Agradecemos SIGA Technologies (Corvallis, OR) para a prestação de ST-246. DKR foi apoiado por uma bolsa de formação NIH em imunologia para Boston University (5T32AI 7309). Este trabalho foi financiado em parte pelo P41 086.180, NIH RO1AI1096159-01, e SR3 (a JHC).

1. Placa Células <ol><li> Dissociar células HeLa a partir de chapa e dilua em meio de crescimento (DMEM, 2 mM de L-glut, FBS a 10%) para aproximadamente 2,0 x 10 5 células / ml. Distribuir 100 ^ l / poço de uma, claro placa de 96 poços de fundo plano com paredes pretas (20000 células / poço). </li><li> Incubar as células durante 24 horas até à confluência em uma incubadora a 37 ° C + 5% de CO 2. </li></ol> 2. Infectar as células <ol><li> Diluir vírus e infectar as células. <ol><li> Thaw TRPV (vírus Triplo; Precoce Vênus, Intermediário mCherry, Late TagBFP) e PLV (Promotor-less Vênus) vírus fluorescente e desagregar utilizando sonicação por 5 minutos. Alternativamente, os estoques de vírus em bruto pode ser misturado a 1:1 com 0,25 mg / ml de tripsina em meio de infecção e incubados a 37 ° C durante 30 min. </li><li> Diluir stocks de vírus bruto em 37 ° C media de infecção (DMEM, 2 mM L-glut, 2% FBS). Para infecções de alto MOI (10 UFP / célula), diluir stock de virus para 1,0 x 10 7 PFU / ml assumindo 5 x 10 4células / poço e um volume de inoculo de 50 ul. Plano de infectar 3 poços réplicas para cada tratamento com TRPV e outros 3 poços idênticas para o mesmo tratamento com PLV para explicar a fluorescência de fundo específico para cada tratamento. </li><li> Infect poços por adição de 50 ul / cavidade diluída em meio de infecção do vírus. Esta é definida como a infecção tempo = 0 hr post (0 hpi). </li></ol></li><li> Dilui-se os compostos de tratamento desejados para o meio de infecção. Para todos os tratamentos, uma única diluição 2x mestre mistura deve ser feita e aplicada a replicar poços (volume total de 350 ul, por exemplo para seis placas de 96 poços com 50 mL de cada). <ol><li> Dilui-se os compostos experimentais em meios de infecção. </li><li> Dilui-controlo com o veículo solvente (por exemplo, PBS, DMSO) para o meio de infecção. Nota: parecidos concentrações finais de solventes de controle de veículo deve ser usado como aplicado para compostos experimentais (por exemplo, se o seu estoque IBT é feito com DMSO e utilizado numa concentratio finaisns de 1 ul / ml, em seguida, utilizar apenas DMSO a 1 mL / mL como o controlo do veículo). </li><li> Dilui-se os compostos em meios de controlo de infecção. Compostos de controlo recomendadas incluem: 3,6 uM (1 ug / ml) 1-β-D-arabinofuranosilcitosina (AraC), 50 ^ M (11,7 mg / ml) de isatina β-tiossemicarbazona (TBI), 60 uM (50 ug / ml), rifampicina, ou 5 uM (1,9 ug / ml) de ST-246 8,15,16. Veja resultados representativos para efeitos esperados. Nota: certifique-esta diluição em duas vezes (2x), a concentração final desejada, desde que este é adicionado numa proporção de 1:1 para o volume já no poço. </li></ol></li><li> Imediatamente após a adição do vírus, adicionar 50 ul de meios de infecção contendo tratamentos e controles desejados como diluída no passo 2.2. Nota: Para ensaiar a inibição de expressão antes fase inicial, o composto (s) pode ser adicionado directamente à suspensão de inóculo de vírus diluído (passo 2.1.2) ou a células hospedeiras antes da infecção (antes do passo 2.1.3). </li><li> Incubar 6-24 hr em um 37 &amp; #176; incubadora C + 5% de CO 2. </li></ol> 3. Corrigir Células <ol><li> Fixar as células por adição de 100 ul de paraformaldeido a 8% mídia infecção já em cada poço. Incubar à temperatura ambiente durante 15 minutos ao abrigo da luz. Nota: Recomenda-se adicionar 2x PFA (8%) diretamente para a mídia de infecção para evitar aerosolization de vaccinia infecciosa que pode ocorrer se a mídia de alta titulação é invertida diretamente no prato resíduos previamente à sua fixação. </li><li> Remover fixador invertendo a placa para o prato de resíduos. </li><li> Adicionar 100 ul de PBS temperatura ambiente. </li><li> Selar placa com película adesiva opticamente clara. Nota: As placas podem ser armazenadas a 4 ° C se não for lida imediatamente. Certifique-se de retornar placas seladas à temperatura ambiente antes de ler para evitar a condensação de distorcer as medições espectrofotômetro. </li></ol> 4. Quantificar Vírus Crescimento <ol><li> Meça endpoint fluorescência em 515:530 para Vênus, 587:610 para mCherry, e 415:457 fou TagBFP (Excitação: comprimento de onda de emissão em nm). Quatro medidas devem ser em média por poço usando ajustes de ganho otimizados para cada canal. Nota: O comprimento de onda de emissão apropriado pode ser diferente, dependendo do modelo e do filtro características específicas do seu leitor de placas. Isto pode ser determinado empiricamente através da realização de um comprimento de onda de emissão de verificação para determinar o ponto em que não é a maior diferença entre TRPV e VLP para cada canal. </li><li> Normalizar os dados brutos para facilitar a comparação entre experimentos. <ol><li> Para cada canal, determinar a média de TRPV replicar e poços PLV. </li><li> Subtrair os médios medições de fundo PLV-infectados a partir das medições experimentais médios TRPV infectados para cada tratamento. </li><li> Normalizar os dados pela divisão de cada fundo valor subtraído pelo valor somente veículo bem. </li><li> Uma vez que um experimento foi repetido várias vezes, a análise de um teste de variância (one-way ANOVA) e vários cOMPARAÇÃO pós-teste pode ser executado para determinar se qualquer um dos tratamentos reflecte uma diferença estatisticamente significativa relativamente ao tratamento apenas com veículo, para cada canal. </li></ol></li></ol> . 5 Protocolo Alternativa: Ensaios de Placa cinéticos <ol><li> Execute todas as etapas através da adição de tratamentos (passo 2.3). </li><li> Placa de vedação com película adesiva e coloque na câmara leitor de placas equilibrada a 37 ° C. Nota: Cuidado especial deve ser tomado para manter as placas de forma consistente a 37 ° C para prevenir o stress celular indevida e condensação. Para os ensaios cinéticos, é particularmente importante a utilização de um meio tamponado (bicarbonato de sódio e HEPES) para manter o pH adequado sem a adição de 5% de CO 2. </li><li> Conjunto leitor de placas de ganhar manualmente para evitar a saturação de pontos de tempo posteriores. Nota: optimização ganho exacta pode ser obtida através da realização de ensaios de um ponto de extremidade, sob condições semelhantes. </li><li> Adquirir leituras de hora em hora para 8-24 hpi e normalizar usando simiprocedimento lar como no ensaio endpoint (passo 4.2). </li><li> Os pontos de tempo individuais podem ser analisadas para a significância estatística usando métodos semelhantes como o ponto final do ensaio anteriormente descrito. </li></ol>

