Los autores desean agradecer a varios miembros del laboratorio por sus valiosas sugerencias en el desarrollo de agroinfiltración y en ensayos in vitro de montaje. Este trabajo fue financiado por una beca de la Universidad de California. Este estudio fue apoyado en parte por una beca de la National Science Foundation (CBET-1144237).

1. Material vegetal <ol><li> Plantas de Nicotiana benthamiana para ser utilizados en el ensayo de encapsidación debe estar en fase 4 hojas (plantas semana aproximadamente 3-4 de edad). </li></ol> 2. Entrega y expresión de los componentes funcionales del genoma viral para sembrar las células por agroinfiltración <ol><li> Día 1: La cepa de Agrobacterium (por ejemplo, EH 105 o GV 3101) que alberga el ARN pCass-BMV 1, 2 y ARN BMV BMV ARN 3 1,2 se cultivan en placas de agar LB suplementadas con 50 mg / ml de kanamicina (selecciona para el vector pCASS) y 100mg/ml de rifampicina (selecciona para el Agrobacterium). La incubación se lleva a cabo a 28 ° C durante dos días. </li><li> Día 3: Se inoculan colonia única de la placa de agar LB en 2 ml de caldo LB que contenía 50 mg / ml de kanamicina y 100 mg / ml de rifampicina durante 1 día a 28 ° C en un agitador orbital fijado en 250 RPM. </li><li> Día 4: Se inoculan 50 ml de caldo LB el suplementoed con 50 mg / ml de kanamicina y 100 mg / ml de rifampicina, MES 10 mM pH 5,6 y 100 mM acetosiringona con un ml de cultivo en 250 ml de un matraz Erlenmeyer de 16 horas a 28 ° C en un agitador orbital a 250 rpm . </li><li> Día 5: Traslado de la cultura (OD 600, cuando llegó a 1,0) a un tubo de Oak Ridge o estéril tubo Falcon y se centrifuga durante 10 minutos a 5000 rpm a 4 ° C. </li><li> Disolver el precipitado en 10 ml de MgCl2 10 mM y se centrifuga durante 10 minutos a 5000 rpm a 4 ° C, Repita este paso una vez más para asegurar la eliminación completa del antibiótico. </li><li> Suspender el precipitado en 10 ml de tampón conteniendo 10 mM de MgCl 2 y 10 mM MES (pH 5,6). </li><li> Mida el diámetro exterior 600 por cada cultura de la BMV ARN 1, 2 ARN y ARN 3 y ajustar a 0,1 OD 600 con 10 mM de MgCl 2 y 10 mM MES (pH 5,6). </li><li> Mezclar 1 ml de cada uno de los tres 0.1 OD 600 suspensiones de cultivo. </li><li> Añadir 100 mM de acetosiringona, mezcla de detergentely y mantener la mezcla en reposo a temperatura ambiente durante 3 horas. </li><li> Dibuje la suspensión de la cultura en 1 ml. una jeringa sin aguja. </li><li> Infíltrate en la suspensión del cultivo anterior en el lado abaxial de 2-3 hojas expandidas bien de N. benthamiana (5 hojas etapa, las plantas 3-4 semanas de edad) presionando suavemente la jeringa a una mitad de la superficie abaxial de la hoja. </li><li> Repita este procedimiento con la infiltración a otras hojas. </li><li> Mantenga las plantas se infiltraron en la casa verde con 24 ° C. </li><li> Cosecha infiltrado en las hojas de 3 a 4 días después de la infiltración (ppp). </li></ol> 3. La purificación de los viriones BMV <ol><li> Recoger N. benthamiana deja agroinfiltradas con una mezcla que contiene los tres tipo salvaje agrotransformants BMV. </li><li> Moler las hojas en un almirez y mortero estéril con un volumen igual (w / v) de extracción BMV tampón (0,5 M NaAc; 0,08 M ​​MgAc, pH 4,5 y el volumen 1/100 de β-mercaptoetanol que ha de ser añadido justo antes de su uso) 3. Para maximizar el rendimiento del virus, se recomienda añadir arena lavada ácido (~ 0.5-1 g) que facilita la ruptura celular fácil de molienda y eficiente. </li><li> Filtrar el extracto de la hoja a través de 2-3 capas de tela de muselina y recoger la oleada. </li><li> Nuevamente moler la parte retenida en tela de muselina con un volumen igual de tampón de extracción BMV al peso inicial de material de hoja. </li><li> Repita el procedimiento de filtración con tela de muselina. Estos pasos deben llevarse a cabo a 4 ° C. </li><li> Transferir la solución de filtrado a un