Damos las gracias a las Tecnologías de SIGA (Corvallis, OR) para proporcionar ST-246. DKR fue apoyada por una beca de formación de los NIH en la inmunología de la Universidad de Boston (5T32AI 7309). Este trabajo fue apoyado en parte por P41 086.180, NIH RO1AI1096159-01, y SR3 (para JHC).

1. Células de la placa <ol><li> Disociar las células HeLa de la placa y se diluye en medio de crecimiento (DMEM, 2 mM de L-superabundancia, 10% de FBS) a aproximadamente 2,0 x 10 5 células / ml. Dispense 100 l / pocillo en una placa de negro-amurallado, clara de fondo plano de 96 pocillos (20.000 células / pocillo). </li><li> Se incuban las células durante 24 horas hasta la confluencia en un incubador de 37 º C + 5% de CO 2. </li></ol> 2. Infectar células <ol><li> Diluir el virus e infectar las células. <ol><li> Descongele TRPV (virus Triple; Early Venus, Intermedio mCherry, Abierto TagBFP) y PLV (promotor de menos Venus) virus fluorescente y desagregar utilizando sonicación durante 5 minutos. Alternativamente, las reservas de virus en bruto se pueden mezclar 1:1 con 0,25 mg / ml de tripsina en los medios de comunicación de infección y se incubaron a 37 ° C durante 30 min. </li><li> Diluir las reservas de virus crudo en 37 ° C medios de infección (DMEM, 2 mM L-superabundancia, 2% de SFB). Para las infecciones de alto MOI (10 UFP / célula), diluir de virus a 1,0 x 10 7 PFU / ml suponiendo 5 x 10 4células / pocillo y un volumen de inóculo de 50 l. Plan de infectar en 3 pocillos replicados para cada tratamiento con TRPV y otros 3 pocillos replicados para el mismo tratamiento con PLV para tener en cuenta la fluorescencia de fondo específica para cada tratamiento. </li><li> Infectar pozos mediante la adición de 50 l / pocillo diluida virus en medios de infección. Esta se define como la infección por el tiempo = 0 h después (0 hpi). </li></ol></li><li> Diluir compuestos deseados del tratamiento en los medios de comunicación de infección. Para todos los tratamientos se debe hacer un único maestro dilución mezcla 2x y se aplicó a pocillos por duplicado (por ejemplo, 350 l de volumen total de seis de 96 pocillos con 50 l cada uno). <ol><li> Diluir compuestos experimentales en los medios de comunicación de infección. </li><li> Diluir-control del vehículo disolvente (por ejemplo, PBS, DMSO) en los medios de la infección. Nota: Las concentraciones finales similares de disolventes de control del vehículo deben utilizarse conforme a lo solicitado compuestos experimentales (por ejemplo, si sus acciones IBT se hace con DMSO y se utiliza a una concentratio definitivans de 1 l / ml, a continuación, utilizar DMSO solo a 1 mu l / ml como el control del vehículo). </li><li> Diluir compuestos de control en los medios de comunicación de infección. Compuestos de control recomendados incluyen: 3,6 M (1 mg / ml) 1-β-D-arabinofuranosilcitosina (AraC), 50 M (11,7 g / ml) isatina β-tiosemicarbazona (IBT), 60 M (50 g / ml) rifampicina, o 5 M (1,9 g / ml) ST-246 8,15,16. Ver Resultados representante para efectos esperados. Nota: asegúrese de esta dilución en dos veces (2x) la concentración final deseada ya que este se añade en una proporción de 1:1 para el volumen ya en el pozo. </li></ol></li><li> Inmediatamente después de la adición del virus, añadir 50 l que contenía medios de infección tratamientos deseados y los controles, se diluye en el paso 2.2. Nota: Para ensayar para la inhibición de la expresión antes de etapa temprana, el compuesto (s) puede ser agregada bien directamente al inóculo de virus diluido (etapa 2.1.2) o a las células huésped antes de la infección (antes de la etapa 2.1.3). </li><li> Incubar 6-24 horas en un 37 &amp; #176; incubadora a + 5% de CO 2. </li></ol> 3. Fijar