Este trabalho foi suportado por concessões do NIAID (R01AI074951, U54AI057168) eo Penn Genome Institute Fronteiras para SC, a partir de NIH T32HG000046 a TSM, a partir de NIH T32GM07229 e T32AI007324 de LRS.

Parte 1: RNAi em 384 placas de Bem <ol><li> Padronização dos reagentes utilizados para o rastreio é essencial. Testamos lotes individuais de mídia de Schneider, soro fetal bovino, e reagentes de coloração. Então nós alíquotas e usar o mesmo lote para a tela inteira. </li><li> Para qualquer ensaio, é melhor testar várias linhas de células diferentes para determinar qual é mais receptivo à sua biológico de leitura. Normalmente usamos a linha S2-NUPAD. </li><li> Cultivar células Drosophila a 25 ° C nos meios de comunicação Schneider completo. <ol class="lalpha"><li> Media Schneider </li><li> 10% de calor FBS inativada </li><li> 1X L-glutamina </li><li> 1X Pen / strep antibióticos </li></ol></li><li> Células Drosophila são semi-aderente e são várias passagens sem tripsinização a cada quatro dias por pipetagem as células fora os balões e diluir as células 1:10 em um frasco fresco. As células devem estar em fase de registro para experimentos. </li><li> Placa-to-plate e dia-a-dia variabilidade são minimizados através do uso de manipulação de líquidos automatizado para todas as adições líquidas de multi-lamelas. Nós usamos um WellMate (Matrix). </li><li> Prepare tubulação WellMate. <ol class="lalpha"><li> Spray de capuz e WellMate com etanol 70%. </li><li> Remover tubos de embalagem e de pulverização com etanol 70%. </li><li> Inserir cassete tubulação na dispensação posição no WellMate. </li><li> Tubulação privilegiada, com 25 mL de etanol 70%. </li><li> Esterilizar tubulação em etanol por 15 minutos, então primeiro com outro de 25 mL de etanol. </li><li> Enxágüe tubulação por priming com 50 mL de PBS estéril. </li></ol></li><li> Desalojar células do frasco e contagem. Células pelota para que você tenha 25% a mais do que o necessário (300xg, 5 min). O número de células inoculadas por poço deve ser otimizado de acordo com a duração do ensaio. No âmbito da análise automática de imagem, uma monocamada único sem aglomeração celular é ideal para segmentação de imagem. </li><li> Ressuspender as células peletizada no soro livre de Schneider de mídia em 1.7x10 6 células / mL. </li><li> Realizamos a tela em 384 placas bem pré-aliquotado com uma biblioteca disponível comercialmente de dsRNA em uma concentração de 250ng/well. Vários poços de cada placa são deixados vazios para controles, que são adicionados manualmente. Usamos a luciferase dsRNA como controle negativo. Usamos dsRNA contra Thread (DIAP) como um controle para robusto RNAi. Derrubar resultados deste anti-apoptóticos fator na morte da célula dramática e é uma maneira rápida e visual para avaliar a qualidade do knock down. Por fim, usamos um dsRNA contra o repórter viral, neste caso galacosidase beta, como um controle positivo para validar que pode inibir a infecção viral com o nosso ensaio de leitura. Se detectar dsRNA em placa no momento da semeadura de células, dsRNA alíquota 1 ml para o canto do bem, para facilitar a visualização, em seguida, centrifugar dsRNA em fundo de poço (300xg, 1 min) antes de adicionar células. </li><li> Use WellMate para adicionar 10 mL de células preparadas acima para cada poço. </li><li> Spin-células em placas por centrifugação 300xg, 1 min. </li><li> Incubar 25 ° C em incubadora por 45 minutos. </li><li> Adicionar 20 mL completa Schneider de mídia e centrifugar 300xg, 1 min. </li><li> Placas lugar em recipientes Tupperware umidificado forrado com toalhas de papel embebido em água. O volume de 384 poços é pequena, assim, a evaporação pode ter grandes efeitos sobre a biologia, que por sua vez pode levar a mudanças na artifactual outs leitura dos poços borda. Para superar isso, as placas devem ser mantidos sob alta umidade. </li><li> Incubar durante três dias para permitir a knockdown robusto. Não