<ol>
	<li>Yen, J., Golan, R., Rubins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+infection+&amp;+temporal+analysis+of+virus+gene+expression:+part+1.">Vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1.</a> J Vis Exp. 26 (26), (2009).</li><li>Hansen, S. G., Cope, T. A., Hruby, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BiZyme:+a+novel+fusion+protein-mediating+selection+of+vaccinia+virus+recombinants+by+fluorescence+and+antibiotic+resistance.">BiZyme: a novel fusion protein-mediating selection of vaccinia virus recombinants by fluorescence and antibiotic resistance.</a> BioTechniques. 32 (5), 1182-1187 (2002).</li><li>Popov, S., Mirshahidi, S., Essono, S., Song, R., Wang, X., Ruprecht, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+recombinant+vaccinia+viruses+via+green+fluorescent+protein+selection.">Generation of recombinant vaccinia viruses via green fluorescent protein selection.</a> DNA and Cell Biology. 28 (3), 103-108 (2009).</li><li>Bartee, E., Mohamed, M. R., Lopez, M. C., Baker, H. V., McFadden, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+addition+of+tumor+necrosis+factor+plus+beta+interferon+induces+a+novel+synergistic+antiviral+state+against+poxviruses+in+primary+human+fibroblasts.">The addition of tumor necrosis factor plus beta interferon induces a novel synergistic antiviral state against poxviruses in primary human fibroblasts.</a> Journal of Virology. 83 (2), 498-511 (2009).</li><li>Ward, B. M., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+intracellular+movement+of+vaccinia+virus+virions+containing+a+green+fluorescent+protein-B5R+membrane+protein+chimera.">Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera.</a> Journal of Virology. 75 (10), 4802-4813 (2001).</li><li>Johnson, M. C., Damon, I. K., Karem, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+high-throughput+vaccinia+virus+neutralization+assay+for+testing+smallpox+vaccine+efficacy+based+on+detection+of+green+fluorescent+protein.">A rapid, high-throughput vaccinia virus neutralization assay for testing smallpox vaccine efficacy based on detection of green fluorescent protein.</a> Journal of Virological Methods. 150 (1-2), 14-20 (2008).</li><li>Dower, K., Rubins, K. H., Hensley, L. E., Connor, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Vaccinia+reporter+viruses+for+rapid,+high+content+analysis+of+viral+function+at+all+stages+of+gene+expression.">Development of Vaccinia reporter viruses for rapid, high content analysis of viral function at all stages of gene expression.</a> Antiviral Research. 91 (1), 72-80 (2011).</li><li>Dower, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+pyridopyrimidinone+inhibitor+of+orthopoxviruses+from+a+diversity-oriented+synthesis+library.">Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library.</a> Journal of Virology. 86 (5), 2632-2640 (2012).</li><li>De Clercq, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Potential+of+the+Acyclic+Nucleoside+Phosphonates+Cidofovir,+Adefovir,+and+Tenofovir+in+Treatment+of+DNA+Virus+and+Retrovirus+Infections.">Clinical Potential of the Acyclic Nucleoside Phosphonates Cidofovir, Adefovir, and Tenofovir in Treatment of DNA Virus and Retrovirus Infections.</a> Clinical Microbiology Reviews. 16 (4), 569-596 (2003).</li><li>Prichard, M. N., Kern, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthopoxvirus+Targets+for+the+Development+of+New+Antiviral+Agents.">Orthopoxvirus Targets for the Development of New Antiviral Agents.</a> Antiviral Research. 10, (2012).</li><li>Fenner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+successful+eradication+campaign.