Os autores gostariam de agradecer a vários membros do laboratório para as suas sugestões valiosas para o desenvolvimento de agroinfiltration e em ensaios in vitro de montagem. Este trabalho foi financiado por uma bolsa da Universidade da Califórnia. Este estudo foi financiado em partes por uma concessão do National Science Foundation (CBET-1144237).

1. O material vegetal <ol><li> Plantas de Nicotiana benthamiana a serem utilizados no ensaio de encapsidação deve estar na fase 4 folhas (plantas semana aproximadamente 3-4 de idade). </li></ol> 2. Entrega e expressão de componentes funcionais do genoma viral para plantar células por agroinfiltration <ol><li> Dia 1: A estirpe de Agrobacterium (por exemplo, EH 105 ou GV 3101) abrigando o RNA pCass-BMV 1, BMV RNA 2 e 3 BMV RNA 1,2 são cultivadas em placas de LB agar suplementado com 50 mg / ml de canamicina (selecciona para o vetor pCASS) e 100mg/ml de rifampicina (seleciona para o Agrobacterium). A incubação é realizada a 28 ° C durante dois dias. </li><li> Dia 3: única colónia Inocular a partir da placa de agar LB em 2 ml de caldo LB contendo 50 mg / ml de canamicina e 100 mg / ml de rifampicina durante 1 dia a 28 ° C em agitador orbital fixado em 250 RPM. </li><li> Dia 4: Inocular 50 ml de suplemento de caldo LBed com 50 mg / ml de canamicina e 100 mg / ml de rifampicina, 10 mM de MES pH 5,6 e 100 mM de acetoseringona com um ml de cultura em um 250 ml de Erlenmeyer durante 16 horas a 28 ° C em agitador orbital a 250 rpm . </li><li> Dia 5: Transferência da cultura (quando OD 600 atingiu 1,0) para um tubo Oak Ridge ou tubo Falcon estéril e centrifugar durante 10 minutos a 5000 rpm a 4 ° C. </li><li> Dissolve-se o pellet em 10 ml de 10 mM de MgCl2 e centrifuga-se durante 10 minutos a 5000 rpm a 4 ° C, Repetir este passo mais uma vez para assegurar a remoção completa do antibiótico. </li><li> Suspender o sedimento em 10 ml de tampão contendo 10 mM de MgCl2 e 10 mM de MES (pH 5,6). </li><li> Medir a OD 600 para cada cultura de BMV RNA 1, 2 e RNA do RNA 3 e ajusta-se 0,1 OD 600 utilizando 10 mM de MgCl2 e 10 mM de MES (pH 5,6). </li><li> Misturar 1 ml cada todos os três 0,1 OD 600 suspensões de cultura. </li><li> Adicionar 100 mM de acetoseringona, mix gently e manter a mistura não perturbada à temperatura ambiente durante 3 horas. </li><li> Desenhar a suspensão de cultura em 1 ml. seringa sem agulha. </li><li> Infiltrar-se na suspensão cultura acima no lado abaxial de 2-3 bem expandidos folhas de N. benthamiana (5 folhas, as plantas fase de 3-4 semanas de idade), pressionando cuidadosamente a seringa para uma metade da superfície abaxial de folha. </li><li> Repita este procedimento de infiltração para outras folhas. </li><li> Mantenha as plantas infiltradas na casa verde com 24 ° C. </li><li> Colheita infiltrada folhas de 3 a 4 dias depois da infiltração (dpi). </li></ol> 3. Purificação de viriões BMV <ol><li> Colete N. benthamiana deixa agroinfiltrated com uma mistura contendo todos os três agrotransformants tipo selvagem BMV. </li><li> Moer folhas em um pilão e almofariz estéril com um volume igual (w / v) de tampão de extracção BMV (0,5 M NaAc; 0,08 M ​​MgAc, pH 4,5 e volume 1/100 de β-mercaptoetanol, que é para ser adicionado imediatamente antes da utilização) 3. Para maximizar o rendimento do vírus, é recomendável adicionar areia lavada ácido (~ 0,5-1 g) que facilita rompimento celular fácil trituração e eficiente. </li><li> Filtrar o extrato da folha através de 2-3 camadas de musselina e recolher a enxurrada. </li><li> Novamente moer a parte retida sobre o pano de musselina com igual volume de tampão de extracção BMV ao peso inicial do material de folha. </li><li> Repita o procedimento de filtração, utilizando pano de musselina. Estes passos devem ser