Los autores agradecen las contribuciones de Greg Smith, Lisa Pomeranz, Lyman Matt, Eldridge Marlies, Halina Staniszewska Goraczniak, y los miembros del laboratorio de Enquist en el perfeccionamiento de este protocolo.

1. Primer Día: infección viral y preparación de tampones <ol><li> Preparar platos 50-10 (15 cm de diámetro) de las células de cultivo de tejidos para la infección, por ejemplo, células PK-15 para el virus de la pseudorrabia (PRV) o de células Vero de virus del herpes simple (VHS). </li><li> Cuando las células son de 95 - 100% confluentes, que infectan a un (MOI) de 5.10. Para ello, vacunar a cada plato con stock de virus en un volumen total de 4 ml por placa, y luego se incuban las placas durante 1 hora a 37 ° C. Placas de roca suavemente cada 15 minutos para asegurarse de que la monocapa celular sigue siendo totalmente recubierto por el inóculo de virus. Mientras tanto, calentar el medio para el siguiente paso. </li><li> Después de una hora de la infección, aspiración de inóculo viral de las placas, agregar 15 ml de medio caliente, y se incuba a 37 ° C durante 12-20 horas en un incubador humidificado. </li><li> Preparar buffers LCM (ver Tabla 1) y el rock la noche a 4 ° C para la mezcla completa. Prepare TNE para la resuspensión de ADN viral (ver Tabla 2). </li></ol> 2. Segundo día, Fase 1: lisis celular y ultracentrifugación <ol><li> Confirmar visualmente que la infección de las células ha causado un efecto uniforme citopático (CPE). Permitir la infección de progreso hasta que todas las células tienen rodeados, pero no han quitado la placa. </li><li> Raspar las células infectadas en el medio, y se combinan células y el medio en tubos de 50 ml cónicos. El número de tubos cónicos necesario para este paso dependerá de la cantidad de los platos utilizados para la infección en el Paso 1.2, por ejemplo, un tubo de 50 ml cónicos se puede utilizar para precipitar un máximo de tres platos de medio de 15 ml cada uno. Opcional: Enjuague el placas con 10 ml de PBS y se combinan con las células y medianas empresas. </li><li> Centrifugue las células y medio durante 10 minutos a 2000 la fuerza relativa centifugal (FCR) a temperatura ambiente, y aspirar el sobrenadante y resuspender cada pellet en 10 ml de PBS. Si el sedimento de una infección viral se extendió en varios tubos cónicos en el Paso 2.2, estos se pueden recombinar en un tubo cónico en este paso. Repetir la centrifugación y la aspiración. Opcional: Esta pastilla puede ser almacenado a -20 ° C y el protocolo seguido en un momento posterior. </li></ol> 3. Segundo día, Fase 2: Ultracentrifugación Mantener el sedimento celular y tampones LCM en hielo siempre que sea posible durante los siguientes pasos. <ol><li> Añadir β-mercaptoetanol al glicerol 0% - búfer LCM. Empieza a enfriar a la ultracentrífuga (para el paso por debajo de 3,9) mediante el establecimiento a 4 ° C y girando en el vacío. </li><li> Resuspender el botón celular en 5 ml de glicerina de 0% - búfer LCM hasta que ya no es abultada. Si es necesario, el tubo de vórtice para romper cualquier disolver el sedimento celular. </li><li> Extraer mediante la adición de 1,5 ml de freón (1,1,2-tricloro-1 ,2,2-trifluoro) y agitación vigorosa. Inmediatamente se centrifuga durante 10 minutos a 2000 fcr a una temperatura de 4 ° C. Recoger la capa superior con una punta de pipeta de gran calibre (evitando la interfaz) y la transferencia a un nuevo tubo. Opcional: Si el pellet es un sólido gelatinoso, usted puede verter la capa superior en un nuevo tubo. </li><li> Repetir la extracción de freón y centrifugar de nuevo. </li><li> Recoger la capa superior (~ 5 ml) y transferir a un tubo de ultracentrífuga polyallomer nuevo. Añadir β-mercaptoetanol al glicerol dos restantes - buffers LCM. </li><li> Preparar un gradiente en el tubo de centrífuga polyallomer underlaying por el extracto de freón (capa superior) con 3 ml de glicerol al 5% - LCM (capa media), y luego underlaying de nuevo con 2,5 ml de 45% de glicerol - LCM (capa inferior). Use una pipeta fina para evitar el desbordamiento de los