La publicación de este artículo fue financiada por una beca de la Oficina de Investigación de la Universidad de Tennessee. Los autores agradecen a la anterior generación de estudiantes e investigadores que han trabajado para perfeccionar esos procedimientos. La investigación fue financiada por becas de la Fundación Nacional de Ciencia (NSF-0851113, NSF-0825405 y 0550485, NSF).

1. Ultrafiltración de agua de mar para generar &quot;libre de virus&quot; de agua (Wilhelm y Poorvin 2001) <ol><li> Aproximadamente 20 litros de agua de mar / agua del lago se recoge como asépticamente posible. </li><li> El agua es prefiltrado en serie a 142 mm de diámetro de policarbonato 0,8 micras filtros que se pueden guardar a -20 ° C para el análisis de la comunidad. Los filtros más grandes de tamaño de poro se puede utilizar antes de este paso para los sistemas muy productivos. </li><li> Para obtener agua ultrafiltrada de un sistema de M12 Amicon (Millipore) se utiliza un 30 kDa de corte de cartucho espiral de excluir a todos los virus, incluso los pequeños virus de ARN. </li><li> Las muestras se procesan y se concentran en la velocidad de ~ 25% ~ 15 a 16 kPa de contrapresión. </li><li> El resto de la muestra de ~ 500 ml de agua contenga una comunidad virus concentrado (que se pueden guardar para otros experimentos), mientras que el restante 19,5 L de agua libre de virus serán utilizados para los ensayos de la producción viral. </li><li> Después de cada día de uso, el Amicon M12 sistema debe ser limpiado para prevenir el daño a la membrana del cartucho de filtro. </li><li> Si está trabajando con el mar, lave la membrana con por lo menos 6L de agua Milli-Q seguido de un lavado con una solución de NaOH 0,1 N durante 30-45 minutos. </li><li> Otra vez aclarado el cartucho con un mínimo de 6 L de agua Milli-Q. </li><li> Cuando termine de usar el sistema de M12, el cartucho espiral debe ser almacenado en un 0.05MH 3 PO 4 solución a 4 ° C. </li></ol> 2. Virus Método de reducción de la producción viral (Wilhelm et al. 2002) <ol><li> Hasta 500 ml de la muestra de agua de mar / agua del lago con el host y los virus se obtiene y se coloca en una unidad sterifilter con un 0,2 micras de tamaño de poro nominal baja unión a proteínas de filtro (por ejemplo, Durapore) en el mismo. </li><li> La muestra es ligeramente vacío presión a &lt;200 mmHg, mientras que continuamente resuspender la muestra con una pipeta de transferencia estéril para inhibir las células bacterianas de concentrarse en el filtro. </li><li> Poco a poco los tres volúmenes de ultrafiltrado se añaden a la suspensión de bacterias para reducir significativamente el número de virus libre en la muestra. </li><li> La fracción bacteriana, se diluye de nuevo a 500 ml con agua libre de virus y se divide en tres repeticiones de 150 ml cada uno y se colocan en botellas de policarbonato transparente de 250 ml. </li></ol> 3. Filtración tangencial de flujo (TFF) Método para la producción viral (Weinbauer et al. 2002, Winget et al. 2005) TFF representa un enfoque alternativo para el método de reducción viral. <ol><li> Aproximadamente 500 ml de muestra natural se obtiene como se describe anteriormente. </li><li> Esta muestra se concentra con un 0,2 micras de tamaño de poro nominal del sistema de filtración tangencial de flujo. </li><li> Cuando la fracción bacteriana se reduce a aproximadamente 10-15 ml, ultrafiltrado, libre de virus se añade agua y se distribuye de