Celdas <ol><li> Fijar las células mediante la adición de 100 l 8% de paraformaldehído a los medios de comunicación ya la infección en cada pocillo. Se incuba a temperatura ambiente durante 15 min protegidos de la luz. Nota: Se recomienda añadir PFA 2x (8%) directamente a los medios de comunicación para prevenir la infección por aerosolización de vaccinia infecciosa que puede ocurrir si los medios de comunicación de alto título se invierte directamente en la fuente de residuos antes de la fijación. </li><li> Quite el fijador invirtiendo la placa en el plato de residuos. </li><li> Añadir 100 l de PBS temperatura ambiente. </li><li> Sellar la placa con película adhesiva ópticamente transparente. Nota: Las placas pueden guardarse a 4 º C si no se lee inmediatamente. Asegúrese de volver placas selladas a temperatura ambiente antes de la lectura para evitar la condensación de distorsionar las mediciones del espectrómetro. </li></ol> 4. Cuantificar el crecimiento del virus <ol><li> Medir la fluorescencia de punto final a 515:530 para Venus, 587:610 para mCherry y 415:457 fo TagBFP (Excitación: longitud de onda de emisión en nm). Cuatro mediciones deben ser promediados por pocillo usando ajustes de ganancia óptimos para cada canal. Nota: La longitud de onda de emisión apropiado puede ser diferente en función de los modelos y de filtro características específicas de su lector de placas. Esto se puede determinar empíricamente mediante la realización de una exploración de longitud de onda de emisión para determinar el punto donde existe la mayor diferencia entre TRPV y PLV para cada canal. </li><li> Normalizar los datos en bruto para facilitar la comparación entre los experimentos. <ol><li> Para cada canal, determinar la media de TRPV réplica y pozos PLV. </li><li> Restar las mediciones de fondo infectadas por el PLV medias de las mediciones experimentales infectados-TRPV medios para cada tratamiento. </li><li> Normalizar los datos dividiendo cada fondo restado valor por el valor del vehículo sólo así. </li><li> Una vez que un experimento ha sido repetido varias veces, un análisis unidireccional de la varianza (prueba de ANOVA de una vía) y múltiples COMPARACIÓN después de la prueba se puede realizar para determinar si cualquiera de los tratamientos refleja una diferencia estadísticamente significativa del tratamiento-vehículo sólo para cada canal. </li></ol></li></ol> . 5 Protocolo Alternativo: Ensayos de placas cinético <ol><li> Realice todos los pasos a través de la adición de los tratamientos (paso 2.3). </li><li> Placa de sello con lámina adhesiva y colocar en la cámara lector de placas equilibró a 37 º C. Nota: El cuidado especial se debe tomar para mantener las placas constantemente a 37 º C para evitar el estrés celular y la condensación excesiva. Para los ensayos cinéticos, es particularmente importante usar un medio tamponado (bicarbonato sódico y HEPES) para mantener el pH adecuado sin la adición de 5% de CO 2. </li><li> Lector Set placa ganar manualmente para evitar la saturación de los puntos de tiempo posteriores. Nota: la optimización de la ganancia exacta se puede obtener mediante la realización de un análisis de punto final, en condiciones similares. </li><li> Adquirir valores horarios de 8 a 24 hpi y normalizar el uso de simiprocedimiento lar como en el ensayo de punto final (paso 4.2). </li><li> Puntos de tiempo individuales pueden ser analizados para la significación estadística usando métodos similares como el ensayo de punto final descrito anteriormente. </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>

Los autores no tienen nada que revelar, y no a las patentes están pendientes en busca de virus o métodos de detección.

