificate pellet in un volume desiderato di virci sospensione tampone. </li><li> Misurare la concentrazione del virione (mg / ml) mediante spettrofotometro a OD 260. </li></ol><img alt="Equazione 1" src="/files/ftp_upload/3645/3645eq1.jpg" /> Nota: in base al contenuto di RNA, il coefficiente di estinzione per la BMV è 5 4. <ol start="19"><li> Verificare la purezza di virioni con la visualizzazione in TEM (Fig. A) </li></ol> 4. Preparazione di subunità proteiche del capside di assemblaggio in vitro <ol><li> Preparare 1 litro di tampone di dissociazione 1x virus (0,5 M di CaCl 2, 50 mM Tris HCl, pH 7,5, 1,0 mM EDTA, 1,0 mM DTT e 0,5 mM PMSF) 5. </li><li> Preparare membrana di dialisi secondo Sambrook et al 6. </li><li> Dispensare concentrazione richiesta del virus purificato in un sacchetto di dialisi. </li><li> Posizionare il sacchetto contenente dialisi virus in un becher contenente il virus 1x tampone di dissociazione. </li><li> Dializzare 24 ore a 4 ° C whille e agitazione. </li><li> Raccogliere la soluzione dalla sacca dialisi dopo 24 ore e centrifugare a 12.000 xg per 15 min a 4 ° C. Ciò separare il RNA BMV dai virioni dissociate. </li><li> Raccogliere il supernatante e centrifugare a 35.000 rpm in centrifuga Beckman per 3 ore a 4 ° C a pellet un non-dissociato particella virale. </li><li> Raccogliere il surnatante. </li><li> In questa fase è indispensabile per rimuovere eventuali contaminanti RNA virioni dissociate da subunità proteiche del capside. A tale scopo, il supernatante dal passaggio 8 devono essere sottoposti ad un altro ciclo di notte su riassemblaggio di subunità proteiche capside senza aggiungere alcun RNA (in tampone RNA 1x assemblaggio, vedi sotto) seguito da una centrifugazione ad alta velocità (30.000 rpm per 3 ore a 4 ° C) a pellet eventuali virioni assemblati. Dopo questa fase di rimontaggio, il surnatante contenente subunità proteiche cappotto sarebbe priva di RNA residui contaminanti. Tuttavia, per assicurare questo, è consigliabile eseguire un ulteriore vitro rimontaggio con subunità solo proteina di rivestimento utilizzando RNA tampone di montaggio 1x. Dopo una notte rimontaggio, la preparazione deve essere privo di virioni assemblati (verificare con TEM). </li><li> Determinare la concentrazione di subunità proteiche capside misurando OD 254 e 280 nm o da altri metodi come saggio Bradford. </li><li> Eseguire 12-15% SDS-PAGE seguita da Western blot per determinare l&#39;integrità delle subunità dissociate proteina del capside. </li></ol> 5. In vitro montaggio di RNA contenente virioni <ol><li> Preparare trascritti di RNA di essere ri-assemblati in virioni e calcolare la concentrazione 7. </li><li> Mescolare subunità proteina del capside e RNA trascritto in un rapporto di 1:5 (peso / peso) 5. </li><li> Versare il composto sopra un sacchetto di dialisi e adeguatamente sicuro per evitare eventuali perdite. </li><li> Preparare 1000 ml. di RNA 1x tampone di montaggio (50 mM NaCl, 50 mM