tubo de centrífuga y añadir volumen igual de pre-enfriada cloroformo y agitar (o batido) durante 5 minutos a la temperatura ambiente hasta que el color de la suspensión se vuelve de color verde claro. </li><li> Centrifugar la solución emulsionada durante 15 minutos a 12.000 rpm a 4 ° C. </li><li> Recoger la fase acuosa y se agita durante 30 minutos en un agitador magnético para eliminar cualquier cloroformo residual. </li><li> Transferir la suspensión a un tubo de ultracentrífuga estéril. </li><li&gt; Debajo De 1 ml. 10% de sacarosa (preparada en tampón de virus de la extracción) como un cojín 3. </li><li> Centrifugar a 30.000 rpm durante 3 horas en una ultracentrífuga Beckman a 4 ° C. </li><li> Se desecha el sobrenadante y suspender el pellet en volumen deseado de tampón de suspensión BMV (para preparar tampón de suspensión diluida BMV tampón de extracción a 1/10 con agua destilada estéril). </li><li> La preparación virión parcialmente purificada de la etapa anterior se somete a centrifugación en gradiente de densidad de sacarosa 10-40% para 150 min a 28000 rpm a 4 ° C. </li><li> Recoger la banda de virus, ya sea manualmente o por un fraccionador. La banda de virus aparecen de color azul con iluminación de luz blanca. </li><li> Se diluye la solución de sacarosa que contiene muestras de virus por lo menos 50% con tampón de virus de la suspensión. </li><li> Centrifugar virus diluido que contenía solución de sacarosa durante 3 horas a 30.000 rpm en una ultracentrífuga Beckman a 4 ° C. </li><li> Finalmente suspender el sedimento de virus altamente purificado en un volumen deseado de virnos suspensión de amortiguación. </li><li> Medir la concentración del virión (mg / ml) usando un espectrofotómetro a OD 260. </li></ol><img alt="Ecuación 1" src="/files/ftp_upload/3645/3645eq1.jpg" /> Nota: Basado en el contenido de ARN, el coeficiente de extinción para la BMV es 5 4. <ol start="19"><li> Verifique la pureza de los viriones por ver en TEM (Fig. A) </li></ol> 4. Preparación de las subunidades de proteína de la cápside para montaje in vitro <ol><li> Preparar 1 litro de tampón 1x virus de disociación (0,5 M CaCl 2; 50 mM Tris HCl, pH 7,5; 1,0 mM de EDTA; 1,0 mM de DTT y 0,5 mM PMSF) 5. </li><li> Preparar membrana de diálisis de acuerdo con Sambrook et al 6. </li><li> Dispensación concentración requerida de virus purificado en una bolsa de diálisis. </li><li> Coloque el virus que contiene la bolsa de diálisis en un vaso que contiene el virus 1x tampón de disociación. </li><li> Dializar 24 horas a 4 ° C whilagitación e. </li><li> Recoger la solución de la bolsa de diálisis después de 24 horas y centrifugar a 12.000 xg durante 15 min a 4 ° C. Esto separar el ARN BMV de los viriones disociados. </li><li> Recoger el sobrenadante y se centrifuga a 35.000 rpm en centrífuga Beckman durante 3 horas a 4 ° C para sedimentar la no-disociado partícula del virus. </li><li> Recoger el sobrenadante. </li><li> En esta etapa es imprescindible para eliminar los contaminantes del ARN del virión disociados subunidades de la proteína de cápside. Para hacer esto, el sobrenadante del paso 8 debe ser sometido a otra ronda de noche sobre el reensamblaje de las subunidades de proteína de la cápside sin añadir ningún ARN (ARN en 1x tampón de montaje, véase más adelante), seguido por una centrifugación a alta velocidad (30.000 rpm durante 3 horas a 4 ° C) para que sedimenten los viriones ensamblados. Después de este paso reensamblaje, el sobrenadante subunidades que contienen proteína de la cubierta estaría libre de cualquier ARN residuales contaminantes. Sin embargo, para asegurar esto, es conveniente realizar una adicional en vitro reensamblaje con sólo subunidades capa de proteínas utilizando ARN tampón ensamblado 1x. Después de un día para el montaje, la preparación debe estar libre de viriones ensamblados (verificar mediante TEM). </li><li> Determinar la concentración