realizadas a 4 ° C. </li><li> Transferir a solução de filtrado para um tubo de centrífuga e adicionar volume igual de pré-refrigeradas clorofórmio e vórtice (ou agitação) durante 5 minutos à temperatura ambiente até que a cor da suspensão vira verde claro. </li><li> Centrifugar a solução emulsionada durante 15 minutos a 12.000 rpm a 4 ° C. </li><li> Recolher a fase aquosa e agita-se durante 30 minutos em um agitador magnético para remover qualquer clorofórmio residual. </li><li> Transferir a suspensão para um tubo de ultracentrifuga estéril. </li><li&gt; Underlay 1 ml. Sacarose a 10% (preparada no tampão de extracção de vírus) como um 3 almofada. </li><li> Centrifugar a 30.000 rpm durante 3 horas numa ultracentrífuga Beckman a 4 ° C. </li><li> Descartar o sobrenadante e suspender o pellet em volume desejado de BMV tampão de suspensão (para preparar a suspensão de tampão de tampão de extracção diluído BMV a 1/10 com água destilada estéril). </li><li> A preparação do virião parcialmente purificada a partir do passo acima é submetida a 10-40% centrifugação em gradiente de sacarose de densidade para 150 min a 28000 rpm a 4 ° C. </li><li> Coletar a banda de vírus manualmente ou por um fraccionamento. A banda vírus aparecem em azul sob iluminação de luz branca. </li><li> Dilui-se a solução de sacarose contendo amostra de vírus pelo menos 50% com tampão de suspensão de vírus. </li><li> Centrifugar diluída vírus solução de sacarose contendo durante 3 horas à 30.000 rpm numa ultracentrífuga Beckman a 4 ° C. </li><li> Finalmente suspender o vírus altamente purificada pelete num volume desejado de Virnos suspensão buffer. </li><li> Medir a concentração do virião (mg / ml) usando espectrofotómetro a OD 260. </li></ol><img alt="Equação 1" src="/files/ftp_upload/3645/3645eq1.jpg" /> Nota: Com base no conteúdo de ARN, o coeficiente de extinção para BMV é 5 4. <ol start="19"><li> Verificar a pureza de viriões por visualização sob MET (Fig. A) </li></ol> 4. Preparação de subunidades de proteínas da cápside de montagem in vitro <ol><li> Prepare 1 litro de tampão 1x vírus dissociação (0,5 M de CaCl2, 50 mM Tris HCl, pH 7,5; 1,0 mM de EDTA, 1,0 mM de DTT e 0,5 mM de PMSF) 5. </li><li> Preparar membrana de diálise de acordo com Sambrook et ai 6. </li><li> Pipetar concentração necessária de vírus purificado em um saco de diálise. </li><li> Coloque o vírus saco de diálise contendo a um copo contendo o vírus 1x tampão de dissociação. </li><li> Dialisar 24 horas a 4 ° C whilagitação e. </li><li> Recolher a solução a partir do saco de diálise, após 24 horas e centrifugar a 12.000 xg durante 15 min a 4 ° C. Isto irá separar o ARN a partir dos viriões BMV dissociados. </li><li> Recolher o sobrenadante e centrifugar a 35.000 rpm em centrífuga Beckman durante 3 horas a 4 ° C para sedimentar quaisquer partículas de vírus un-dissociados. </li><li> Recolher o sobrenadante. </li><li> Nesta fase é fundamental para remover todos os contaminantes a partir de RNA virion dissociados subunidades da proteína do capsídeo. Para fazer isto, o sobrenadante do passo 8 deve ser submetido a outro ciclo de noite sobre a remontagem das subunidades de proteínas da cápside, sem adicionar qualquer RNA (em tampão de montagem RNA 1x, ver abaixo), seguido de uma centrifugação a alta velocidade (30.000 rpm, durante 3 horas à 4 ° C) para sedimentar quaisquer viriões montadas. Após este passo remontagem, o sobrenadante contendo subunidades de proteína revestimento seria livre de quaisquer contaminantes residuais RNA. No entanto, para garantir que esta, é aconselhável realizar um adicional em vitro remontagem com subunidades da proteína usando casaco apenas RNA tampão 1x montagem. Depois de durante a noite remontagem, a preparação deve estar livre do vírus da