+Global+eradication+of+smallpox.">A successful eradication campaign. Global eradication of smallpox.</a> Reviews of Infectious Diseases. 4 (5), 916-930 (1982).</li><li>Breman, J. G., Henderson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+dilemmas--monkeypox,+smallpox,+and+biologic+terrorism.+The+New+England.">Poxvirus dilemmas--monkeypox, smallpox, and biologic terrorism. The New England.</a> Journal of Medicine. 339 (8), 556-559 (1998).</li><li>Baker, R. O., Bray, M., Huggins, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+antiviral+therapeutics+for+smallpox,+monkeypox+and+other+orthopoxvirus+infections.">Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections.</a> Antiviral Research. 57, 1-2 (2003).</li><li>Rimoin, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+increase+in+human+monkeypox+incidence+30+years+after+smallpox+vaccination+campaigns+cease+in+the+Democratic+Republic+of+Congo.">Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo.</a> Proceedings of the National Academy of Sciences of the U S A. 107 (37), 16262-167 (2010).</li><li>Sodeik, B., Griffiths, G., Ericsson, M., Moss, B., Doms, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+vaccinia+virus:+effects+of+rifampin+on+the+intracellular+distribution+of+viral+protein+p65.">Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.</a> Journal of Virology. 68 (2), 1103-1114 (1994).</li><li>Grosenbach, D. W., Jordan, R., Hruby, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+small-molecule+antiviral+ST-246+as+a+smallpox+therapeutic.">Development of the small-molecule antiviral ST-246 as a smallpox therapeutic.</a> Future Virology. 6 (5), 653-671 (2011).</li><li>Broyles, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+transcription.">Vaccinia virus transcription.</a> Journal of General Virology. 84 (9), 2293-2303 (2003).</li><li>Condit, R. C., Niles, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+viral+transcription+elongation+and+termination+during+vaccinia+virus+infection.">Regulation of viral transcription elongation and termination during vaccinia virus infection.</a> Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1577 (2), 325-336 (2002).</li><li>D&#237;az-Guerra, M., Rivas, C., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+expression+of+the+2-5A+synthetase/RNase+L+system+results+in+inhibition+of+vaccinia+virus+replication.">Inducible expression of the 2-5A synthetase/RNase L system results in inhibition of vaccinia virus replication.</a> Virology. 227 (1), 220-228 (1997).</li><li>Cohrs, R. J., Condit, R. C., Pacha, R. F., Thompson, C. L., Sharma, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+ppp(A2&#8217;p)nA-dependent+RNase+by+a+temperature-sensitive+mutant+of+vaccinia+virus.">Modulation of ppp(A2&#8217;p)nA-dependent RNase by a temperature-sensitive mutant of vaccinia virus.</a> Journal of Virology. 63 (2), 948-951 (1989).</li><li>S&#225;nchez-Puig, J. M., S&#225;nchez, L., Roy, G., Blasco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+different+leukocyte+cell+types+to+Vaccinia+virus+infection.">Susceptibility of different leukocyte cell types to Vaccinia virus infection.</a> Virology Journal. 1 (1), (2004).</li><li>Bengali, Z., Satheshkumar, P. S., Yang, Z., Weisberg, A. S., Paran, N., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+S2+cells+are+non-permissive+for+vaccinia+virus+DNA+replication+following+entry+via+low+pH-dependent+endocytosis+and+early+transcription.">Drosophila S2 cells are non-permissive for vaccinia virus DNA replication following entry via low pH-dependent endocytosis and early transcription.</a> PLoS One. 6 (2), (2011).</li></ol>