há necessidade de alterar a mídia ou abrir recipientes umidificado durante este tempo. </li></ol> Parte 2: Infectando com Vaccinia Virus <ol><li> Esterilizar canal multi-aspirador em Quatricide por 15 minutos, em seguida, lavar em etanol 70%. </li><li> Prepare WellMate como descrito acima. </li><li> Prepare media Schneider, com 2% de SFB para a infecção por diluindo completa media 1:05 em soro de mídia livre. </li><li> Estoque de vírus filtro de crude através de seringa 0,8 hum filtro para remover os restos celulares ou uso de vírus purificado. </li><li> Diluir vírus vaccinia em 2% de soro Schneider de mídia para obter multiplicidade de infecção (MOI) de 2. </li><li> Remover PBS a partir de tubos WellMate e privilegiada, com vírus diluído até a tubulação está livre de bolhas de ar. </li><li> Use esterilizados multi-canal de aspiração e coletor de pó para remover suavemente media das placas de células dsRNA tratados com Drosophila. </li><li> Dispense 30 mL por poço de vírus em placas usando o WellMate. </li><li> Centrífuga placas de 1 minuto a 300xg. </li><li> Esterilizar tubulação WellMate. Prime com 25 mL de blea 10%ch, aguarde 10 minutos, e privilegiada, com outra água sanitária de 25 mL de 10%. </li><li> Tubulação Prime com 50 mL de água seguida de 50 mL de etanol a 70%. Retorno tubulação para sua embalagem. </li><li> Retorno placas para recipiente Tupperware umidificado. Incubar a 25 ° C por 48 horas. </li></ol> Parte 3: Placas de coloração <ol><li> Fixar placas em 15 mL de formaldeído 4% / PBS por 10 minutos. Líquido é removido usando um aspirador multi-canal e distribuidor de vácuo em cada etapa. Uma vez que as placas foram fixadas, equipamentos esterilizados e os reagentes não são mais necessárias. </li><li> Remover corrigir e use WellMate para adicionar 30 mL PBST (PBS + 0,1% Triton-X). Incubar 10 minutos e repita. WellMate operar como descrito acima, exceto tubos não precisam ser esterilizados. </li><li> Para detectar a infecção vaccinia usamos um repórter β-galactosidase dirigido por um promotor vaccinia cedo / tarde. </li><li> Remover as células líquido e incubar em bloco (PBST com BSA 2%) por 10 minutos. </li><li> Diluir o anticorpo primário em bloco, remover o líquido e adicionar 15 mL de anticorpos para cada um pipetador de repetição bem usando uma multi-canal (Matrix). </li><li> Spin down placas (300xg, 1 min), cubra com um adesivo claro, e incubar a 4 ° C durante a noite. </li><li> Remover o anticorpo primário, e usar o WellMate para lavar poços em PBST 3 vezes, 10 minutos cada. </li><li> Diluir fluorescente etiquetado anticorpo secundário (FITC) e nuclear mancha em bloco, e adicionar 15 mL de poços usando um multi-canal de pipetador de repetição. </li><li> Incubar as placas de uma hora à temperatura ambiente, protegido da luz. </li><li> Lavar os poços no PBST 3 vezes, 10 minutos cada um usando o WellMate. </li><li> Seal placa com adesivo transparente, capa, e armazenar a 4 ° C até três semanas. </li></ol> Parte 4: Imagem e Análise de Imagem <ol><li> Use um microscópio automatizado com ampliação de 20x para placas de imagem. </li><li> Capturar imagens de núcleos total (DAPI) e as células infectadas (FITC). Otimizar o tempo de exposição para cada canal, individualmente, a fim de maximizar o alcance dinâmico. </li><li> Ajustar a função de foco automático, tendo uma pilha de imagens Z, 1um espaçadas, que abrangem os aviões acima e abaixo do plano previsto de foco. Assim que o avião ideal focal está localizado, ajustar as configurações de focagem automática em conformidade. </li><li> Imagem da placa inteira, captura de pelo menos três imagens por bem, tanto no canal de Hoechst (DAPI) e do canal de vírus (FITC). Escolha sites de várias regiões diferentes dentro de um poço, a fim de capturar imagens que representam o bem como um todo. </li><li> Use MetaXpress software para escrever um diário para o segmento de imagem e usar módulos Núcleos Contar para calcular o