arriba. </li><li> Las botellas se incuban a replicar en condiciones in situ con cámaras ambientales. <ol><li> Los niveles de luz se alteran las condiciones de la superficie mediante el uso de tinte azul acrílico acrílico o con clara proyección neta para disminuir la intensidad de la luz. </li><li> La temperatura ambiente de superficie se obtienen a menudo mediante el uso de agua de mar que fluye cubierta incubadora. </li></ol></li><li> Las muestras para las estimaciones de abundancia bacteriana y viral se toman en el momento 0, con una concentración final de 2,0 a 2,5% de glutaraldehído estéril añadido en crioviales. Estas muestras son inmediatamente flash congelado con nitrógeno líquido y se almacenan congelados hasta su transformación. <ol><li> Si el nitrógeno líquido no está disponible, portaobjetos se pueden preparar y procesar inmediatamente (consulte el siguiente procedimiento) </li></ol></li><li> Submuestras se recogen cada 2,5 horas por lo menos 10 horas por el método descrito anteriormente. <ol><li> En este tiempo el agua puede ser recogida para análisis de PCR cuantitativa. Hasta 5 ml de la muestra se pueden añadir a un criovial con ningún agente fijador con flash que congela inmediatamente en nitrógeno líquido. </li></ol></li></ol> 4. Microscopía de producción viral (Noble y Fuhrman 1998, Wen et al. 2004) <ol><li> Las muestras congeladas a ser de 0,02 m filtrada para microscopía se deben descongelar en hielo. </li><li> Prepare una solución de reserva de SYBR Green por dilución de la solución madre 1:10 con agua estéril. A continuación, a partir de la solución madre, preparar una solución de trabajo mediante la adición de 1 ml de la solución a 39 ml de agua estéril. Un 50% de glicerol, el 50% de tampón fosfato soluciones salinas (PBS, 0,05 M Na 2 HPO 4, 0,85% de NaCl, pH 7,5) y la nueva solución al 2,5% de valores de p-fenilendiamina también deben estar preparados antes de empezar. Mantener el 50% glycerol/50% de solución PBS a 4 ° C y la phenylenediam p-INE acciones a -20 ° C en la oscuridad hasta que arranque. Justo antes de agregar el filtrado de p-fenilendiamina al PBS 50% glycerol/50% hasta una concentración final de 0,1% para ser usado como la solución Antifade. </li><li> Lugar de 25 mm de 0,02 micras filtro Anodisc en la parte superior de un MicronStep de 0,45 micras, filtro de respaldo de celulosa. Añadir 850 l de la muestra fijada a la parte superior de la Anodisc y vacío a 20 pKa hasta que esté completamente seca. Colocar 100 L de solución de SYBR Verde de trabajo a una placa de Petri estéril y, con el vacío en pie, retire con cuidado la Anodisc de la torre de filtro y el lugar en el SYBR Green. Incubar las muestras en la oscuridad a temperatura ambiente durante 20 minutos. Retire con cuidado el filtro de la solución de SYBR Green y la mecha de la parte posterior del filtro con un Kimwipe para eliminar todo el colorante residual. Si lo desea, el filtro de retorno a la torre y pasar hasta 800 l de agua filtrada de 0,02 micras, o los medios de comunicación a través del filtro estéril para enjuagar el exceso de colorante. </li><li> Añada una pequeña gota de solución Antifade a un portaobjetos de microscopio y colocar una hoja de cubierta en la parte superior. Retire la hoja de la cubierta y agregar el filtro se seca a la húmeda portaobjetos con la solución Antifade. Una vez más, añadir una pequeña cantidad de solución Antifade a la hoja de la cubierta y poco a poco lugar