contenidos del tubo. </li><li> Si es necesario, prepare un tubo de equilibrio equivalente con las mismas proporciones de búferes de LCM. </li><li> La transferencia de los tubos en los cubos de ultracentrifugación. Balance de cubos de 0,1 g de cada uno, añadiendo lentamente glicerol extra 0% - búfer LCM a la capa superior (~ 50 a 100 l en un momento). </li><li> Usando una ultracentrífuga frío con un rotor de cubeta horizontal o basculante, girar las muestras equilibrada durante 1 hora a 77.000 RCF (por ejemplo, 25.000 rpm para un rotor SW41Ti) a una temperatura de 4 ° C. </li><li> Durante la ultracentrifugación, hacer un gancho de vidrio para capturar el ADN flotante en la etapa de precipitación con etanol (4.6). Sostenga los dos extremos de una pipeta Pasteur de vidrio y la suspensión de la sección media sobre una llama. Cuando una sección del vaso está casi fundido, tire suavemente para estirar el vidrio, doble suavemente para crear un gancho fuertemente inclinadas (&lt;90 grados), y tirar fuertemente para sellar el final. Si la punta del gancho de vidrio está abierta, mantenga la punta sobre la llama para que el vidrio se funde lo suficiente como para cerrarla. </li><li> Después de la ultracentrifugación, debería ver un fino precipitado opaco, con una mancha oscura en el centro. Con cuidado aspirar el líquido del tubo, incluyendo grasa a lo largo de los lados, pero evitando los pellets en la parte inferior del tubo. </li><li> Añadir 0,5 ml TNE a temperatura ambiente. </li><li> Deje que el pellet se siente por lo menos 10 minutos para permitir la hidratación de la pastilla. Opcional: La pastilla también podría volver a suspender en TNE noche a la mañana, si se conserva a 4 ° C. </li></ol> 4. Tercer Día: &quot;Ghost&quot; ADN Precipitaciones <ol><li> Romper el pellet con la pipeta con una punta de pipeta de pequeño calibre (por ejemplo, una punta de la pipeta p200). No te preocupes por corte en este paso, ya que el ADN viral es todavía contenida en cápsides en este momento. Añadir los siguientes reactivos al tubo que contiene el virus de pellets, a temperatura ambiente, para destruir cápsides: <ul><li> 4,25 ml TNE </li><li> 0,25 ml de 10% SDS </li><li> 2 mg de proteinasa K </li></ul> Agite suavemente para mezclar los reactivos. Debe haber un ligero aumento de la viscosidad. </li></ol> Tenga cuidado con los de corte a partir de ahora, por ejemplo, el uso de gran calibre pipetas y no agite el material viral <ol start="2"><li> Extracto de proteínas virales mediante la adición de 5 ml de fenol-cloroformo y empezar inmediatamente a invertir o agitación para mantener una emulsión. Mezclar durante 10 segundos, centrifugar la emulsión durante 5 minutos a 2000 fcr a temperatura ambiente. Recoger la capa superior con una punta de gran calibre la pipeta, evitando la interfaz, y la transferencia a un nuevo tubo. Opcional: Use la fase de bloqueo de gel (PLG) en los tubos de este paso para evitar la contaminación de la interfaz. </li><li> Repetir la extracción con fenol-cloroformo y centrifugar de nuevo. </li><li> Después de la segunda extracción con fenol-cloroformo, recoger la capa superior en un tubo de 30 ml de cristal Corex y enfriar el tubo a -20 ° C durante 10-15 minutos, teniendo cuidado de que no se congela. </li><li> Añadir 10 ml de etanol helado, cubrir el tubo (por ejemplo, se extiende un trozo de Parafilm M en la parte superior), e invierta para mezclar inmediatamente. Esté atento a un ADN &quot;fantasma&quot; que aparecen, que consiste en un precipitado viscoso delgada. Invertir el tubo suavemente para mezclar aún más y hacer que el ADN pegajoso precipita a agruparse. </li><li> Utilice un gancho de vidrio para capturar el fantasma del ADN (s). Cuidadosamente blot el ADN para eliminar el exceso de líquido, a continuación, coloque el gancho (la punta hacia abajo) en un tubo de 1.5 ml y dejar secar. Añadir 0,25 a 0,5 ml de TE para disolver el fantasma del ADN. Permitir la resuspensión de ADN para proceder de al menos una hora. ADN se almacenan a 4 ° C. Opcional: Para maximizar el rendimiento de ADN, se