número total de células positivas para a coloração nuclear (células total) e infecção (FITC) para cada prato. </li><li> Calcular a infecção por cento (100 células * FITC positivo / total) e log transformar. </li><li> Para identificar os candidatos que usamos uma pontuação Z robusto baseado na mediana e intervalo interquartil de cada prato. Este método é insensível a outliers e dados não simétrica, proporcionando assim maior poder na identificação ainda bate fraca ou moderada 10. Para cada placa, a mediana é subtraído os dados log-transformados. Em seguida, os valores resultantes são divididos por 0,74 vezes o intervalo interquartil (diferença entre os valores do percentil 75 eo percentil 25). O fator 0,74 é utilizado para aproximar uma distribuição normal padrão. </li><li> A pontuação Z robusta é uma medida de distância em desvios-padrão da mediana da placa. Por exemplo, um robusto Z-score de 2 indica a infecção% do poço é de aproximadamente 2 desvios-padrão da mediana placa ou p &lt;0,05. </li><li> Analisar o número de células para identificar candidatos citotóxicos. Calcular o escore Z robusta de contagens nuclear. Candidatos com um robusto Z &lt;-2 em telas duplicadas são considerados citotóxicos. </li><li> Poços que têm uma pontuação Z robusta para a infecção por cento dos&gt; 2 ou &lt;-2 em telas duplicados (p &lt;0,0025), sem citotoxicidade são considerados candidatos potenciais a partir do ecrã principal. </li><li> Candidatos positivos são validados usando reagentes independentes. dsRNAs são gerados contra os genes de interesse de outra região do mRNA e testado como descrito acima. </li><li> Os genes para os quais duas dsRNAs independentes têm o mesmo fenótipo podem ser considerados para um estudo mais aprofundado. </li></ol> Parte 5: Imagens Representante e Interpretação das taxas de infecção <img src="/files/ftp_upload/2137/2137fig1.jpg" alt="Figura 1" /> Figura 1. Poços não infectadas não apresentam qualquer coloração para proteínas do vírus vaccinia, enquanto um representante infectados poço contém células coloração positiva para vaccinia-expressa a proteína beta-galactosidase (βgal), medido por microscopia de imunofluorescência.Virus = verde, núcleos = blue. <img src="/files/ftp_upload/2137/2137fig2.jpg" alt="Figura 2" /> Figura 2. Software de análise automatizada de imagem quantifica infecção em cada imagem usando parâmetros definidos pelo usuário. Em uma imagem representativa, o número total de núcleos celulares eo número de células que expressam o antígeno viral são contados com base no tamanho e da intensidade da coloração acima da coloração de fundo local. <img src="/files/ftp_upload/2137/2137fig3.jpg" alt="Figura 3" /> Figura 3. Knockdown de thread (DIAP) serve como um controle positivo para o esgotamento RNAi robusto de genes-alvo. Knockdown deste fator anti-apoptótico leva à morte celular dramático. Núcleos = blue. <img src="/files/ftp_upload/2137/2137fig4.jpg" alt="Figura 4" /> Figura 4. Knockdown da luciferase serve como um controle negativo para o efeito de double-stranded RNA tratamento sobre a infecção. Esgotamento de luciferase não tem nenhum efeito sobre a infecção em relação às células não tratadas. Virus = verde, núcleos = blue. <img src="/files/ftp_upload/2137/2137fig5.jpg" alt="Figura 5" /> Figura 5. Knockdown de proteína βgal serve como um controle positivo para a redução da infecção, já que o ensaio utiliza níveis de proteína βgal como uma leitura de infecção. Esgotamento dos resultados βgal em uma diminuição na porcentagem de células infectadas. Virus = verde, núcleos = blue. <img src="/files/ftp_upload/2137/2137fig6.jpg" alt="Figura 6" /> Figura 6. Knockdown de celulares fator Rab5 resulta em uma diminuição da percentagem de células infectadas. Desde Rab5 é conhecido por participar de endocitose, este fator provavelmente contribui para a entrada do vírus vaccinia. Isto representa um outlier exemplo da tela. Virus = verde, núcleos = blue.