Tris-HCl, pH 7,2, 10 mM KCl, 5 mM MgCl 2, 1,0 mM DTT) 5. </li><li> Posizionare il sacchetto di dialisi contenente la miscela di reazione contro il tampone di montaggio 1x nel bicchiere e mescolate. </li><li> Dializzare il riassemblaggio reazione a 4 ° C per 24 ore di agitazione. </li><li> Raccogliere la miscela dalla sacca dialisi dopo 24 ore e aggiungere 1,5 ml di tampone RNA montaggio. </li><li> Passare tale miscela attraverso una colonna Centricon-100 mediante centrifugazione a bassa velocità (2.000 xg) per 30 minuti. </li><li> Lavare la colonna volta con 1,5 ml di tampone gruppo a 4 ° C per 30 min. </li><li> Ripetere il passaggio precedente di lavaggio due volte. </li><li> Infine, eluire i ri-assemblati virioni mediante centrifugazione a 10.000 g a 4 ° C per 5 min. </li><li> Stimare la concentrazione virione a OD 260 nm. </li></ol> 6. Fabbricazione di ottiche Ghosts virali (OVGs) <ol><li> Aggiungere verde indocianina (ICG) 8 per la proteina purificata capside a rapporto di concentrazione desiderata (il rapporto in peso di proteina verde indocianina e capside usato in questa relazione è stata 1:10) uno<html> d mescolare accuratamente pipettando. </li><li> Dializzare la proteina del capside e soluzione ICG contro il tampone gruppo OVG (1 M NaCl, 50 mM NaAc, 1 mM EDTA, e 1 mM DTT, pH4.8) a 4 ° C per 24 ore. </li><li> Dopo 24 ore, raccogliere la soluzione dalla sacca di dialisi (12 kDa dimensione dei pori) e centrifugare a 90.000 rpm per 1 ora a 4 ° C (Fig. B) </li><li> Rimuovere il surnatante e sospendere la risospeso nel tampone sospensione BMV vortexando o permettendogli di sospensione per sé in sospensione tampone BMV notte a 4 ° C. </li><li> Verificare la morfologia del OVGs da TEM (Fig. C). </li><li> Misurare l&#39;assorbanza da 240 nm a 950 nm con spettrofotometro (Cary 50, Varian Inc.), la presenza di ICG è confermata dalla firma picchi di assorbanza circa 700 nm e 790 nm 8. (Fig. D) </li><li> I tipici picchi ICG fluorescenza a ~ 700 nm e ~ 800 nm può essere visto su 620 nm di eccitazione della soluzione OVG (Fig. D) con spettrofluorimetro (Fluorolog 3, Jobin-Yvon). </li></ol>_title &quot;&gt; 7. rappresentativi Risultati <img alt="Figura A" src="/files/ftp_upload/3645/3645figA.jpg" /> Figura A. immagine TEM di colorazione negativa BMV virioni purificati da N. benthamiana agroinfiltrated con un misto di tutte e tre le agroconstructs wild type BMV (barra della scala = 100 nm). <img alt="Figura B" src="/files/ftp_upload/3645/3645figB.jpg" /> Figura B. di pellet in vitro assemblato ICG OVGs contenenti dopo centrifugazione ad alta velocità. <img alt="Figura C" src="/files/ftp_upload/3645/3645figC.jpg" /> Figura C. immagine TEM di colorazione negativa OVGs contenenti ICG (barra della scala = 100 nm). <img alt="Figura D" src="/files/ftp_upload/3645/3645figD.jpg" /> Figura D. Assorbanza (Top) e la fluorescenza (in basso) spettri di OVGs. La lunghezza d&#39;onda di eccitazione utilizzato per OVG emissione wdi 620 nm.