rompe el gancho de vidrio en el tubo de 1.5 ml, de modo que la resuspensión de los fragmentos de vidrio pueden seguir el paso del tiempo. </li></ol> 5. Resultados representante Con un alto MOI suficiente, las células deben lograr la plena CPE dentro de ~ 18 horas post-infección (hpi) para cepas de tipo salvaje PRV, y ~ 24 hpi de tipo salvaje cepas de HSV-1 (Figura 1). Una vez que el pellet de células infectadas se cosecha, el resto de pasos del protocolo se puede completar en un día largo o llevado a cabo durante dos días (Figura 2). En la preparación de los ganchos de vidrio para capturar el fantasma del ADN, es importante que el ángulo de gancho de ser inferior a 90 grados, para que después pueda caber en la base de un tubo de 1.5 ml (Figura 3). Sellado de la punta de este gancho previene la pérdida de ADN dentro de la pipeta. Los fantasmas de ADN se forman durante la etapa de precipitación y aparecerá como hilos blancos, viscosa (Figura 4). Estos agregados y se adhieren entre sí y / o el gancho de vidrio utilizado para capturar el ADN. Así, el gancho de vidrio mismo se puede utilizar para reunir varios hilos de ADN dentro de la solución de precipitación. <table border="1"><tbody><tr><td> Elija columna basada en el volumen deseado: </td><td> 10 ml </td><td> 15 ml </td><td> 20 ml </td><td> 40 ml </td></tr><tr><td> 1M KCl </td><td> 1,3 ml </td><td> 1,9 ml </td><td> 2,5 ml </td><td> 5 ml </td></tr><tr><td> 1M Tris (pH 7,4) </td><td> 300 l </td><td> 450 l </td><td> 600 l </td><td> 1,2 ml </td></tr><tr><td> MgCl2 1M </td><td> 50 l </td><td> 75 l </td><td> 100 l </td><td> 200 l </td></tr><tr><td> 0,5 EDTA </td><td> 10 l </td><td> 15 l </td><td> 20 l </td><td> 40 ml </td></tr><tr><td> NP40/IGEPAL </td><td> 50 l </td><td> 75 l </td><td> 100 l </td><td> 200 l </td></tr><tr><td> β-mercaptoetanol (agregue inmediatamente antes del uso) </td><td> 4,3 l </td><td> 6,5 l </td><td> 8,6 l </td><td> 17,2 l </td></tr><tr><td colspan="5"></td></tr><tr><td colspan="5"> Usar proporciones para el glicerol por ciento deseado (0, 5, o 45%) </td></tr><tr><td> 0% de glicerol </td><td> Ninguno </td><td> Ninguno </td><td> Ninguno </td><td> Ninguno </td></tr><tr><td> de agua </td><td> 8,3 ml </td><td> 12,5 ml </td><td> 16,7 ml </td><td> 33,4 ml </td></tr><tr><td> 5% de glicerol </td><td> 0,5 ml </td><td> 0,8 ml </td><td> 1,0 ml </td><td> 2,0 ml </td></tr><tr><td> de agua </td><td> 7,8 ml </td><td> 11,7 ml </td><td> 15,7 ml </td><td> 31,4 ml </td></tr><tr><td> 45% glycerol </td><td> 4,5 ml </td><td> 6,8 ml </td><td> 9,0 ml </td><td> 18,0 ml </td></tr><tr><td> de agua </td><td> 3,8 ml </td><td> 5,7 ml </td><td> 7,7 ml </td><td> 15,4 ml </td></tr></tbody></table> Tabla 1. Recetas para la preparación de tampones LCM <table border="1"><tbody><tr><td> Por volumen de 50 ml </td></tr><tr><td> 5 ml de NaCl 1M </td></tr><tr><td> 2,5 ml de 1M Tris, pH 7,5 </td></tr><tr><td> Una de 0,5 ml con EDTA </td></tr><tr><td> 41,5 ml de H 2 O </td></tr></tbody></table> Tabla 2. Preparación de la TNE (0,1 M NaCl, 50 mM Tris, pH 7,5, 10 mM EDTA) <table border="1"><tbody><tr><td> Para el volumen 1 L </td></tr><tr><td> 0,2 g de KCl </td></tr><tr><td> 0,2 g de KH 2 PO 4 </td></tr><tr><td> 8 g de NaCl </td></tr><tr><td> 1,15 g de Na 2 HPO 4 </td></tr><tr><td> H 2 O a 1 litro </td></tr></tbody></table> Tabla 3. Preparación de PBS (2,7 mM KCl, 1,5 mM KH 2 PO 4, 137 mM NaCl, 8,1 mM Na 2 HPO 4 pH 7,0) <img alt="Figura 1" src="/files/ftp_upload/3151/3151fig1.jpg" /> Figura 1. PK15 células (A) antes de la infección, (B) a mitad de la infección, y (C) en el efecto citopático (CPE), después de una multiplicidad de infección. <img alt="Figura 2" src="/files/ftp_upload/3151/3151fig2.jpg" /> Figura 2. Descripción general del protocolo. <img alt="Figura 3" src="/files/ftp_upload/3151/3151fig3.jpg" /> Figura 3. Tres ejemplos representativos de vidrio anzuelos utilizados para recoger los fantasmas del ADN viral. <img alt="Figura 4" src="/files/ftp_upload/3151/3151fig4.jpg" /> Figura 4. Ejemplo representativo de un ADN bien formado y abundante &quot;fantasma&quot;.