Os autores não têm nada a divulgar e sem patentes estão pendentes de vírus ou métodos de rastreio.

Aqui, nós descrevemos o uso prático de um multi-estágio vírus vaccinia repórter (TRPV), que fornece em tempo real reprodutíveis medidas, feedback de replicação do vírus. Ao utilizar inibidores poxvírus bem definidos, fomos capazes de mostrar que TRPV responde de uma maneira consistente com o mecanismo entendido de ação para cada inibidor. Embora o vírus TRPV fornece as informações mais abrangentes sobre vírus progressão etapa, em duas etapas (iRev, LREV) e de fase única (EV, IV, LV) vírus também pode...

Tradicionalmente, a expressão do vírus é estudada, utilizando técnicas de biologia molecular (por exemplo, transferência de Northern, transferência de western, de microarray de hibridação, etc) 1. Enquanto estes métodos são capazes de fornecer a informação detalhada com respeito à categorização mudanças de mRNAs individuais ou de proteínas de expressão, que não são normalmente passíveis de processos em tempo real e de alto rendimento. Abordagens alternativas usando os repórteres à base de fluorescência foram aplicadas anteriormente ao trabalhar com poxvírus; no entanto, o seu desenvolvimento e uso foi motivado por objetivos variados. Vários destes métodos foram concebidos para a selecção de vírus recombinantes 2,3. Estas técnicas expressar EGFP com a incorporação apropriada e expressão de ADN exógena a partir de clones de vaccinia recombinantes. Da mesma forma, várias estirpes de vaccinia que expressam estavelmente EGFP solúveis ou proteínas GFP expressos sob um promotor de vacina nativa tem sido amplamente usado. Estes são typcamente utilizadas para quantificar a entrada do vírus e replicação durante ensaios de neutralização com base em anticorpos, inibição química, ou a comparação da eficácia antiviral 4-6. Embora esses vírus têm se mostrado útil, eles são limitados em sua capacidade de fornecer informações detalhadas sobre o ponto de inibição devido ao seu uso de um único promotor viral início / final ambíguo. Métodos anteriores também fizeram uso da proteína EGFP com características sub-ótimos sinal-ruído. Devido à complexidade da replicação de poxvírus ea falta de ferramentas existentes disponíveis para analisar mudanças em tempo real na expressão genética viral, desenvolvemos um conjunto de vírus repórter fase única e multi 7,8. Conforme descrito em publicações anteriores, esses vírus expressar um, dois, ou três proteínas fluorescentes espectralmente distintas de promotores vaccinia nativas durante o início, etapas intermediárias, ou no final infecção. Esses vírus podem ser nósed como indicadores de andamento da replicação do vírus, utilizando um microscópio de fluorescência e que são igualmente adequados para a placa-e-leitor de ensaios à base de alto rendimento. Estes vírus são fáceis de usar no lugar do vírus de tipo selvagem, crescendo com cinética similar para alcançar títulos equivalentes 7. Durante o planejamento e criação desses vírus, muito cuidado foi tomado na seleção de fluoróforos que têm altas características de sinal-para-fundo com eficiência dobradura superiores para facilitar a quantificação confiável, com feedback rápido às mudanças na expressão (Vênus, mCherry e TagBFP). Além disso, as combinações de promotores virais foram escolhidos que produzem alta fidelidade, a expressão específica do estágio inequívoca, fornecendo informações sobre a gama completa de início (C11R promotor), intermediária (promotor G8R) e tardia (F17R promotor) a expressão do gene, em contraste com a ambígua promotores de início / final. Estes vírus podem ser utilizados como uma ferramenta para a investigating o ciclo de vida do poxvírus, vírus vaccinia prototípico. Muito ainda não se sabe sobre a interação hospedeiro-vírus, apesar da sua longa história de estudo. Vaccinia é complexo, produzindo mais de 200 proteínas específicas, muitas das quais são imunomoduladores