<ol>
	<li>Annamalai, P., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-independent+expression+of+genome+components+and+capsid+protein+of+brome+mosaic+virus+in+planta:+a+functional+role+for+viral+replicase+in+RNA+packaging.">Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging.</a> Virology. 338, 96-111 (2005).</li><li>Annamalai, P., Rofail, F., Demason, D. A., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-coupled+packaging+mechanism+in+positive-strand+RNA+viruses:+synchronized+coexpression+of+functional+multigenome+RNA+components+of+an+animal+and+a+plant+virus+in+Nicotiana+benthamiana+cells+by+agroinfiltration.">Replication-coupled packaging mechanism in positive-strand RNA viruses: synchronized coexpression of functional multigenome RNA components of an animal and a plant virus in Nicotiana benthamiana cells by agroinfiltration.</a> J. Virol. 82, 1484-1495 (2008).</li><li>Annamalai, P., Rao, A. L., Foster, N., Johansen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Virology Protocols. 451, 251-264 (2008).</li><li>Lane, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bromoviruses.">The bromoviruses.</a> Adv. Virus Res. 19, 151-220 (1974).</li><li>Choi, Y. G., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+studies+on+bromovirus+capsid+protein.+VII.+Selective+packaging+on+BMV+RNA4+by+specific+N-terminal+arginine+residuals.">Molecular studies on bromovirus capsid protein. VII. Selective packaging on BMV RNA4 by specific N-terminal arginine residuals.</a> Virology. 275, 207-217 (2000).</li><li>Sambrrok, J., Russel, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. A Laboratory Manual. , (2001).</li><li>Dreher, T. W., Rao, A. L., Hall, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+in+vivo+of+mutant+brome+mosaic+virus+RNAs+defective+in+aminoacylation.">Replication in vivo of mutant brome mosaic virus RNAs defective in aminoacylation.</a> J. Mol. Biol. 206, 425-438 (1989).</li><li>Jung, B., Rao, A. L., Anvari, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+nano-constructs+composed+of+genome-depleted+brome+mosaic+virus+doped+with+a+near+infrared+chromophore+for+potential+biomedical+applications.">Optical nano-constructs composed of genome-depleted brome mosaic virus doped with a near infrared chromophore for potential biomedical applications.</a> A.C.S. Nano. 5, 1243-1252 (2011).</li><li>Voinnet, O., Lederer, C., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+viral+movement+protein+prevents+spread+of+the+gene+silencing+signal+in+Nicotiana+benthamiana.">A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana.</a> Cell. 103, 157-167 (2000).</li><li>Wroblewski, T., Tomczak, A., Michelmore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Agrobacterium-mediated+transient+assays+of+gene+expression+in+lettuce,+tomato+and+Arabidopsis.">Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis.</a> Plant Biotechnol. J. 3, 259-273 (2005).</li></ol>