<ol>
	<li>Szpara, M. L., Parsons, L., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+variability+in+clinical+and+laboratory+isolates+of+herpes+simplex+virus+1+reveals+new+mutations.">Sequence variability in clinical and laboratory isolates of herpes simplex virus 1 reveals new mutations.</a> J Virol. 84, 5303-5313 (2010).</li><li>Banfield, B. W., Kaufman, J. D., Randall, J. A., Pickard, G. 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+pseudorabies+virus+strains+expressing+red+fluorescent+proteins:+new+tools+for+multisynaptic+labeling+applications.">Development of pseudorabies virus strains expressing red fluorescent proteins: new tools for multisynaptic labeling applications.</a> J Virol. 77, 10106-10112 (2003).</li><li>Kobiler, O., Lipman, Y., Therkelsen, K., Daubechies, I., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpesviruses+carrying+a+Brainbow+cassette+reveal+replication+and+expression+of+limited+numbers+of+incoming+genomes.">Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes.</a> Nat Commun. 1, 146-146 (2010).</li><li>Enquist, L. W., Madden, M. J., Schiop-Stanley, P., Vande Woude, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+herpes+simplex+type+1+DNA+fragments+in+a+bacteriophage+lambda+vector.">Cloning of herpes simplex type 1 DNA fragments in a bacteriophage lambda vector.</a> Science. 203, 541-544 (1979).</li><li>Smith, G. A., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+transposon+mutagenesis+in+Escherichia+coli+of+a+full-length+infectious+clone+of+pseudorabies+virus,+an+alphaherpesvirus.">Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus.</a> J Virol. 73, 6405-6414 (1999).</li><li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of virology. , (2008).</li><li>Pomeranz, L. E., Reynolds, A. E., Hengartner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pseudorabies+virus:+impact+on+neurovirology+and+veterinary+medicine.">Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine.</a> Microbiol Mol Biol Rev. 69, 462-500 (2005).</li><li>Roizman, B., Pellett, P. E., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology. , 2381-2397 (2001).</li><li>Mendez, I. I., Hermann, L. L., Hazelton, P. R., Coombs, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+analysis+of+freon+substitutes+in+the+purification+of+reovirus+and+calicivirus.">A comparative analysis of freon substitutes in the purification of reovirus and calicivirus.</a> J Virol Methods. 90, 59-67 (2000).</li><li>Gharabaghi, F., Aymard, M., Trotemann, P., Gerdil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+simplified+micromethod+for+subtyping+varicella-zoster+virus.">A rapid and simplified micromethod for subtyping varicella-zoster virus.</a> J Med Virol. 31, 129-134 (1990).</li><li>Granstedt, A. E., Szpara, M. L., Kuhn, B., Wang, S. S., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+monitoring+of+in+vivo+neural+activity+using+a+circuit-tracing+pseudorabies+virus.">Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus.</a> PLoS One. 4, e6923-e6923 (2009).</li></ol>