<ol>
	<li>Ramadan, N., Flockhart, I., Booker, M., Perrimon, N., Mathey-Prevot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+high-throughput+RNAi+screens+in+cultured+Drosophila+cells.">Design and implementation of high-throughput RNAi screens in cultured Drosophila cells.</a> Nat Protoc. 2, 2245-2264 (2007).</li><li>Chotkowski, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus+infection+of+Drosophila+melanogaster+induces+a+protective+RNAi+response.">West Nile virus infection of Drosophila melanogaster induces a protective RNAi response.</a> Virology. 377, 197-206 (2008).</li><li>Blondel, D., Petitjean, A. M., Dezelee, S., Wyers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+in+Drosophila+melanogaster+cells:+regulation+of+viral+transcription+and+replication.">Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication.</a> J Virol. 62, 277-284 (1988).</li><li>Sessions, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+insect+and+human+dengue+virus+host+factors.">Discovery of insect and human dengue virus host factors.</a> Nature. 458, 1047-1050 (2009).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+RNAi+screening+in+Drosophila+S2+cells:+what+have+we+learned+about+host-pathogen+interactions?.">Genomic RNAi screening in Drosophila S2 cells: what have we learned about host-pathogen interactions?.</a> Curr Opin Microbiol. 11, 262-270 (2008).</li><li>Hao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+RNAi+screen+identifies+host+genes+important+for+influenza+virus+replication.">Drosophila RNAi screen identifies host genes important for influenza virus replication.</a> Nature. 454, 890-893 (2008).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+activity+coupled+with+fatty+acid+biosynthesis+is+required+for+viral+replication.">COPI activity coupled with fatty acid biosynthesis is required for viral replication.</a> PLoS Pathog. 2, e102-e102 (2006).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+reveals+a+specific+sensitivity+of+IRES-containing+RNA+viruses+to+host+translation+inhibition.">Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition.</a> Genes Dev. 19, 445-452 (2005).</li><li>Moser, T. S., Jones, R. G., Thompson, C. B., Coyne, C. B., Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Kinome+RNAi+Screen+Identified+AMPK+as+Promoting+Poxvirus+Entry+through+the+Control+of+Actin+Dynamics.">A Kinome RNAi Screen Identified AMPK as Promoting Poxvirus Entry through the Control of Actin Dynamics.</a> PLoS Pathog. 6, e1000954-e1000954 (2010).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4, 67-73 (1999).</li><li>Echeverri, C. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+in+cultured+cells:+a+user's+guide.">High-throughput RNAi screening in cultured cells: a user's guide.</a> Nat Rev Genet. 7, 373-384 (2006).</li></ol>