Non abbiamo nulla da rivelare.

Agroinfiltration approccio qui presentate può essere ampiamente applicabile a un&#39;ampia gamma di virus vegetali. Un elemento caratterizzante di questo approccio è sincronizzata consegna di molteplici agroconstruct alla stessa cella, un grave inconveniente comunemente associato solitamente usata inoculazione meccanica di virus vegetali. In vivo e in vitro studi di assemblaggio utilizzando virus del mosaico del bromo come modello può essere condotto in modo efficiente seguono alcuni suggerimenti. (...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> MES sali di sodio </td><td> Sigma-Aldrich </td><td> M2993 </td></tr><tr><td> Verde di indocianina </td><td> Sigma-Aldrich </td><td> I2633 </td></tr><tr><td> Beckman ultracentrifuga </td><td> Beckman Coulter </td><td> Modello: L8-70M </td></tr><tr><td> Centricon-100 colonna </td><td> Millipore </td><td> YM-100 </td></tr><tr><td> Spettrofotometro </td><td> Cary 50, Varian Inc. </td><td> Codice 10068900 </td></tr><tr><td> Spettrofluorimetro </td><td> Fluorolog 3, Jobin-Yvon. </td><td> Codice FL3-21 </td></tr></tbody></table>

simple and robust in vivo and in vitro approach for studying virus assembly

In positivo-virus con RNA genomi senso patogeno per gli esseri umani, animali e piante, incapsidazione progenie in virioni maturi e stabili è una fase cardinale durante la creazione di infezione in un determinato host. Di conseguenza, lo studio delle incapsidazione decifra le informazioni riguardanti il ​​know-how del meccanismo di regolazione di assemblaggio del virus per formare virioni infettivi. Tale informazione è di vitale importanza nella formulazione di nuovi metodi di arginare diffusione del virus e il controllo delle malattie. Incapsidamento Virus possono essere studiati in vivo e in vitro. Incapsidazione Genoma in vivo è un processo altamente regolato selettiva che le interazioni macromolecolari e compartimentazione subcellulare. Pertanto, lo studio porta a sezionare gli eventi che comprendono incapsidazione virus in vivo avrebbe fornito le conoscenze di base per comprendere come i virus proliferano e assemblare. Recentemente incapsidazione in vitro è stata sfruttata per la ricerca nel campo della biomedica imaging e applicazioni terapeutiche. Non capsulati virus delle piante stanno molto più avanti nella joint venture di incapsidazione in vitro del materiale caricato negativamente straniera. Virus del mosaico Brome (BMV), privi di involucro multicomponente RNA virus patogeno per le piante, è stato utilizzato come sistema modello per lo studio confezionamento genoma in vivo e in vitro. Per i saggi di incapsidamento in Nicotiana benthamiana, mediata da Agrobacterium espressione transiente, si riferiscono come agroinfiltration, è una tecnica efficiente e robusto per la consegna sincronizzata e l&#39;espressione di più componenti alla stessa cella. In questo approccio, una sospensione di cellule di Agrobacterium tumefaciens che trasportano binari vettori plasmidici che trasportano cDNA di mRNA desiredviral è infiltrata nella foglia spazio intercellulare withina utilizzando niente di più sofisticato di una siringa monouso da 1 ml (senza ago). Questo processo determina il trasferimento di inserto di DNA nelle cellule vegetali, il T-DNAInserto rimane transitoriamente nel nucleo e viene quindi trascritto dall&#39;host polimerasi II, con conseguente espressione transiente. La trascrizione mRNA risultante (livellata e poliadenilato) viene poi esportato nel citoplasma per la traduzione. Dopo circa 24 a 48 ore di incubazione, sezioni di foglie infiltrati possono essere prelevati per le analisi biochimiche microscopyor. Agroinfiltration permette grandi numeri (centinaia di migliaia) di cellule trasfettate da simultaneamente. Per incapsidazione in vitro, virioni purificati di BMV sono dissociate in proteina del capside di dialisi contro tampone di cloruro di calcio dissociazione contenente seguita dalla rimozione di RNA e non-virioni dissociate mediante centrifugazione. Genome subunità proteiche del capside impoverito vengono poi riassemblati con desiderati componenti del genoma virale o non virale componenti come indocianina colorante.

Watch this Scientific Journal Video about Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly at JoVE.com

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

Un modo semplice, efficace e robusto per sincronizzare l&#39;erogazione di più componenti virali di cellule vegetali tramite<em&gt; Agrobacterium</em&gt;-Mediata espressione transiente è descritto. Questo approccio è suscettibile per studiare la replicazione, seguita da incapsidamento<em&gt; In vitro</em&gt; Riassemblaggio di non virali componenti nel genoma impoverito ottici fantasmi virali adatti per applicazioni biomediche.

Semplice e robusta In vivo E In vitro</emApproccio> per lo Studio Assemblea Virus

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

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Immunology and Infection

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

12.1K Views.  University of California, Riverside. Un modo semplice, efficace e robusto per sincronizzare l'erogazione di più componenti virali di cellule vegetali tramite Agrobacterium-Mediata espressione transiente è descritto. Questo approccio è suscettibile per studiare la replicazione, seguita da incapsidamento In vitro Riassemblaggio di non virali componenti nel genoma impoverito ottici fantasmi virali adatti per applicazioni biomediche.

Video: Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

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 ...

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Immunologia e infezioni

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

In Vivo Imaging Systems (IVIS) il rilevamento di un neuro-invasiva Virus encephalitic

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; Modello per lo studio delle interazioni tra l&#39;uomo di sangue intero e l&#39;endotelio

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

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

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

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

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

In Analisi Vitro di Myd88 mediata Cellular Immune Response to West Nile virus mutante Strain Infezione

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

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

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

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

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 ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

In Vitro Smontaggio del virus influenzale A capsids per centrifugazione in gradiente

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

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 ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Metodi in vitro per il confronto obiettivo vincolante e CDC induzione Tra terapeutiche Anticorpi: Applicazioni nel Analisi Biosimilarity

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Un metodo semplice ed efficace per stabilire NK e linee a cellula T da pazienti con infezione attiva cronica del Virus di Epstein - Barr

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Base di un semplice flusso Cytometry Assay per determinare In Vitro dell'anticorpo dipendente valorizzazione del Virus di febbre rompiossa utilizzando siero convalescente Zika Virus

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Semplice e robusta In vivo E In vitro per lo Studio Assemblea Virus