montados (verificar usando MET). </li><li> Determinar a concentração de subunidades de proteína da cápside por medição em OD 254 e 280 nm ou por outros métodos, como ensaio de Bradford. </li><li> Realizar 12-15% de SDS-PAGE seguido por Western blot para determinar a integridade dos dissociados subunidades da proteína da cápside. </li></ol> 5. In vitro montagem de RNA contendo os viriões <ol><li> Preparar transcritos de RNA de ser re-montados em viriões e calcular a concentração de 7. </li><li> Misturar subunidades da proteína da cápside e RNA transcrito a uma razão de 1:5 (peso / peso) 5. </li><li> Distribua a mistura acima para um saco de diálise e garantir de forma adequada para evitar vazamentos. </li><li> Prepare 1.000 ml. de RNA 1x montagem tampão (50 mM NaCl; 50 mM Tris-HCl, pH 7,2; 10 mM de KCl, 5 mM de MgCl2, 1,0 mM de DTT) 5. </li><li> Coloque o saco de diálise contendo a mistura de reacção contra a 1x tampão de montagem para o copo e agitar. </li><li> Dialisar a reacção a montagem, a 4 ° C durante 24 horas por agitação suave. </li><li> Recolher-se a mistura a partir do saco de diálise, após 24 horas e adicionar 1,5 ml de tampão de RNA de montagem. </li><li> Passa esta mistura através de uma coluna Centricon-100 por centrifugação a baixa velocidade (2000 xg) durante 30 minutos. </li><li> Lavar a coluna uma vez com 1,5 ml de tampão de montagem, a 4 ° C durante 30 min. </li><li> Repetir o passo de lavagem acima duas vezes. </li><li> Finalmente, eluir os viriões re-montado por centrifugação a 10.000 g, a 4 ° C durante 5 min. </li><li> Estimar a concentração de OD virion 260 nm. </li></ol> 6. Fabricação de óptica Ghosts Viral (OVGs) <ol><li> Adicionar verde de indocianina (ICG) 8 para a proteína purificada da cápside em rácio de concentração desejada (a proporção em peso de Indocianina proteína verde e cápside utilizado neste relatório foi 1:10) um<html> d misture bem por pipetagem. </li><li> Dialisar a proteína da cápside e solução ICG contra o tampão de montagem OVG (1 M de NaCl, 50 mM de NaAc; 1 mM de EDTA e 1 mM de DTT, pH4.8), a 4 ° C durante 24 horas. </li><li> Após 24 horas, recolher a solução a partir do saco de diálise (12 kDa tamanho de poro) e centrifugar a 90.000 rpm durante 1 hora a 4 ° C (Fig. B) </li><li> Remover o sobrenadante e suspender o sedimento em tampão BMV suspensão em vórtice ou permitindo que a suspensão, por si só em tampão BMV suspensão durante a noite a 4 ° C. </li><li> Verifique a morfologia de OVGs por TEM (Fig. C). </li><li> Medir a absorvância de 240 nm a 950 nm utilizando espectrofotômetro (Cary 50, Varian Inc.); a presença de ICG é confirmada por picos de absorvância de assinatura cerca de 700 nm e 790 nm 8. (Fig. D) </li><li> Os típicos ICG picos de fluorescência em ~ 700 nm e ~ 800 nm pode ser visto sobre 620 nm de excitação de solução OVG (Fig. D) utilizando espectrofluorímetro (Fluorolog 3, Jobin-Yvon). </li></ol>_title &quot;&gt; 7. resultados representativos <img alt="Figura A" src="/files/ftp_upload/3645/3645figA.jpg" /> Figura A. Imagem TEM de viriões coradas negativamente VMB purificadas de N. benthamiana plantas agroinfiltrated com uma mistura de todos os três agroconstructs tipo selvagem BMV (Barra de escala = 100 nm). <img alt="Figura B" src="/files/ftp_upload/3645/3645figB.jpg" /> Figura B. Pellet da in vitro montado ICG OVGs contendo sequência de alta velocidade de centrifugação. <img alt="Figura C" src="/files/ftp_upload/3645/3645figC.jpg" /> Figura C. imagem TEM de coradas negativamente ICG OVGs contendo (barra de escala = 100 nm). <img alt="Figura D" src="/files/ftp_upload/3645/3645figD.jpg" /> Figura Absorbância D. (Top) e fluorescência de espectros (fundo) de OVGs. O comprimento de onda de excitação utilizado para OVG emissão wcomo 620 nm.