Não há conflitos de interesse declarados.

Genoma de despistagem RNAi em Drosophila fornece um método robusto e eficiente para a análise da componente celular da infecção viral. Um número de vírus infectam as células de mamíferos Drosophila, que pode ser usado para identificar os componentes da máquina de resposta intrínseca imunológico e fatores do hospedeiro proteínas necessárias para a replicação viral que pode representar novos alvos terapêuticos. Antes de executar uma tela, o ensaio deve ser cuidadosamente otimizado para o tipo de célula, número de células, e nível de infecção, e deve ser bem controlada, incluindo tanto positivas como negativas viral e alvos celulares 11. É importante garantir a separação suficiente entre os controles positivos e negativos antes de triagem para maximizar a gama dinâmica do ensaio. Uma vez que a tela foi realizada, os candidatos podem ser divididos em categorias funcionais para determinar quais os mecanismos de acolhimento contribuem para a infecção. Em busca de vírus vaccinia mamíferos, tais como, a contribuição dos homólogos mamíferos por RNAi deve ser determinada como parte da análise secundária. Juntos, esses métodos de triagem robusta nos permitirá ganhar a introspecção em mecanismos complexos pelos quais os vírus interagem com as células do hospedeiro.

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<td>Cell culture</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Schneider&#x2019;s media</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11720</td>
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<td>GlutaMAX 100X</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>35050</td>
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<td>Penicillin/streptomycin (pen/strep)</td>
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<td>GIBCO</td>
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<td>Fetal Bovine Serum (FBS)</td>
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<td>Genome-wide dsRNA library</td>
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<td>384 well plates, black with clear bottom</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>B2261</td>
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<td>Fluorescently-labeled secondary antibodies</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
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<td>ImageXpressMicro automated microscope</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
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<td>MetaXpress image analysis software</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
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rnai screening for host factors involved in vaccinia virus infection using drosophila cells

Patógenos virais representam uma ameaça de saúde pública, não só pode causar epidemias vírus natural da doença humana, mas o seu uso potencial em bioterrorismo também é uma preocupação. Uma melhor compreensão dos fatores celulares que a infecção impacto seria facilitar o desenvolvimento de muito necessária terapêutica. Os recentes avanços na tecnologia de interferência de RNA (RNAi) juntamente com o seqüenciamento do genoma completo de vários organismos levou para a otimização do genome-wide, baseada em células de perda de função-telas. Células Drosophila são particularmente passíveis de genoma escala de telas por causa da facilidade e eficiência de RNAi neste sistema 1. Importante, uma grande variedade de vírus podem infectar as células Drosophila, incluindo um número de vírus de mamíferos de importância médica e agrícola 2,3,4. Telas anteriores RNAi em Drosophila identificou factores do hospedeiro que são necessários para as várias etapas na infecção pelo vírus, incluindo entrada, tradução e replicação de RNA 5. Além disso, muitos dos fatores celulares necessárias para a replicação viral em cultura de células Drosophila também são limitantes nas células humanas infectadas com estes vírus 4,6,7,8, 9. Portanto, a identificação de fatores do hospedeiro cooptados durante a infecção viral apresenta novos alvos para a terapêutica antiviral. Aqui apresentamos um protocolo generalizada para uma tela de RNAi de alto rendimento para identificar os fatores celulares envolvidos na infecção viral, usando vírus vaccinia como um exemplo.

Watch this Scientific Journal Video about RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells at JoVE.com

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novos fatores hospedeiro envolvidos na infecção viral podem ser identificados através de células baseado em perda de todo o genoma da função de triagem RNAi. A<em&gt; Drosophila</em&gt; Modelo de cultura de células é particularmente receptivo a esta abordagem, devido à facilidade e eficiência de RNAi. Aqui demonstramos esta técnica usando<em&gt; Vaccinia</em&gt; Vírus como um exemplo.

RNAi Triagem para Fatores Hospedeiros Envolvidos na Vaccinia Infecção pelo Vírus usando Drosophila Células

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

rnai-screening-for-host-factors-involved-vaccinia-virus-infection

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JoVE Journal

Immunology and Infection

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

9.4K visualizações.  University of Pennsylvania. Novos fatores hospedeiro envolvidos na infecção viral podem ser identificados através de células baseado em perda de todo o genoma da função de triagem RNAi. A Drosophila Modelo de cultura de células é particularmente receptivo a esta abordagem, devido à facilidade e eficiência de RNAi. Aqui demonstramos esta técnica usando Vaccinia Vírus como um exemplo.

Vídeo: RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Dissecando interação hospedeiro-vírus em replicação lítica de um herpesvírus modelo

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Imunologia e Infecção

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS Mediada frente Triagem Genética para identificação de genes envolvidos na resistência não hospedeiras


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Metodologia para a Geração Eficiente de Proteínas do Vírus da Vaccinia marcadas fluorescentes

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

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 ...

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 ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Os ensaios baseados em microscopia para high-throughput screening de Fatores Hospedeiros Envolvidos na<em&gt; Brucella</em&gt; A infecção de células HeLa

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

Ensaios de caracterização de romance endoteliais reguladores envolvidos na resposta inflamatória de triagem

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

Arbovírus infecções como ferramentas de triagem para a identificação de fatores Antiviral Viral de imunomoduladores e Host

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Interação de célula de imagem latente na Mucosa traqueal durante a infecção de vírus de Influenza usando microscopia Intravital dois fotões

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Estabelecimento de infecção Viral e análise da interação vírus-hospedeiro em Drosophila Melanogaster.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...