en la parte superior del filtro, asegurándose de eliminar todas las burbujas que se forman. </li><li> Inmediato congelamiento de los portaobjetos a -20 ° C hasta su utilización (estos deben ser utilizados en varios meses para evitar decoloración y la baja cuenta de virus) </li><li> Los virus se enumeran utilizando microscopía de fluorescencia (en nuestro caso un microscopio Leica DMRXA) con una amplia gama de conjunto filtro azul (λ Ex = 450 a 490 nm, λ Em = 510 nm con un filtro de supresión en λ = 510 nm). Cada filtro tendrá por lo menos 20 campos de visión en cuenta, asegurándose de cuantificar virus total de cada red de campo para asegurar una distribución uniforme de los virus a través de la membrana del filtro. </li><li> Las tasas promedio de recurrencia del virus de la independiente de tres repeticiones se calculan y una desviación estándar se determina a partir de las tasas de producción. </li></ol> 5. Resultados representante Los datos originales recopilados por el investigador requiere un mínimo de procesamiento matemático para generar tasas de recurrencia de la abundancia de virus. El conjunto de datos primarios resultantes de este estudio es la tasa de recurrencia de la abundancia de virus en las sub-muestras de la incubación. Estos resultados forman regresiones independientes del tiempo frente a virus de la abundancia para cada una de las muestras. Para cada muestra, las incubaciones individuales actúan como un tratamiento, por lo que al completar tres replicar-incubación, el investigador puede calcular las tasas, así como una estimación de la variación (por ejemplo, la desviación estándar) (ver Figura 1). Una advertencia de este proceso es que la reducción en la abundancia invariable virus lleva a una reducción en las células huésped en la muestra que se están llevando la carga de virus. Para compensar esta pérdida, la enumeración de la abundancia de bacterias de la fuente (agua de mar no filtrada o agua del lago) y la T = 0 muestra de incubación son necesarios. Esta información puede ser utilizada para tener en cuenta el porcentaje de células perdidas: en el supuesto de que el proceso de reducción de la abundancia de virus no es selectivo a favor o en contra de los miembros de la comunidad microbiana, este factor puede ser utilizado para estimar la tasa de producción in situ de los virus . <img src="/files/ftp_upload/2196/2196fig1.jpg" alt="Figura 1" /> Figura 1. La producción de las muestras de partículas similares al virus más de una incubación de 10 horas en condiciones in situ mediante microscopía de epifluorescencia. Fueron recogidos durante una floración de fitoplancton en las costas de Nueva Zelanda en septiembre de 2008. <img src="/files/ftp_upload/2196/2196fig2.jpg" alt="Figura 2" /> Figura 2. Diagrama esquemático del proceso de flujo de trabajo para el ensayo de producción de virus. El proceso comienza con la ultrafiltración de la muestra de agua para generar agua libre de virus. Esto se completa con un sistema de ultrafiltración. En muestras de agua en paralelo se recogen en el mismo sitio y el virus sin pasar por un filtro, mientras que la comunidad microbiana (que contiene una mezcla de las células infectadas y no infectadas) se mantiene. Esta comunidad es luego resuspendido en agua libre de virus y se incubaron en condiciones in situ. La tasa de recurrencia de los virus es de cerca por las próximas 10 horas para determinar las tasas de producción de virus.