Aquí, hemos descrito el uso práctico de un multi-etapa reportero virus vaccinia (TRPV), que proporciona medidas de retroalimentación en tiempo real reproducibles de la replicación del virus. Mediante el uso de inhibidores de poxvirus bien definidos, hemos sido capaces de demostrar que TRPV responde de una manera consistente con el mecanismo entendido de acción para cada inhibidor. Mientras que el virus TRPV proporciona la información más completa acerca de la progresión de la fase de virus, de dos etapas (iRev, ...

Tradicionalmente, la expresión del virus se estudia el uso de técnicas de biología molecular (por ejemplo, transferencia de Northern, Western Blot, microarrays de hibridación, etc) 1. Si bien estos métodos son capaces de proporcionar información detallada con respecto a los cambios de expresión de ARNm o proteínas individuales categorizar, por lo general no son susceptibles de procesos en tiempo real y de alto rendimiento. Los enfoques alternativos que utilizan los periodistas basados ​​en fluorescencia se han aplicado anteriormente al trabajar con poxvirus; Sin embargo, su desarrollo y su uso ha sido motivado por diversos objetivos. Varios de tales métodos se diseñaron para la selección de virus recombinantes 2,3. Estas técnicas expresan EGFP después de la incorporación y expresión del ADN exógeno a partir de clones de vaccinia recombinantes adecuada. Del mismo modo, varias cepas de vaccinia que expresan establemente EGFP solubles o proteínas GFP-etiquetados expresadas bajo un promotor de vaccinia nativo han sido ampliamente utilizados. Estos son typcamente utiliza para cuantificar la entrada del virus y la replicación durante los ensayos de neutralización a base de anticuerpos, inhibición química, o la comparación de la eficacia antiviral 4-6. Si bien estos virus han demostrado ser útiles, están limitados en su capacidad para proporcionar información detallada sobre el punto de inhibición debido a su uso de un único promotor viral temprano / tardío ambigua. Los métodos anteriores también han hecho uso de la proteína EGFP con características óptimas de señal a ruido. Debido a la complejidad de la replicación del virus de la viruela y la falta de herramientas existentes disponibles para ensayar los cambios en tiempo real en la expresión génica viral, hemos desarrollado una serie de virus reportero sola etapa y de múltiples 7,8. Como se ha descrito en publicaciones anteriores, estos virus expresan uno, dos, o tres proteínas fluorescentes espectralmente distintos promotores de vaccinia nativos durante principios, etapas intermedias o tardías de la infección. Estos virus pueden ser nosotrosed como indicadores de progreso de la replicación del virus utilizando un microscopio de fluorescencia y son igualmente adecuados para los ensayos de placa-y-lector basado en alto rendimiento. Estos virus son fáciles de usar en lugar del virus de tipo salvaje, creciendo con una cinética similar para alcanzar títulos equivalentes 7. Durante la planificación y creación de estos virus, mucho se tuvo cuidado en la selección de fluoróforos que tienen altas características de señal a fondo con la eficiencia de plegado superiores para facilitar la cuantificación fiable con retroalimentación rápida a los cambios en la expresión (Venus, mCherry y TagBFP). Además, se eligieron combinaciones de promotores virales que producen alta fidelidad, inequívoca expresión-etapa específica, el suministro de información sobre la gama completa de temprana (promotor C11R), intermedio (promotor G8R) y tardía (promotor F17R) la expresión de genes, en contraste con ambigua principios / finales de los promotores. Estos virus pueden ser utilizados como una herramienta para la investigating el ciclo de vida del virus de la viruela, virus de la vacuna prototípica. Gran parte todavía no se sabe acerca de la interacción huésped-virus a pesar de su larga historia de estudio. Vaccinia es complejo, produciendo más de 200 proteínas únicas, muchas de las cuales son inmunomoduladores y albergan antagónica. Tras la infección, el virus vaccinia se inicia inmediatamente la transcripción de ARNm temprano. Esto es facilitado por una ARN polimerasa y los factores