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	<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>

Não temos nada a divulgar.

Abordagem Agroinfiltration aqui apresentadas podem ser amplamente aplicável a uma ampla gama de vírus de plantas. Uma característica marca desta abordagem é sincronizado entrega de agroconstruct múltipla para a célula-a mesma desvantagem importante geralmente associado com rotineiramente utilizado inoculação mecânica de vírus de plantas. In vivo e in vitro de montagem, utilizando brome vírus do mosaico como um modelo pode ser conduzida de forma eficiente pela a seguir algumas dicas. (i) para...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> Sódio sais MES </td><td> Sigma-Aldrich </td><td> M2993 </td></tr><tr><td> Indocianina verde </td><td> Sigma-Aldrich </td><td> I2633 </td></tr><tr><td> Ultracentrífuga Beckman </td><td> Beckman Coulter </td><td> Modelo: L8-70M </td></tr><tr><td> Centricon-100 coluna </td><td> Millipore </td><td> YM-100 </td></tr><tr><td> Espectrofotômetro </td><td> Cary 50, Varian Inc. </td><td> Número da peça 10068900 </td></tr><tr><td> Espectrofluorímetro </td><td> Fluorolog 3, Jobin-Yvon. </td><td> Número da peça FL3-21 </td></tr></tbody></table>

