Les auteurs tiennent à remercier plusieurs membres du laboratoire pour leurs précieuses suggestions dans le développement de agroinfiltration et dans des essais in vitro de montage. Ce travail a été financé par une subvention de l&#39;Université de Californie. Cette étude a été soutenue dans les régions par une subvention de la National Science Foundation (CBET-1144237).

1. Le matériel végétal <ol><li> Nicotiana benthamiana à être utilisés dans le test d&#39;encapsidation doivent être au stade de 4 feuilles (plantes semaine environ 3-4 ans). </li></ol> 2. La livraison et l&#39;expression de composants fonctionnels du génome viral à planter des cellules par agroinfiltration <ol><li> Jour 1: La souche d&#39;Agrobacterium (par exemple EH 105 ou GV 3101) hébergeant l&#39;ARN pCass-BMV 1, 2 et BMV ARN BMV ARN 3 1,2 sont cultivés sur des plaques d&#39;agar LB supplémenté avec 50 mg / ml de kanamycine (sélectionne pour le vecteur pCASS) et de la rifampicine 100mg/ml (sélectionne pour la Agrobacterium). L&#39;incubation est effectuée à 28 ° C pendant deux jours. </li><li> Jour 3: Inoculer seule colonie de la plaque de gélose LB dans 2 ml de bouillon LB contenant 50 mg / ml de kanamycine et 100 mg / ml de rifampicine pendant 1 jour à 28 ° C dans un agitateur orbital réglé à 250 RPM. </li><li> Jour 4: Inoculer 50 ml de bouillon LB le supplémented avec 50 mg / ml de kanamycine et 100 mg / ml de rifampicine, 10 mM MES pH 5,6 et 100 mM acétosyringone avec un ml de culture dans un 250 ml de la fiole Erlenmeyer de 16 heures à 28 ° C sur un agitateur orbital à 250 RPM . </li><li> Jour 5: Transfert de la culture (quand DO 600 a atteint 1,0) à un tube Oak Ridge ou stériles tube Falcon et centrifuger pendant 10 minutes à 5000 rpm à 4 ° C. </li><li> Dissoudre le culot dans 10 ml de 10 mM MgCl 2 et centrifuger pendant 10 minutes à 5000 rpm à 4 ° C, Répéter cette étape une fois de plus pour assurer l&#39;élimination complète de l&#39;antibiotique. </li><li> Suspendre le culot dans 10 ml de tampon contenant 10 mM MgCl 2 et 10 mM de MES (pH 5,6). </li><li> Mesurer la DO 600 pour chaque culture de BMV ARN 1, 2 et ARN ARN 3 et ajuster à 0,1 DO 600 en utilisant 10 mM de MgCl 2 et 10 mM MES (pH 5,6). </li><li> Mélanger 1 ml chacun trois tous les 0,1 suspensions OD culture 600. </li><li> Ajouter 100 mM de acétosyringone, mélange gentment et de garder intact le mélange à température ambiante pendant 3 heures. </li><li> Dessiner la suspension de la culture dans 1 ml. seringue sans aiguille. </li><li> Infiltrez la suspension de culture ci-dessus dans le côté abaxial de 2-3 bien élargis feuilles de N. benthamiana (5 feuilles de scène, les plantes semaine 3-4 ans) en appuyant doucement sur ​​la seringue à la moitié de la surface abaxiale de la feuille. </li><li> Répétez cette procédure pour l&#39;infiltration d&#39;autres feuilles. </li><li> Gardez les plantes infiltrés dans la maison verte à 24 ° C. </li><li> Récolte infiltré feuilles de 3 à 4 jours après l&#39;infiltration (dpi). </li></ol> 3. Purification des virions BMV <ol><li> Recueillir N. benthamiana laisse agroinfiltrated avec un mélange contenant toutes les trois de type sauvage agrotransformants BMV. </li><li> Meuler feuilles dans un pilon stérile et mortier avec un volume égal (p / v) d&#39;extraction BMV tampon (0,5 M NaAc; 0,08 M ​​MgAc, pH 4,5 et volume de 1/100 de β-mercaptoéthanol, qui doit être ajouté juste avant l&#39;emploi) 3. Pour maximiser le rendement de virus, il est recommandé d&#39;ajouter du sable lavé à l&#39;acide (~ 0,5-1 g) qui facilite la désintégration cellulaire facile de broyage et efficace. </li><li> Filtrer l&#39;extrait de feuilles par 2-3 couches de mousseline et de recueillir la bourrasque. </li><li> Encore une fois broyer la partie retenue sur un tissu en mousseline avec un volume égal de tampon d&#39;extraction BMV au poids initial de matériel foliaire. </li><li> Répétez la procédure de filtration d&#39;utiliser un chiffon de mousseline. Ces mesures devraient