de las subunidades de proteína de la cápside midiendo a OD 254 nm y 280 o por otros métodos tales como ensayo de Bradford. </li><li> Realizar 12-15% de SDS-PAGE seguida por Western blot para determinar la integridad de las subunidades de la proteína de cápside disociados. </li></ol> 5. In vitro montaje de ARN que contiene viriones <ol><li> Preparar las transcripciones de ARN para ser re-ensamblados en viriones y calcular la concentración 7. </li><li> Mezclar subunidades cápside de proteínas y ARN transcrito en una proporción de 1:5 (peso / peso) 5. </li><li> Dispense la mezcla anterior a una bolsa de diálisis y asegurar apropiadamente para evitar cualquier fuga. </li><li> Preparar 1.000 ml. de ARN 1x tampón de montaje (50 mM de NaCl; 50 mM Tris-HCl, pH 7,2; 10 mM de KCl; 5 mM de MgCl 2, 1,0 mM de DTT) 5. </li><li> Coloque la bolsa de diálisis que contiene la mezcla de reacción frente a tampón de montaje 1x en el vaso y remover. </li><li> Dializar la reacción de re-ensamblaje a 4 ° C durante 24 horas por agitación suave. </li><li> Recoger la mezcla de la bolsa de diálisis después de 24 horas y añadir 1,5 ml de ARN tampón ensamblado. </li><li> Pase esta mezcla a través de un Centricon-100 en la columna por centrifugación a baja velocidad (2.000 xg) durante 30 minutos. </li><li> Lavar la columna una vez con 1,5 ml de tampón de montaje a 4 ° C durante 30 min. </li><li> Repita el paso por encima de lavado dos veces. </li><li> Finalmente, eluir los viriones de re-ensambladas por centrifugación a 10.000 ga 4 ° C durante 5 min. </li><li> Estimación de la concentración en el virión de DO 260 nm. </li></ol> 6. La fabricación de ópticas fantasmas virales (OVGs) <ol><li> Añadir verde de indocianina (ICG) 8 a la proteína purificada cápside en el coeficiente de concentración deseada (la relación en peso de la proteína verde de indocianina y la cápside utilizada en este informe fue 1:10) una<html> d mezclar bien con la pipeta. </li><li> Dializar la proteína de la cápside y la solución ICG contra el tampón de montaje OVG (1 M de NaCl; 50 mM de NaAc, 1 mM EDTA y DTT 1 mM, pH4.8) a 4 ° C durante 24 horas. </li><li> Después de 24 horas, recoger la solución de la bolsa de diálisis (12 kDa de tamaño de poro) y se centrifuga a 90.000 rpm durante 1 hora a 4 ° C (Fig. B) </li><li> Eliminar el sobrenadante y suspender el sedimento en tampón de suspensión BMV por agitación o de lo que le permite suspender por sí misma en un tampón de suspensión de la BMV la noche a 4 ° C. </li><li> Verifique que la morfología de OVGs por TEM (Fig. C). </li><li> Medir la absorbancia de 240 nm a 950 nm utilizando un espectrofotómetro (Cary 50, Varian Inc.), la presencia de ICG es confirmado por los picos de absorción de la firma alrededor de 700 nm y 790 nm 8. (Fig. D) </li><li> Los típicos picos de fluorescencia ICG a ~ 700 nm y ~ 800 nm se puede ver a la excitación de 620 nm de la solución de OVG (Fig. D), utilizando espectrofluorómetro (Fluorolog 3, Jobin-Yvon). </li></ol>_title &quot;&gt; 7. Resultados representativos <img alt="La figura A" src="/files/ftp_upload/3645/3645figA.jpg" /> Figura A. TEM imagen de viriones BMV tinción negativa purificados de N. benthamiana plantas agroinfiltradas con una mezcla de las tres de tipo salvaje agroconstructs BMV (barra de escala = 100 nm). <img alt="Figura B" src="/files/ftp_upload/3645/3645figB.jpg" /> Figura B. Pellet in vitro montado ICG OVGs que contienen después de centrifugación a alta velocidad. <img alt="Figura C" src="/files/ftp_upload/3645/3645figC.jpg" /> Figura C. TEM imagen de tinción negativa del ICG OVGs que contienen (barra de escala = 100 nm). <img alt="Figura D" src="/files/ftp_upload/3645/3645figD.jpg" /> Figura D. Absorbancia (Arriba) y fluorescencia (abajo) los espectros de OVGs. La longitud de onda de excitación utilizados para OVG emisión wcomo 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>