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

Em vírus com sentido positivo patogênico genomas RNA de seres humanos, animais e plantas, de encapsidação progênie em viriões maduros e estáveis ​​é uma fase cardeal durante o estabelecimento da infecção em um determinado host. Consequentemente, o estudo de encapsidação decifra as informações sobre o know-how do mecanismo de regulação montagem do vírus para formar virions infecciosos. Essa informação é vital na formulação de novos métodos de contenção propagação do vírus e controle da doença. Encapsidação vírus pode ser estudada in vivo e in vitro. Encapsidação Genoma in vivo é um processo altamente regulado seletiva envolvendo interações macromoleculares e compartimentalização subcelular. Portanto, estudar levando a dissecar eventos abrangendo encapsidação vírus in vivo seria fornecer conhecimentos básicos para compreender como os vírus se proliferam e montar. Recentemente, em encapsidação vitro tem sido explorada para a pesquisa na área de biomédica imaging e aplicações terapêuticas. Vírus não-envelopados plantas estão muito à frente no empreendimento de encapsidação em vitro da matéria carregada negativamente estrangeira. Brome vírus do mosaico (BMV), um não encapsulado multicomponente patogenicidade do vírus de RNA para plantas, tem sido utilizado como um sistema modelo para estudar embalagem genoma in vivo e in vitro. Para os ensaios de encapsidação em plantas de Nicotiana benthamiana, mediada por Agrobacterium de expressão transiente, se referem como agroinfiltration, é uma técnica eficiente e fiável para a entrega sincronizado e expressão de componentes múltiplos para a mesma célula. Nesta abordagem, uma suspensão de células de Agrobacterium tumefaciens que transportam binários vectores plasmídicos transportando cDNAs de mRNAs desiredviral é infiltrado no espaço intercelular folha withina usando nada mais sofisticado que uma seringa de 1 ml descartável (sem agulha). Este processo resulta na transferência de inserção de ADN em células vegetais; o T-DNAinserto permanece transientemente no núcleo e, em seguida, é transcrito pelo hospedeiro polimerase II, que conduz à expressão transiente. A transcrição do mRNA resultante (nivelado e poliadenilado) é então exportado para o citoplasma para a tradução. Após aproximadamente 24 a 48 horas de incubação, as secções de folhas infiltradas pode ser amostrada para microscopyor análises bioquímicas. Agroinfiltration permite um grande número (centenas a milhares) de células a serem transfectadas simultaneamente. Para na encapsidação vitro, partículas virais purificadas de BMV são dissociados em proteína da cápside por diálise contra a dissociação de cloreto de cálcio tampão contendo seguido por remoção de RNA e un-dissociados viriões por centrifugação. Genoma empobrecido subunidades da proteína da cápside são reagrupados com desejados componentes do genoma viral ou não virais, tais como componentes indocianina corante.

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

Uma maneira simples, eficiente e robusta para sincronizar a entrega de múltiplos componentes virais para as células vegetais através<em&gt; Agrobacterium</em&gt; Mediada expressão transiente é descrito. Esta abordagem é favorável para estudar a replicação, encapsidação seguido por<em&gt; In vitro</em&gt; Remontagem de componentes não-virais do genoma empobrecido ópticos fantasmas virais adequados para aplicações biomédicas.

Simples e robusto In vivo E In vitro Abordagem para o Estudo Assembléia 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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JoVE Journal

Immunology and Infection

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

12.1K visualizações.  University of California, Riverside. Uma maneira simples, eficiente e robusta para sincronizar a entrega de múltiplos componentes virais para as células vegetais através Agrobacterium Mediada expressão transiente é descrito. Esta abordagem é favorável para estudar a replicação, encapsidação seguido por In vitro Remontagem de componentes não-virais do genoma empobrecido ópticos fantasmas virais adequados para aplicações biomédicas.

Vídeo: 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.

Visualizando Vírus da Dengue por meio 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 ...

Imunologia e Infecção

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 Detecção (IVIS) de um vírus de Neuro-invasiva encefalítico

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; Modelo para estudar as interações entre a Whole Humano Sangue e endotélio

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 Vitro Análise do mediada por Myd88 Cellular resposta imune a vírus do Nilo Ocidental 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 ...

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.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

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 A desmontagem do vírus influenza A capsï¿½eos por centrifugação em 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.

Uma abordagem simples e eficiente para construir vectores de vírus mutante Vaccinia

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.

Os métodos in vitro para Comparando meta vinculativa e CDC indução Entre anticorpos terapêuticos: Aplicações em Análise 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.

Um método simples e eficiente para estabelecer linhas de células T de pacientes com infecção crônica ativa vírus Epstein - Barr e 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.

A citometria de fluxo simples com base em ensaio para determinar In Vitro anticorpo dependente do realce do vírus da Dengue, usando soro convalescente vírus Zika

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