être effectuées à 4 ° C. </li><li> Transférer la solution de filtrat dans un tube de centrifugeuse et ajouter un volume égal de pré-réfrigérés chloroforme et vortex (ou secouer) pendant 5 minutes à la température ambiante jusqu&#39;à ce que la couleur de la suspension devient vert clair. </li><li> Centrifuger la solution émulsionnée pendant 15 minutes à 12000 rpm à 4 ° C. </li><li> Recueillir la phase aqueuse et remuer pendant 30 minutes sur un agitateur magnétique pour retirer tout le chloroforme résiduel. </li><li> Transférer la suspension à un tube d&#39;ultracentrifugation stérile. </li><li&gt; Sous-couche de 1 ml. 10% de saccharose (préparée dans un tampon d&#39;extraction du virus) en tant que coussin 3. </li><li> Centrifuger à 30000 rpm pendant 3 heures dans une ultracentrifugeuse Beckman à 4 ° C. </li><li> Jeter le surnageant et suspendre le culot dans de volume souhaité de tampon de suspension BMV (pour préparer un tampon de suspension de tampon d&#39;extraction dilué BMV à 1/10 avec de l&#39;eau distillée stérile). </li><li> La préparation virion partiellement purifié provenant de l&#39;étape ci-dessus est soumis à une centrifugation saccharose gradient de densité de 10-40% pour 150 min à 28 000 tpm à 4 ° C. </li><li> Recueillir le bande de virus, soit manuellement ou par une colonne de fractionnement. La bande de virus apparaissent en bleu sous éclairage en lumière blanche. </li><li> Diluer la solution contenant du saccharose échantillon de virus d&#39;au moins 50% avec un tampon suspension virale. </li><li> Centrifuger dilué solution de saccharose contenant le virus pendant 3 heures à 30.000 tours par minute dans une ultracentrifugeuse Beckman à 4 ° C. </li><li> Enfin suspendre le virus de culot hautement purifié dans un volume souhaité de virnous suspension tampon. </li><li> Mesurer la concentration du virion (mg / ml) en utilisant un spectrophotomètre à une DO 260. </li></ol><img alt="L&#39;équation 1" src="/files/ftp_upload/3645/3645eq1.jpg" /> Nota: Basé sur la teneur en ARN, le coefficient d&#39;extinction pour BMV est 5 4. <ol start="19"><li> Vérifiez la pureté de virions en regardant sous TEM (Fig. A) </li></ol> 4. Préparation de sous-unités protéiques capside pour assemblage in vitro <ol><li> Préparer 1 litre de 1x virus tampon de dissociation (0,5 M CaCl 2, 50 mM Tris HCl, pH 7,5, 1,0 mM d&#39;EDTA, 1,0 mM de DTT et 0,5 mM PMSF) 5. </li><li> Préparer membrane de dialyse selon Sambrook et al 6. </li><li> Répartir la concentration nécessaire de virus purifié dans un sac de dialyse. </li><li> Placez le sac de dialyse contenant le virus dans un bêcher contenant le virus 1x tampon de dissociation. </li><li> Dialyser 24 heures à 4 ° C tout en recueillante agitation. </li><li> Recueillir la solution de la poche de dialyse après 24 heures et centrifuger à 12000 xg pendant 15 min à 4 ° C. Ce sera séparer l&#39;ARN des virions BMV dissociées. </li><li> Recueillir le surnageant et centrifuger à 35000 rpm dans une centrifugeuse Beckman pendant 3 heures à 4 ° C à granulés toute particule virale non dissociée. </li><li> Recueillir le surnageant. </li><li> A ce stade, il est impératif d&#39;éliminer toutes les ARN des virions contaminant à partir dissociées sous-unités protéiques de capside. Pour ce faire, le surnageant de l&#39;étape 8 devraient être soumis à une autre ronde de nuit au-dessus du remontage de sous-unités protéiques de capside, sans ajouter de l&#39;ARN (l&#39;ARN 1x assemblage de tampons, voir ci-dessous) suivie d&#39;une centrifugation à grande vitesse (30.000 rpm pendant 3 heures à 4 ° C) à culot des virions assemblés. Après cette étape le remontage, le surnageant contenant sous-unités protéiques manteau serait libre de tout ARN contaminantes résiduelles. Toutefois, pour ce faire, il est conseillé d&#39;effectuer une supplémentaire en vitro remontage avec seulement l&#39;aide de sous-unités protéiques manteau tampon ARN assemblage 1x. Après le remontage pendant la nuit, la préparation doit