de transcripción que se cargaron en el genoma viral durante el envasado del virión y se mantienen en un estado de pausa hasta que la infección subsiguiente. Esta expresión temprana produce principalmente proteínas necesarias para la supresión del sistema inmune del huésped (ARNm decapping, el secuestro de dsRNA, y las proteínas de los receptores de señuelo, así como inhibidores de la apoptosis, la respuesta al estrés, y peaje, IL, y la señalización de NF-kappa B) y la replicación del genoma. Expresión temprana también produce factores de transcripción necesarios para la expresión intermedia. Expresión intermedia incluye la expresión de factores de transcripción etapa tardía. Estecascada de expresión conduce a la producción de proteínas del virus estructurales y enzimáticas durante las etapas tardías de la infección, que son necesarios para el montaje completo de la vaccinia virion maduro. Nuestro conjunto de virus reportero fluorescentes permite un rápido progreso que se hará en la comprensión de la biología de los poxvirus. Uno de los métodos más comunes y que requieren mucho tiempo en el campo de la virología es el ensayo de crecimiento. Normalmente, esto implica la infección de células, la promulgación de una serie de tratamientos, el virus de la cosecha mediante la lisis de las células infectadas, y cuantificar el título viral resultante mediante ensayo en placa. Uso de los virus descritos aquí reportero permite la medición en tiempo real de crecimiento del virus que puede ser ensayada y se comparó entre los numerosos tratamientos realizados en paralelo fácilmente. Prevemos este conjunto de reactivos se utiliza en diversos protocolos para la identificación de alteraciones en la expresión génica viral en respuesta al tratamiento con fármacos, el ARNi knockdown o restricción gama de huéspedes. Este método también permite el análisis de alto contenido en una escala mayor que la disponible anteriormente, lo que permite alto rendimiento pantallas de drogas antivirales para las etapas de destinatarios definidos específicamente-de interés. Aunque se han identificado numerosos tratamientos potenciales para combatir la infección por poxvirus, el único aprobado por la FDA terapias eficaces para el tratamiento de la infección por virus de la viruela son el nucleósido acíclico fosfonato, cidofovir, y el tratamiento con la vacuna inmune 9,10 globulina. A pesar de la erradicación de la viruela en 1977 11, poxvirus siguen siendo una amenaza considerable para la salud humana 12. El cese de la vacunación generalizada contra el virus de la viruela ha llevado a un aumento de la susceptibilidad a otros poxvirus 13. Por ejemplo, las regiones de África central que antes estaban protegidos por la vacunación están experimentando un aumento de las infecciones por virus del simio 14. Preocupación significativa también ha sidoplanteadas en relación con la susceptibilidad latente para la liberación intencional de virus de la viruela. Debido a las terapias limitadas disponibles actualmente, existe una necesidad urgente para el desarrollo de nuevos tratamientos. Estos virus informadores permiten la detección inhibidor de molécula pequeña rápida y de alto rendimiento para la inhibición de una etapa específica de la replicación viral. Identificación de inhibidores dirigidos a las etapas de expresión viral en la actualidad no es el objetivo de la inhibición facilitará el desarrollo de las terapias combinadas con una mayor potencia. Mucho se puede extraer acerca de la biología celular de la vacuna mediante la observación de los cambios en la expresión génica de cada etapa. Atenuación por manipulación química o genética de una célula huésped infectada se expresa normalmente en términos de reducción de los títulos virales. Sin embargo, mediante la comparación de los cambios en cada etapa de la cascada de expresión viral, se puede obtener una comprensión más completa de cómo la aptitud virus es impactoed por un tratamiento en particular. Estos datos han sido demostrado que se correlaciona bien con la salida de título de virus tradicional, pero proporcionar información mecanicista más detallada, así como capacidades de alto rendimiento 7.