<ol>
	<li>Higgins, J. L., Kudo, I., Nishioka, J., Tsuda, A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+the+virus+community+to+a+mesoscale+iron+fertilization+in+the+sub-Arctic+Pacific+Ocean.">The response of the virus community to a mesoscale iron fertilization in the sub-Arctic Pacific Ocean.</a> Deep-Sea Res II. 56, 2788-2795 (2009).</li><li>Mioni, C. E., Poorvin, L., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+and+siderophore-mediated+transfer+of+available+Fe+between+heterotrophic+bacteria:+characterization+using+a+Fe-specific+bioreporter.">Virus and siderophore-mediated transfer of available Fe between heterotrophic bacteria: characterization using a Fe-specific bioreporter.</a> Aquat Microb Ecol. 41, 233-245 (2005).</li><li>Noble, R. T., Fuhrman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+SYBR+Green+I+for+rapid+epifluorescence+counts+of+marine+viruses+and+bacteria.">Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.</a> Aquat Microb Ecol. 14, 113-118 (1998).</li><li>Parada, V., Herndl, G., Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+burst+size+of+heterotrophic+prokaryotes+in+aquatic+systems.">Viral burst size of heterotrophic prokaryotes in aquatic systems.</a> J Mar Biol Assoc U K. 86, 613-621 (2006).</li><li>Poorvin, L., Rinta-Kanto, J. M., Hutchins, D. A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+release+of+Fe+and+its+bioavailability+to+marine+plankton.">Viral release of Fe and its bioavailability to marine plankton.</a> Limnol Oceanogr. 49, 1734-1741 .</li><li>Rowe, J. M., Saxton, M. A., Cottrell, M. T., DeBruyn, J. M., Berg, G. M., Kirchman, D. L., Hutchins, D. A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constraints+on+virus+production+in+the+Sargasso+Sea+and+North+Atlantic.">Constraints on virus production in the Sargasso Sea and North Atlantic.</a> Aquat Microb Ecol. 52, 233-244 (2008).</li><li>Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+of+prokaryotic+viruses.">Ecology of prokaryotic viruses.</a> FEMS Microbiol Rev. 28, 127-181 (2004).</li><li>Weinbauer, M. G., Peduzzi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency,+size+and+distribution+of+bacteriophages+in+different+marine+morphotypes.">Frequency, size and distribution of bacteriophages in different marine morphotypes.</a> Mar Ecol Prog Ser. 108, 11-20 (1994).</li><li>Weinbauer, M. G., Winter, C., H&amp;ouml, f. l. e., G, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconsidering+transmission+electron+microscopy+based+estimates+of+viral+infection+of+bacterioplankton+using+conversion+factors+derived+from+natural+communities.">Reconsidering transmission electron microscopy based estimates of viral infection of bacterioplankton using conversion factors derived from natural communities.</a> Aquat Microb Ecol. 27, 103-110 (2002).</li><li>Wen, K., Ortmann, A. C., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+estimation+of+viral+abundance+by+epifluorescence+microscopy.">Accurate estimation of viral abundance by epifluorescence microscopy.</a> Appl Environ Microbiol. 70, 3862-3867 (2004).</li><li>Wilhelm, S. W., Brigden, S. M., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dilution+technique+for+the+direct+measurement+of+viral+production:+a+comparison+in+stratified+and+tidally+mixed+coastal+waters.">A dilution technique for the direct measurement of viral production: a comparison in stratified and tidally mixed coastal waters.</a> Microb Ecol. 43, 168-173 (2002).</li><li>Wilhelm, S. W., Matteson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freshwater+and+marine+virioplankton:+a+brief+overview+of+commonalities+and+differences.">Freshwater and marine virioplankton: a brief overview of commonalities and differences.</a> Freshw Biol. 53, 1076-1089 (2008).</li><li>Wilhelm, S. W., Poorvin, L., Paul, 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=.">.</a> Quantification of algal viruses in marine samples. , 53-66 (2001).</li><li>Wilhelm, S. W., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viruses+and+nutrient+cycles+in+the+sea.">Viruses and nutrient cycles in the sea.</a> Bioscience. 49, 781-788 (1999).</li><li>Wilhelm, S. W., Weinbauer, M. G., Suttle, C. A., Jeffrey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+sunlight+in+the+removal+and+repair+of+viruses+in+the+sea.">The role of sunlight in the removal and repair of viruses in the sea.</a> Limnol Oceanogr. 43, 586-592 (1998).</li><li>Winget, D. M., Williamson, K. E., Helton, R. R., Wommack, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tangential+flow+diafiltration:+an+improved+technique+for+estimation+of+virioplankton+production.">Tangential flow diafiltration: an improved technique for estimation of virioplankton production.</a> Aquat Microb Ecol. 41, 221-232 (2005).</li></ol>