être exempte de virions assemblés (vérifier à l&#39;aide TEM). </li><li> Déterminer la concentration de sous-unités protéiques de capside par la mesure à une DO 254 et 280 nm ou par d&#39;autres méthodes telles que dosage de Bradford. </li><li> Effectuer 12-15% SDS-PAGE suivie par western blot pour déterminer l&#39;intégrité des sous-unités dissociées protéine de capside. </li></ol> 5. In vitro l&#39;assemblage de l&#39;ARN des virions contenant <ol><li> Préparer des transcrits d&#39;ARN à être ré-assemblés dans les virions et de calculer la concentration 7. </li><li> Mélanger sous-unités protéiques de capside et l&#39;ARN transcrit à un ratio de 1:5 (poids / poids) 5. </li><li> Répartir le mélange ci-dessus pour un sac de dialyse et de sécuriser convenablement pour éviter toute fuite. </li><li> Préparer 1000 ml. de l&#39;ARN 1x tampon d&#39;assemblage (50 mM de NaCl, 50 mM Tris-HCl, pH 7,2, 10 mM de KCl, 5 mM MgCl 2, 1,0 mM DTT) 5. </li><li> Placez le sac de dialyse contenant le mélange réactionnel contre un tampon de montage 1x dans le bécher et agiter. </li><li> Dialyser la réaction ré-assemblage à 4 ° C pendant 24 heures par agitation douce. </li><li> Collecter le mélange de la poche de dialyse après 24 heures et ajouter 1,5 ml de l&#39;ARN ensemble tampon. </li><li> Passez ce mélange à travers une colonne Centricon-100 par centrifugation à basse vitesse (2000 xg) pendant 30 minutes. </li><li> Laver la colonne une fois avec 1,5 ml de tampon d&#39;assemblage à 4 ° C pendant 30 min. </li><li> Répétez l&#39;étape de lavage ci-dessus deux fois. </li><li> Enfin, éluer les virions re-assemblés par centrifugation à 10.000 g à 4 ° C pendant 5 min. </li><li> Estimer la concentration virion à OD 260 nm. </li></ol> 6. Fabrication de fantômes optiques virales (OVGs) <ol><li> Ajouter au vert d&#39;indocyanine (ICG) 8 à la protéine de capside purifié à rapport de concentration désiré (le rapport pondéral de la protéine d&#39;indocyanine vert et capside utilisé dans ce rapport est 01:10) une<html> d mélanger par pipetage. </li><li> Dialyser la solution de protéine de capside et ICG contre le tampon de montage OVG (1 M de NaCl; 50 NaAc mM; EDTA 1 mM, et 1 mM, DTT pH4.8) à 4 ° C pendant 24 heures. </li><li> Après 24 heures, recueillir la solution de la poche de dialyse (12 kDa taille des pores) et centrifuger à 90000 rpm pendant 1 heure à 4 ° C (fig. B) </li><li> Eliminer le surnageant et suspendre le culot dans un tampon de suspension par vortex BMV ou lui permettant de suspendre par lui-même dans un tampon de suspension BMV nuit à 4 ° C. </li><li> Vérifiez la morphologie de OVGs par TEM (Fig. C). </li><li> Mesurer l&#39;absorbance de 240 nm à 950 nm en utilisant un spectrophotomètre (Cary 50, Varian Inc), la présence de l&#39;ICG est confirmée par la signature pics d&#39;absorption à 700 nm environ et 790 nm 8. (Fig. D) </li><li> Les pics de fluorescence typiques du GIC à ~ 700 nm et environ 800 nm peut être vu sur 620 nm d&#39;excitation de la solution OVG (Fig. D) en utilisant spectrofluorimètre (Fluorolog 3, Jobin-Yvon). </li></ol>_title &quot;&gt; Résultats représentant 7. <img alt="Figure A" src="/files/ftp_upload/3645/3645figA.jpg" /> Figure A. TEM image de virions BMV coloration négative purifiés à partir de N. benthamiana agroinfiltrated avec un mélange de tous les trois sauvages agroconstructs BMV type (barre d&#39;échelle = 100 nm). <img alt="Figure B" src="/files/ftp_upload/3645/3645figB.jpg" /> Figure B. Pellet in vitro assemblé ICG OVGs contenant la suite de centrifugation à grande vitesse. <img alt="Figure C" src="/files/ftp_upload/3645/3645figC.jpg" /> Figure C. TEM image de coloration négative ICG OVGs contenant (barre d&#39;échelle = 100 nm). <img alt="Figure D" src="/files/ftp_upload/3645/3645figD.jpg" /> Figure D. Absorbance (Haut) et la fluorescence (en bas) des spectres de OVGs. La longueur d&#39;onde d&#39;excitation utilisée pour OVG émissions wque 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>

