Deseamos agradecer a M. y R. Scullion Crujiente de las Tecnologías de Idaho para el apoyo técnico y el USGS Western Centro de Investigaciones Ecológicas de financiación (S. Schwarzbach) y asistencia (K. Spragens, T. Graham). Esta investigación se realizó bajo los auspicios del Centro para la Innovación Tecnológica - Instituto para la Defensa y Seguridad Nacional (www.idhs.org), en apoyo del Departamento de Defensa y la Fuerza Aérea Laboratorio de Investigación. Manejo de los animales siguieron los protocolos aprobados por el Cuidado de Animales y el empleo de los EE.UU. Geological Survey Centro Occidental de Investigación Ecológica y los permisos de las Aves Servicio Geológico de EE.UU. Laboratorio de Anillamiento. Cualquier uso de nombres comerciales, productos, o de una empresa en esta publicación es sólo con fines descriptivos y no implica reconocimiento alguno por parte del gobierno de EE.UU..

1. La captura de aves silvestres con redes de niebla <ol><li> Para la captura de aves playeras, establecer redes de niebla en un sitio activo alimentación como un pantano, del litoral o de barro plana. </li><li> Deslice la línea de trasmallo bucles de un extremo de la red de niebla alrededor del poste y poste de inserción vertical en el barro. </li><li> Extiende la red, inserte el segundo polo a través de lazos de trasmallo en el otro extremo de la red de niebla y verticalmente insertar pole en el barro, asegurándose de que las líneas de trasmallo se les enseña. </li><li> Una vez que el pájaro es capturado, el extracto de las aves de la red y volver a la estación de anillamiento. </li></ol> 2. Hisopos cloacales de muestreo <ol><li> Recoger hisopos cloacales inmediatamente después de la captura </li><li> Localizar la cloaca y el uso de los dedos para hacer retroceder a las plumas de los alrededores </li><li> Separar un Dacron o estériles de poliéster hisopo, madre primer extremo, y humedecer la punta estéril con medio de transporte viral para una mayor facilidad en el hisopado. Después de humedecer, toda la cabeza del hisopo es insertado en la cloaca. Limpie la circunferencia interior de la cloaca girando lentamente el hisopo y retire el hisopo de cloaca. </li><li> La inserción de la punta del hisopo directamente en el vial de la muestra de transporte viral medios de comunicación; girar el hisopo entre el pulgar y el índice mientras el aplicador está en los medios de comunicación. Tener un asistente de completar el procedimiento tirando de la punta de la espalda hisopo de la parte inferior del vial aproximadamente 1 cm y cortar el eje del hisopo con un par de tijeras para que la punta del hisopo se queda en el vial y el eje no interfiere con tapa de cierre. Tapón del vial con fuerza. Desinfectar las tijeras con alcohol en el corte entre los ejes hisopo. </li><li> Colocar los frascos en una caja de congelador que contienen hielo seco o hielo. </li></ol> 3. Orofaríngea técnica de hisopo <ol start="1"><li> Abra cuidadosamente el pico del pájaro, de modo que la ranura de coanas es visible </li><li> Separar un hisopo limpio del paquete, el tallo primero, y humedecer la punta con los medios de transporte viral para una mayor facilidad en el hisopado </li><li> Sin tocar la punta a cualquier otras superficies, se insertan en la boca y el hisopo de la apertura de coanas en el techo de la boca y volver de nuevo hacia la región orofaríngea o la garganta </li><li> Coloque la punta del hisopo directamente en el transporte viral vial medios de comunicación. Gire el hisopo entre el pulgar y el índice mientras el aplicador está en los medios de comunicación. Como el anterior, tiene un asistente tirar de la punta de la parte de atrás hisopo de la parte inferior del vial aproximadamente 1 cm y cortar el eje de la esponja con un par de tijeras para que la punta del hisopo se queda en el vial y el eje no interfiera con la tapa cierre. Tapón del vial con fuerza. Desinfectar las tijeras con alcohol en el corte entre los ejes hisopo. </li><li> Colocar los frascos en una caja que contiene el congelador hielo seco o hielo. </li></ol> 4. Manipulación y la liberación de aves <ol start="1"><li> Retorno de aves en el lugar de captura y liberación en el momento oportuno (&lt;20 minutos). Manipuladores deben ser conscientes de los principios de bienestar animal y estar alerta para detectar signos de sufrimiento durante el muestreo de aves (abrasiones, es decir, la captura de miopatía e hipo o hipertermia). Un botiquín de primeros auxilios deben ser incluidos en la lista de equipo. </li></ol> 5. Instalaciones de prueba <ol start="1"><li> Las muestras se pueden probar en cualquier espacio cerrado, como un remolque de investigación o tienda de campaña, con una fuente de alimentación (voltaje