No tenemos nada que revelar.

Enfoque agroinfiltration que aquí se presenta ampliamente puede ser aplicable a una amplia gama de virus de plantas. Una característica distintiva de este enfoque se sincroniza la entrega de múltiples agroconstruct a la celda-un mismo inconveniente importante comúnmente asociada con la inoculación utiliza rutinariamente mecánica de virus de plantas. In vivo e in vitro en los estudios de montaje utilizando virus del mosaico del bromo como un modelo puede llevar a cabo eficientemente por siguientes...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> MES sales de sodio </td><td> Sigma-Aldrich </td><td> M2993 </td></tr><tr><td> Verde de indocianina </td><td> Sigma-Aldrich </td><td> I2633 </td></tr><tr><td> Beckman ultracentrífuga </td><td> Beckman Coulter </td><td> Modelo: L8-70M </td></tr><tr><td> Centricon-100 columna </td><td> Millipore </td><td> YM-100 </td></tr><tr><td> Espectrofotómetro </td><td> Cary 50, Varian Inc. </td><td> Número de referencia 10068900 </td></tr><tr><td> Espectrofluorómetro </td><td> Fluorolog 3, Jobin-Yvon. </td><td> Pieza número FL3-21 </td></tr></tbody></table>

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

En los virus con ARN de sentido positivo genomas patógenos para los seres humanos, animales y plantas, la progenie de encapsidación en viriones maduros y estables es una fase de cardenal durante el establecimiento de la infección en un equipo dado. En consecuencia, el estudio de encapsidación descifra la información relativa a los conocimientos del mecanismo de regulación de ensamblaje del virus para formar viriones infecciosos. Dicha información es de vital importancia en la formulación de nuevos métodos de frenar la propagación del virus y control de la enfermedad. Encapsidación del virus puede ser estudiado in vivo e in vitro. Encapsidación del genoma en vivo es un proceso altamente regulado selectiva que implica interacciones macromoleculares y compartimentación subcelular. Por lo tanto, el estudio que lleva a diseccionar los acontecimientos que abarcan encapsidación del virus in vivo proporcionará los conocimientos básicos para comprender cómo los virus proliferan y se ensamblan. Recientemente, en la encapsidación in vitro ha sido explotada para la investigación en el área de la investigación biomédica imaging y aplicaciones terapéuticas. Virus no envueltos de plantas destacan muy por delante en la aventura de en la encapsidación in vitro de la materia con carga negativa externa. Virus de mosaico Brome (BMV), un no envueltos multicomponente patógeno virus ARN a las plantas, ha sido utilizado como un sistema modelo para estudiar embalaje genoma in vivo e in vitro. Para los ensayos de encapsidación en plantas de Nicotiana benthamiana, mediada por Agrobacterium expresión transitoria, se refieren como agroinfiltración, es una técnica eficaz y sólido para la entrega sincronizada y la expresión de varios componentes para la misma célula. En este enfoque, una suspensión de Agrobacterium tumefaciens que llevan las células binarias de vectores plásmidos portadores de ADNc de ARNm desiredviral se infiltra en el espacio intercelular de la hoja withina usando nada más sofisticado que el de una jeringa desechable de 1 ml (sin aguja). Este proceso resulta en la transferencia de inserción de DNA en células vegetales; el T-DNAinserto se mantiene transitoriamente en el núcleo y luego se transcribe por el huésped polimerasa II, que conduce a la expresión transitoria. La transcripción del ARNm resultante (nivelado y poliadenilado) luego se exporta al citoplasma para su traducción. Después de aproximadamente 24 a 48 horas de incubación, las