Un componente fundamental en la comprensión de cómo los virus de la influencia marina comunidades microbianas es determinar la velocidad a la que se producen partículas de virus. Dado que la abundancia es (más o menos) en la mayoría de los sistemas estáticos (Wilhelm y Suttle 1999, Weinbauer 2004), y que los virus son eliminados o convertidos en no infecciosos rápidamente en los sistemas acuáticos (Wilhelm et al. 1998), entonces las tasas de producción debe ser relativa rapidez para sustituir a las partículas perdidas. Estimación de los virus de la mortalidad por cualquier causa a la comunidad microbiana requiere un conocimiento de cómo muchos de los virus se producen cada vez que un virus de la lisis de una célula (el &quot;tamaño de la ráfaga&quot;). Tamaños virus irrumpió en las muestras naturales pueden variar mucho. Tamaños de ráfaga puede ser determinada directamente por microscopía electrónica de transmisión (por ejemplo, Weinbauer Peduzzi y 1994), pero esto es a menudo más allá de las capacidades de un determinado laboratorio o no siempre es práctico. En situaciones en las que no puede ser determinada empíricamente, los valores de la literatura de los 24 virus por evento líticas pueden ser utilizados para los sistemas marinos y 34 para los sistemas de agua dulce (Parada et al. 2006). Si la tasa de producción de virus se divide por este número, el resultado es la abundancia de células por unidad de volumen destruido por el virus en una base diaria. Los microbios lisadas valor puede ser dividido por la población permanente de la abundancia bacteriana que resulta en la mortalidad inducida por el virus para el sistema en cuestión: las estimaciones van existentes de porcentaje a casi toda la población y son a menudo dependen de otros factores en el sistema en cuestión (Wilhelm y Matteson 2008). Para determinar el porcentaje de la mortalidad total, este número se multiplica por dos veces (de trabajo a partir de la suposición de que el 50% de las células van a reproducir y el 50% de las células se pierden, Weinbauer 2004). Dado que la biodisponibilidad de nutrientes y elementos traza (por ejemplo, N, P, Fe) puede limitar la tasa de productividad primaria, y como tal el flujo de carbono, a través de los sistemas acuáticos, y la comprensión del papel de la mortalidad microbiana virus basada en este proceso se ha convertido en de interés para los geoquímicos marinos. Varias estimaciones sugieren que ahora existen los virus de liberar una concentración significativa de los elementos nutrientes de vuelta a la columna de agua sobre una base diaria (Rowe et al. 2008, Higgins et al. 2009) y que estos elementos son rápidamente asimilados por la comunidad microbiana (Poorvin et al. 2004, Mioni et al. 2005). La tasa de flujo de nutrientes al medio ambiente se puede determinar multiplicando el número de células destruidas por la cantidad de nutrientes por la célula (denominado &quot;cuotas&quot;). Esta información puede ser un componente crítico para nuestra comprensión de cómo las redes microbiana de alimentos funcionan a través de los sistemas acuáticos. La evolución en curso: Los esfuerzos actuales por una serie de grupos de investigación incluyen la adaptación de la estrategia anterior para enumerar los virus específicos de la comunidad y, como tal, para determinar cómo los organismos específicos están influidos por la actividad del virus. Para ello los investigadores utilizan la reacción en cadena de polimerasa cuantitativa (qPCR) para estimar la abundancia de los grupos de virus específicos o familias de forma paralela a las estimaciones de la comunidad total de virus. Los resultados son de aplicación directa para proporcionar estimaciones de la mortalidad del virus, ciclos de nutrientes, etc para grupos específicos de plancton. Este nuevo enfoque permitirá a los investigadores de gran alcance en los próximos años para excavar más profundamente en los procesos relacionados con la ecología de los virus y, por primera vez, para cuantificar las interacciones específicas de virus-huésped comunidades más allá de las limitaciones de los sistemas de laboratorio.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2.5% p-phenylenediamine</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>130575000</td>
<td>Stock for Antifade</td>
</tr>
<tr>
<td>Amicon Proflux M12 system</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>N/A</td>
<td>Any ultrafiltration device may be used for this step</td>
</tr>
<tr>
<td>SYBR Green I nucleic acid gel stain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S-7563</td>
<td>&nbsp;</td>
<tr>
<td>Helicon S10 30kDa Filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>CDUF010LT</td>
<td>&nbsp;</td>
<tr>
<td>Pelicon XL filters 0.22 &mu;m</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>PXGVPPC50</td>
<td>&nbsp;</td>
<tr>
<td>GE 0.2 &mu;m PCTE membrane filters (47mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-732-35</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Labscale Tangential Flow Filtration System</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>XX42LSS11</td>
<td>Other TFF systems may be used for this step</td>
</tr>
<tr>
<td>0.45 &mu;m Micronstep, Cellulosic, white plain filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>E04WP02500</td>
<td>&nbsp;</td>
<tr>
<td>GE Whatman 0.02 &mu;m Anodisc filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>68-09-6002</td>
<td>&nbsp;</td>
<tr>
<td>Leica DMRXA microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Any epifluorescence microscope with a blue filter set may be used</td>
</tr>
<tr>
<td>20L polycarbonate carboys</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP229-4</td>
<td>&nbsp;</td>
<tr>
<td>PBS (0.05 M Na2HPO4, 0.85% NaCl, pH 7.5)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP329-500, S640-500</td>
<td>&nbsp;</td>
<tr>
<td>50% Glutaraldehyde </td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G151-1</td>
<td>&nbsp;</td>
<tr>
<td>Corning 2 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-71</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td>Corning 5 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-74</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td> 85% H3PO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A242-212</td>
<td>Spiral Membrane storage</td>
</tr>
<tr>
<td>NaOH pellets</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>S318-3</td>
<td>M12 cleaning</td>
</tr>
<tr>
<td>Graduated Cylinders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopore 0.8-&mu;m pore-size membrane filter (142mm)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ATTP14250</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Stainless Steel Pressure Filter Holder (142mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>YY30 090 00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