regulado generador de energía) para ejecutar la unidad de PCR y el ordenador conectado. </li></ol> 6. La extracción de RNA <ol start="1"><li> Extracción de RNA se realizó con el kit RNeasy Mini con modificaciones menores a las instrucciones del fabricante después de Spackman et al 1. </li><li> Vortex el hisopo medio de 5.3 s, y la transferencia de 350 l con un tubo de microcentrífuga de 2 ml. Nota: Mantenga la muestra restante en el hielo en el caso de PCR muestras positivas deben ser controlados de nuevo o probar H5 necesita ser ejecutado. </li><li> Añadir 350 l de tampón RLT (con β-me) y agitar durante 5 s. </li><li> Añadir 350 l de ARN grado de etanol al 70%. </li><li> Se centrifuga a 5.000 g durante 5 min. </li><li> Añadir 600 l del sobrenadante a una columna de centrifugación en un tubo de recogida de 2 ml. Conservar el resto de la muestra en el bastidor de tubo de ensayo. </li><li> Centrifugar durante 20 s a 10.000 xg y descartar el filtrado. </li><li> Repita los pasos 17-18 hasta que la muestra ha sido pasado a través de spin columna. </li><li> Lugar spin columna en un tubo de recogida limpio de 2 ml. </li><li> Añadir 700 l de buffer RW1 a la columna de centrifugación y centrifugar 25 s de 10.000 x g. Deseche el flujo continuo. </li><li> Añadir 500 l buffer RPE a la columna de centrifugación y centrifugar durante 25 s de 10.000 x g. Deseche el flujo continuo. </li><li> Repita el paso 22 para un total de dos lavados con buffer RPE. </li><li> Centrifugar la columna de centrifugación durante 2 minutos a 14.000 x g. Deseche el tubo de recogida. </li><li> Colocar la columna de giro en un tubo de microcentrífuga limpio de 1.5 ml y añadir 50μl de agua libre de nucleasa. Incubar a temperatura ambiente durante 60 s. </li><li> Eluyen ARN girando durante 60 s de 10.000 xgfo 60 s. Lugar purificado de la muestra en hielo. </li></ol> 7. Preparación de los controles negativos <ol start="1"><li> Reactivos liofilizados fueron preparados para su utilización en el ensayo de PCR de acuerdo a las instrucciones de las Tecnologías de Idaho liofilizado kit de detección de reactivos para la gripe sondas TaqMan Un objetivo 1 (Asay-ASY-0109). </li><li> Centrífuga vial reactivo de control negativo (rojo) durante 3 s para asegurar pellets se encuentran en la parte inferior. </li><li> Quite la tapa, visualmente hacer bolitas que están en el fondo. </li><li> Añadir 20 l de tampón de reconstitución X 2 y 20 l de agua de grado reactivo. </li><li> Recapitulación y agitar durante 5 s en velocidad máxima. </li><li> Centrifugar durante 3 s para llevar líquido a la parte inferior del vial. </li><li> Compruebe visualmente el pellet se rehidrata, si no, repita los pasos 31-32. </li><li> Pipeta 19 ul de la mezcla hidratada en un tubo capilar LightCycler, repita el paso a una segunda pipeta capilar para obtener un duplicado. </li></ol> 8. Preparación de muestras de ensayo <ol start="1"><li> Centrifugar el vial desconocido (azul) durante 3 segundos para asegurarse de pellets se encuentran en la parte inferior. </li><li> Quite la tapa, visualmente hacer bolitas que están en el fondo. </li><li> Añadir 40 ml de ARN purificado. </li><li> Recapitulación y agitar durante 5 s en velocidad máxima. </li><li> Centrifugar durante 3 s para llevar líquido a la parte inferior del vial. </li><li> Compruebe visualmente el pellet se rehidrata, si no, repita los pasos 38-39. </li><li> Pipeta de 19 l de mezcla hidratada en un tubo capilar LightCycler, repita el paso a una segunda pipeta capilar para obtener un duplicado. </li></ol> 9. Preparación de controles positivos <ol start="1"><li> Centrifugar el vial de control reactivo positivo (verde) durante 3 s para asegurarse de pellets se encuentran en la parte inferior. </li><li> Quite la tapa, visualmente hacer bolitas que están en el fondo. </li><li> Añadir 20 l de tampón de reconstitución X 2 y 20 l de agua de grado reactivo. </li><li> Recapitulación y agitar durante 5 s en velocidad máxima. </li><li> Centrifugar durante 3 s para llevar líquido a la parte inferior del vial. </li><li> Compruebe visualmente el pellet se rehidrata, si no, repita los pasos 45-46. </li><li> Pipeta 19 ul de la mezcla hidratada en un tubo capilar LightCycler, repita el paso a una segunda pipeta capilar para obtener un duplicado. </li></ol> 10. Centrifugar los tubos capilares <ol start="1"><li> Una vez que todos los tubos capilares para el lote ha sido preparado cargarlos en el mini-centrifugadora equipados con adaptadores capilar. </li><li> Centrifugadoras en el nivel más bajo manteniendo pulsado el botón de pulso durante 1 s. Esta transferencia de líquido hacia el fondo en la preparación para la prueba. </li></ol> 11. Funcionamiento del RAPID <ol start="1"><li> Crear un nuevo archivo. Etiqueta