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Approche agroinfiltration présentée ici peut largement s&#39;appliquer à un large éventail de virus de plantes. Un des traits caractéristiques de cette approche est synchronisé livraison de multiples agroconstruct à la cellule, un même inconvénient majeur souvent associée à l&#39;inoculation mécanique couramment utilisés des virus des plantes. In vivo et dans les études d&#39;assemblage in vitro utilisant des virus de la mosaïque du brome comme un modèle peut être menée effic...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Le numéro de catalogue </td></tr><tr><td> Les sels de sodium MES </td><td> Sigma-Aldrich </td><td> M2993 </td></tr><tr><td> Le vert d&#39;indocyanine </td><td> Sigma-Aldrich </td><td> I2633 </td></tr><tr><td> Ultracentrifugeuse Beckman </td><td> Beckman Coulter </td><td> Modèle: L8-70M </td></tr><tr><td> Centricon-100 colonne </td><td> Millipore </td><td> YM-100 </td></tr><tr><td> Spectrophotomètre </td><td> Cary 50, Varian Inc </td><td> Numéro de la pièce 10068900 </td></tr><tr><td> Spectrofluorometer </td><td> Fluorolog 3, Jobin-Yvon. </td><td> Numéro de la pièce FL3-21 </td></tr></tbody></table>