No hay conflictos de interés declarado.

Algunas partes de este protocolo fueron desarrollados originalmente para el aislamiento de ADN viral en BSL4 condiciones, pero se adapta igualmente bien a los no-BSL condiciones. 4 Se suelen usar este protocolo para aislar el ADN de la PRV alfa-herpesvirus y el VHS-1, que tienen genomas de ADN encerrado en una cápside proteica y rodeado por una envoltura lipídica. 7,8 Sin embargo, es probable que directamente adaptables a otras grandes virus de ADN, incluyendo beta-y gamma-herpesvirus y adenoviru...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Freón (1,1,2-tricloro-1 ,2,2-trifluoro) </td><td> Pescador </td><td> T178-4 </td><td> Verifique con su institución para las instrucciones sobre la eliminación adecuada de los desechos que contengan freón, o ver Mendez et al. alternativas posibles freón. 9 </td></tr><tr><td> Fase de bloqueo de los tubos de gel, la capacidad de 15ml pesado </td><td> 5 PRIME </td><td> 2302850 </td><td> Opcional </td></tr><tr><td> Polyallomer tubos de ultracentrífuga </td><td> Beckman * </td><td> 331372 * </td><td> * Seleccione los tubos adecuados para su propia ultracentrífuga, los cuales se incluyen a título de ejemplo </td></tr><tr><td> NP-40 / IGEPAL </td><td> Sigma </td><td> I-3021 </td><td></td></tr><tr><td> Células PK-15 </td><td> ATCC </td><td> CCL-33 </td><td></td></tr><tr><td> Las células Vero </td><td> ATCC </td><td> CCL-81 </td><td></td></tr><tr><td> PBS </td><td> HyClone </td><td> SH30028.03 </td><td></td></tr></tbody></table>