estimating virus production rates in aquatic systems

Los virus son los componentes generalizada de los sistemas marinos y de agua dulce, y se sabe que son importantes agentes de la mortalidad microbiana. El desarrollo de estimaciones cuantitativas de este proceso es fundamental, ya que se puede desarrollar mejores modelos de estructura de la comunidad microbiana y la función, así como avanzar en nuestra comprensión de cómo los virus de trabajo para alterar los ciclos biogeoquímicos acuáticos. La técnica de reducción de virus permite a los investigadores estimar la velocidad a la que las partículas de virus son liberados de la comunidad microbiana endémicas. En resumen, la abundancia de libre (extracelular) virus se reduce en una muestra, mientras que la comunidad microbiana se mantiene en la concentración ambiente cerca. La comunidad microbiana se incuba en la ausencia de virus libre y la velocidad a la que volver a aparecer los virus en la muestra (a través de la lisis de los miembros que ya están infectadas de la comunidad) se puede cuantificar mediante microscopía de epifluorescencia o, en el caso de los virus específicos, cuantitativos PCR. Estas tarifas se pueden utilizar para estimar la tasa de mortalidad microbiana, debido a la lisis celular mediada por el virus.

Watch this Scientific Journal Video about Estimating Virus Production Rates in Aquatic Systems at JoVE.com

Estimating Virus Production Rates in Aquatic Systems

La tasa de rotación de los virus en los sistemas marinos y de agua dulce puede ser estimado por una reducción de la recurrencia y la técnica. Los datos permiten a los investigadores a deducir las tasas de mortalidad microbiana mediada por el virus en los sistemas acuáticos.

Estimación de las tasas de producción de virus en los sistemas acuáticos

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

estimating-virus-production-rates-in-aquatic-systems

Research

JoVE Journal

Immunology and Infection

12.6K vistas.  University of Tennessee. La tasa de rotación de los virus en los sistemas marinos y de agua dulce puede ser estimado por una reducción de la recurrencia y la técnica. Los datos permiten a los investigadores a deducir las tasas de mortalidad microbiana mediada por el virus en los sistemas acuáticos.

Video: Estimating Virus Production Rates in Aquatic Systems

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualización de virus del dengue a través de Alexa Fluor etiquetado

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

Investigación

Inmunología e Infección

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Producción eficiente de parvovirus recombinante con la ayuda de derivados de adenovirus-Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

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

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

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

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Evaluación de la eficacia y la toxicidad de los ARN del VIH-1 Orientación de la producción para uso en terapia génica o de drogas

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Extracción rápido, seguro y sencillo Manual cabecera de ácido nucleico para la detección del Virus en muestras de sangre entera

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identificación de la codificación y las clases de ARN no codificante expresado en cerdos sangre entera

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

Medición de tasas de desconfianza micobacterianas específicas con sistemas de reportera de ganancia de función

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Prueba IN Vitro ELISA para evaluar la potencia de la vacuna contra la rabia

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

Análisis de interacciones entre endobióticos y microbiota de tripa humana utilizando sistemas de fermentación de bañoin in vitro

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

Methods Article

Estimación de las tasas de producción de virus en los sistemas acuáticos