e hospedar antagônicos. Após a infecção, o vírus vaccinia imediatamente começa a transcrição do mRNA precoce. Isto é facilitado por uma RNA polimerase e transcrição fatores que foram carregados no genoma viral durante a embalagem virion e mantidas em um estado pausado até que a infecção subseqüente. Esta expressão precoce produz principalmente proteínas necessárias para a supressão do sistema imunológico hospedeiro (decapping mRNA, dsRNA seqüestro e proteínas receptoras chamariz, bem como inibidores de apoptose, resposta ao estresse, e Toll, IL, e sinalização NF-kB) e replicação do genoma. A expressão precoce também produz fatores necessários para a expressão intermediário de transcrição. Expressão intermédio inclui a expressão de fatores de transcrição fase final. Esteexpressão cascata leva à produção de proteínas de vírus estruturais e enzimáticas durante a fase tardia da infecção, que são necessários para a montagem completa do virião maduro vaccinia. Nosso conjunto de vírus repórter fluorescentes permite um rápido progresso a ser feito na compreensão da biologia poxvírus. Um dos métodos mais comuns e demoradas no campo da virologia é o ensaio de crescimento. Isto tipicamente envolve a infecção de células, promulgação de uma série de tratamentos, o vírus da colheita por lise das células infectadas, e quantificando o título viral resultante por ensaio de placas. Usando os vírus repórter descritos aqui permite a medição em tempo real do crescimento do vírus que pode ser facilmente ensaiada e comparada entre os vários tratamentos realizados em paralelo. Prevemos este conjunto de reagentes serão utilizados em vários protocolos para a identificação de alterações na expressão de genes virais, em resposta a tratamento medicamentoso, o RNAi knockdown ou restrição gama de hospedeiros. Este método também permite a análise de alto conteúdo em uma escala maior do que anteriormente disponível, permitindo a de alto rendimento telas de drogas anti-virais para estágios alvo bem definidas de interesse. Enquanto numerosos potenciais tratamentos para combater a infecção poxvírus têm sido identificadas, a FDA aprovou apenas terapias eficazes para o tratamento de infecção por poxvírus são o nucleosídeo acíclico fosfonato, Cidofovir, e tratamento com vaccinia imune 9,10 globulina. Apesar da erradicação da varíola em 1977 11, poxvírus continuam a ser uma ameaça significativa para a saúde humana 12. Cessação da vacinação generalizada contra o vírus da varíola levou a um aumento da susceptibilidade a outros poxvírus 13. Por exemplo, as regiões da África Central anteriormente protegidos pela vacinação está enfrentando um surto de infecções por vírus Monkeypox 14. Preocupação significativa também foilevantadas em relação a susceptibilidade latente a libertação intencional de vírus da varíola. Devido às terapias limitados actualmente disponíveis, há uma necessidade urgente de desenvolvimento de novos tratamentos. Estes vírus repórter permitir rastreio pequena molécula inibidora rápido e de alta produtividade para a inibição de um determinado estádio da replicação viral. Identificação de inibidores que visam estágios de expressão virais atualmente não alvo de inibição irá facilitar o desenvolvimento de terapias de combinação com o aumento da potência. Muito pode ser adquirida sobre biologia celular vaccinia observando mudanças na expressão gênica de cada etapa. Atenuação por manipulação química ou genética de uma célula hospedeira infectada é normalmente expressa em termos de redução dos títulos virais. No entanto, comparando as alterações em cada fase da cascata de expressão viral, pode-se obter uma compreensão mais completa da forma como a aptidão do vírus é impactoed por um tratamento em particular. Estes dados foram mostrados para correlacionar bem com a saída título do vírus tradicional, mas fornecer informações mecanicista mais detalhada, bem como capacidades de alto rendimento 7.