preparation of viral dna from nucleocapsids

Los virus son parásitos obligados celular, y por lo tanto el estudio de su ADN requiere aislar material viral de distancia de los contaminantes de la célula huésped y el ADN. Varias aplicaciones posteriores requieren grandes cantidades de ADN viral pura, que es proporcionada por este protocolo. Estas aplicaciones incluyen la secuenciación del genoma viral, donde la extracción de ADN del huésped es crucial para optimizar la salida de datos de secuencias virales, y la producción de nuevas cepas virales recombinantes, 1,2, donde la co-transfección de ADN purificado viral y plásmido lineal facilita la recombinación., 3 Este procedimiento utiliza una combinación de extracción y la densidad basado en la centrifugación para aislar ADN purificado herpesvirus nucleocápside lineal de las células infectadas. 4,5 Los pasos de la purificación inicial objetivo de aislar a cápsidas virales purificados, que contienen y protegen el ADN viral en las extracciones y los pasos de centrifugación que eliminan las proteínas celulares y el ADN. Lisis de la nucleocápside libera el ADN viral, y dos finales de fenol-cloroformo pasos eliminar las proteínas restantes. El final de ADN capturado de la solución está muy concentrado y puro, con un promedio de 260/280 OD de 1,90. Dependiendo de la cantidad de células infectadas, los rendimientos de la gama de ADN viral 150 a 800 mg o más. La pureza de este ADN hace que sea estable durante el almacenamiento a largo plazo a 4 ° C. Este ADN es por lo tanto ideal para el alto rendimiento de secuenciación de alta fidelidad de las reacciones de PCR, y transfección. Antes de comenzar el protocolo, es importante conocer el número promedio de células por plato (por ejemplo, un promedio de 8 x 10 6 células PK-15 en un plato cm confluentes 15), y el título de la acción viral para ser utilizado ( por ejemplo, 1 x 10 8 unidades formadoras de placas por ml). Estos son necesarios para calcular la multiplicidad apropiado de la infección (MOI) para el protocolo. 6 Por ejemplo, para infectar a un plato de 15 cm de células PK-15 con la acción viral por encima, en una MOI de 5, se utiliza 400 l de stock viral y la mezclan con 3,6 ml de medio (volumen de inoculación total de 4 ml para una placa de 15 cm). Múltiples preparaciones de ADN viral se puede preparar al mismo tiempo. El número de preparaciones simultáneas está limitado sólo por el número de tubos en poder del rotor ultracentrífuga (uno por virus, consulte el paso 3.9 más adelante). A continuación se describe el procedimiento como si se hace por un virus.

Watch this Scientific Journal Video about Preparation of Viral DNA from Nucleocapsids at JoVE.com

Preparation of Viral DNA from Nucleocapsids

Se describe el proceso de aislamiento de ADN de alta pureza herpesvirus nucleocápside de las células infectadas. El final de ADN capturado de la solución es de alta concentración y pureza, por lo que es ideal para la secuenciación de alto rendimiento, alta fidelidad de las reacciones de PCR, y transfecciones para producir nuevas recombinaciones virales.

Preparación de ADN viral de nucleocápside

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

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Research

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Immunology and Infection

20.8K vistas.  Princeton University. Se describe el proceso de aislamiento de ADN de alta pureza herpesvirus nucleocápside de las células infectadas. El final de ADN capturado de la solución es de alta concentración y pureza, por lo que es ideal para la secuenciación de alto rendimiento, alta fidelidad de las reacciones de PCR, y transfecciones para producir nuevas recombinaciones virales.

Video: Preparation of Viral DNA from Nucleocapsids

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Generación de recombinantes del virus de influenza a partir del ADN plásmido

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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Inmunología e Infección

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

El uso de un ensayo de microarrays Pan-viral (Virochip) a la pantalla de muestras clínicas de los patógenos virales

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

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 ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

La separación de una sola hebra de ADN, el ADN de doble cadena y el ARN de una comunidad ambiental viral mediante cromatografía de hidroxiapatita

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Un protocolo Chelex simple para la extracción de ADN a partir de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

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 ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Un promedio de glicoproteína de la envoltura viral Espigas de Electron cryotomography reconstrucciones utilizando Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

Aislamiento de fracciones Sub-nuclear enriquecido compartimentos viral de replicación de infectados por adenovirus células normales Humanos

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

El desarrollo de la mediación de la Loop liofilizada amplificación isotérmica reactivos para la detección de<em&gt; Coxiella burnetii</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

La medición de la influenza neuraminidasa inhibición títulos de anticuerpos mediante un ensayo de lectina ligado a enzimas

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...