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

Dans les virus avec des génomes ARN de sens positif pathogènes pour les humains, les animaux et les plantes, d&#39;encapsidation descendance dans les virions matures et stable est une phase cardinal lors de l&#39;établissement de l&#39;infection chez un hôte donné. Par conséquent, l&#39;étude de l&#39;encapsidation déchiffre les informations concernant le savoir-faire de l&#39;assemblage du virus mécanisme de régulation pour former des virions infectieux. Cette information est essentielle dans la formulation de nouvelles méthodes de freiner la propagation du virus et de lutte contre les maladies. Encapsidation du virus peut être étudiée in vivo et in vitro. Encapsidation du génome in vivo est un processus hautement régulé sélective impliquant des interactions macromoléculaires et de compartimentation subcellulaire. Par conséquent, l&#39;étude menant à disséquer les événements englobant l&#39;encapsidation du virus in vivo permettrait connaissances de base pour comprendre comment les virus prolifèrent et à assembler. Récemment, dans l&#39;encapsidation in vitro a été exploitée pour la recherche dans le domaine de la recherche biomédicale imaGing et les applications thérapeutiques. Les virus des plantes non-enveloppés situer loin devant dans l&#39;aventure de l&#39;encapsidation in vitro dans de la matière chargée négativement étrangère. Virus de la mosaïque Brome (BMV), un agent pathogène non-enveloppé multicomposant virus à ARN pour les plantes, a été utilisé comme un système modèle pour l&#39;étude du génome dans l&#39;emballage vivo et in vitro. Pour les tests d&#39;encapsidation dans Nicotiana benthamiana, au moyen d&#39;Agrobacterium expression transitoire, reportez-vous comme agroinfiltration, est une technique efficace et robuste pour la livraison synchronisée et l&#39;expression de plusieurs composants à la même cellule. Dans cette approche, une suspension de cellules Agrobacterium tumefaciens portant binaires vecteurs plasmidiques portant des ADNc des ARNm desiredviral est infiltré dans la feuille withina espace intercellulaire en utilisant rien de plus sophistiqué qu&#39;une seringue de 1 ml à usage unique (sans aiguille). Ce processus aboutit à la cession de l&#39;insert d&#39;ADN dans des cellules végétales; l&#39;ADN-Tinsert reste transitoirement dans le noyau et est ensuite transcrit par la polymérase II d&#39;accueil, conduisant à l&#39;expression transitoire. La transcription d&#39;ARNm qui en résulte (plafonnés et polyadénylé) est ensuite exporté vers le cytoplasme pour la traduction. Après environ 24 à 48 heures d&#39;incubation, des sections de feuilles infiltrées peuvent être prélevés pour des analyses biochimiques microscopyor. Agroinfiltration permet un grand nombre (centaines ou milliers) de cellules à transfecter simultanément. À l&#39;encapsidation in vitro, de virions purifiés BMV sont dissociés en protéine de capside par dialyse contre un tampon de dissociation du chlorure de calcium contenant suivie de l&#39;élimination de l&#39;ARN et non dissociés virions par centrifugation. Génome sous-unités appauvries protéine de capside sont ensuite remontés avec souhaités composants du génome viral ou non-viraux tels que les composants indocyanine.

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 moyen simple, efficace et robuste pour synchroniser la livraison de plusieurs composants viraux dans les cellules végétales par le biais<em&gt; Agrobacterium</em&gt; Médiée par l&#39;expression transitoire est décrit. Cette approche est susceptible d&#39;étude de la réplication, l&#39;encapsidation suivie par<em&gt; In vitro</em&gt; Le remontage de la non-virales dans le génome des composants optiques appauvri fantômes virales appropriés pour des applications biomédicales.

Simple et robuste In vivo Et In vitro Démarche pour l&#39;étude de l&#39;assemblage du 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 vues.  University of California, Riverside. Un moyen simple, efficace et robuste pour synchroniser la livraison de plusieurs composants viraux dans les cellules végétales par le biais Agrobacterium Médiée par l'expression transitoire est décrit. Cette approche est susceptible d'étude de la réplication, l'encapsidation suivie par In vitro Le remontage de la non-virales dans le génome des composants optiques appauvri fantômes virales appropriés pour des applications...

Vidéo: 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.

Visualisation virus de la dengue grâce à Alexa Fluor Étiquetage

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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Immunologie et infection

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) Détection d&#39;un neuro-invasive virus encéphalitique

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; Modèle pour l&#39;étude des interactions entre les droits du sang total et de l&#39;endothélium

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.

Analyse in vitro de la réponse immunitaire cellulaire Myd88 médiation au virus du Nil occidental souche mutante 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 ...

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.

Neutrophiles Isolement et analyse pour déterminer leur rôle dans le lymphome cellulaire Sensibilité aux agents thérapeutiques

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 désassemblage du virus grippal A capsides par Centrifugation Gradient

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.

Une approche simple et efficace de construire Mutant Vaccinia vecteurs de 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.

Méthodes in vitro pour la comparaison de liaison cible et CDC induction entre les anticorps thérapeutiques: Applications dans l&#39; analyse biosimilarité

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.

Une méthode Simple et efficace pour établir les NK et les lymphocytes T lignes provenant de Patients avec une Infection chronique Active Virus 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.

Un Simple écoulement Cytometry Based Assay pour déterminer In Vitro anticorps dépendant amélioration du Virus de la Dengue à l’aide de sérum de Convalescent Virus Zika

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