secciones de hojas infiltradas pueden ser degustados por microscopyor análisis bioquímicos. Agroinfiltration permite una gran cantidad (cientos o miles) de las células para ser transfectadas de forma simultánea. Para en la encapsidación in vitro, los viriones purificados de BMV se disocian en proteína de la cápside por diálisis frente a tampón de cloruro de disociación que contiene calcio seguido de la eliminación de ARN y viriones no-disociados por centrifugación. Genoma subunidades de proteína de cápside empobrecido se vuelve a montar con los componentes deseados del genoma viral o no viral de componentes tales como indocianina.

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

Una manera sencilla, eficiente y robusta para sincronizar la entrega de múltiples componentes virales a las células vegetales mediante<em&gt; Agrobacterium</em&gt; Mediada por la expresión transitoria se describe. Este enfoque es susceptible para el estudio de la replicación, encapsidación seguido por<em&gt; In vitro</em&gt; Montaje de los componentes no virales en el genoma agotado ópticas fantasmas virales adecuados para aplicaciones biomédicas.

Simple y robusto En vivo Y In vitro Enfoque para el estudio de ensamblaje del virus

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

simple-robust-vivo-vitro-approach-for-studying-virus

Research

JoVE Journal

Immunology and Infection

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

12.1K vistas.  University of California, Riverside. Una manera sencilla, eficiente y robusta para sincronizar la entrega de múltiples componentes virales a las células vegetales mediante Agrobacterium Mediada por la expresión transitoria se describe. Este enfoque es susceptible para el estudio de la replicación, encapsidación seguido por In vitro Montaje de los componentes no virales en el genoma agotado ópticas fantasmas virales adecuados para aplicaciones biomédicas.

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.

Visualización de virus del dengue a través de Alexa Fluor etiquetado

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

Investigación

Inmunología e Infección

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.

En Vivo Imaging Systems (IVIS) La detección de un virus invasor Neuro-encefálico

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).

Una Novela<em&gt; In vitro</em&gt; Modelo para el estudio de las interacciones entre Whole Sangre humana y del 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.

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

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.

Aislamiento de neutrófilos y análisis para determinar su papel en el linfoma de células sensibles a los agentes terapéuticos

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 El desmontaje de virus de influenza A cápsides por centrifugación en 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 enfoque simple y eficiente de construcción de mutantes de virus vaccinia Vectores

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.

Métodos in vitro para comparar la unión de diana y la inducción de CDC entre anticuerpos terapéuticos: Aplicaciones en análisis de biosimilitud

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 método eficiente y Simple para establecer líneas de células T de pacientes con infección crónica activa Virus Epstein - Barr y NK

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.

Una citometría de flujo Simple basado en análisis para determinar In Vitro anticuerpo dependiente del realce del Virus del Dengue usando suero convaleciente 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 ...