<table border="0" cellpadding="0" cellspacing="0" width="1249">
	<tbody>
		<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

Los poxvirus son una familia de virus de ADN de doble cadena que incluyen patógenos humanos activos tales como viruela de los monos, molusco contagioso, y virus de Contagalo. La familia también incluye el virus de la viruela, de la viruela. Debido a la complejidad de la replicación del virus de la viruela, aún quedan muchas preguntas con respecto a su estrategia de la expresión génica. En este artículo se describe la conceptualización y el uso de los virus vaccinia recombinantes que permiten la medición en tiempo real de las etapas individuales y múltiples de la expresión génica viral en un formato de alto rendimiento. Esto es posible mediante el uso de proteínas fluorescentes espectralmente distintas como reporteros para cada una de tres etapas de la replicación viral. Estos virus ofrecen una alta relación señal-ruido al tiempo que conserva los patrones de expresión específicos de la etapa, los ensayos basados ​​en la placa de habilitación y observaciones microscópicas de la propagación del virus y la replicación. Estas herramientas tienen usos para el descubrimiento antiviral, los estudios de la interacción virus-huésped, y la bio evolutivología.

Como un ejemplo de la utilización típica de estos virus, se utilizó el virus de triple fluorescencia de reportero para comparar el punto de la inhibición de varios inhibidores de poxvirus bien definidos. Las células HeLa se colocaron en placas en cultivo de tejidos claros placas de 96 pocillos de fondo plano de paredes negras tratadas y se incubaron durante la noche. Monocapas confluentes fueron infectadas a una multiplicidad de infección (MOI) de 10 utilizando virus reportero triples ...

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

Se describe el uso de un virus vaccinia reportero fluorescente que permite la medición en tiempo real de la infectividad viral y la expresión génica a través de la expresión de etapa específica de fluoróforos espectralmente distintos reportero. Nos detalle un método basado en la placa para identificar con precisión la etapa en la que la replicación del virus se ve afectada en respuesta a la inhibición pequeña molécula.

Vaccinia Virus Reporter para cuantificar la función viral en todas las etapas de la Expresión Génica

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 vistas.  Boston University School of Medicine.  Se describe el uso de un virus vaccinia reportero fluorescente que permite la medición en tiempo real de la infectividad viral y la expresión génica a través de la expresión de etapa específica de fluoróforos espectralmente distintos reportero. Nos detalle un método basado en la placa para identificar con precisión la etapa en la que la replicación del virus se ve afectada en respuesta a la inhibición pequeña molécula. 

Video: 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.

Generación de células T Multivirus específicos para prevenir / tratar las infecciones virales después de trasplante alogénico de células madre hematopoyéticas

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

Investigación

Inmunología e Infección

'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;Bioluminiscente &#39;reportero fagos para la detección de patógenos bacterianos de la categoría 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.

El análisis cuantitativo de todos Influenza Tipo A Hemaglutininas viral y neuraminidasas el uso de anticuerpos Universal en simples ensayos Slot Blot

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.

Método rentable para el Rastreo de uso microbiana específica virus humanos y animales

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.

El aislamiento de las variantes de los virus de ARN de fidelidad y caracterización de la frecuencia de mutación de virus

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.

El establecimiento de un cultivo líquido cubierto de las vías respiratorias humanas epiteliales polarizadas Calu-3 células para estudiar la respuesta del huésped celular a patógenos respiratorios<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.

Ratón Sistema Cultura Hígado Fetal de diseccionar las funciones de genes objetivo en las primeras y últimas etapas de la Terminal de la eritropoyesis

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.

Administración intranasal de vacunas contra la influenza recombinante en modelos de ratón quimérico para el Estudio de la mucosa Inmunidad

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.

Un enfoque simple y eficiente de construcción de mutantes de virus vaccinia Vectores

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

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.

Desarrollo de un sistema reportero virus de la hepatitis B para supervisar las primeras etapas del ciclo de replicación

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