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		<tr height="24">
			<td height="24" style="height:24px;width:747px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:104px;">11995-065</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fetal Bovine Serum - Optima, Heat Inactivated (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">200 mM L-Glutamine, (L-glut)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3712</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Trypsin, 2X, Sterile, Irradiated</td>
			<td>Worthington Biochemical Corp.</td>
			<td>TRLVMF</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl Sulfoxide, Cell Culture (DMSO)</td>
			<td>American Bioanalytical</td>
			<td>AB03091</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1-&#946;-D-Arabinofuranosylcytosine&#160; (AraC), MW= 280 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>C6645</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">isatin &#946; -thiosemicarbazone (IBT), MW= 234 g/mol</td>
			<td>Fisher Scientific</td>
			<td>NC9075202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Rifampicin, MW= 823 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">ST-246, MW= 376 g/mol</td>
			<td>SIGA Labs, Corvallis, OR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">8% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TempPlate RT optically clear film</td>
			<td>USA Scientific</td>
			<td>2978-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

vaccinia reporter viruses for quantifying viral function at all stages of gene expression

Poxviruses são uma família de vírus de DNA fita dupla que incluem patógenos ativos humanos, tais como macacos, molusco contagiousum e vírus Contagalo. A família também inclui o vírus da varíola, da varíola. Devido à complexidade da replicação de poxvírus, muitas questões ainda permanecem em relação à sua estratégia de expressão gênica. Neste artigo, descreve-se a concepção e utilização de vírus vaccinia recombinantes que permitem a medição em tempo real das fases únicas e múltiplas de expressão de genes virais em um formato de alto rendimento. Esta é activada através da utilização de proteínas fluorescentes espectralmente distintos como repórteres para cada uma das três fases da replicação viral. Estes vírus proporcionar uma razão elevada de sinal-para-ruído de fase, mantendo padrões específicos de expressão, permitindo ensaios baseados em placas e as observações microscópicas de propagação de vírus e de replicação. Estas ferramentas têm usos para descoberta antiviral, os estudos sobre a interação vírus-hospedeiro, e bio evolutivologia.

Como exemplo do uso típico destes vírus, o vírus da fluorescência repórter triplo foi utilizada para comparar o ponto de inibição de vários inibidores de poxvírus bem definidas. As células HeLa foram colocadas em cultura de tecidos tratados claras placas de 96 poços de paredes pretas de fundo plano e incubadas durante a noite. As monocamadas confluentes foram infectadas a uma multiplicidade de infecção (MOI) de 10 usando um triplo vírus repórter (TRPV; Tabela 1)</stron...

Watch this Scientific Journal Video about Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression at JoVE.com

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Descreve-se a utilização de um vírus vaccinia repórter fluorescente que permite a medição em tempo real da infectividade viral e a expressão do gene através da expressão específica da fase de fluoróforos espectralmente distintos repórter. Detalhamos um método baseado em placa para identificar com precisão a fase em que a replicação do vírus é afetada em resposta a pequena inibição molécula.

Repórter Vaccinia vírus para quantificar Função Viral em todas as fases da Expressão Gênica

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

vaccinia-reporter-viruses-for-quantifying-viral-function-at-all

Research

JoVE Journal

Immunology and Infection

11.2K visualizações.  Boston University School of Medicine.  Descreve-se a utilização de um vírus vaccinia repórter fluorescente que permite a medição em tempo real da infectividade viral e a expressão do gene através da expressão específica da fase de fluoróforos espectralmente distintos repórter. Detalhamos um método baseado em placa para identificar com precisão a fase em que a replicação do vírus é afetada em resposta a pequena inibição molécula. 

Vídeo: Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Geração de células T específicas Multivirus para Prevenir / tratar infecções virais após transplante de células-tronco hematopoéticas alogênico

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Imunologia e Infecção

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&quot;Bioluminescent &#39;Phage Reporter para a detecção de patógenos bacterianos Categoria A

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Análise quantitativa de todos Influenza Tipo A Hemaglutininas Virais e neuraminidases usando anticorpos Universal em Ensaios Blot simples slot

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Relação custo-benefício Método de Rastreamento Fonte microbiana, utilizando específicas Vírus Humano e Animal

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Isolamento de variantes de vírus de RNA Fidelity e Caracterização de frequência de mutação de vírus

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Estabelecer uma cultura líquida, coberta de vias respiratórias humanas epiteliais Polarized Calu-3 células para estudar a resposta Cell Host para patógenos respiratórios<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Rato Fetal Liver Sistema de Cultura de dissecar Funções gene alvo nas fases precoces e tardias da Terminal Eritropoiese

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

A administração intranasal de recombinantes Vacinas contra Influenza em quimérico rato Modelos para estudar a imunidade mucosal

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

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 ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Desenvolvimento de um Sistema Reporter vírus da hepatite B de acompanhar as fases iniciais do ciclo de replicação

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...