de control positivo, control negativo, y las muestras (por su número de identificación). </li><li> Programa 1: Ejemplo de ARN Mantenga Preparación <ol><li> Ciclos = 1 </li><li> Modo de análisis = None </li><li> Temperatura objetivo = 40 </li><li> El tiempo de incubación = 30 min </li><li> Temperatura de la tasa de transición = 20 ° C / s (por defecto) </li><li> Objetivo secundario de temperatura = 0 (por defecto) </li><li> Tamaño de paso = 0 (por defecto) </li><li> Retardo de paso = 0 (por defecto) </li><li> Modo de adquisición = None (por defecto) </li></ol></li><li> Programa 2: desnaturalización del ARN <ol><li> Ciclos = 1 </li><li> Modo de análisis = None </li><li> Temperatura objetivo = 94 </li><li> El tiempo de incubación = 120s </li><li> Temperatura de la tasa de transición = 20 ° C / s (por defecto) </li><li> Objetivo secundario de temperatura = 0 (por defecto) </li><li> Tamaño de paso = 0 (por defecto) </li><li> Retardo de paso = 0 (por defecto) </li><li> Modo de adquisición = None (por defecto) </li></ol></li><li> Programa 3: Amplificación del ARN <ol><li> Ciclos = 45 </li><li> Modo de análisis = Auto Análisis </li><li> Temperatura de destino, Segmento 1 = 94, Segmento 2 = 60 </li><li> El tiempo de incubación, Segmento 1 = 0 s, Segmento 2 = 20 s </li><li> Temperatura de la tasa de transición, Seg 1 y 2 = 20 ° C / s (por defecto) </li><li> Temperatura de destino secundario, Seg 1 y 2 = 0 (por defecto) </li><li> Tamaño de paso, Seg 1 y 2 = 0 (por defecto) </li><li> Retardo de paso, Seg 1 y 2 = 0 (por defecto) </li><li> Modo de adquisición, Segmento 1 = Ninguno, Segmento 2 = solo </li></ol></li><li> Los parámetros de fluorescencia <ol><li> Presentación: canal de fluorescencia 2 (CH2 / 1) Ganancias: <ol><li> Ch 1 (F1) = 1 </li><li> Ch 2 (F2) = 8 </li><li> CH 3 (F3) = 1 </li></ol></li><li> Para visualizar los datos de fluorescencia en tiempo real para cada muestra, seleccionar la muestra de interés y haga clic en el botón &quot;Mostrar gráfico&quot; en la parte inferior de la pantalla </li></ol></li><li> Para cada capilar, los resultados son los siguientes: <ul><li> # - Identifica la posición del carrusel del carrusel </li><li> Nombre de la muestra - proporciona el nombre de la muestra </li><li> Control - identifica el tipo de muestra para cada muestra </li><li> Score - calcula utilizando la relación entre la fluorescencia de la muestra y el nivel de fluorescencia de fondo en la muestra </li><li> Resultados - muestra el resultado global de la muestra </li></ul></li></ol> 12. Los resultados representativos: Presente: &quot;presente&quot; Un rojo llamado para una prueba / muestra desconocida indica que el objetivo se identificó en la muestra y los controles fueron exitosos. No detectado: Un verde &quot;no detectado&quot; llamada para una prueba / muestra desconocida indica que el objetivo no fue identificado en la muestra y la prueba de los controles tuvieron éxito. Por favor, repita: A &#39;por favor, repita&#39; indica que el control positivo o negativo no. Un ejemplo de un análisis exitoso por el RAPID 7200 se muestra en la Figura 4. El control positivo se amplifica y se genera fluorescencia detectable (eje Y) a unos 25 ciclos (eje x). Un ciclo umbral (CT)&gt; 35 es el límite acordado para la mayoría de los laboratorios de referencia VIA. Por lo tanto, los controles positivos de este método produce una señal fluorescente en el número de aceptación de los ciclos (0-35) para la detección de VIA. Por el contrario, el control negativo no genera una señal fluorescente, incluso después de 45 ciclos. Del mismo modo, los doce muestras recogidas en el playero occidental no generar una señal fluorescente que indica que las aves fueron negativas para la AIV. Las pruebas de control de todas las muestras positivas en un laboratorio tradicional se anima a asegurar que no los falsos positivos son erróneamente producido. <img src="files/ftp_upload/2829/2829fig1.jpg" alt="Figura 1" /> Figura 1. Aves Playeras de captura con redes de niebla. <img src="files/ftp_upload/2829/2829fig2.jpg" alt="Figura 2" /> Figura 2. Recogida de muestras cloacales y orofaríngeos de por lo menos playeros. <img src="files/ftp_upload/2829/2829fig3.jpg" alt="Figura 3" /> Figura 3. Programación de la RAPID 7200 para la amplificación del ARN. <img src="files/ftp_upload/2829/2829fig4.jpg" alt="Figura 4" /> Figura 4. Fluorogram generado por el portátil RAPID 7200 RT-PCR que muestran cómo los controles positivos y negativos que aparecen en un ensayo de éxito. <a href="http://www.jove.com/files/ftp_upload/2829/2829fig4large.jpg">Haga clic aquí para ver la imagen en tamaño completo</a>

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No hay conflictos de interés declarado.

El método de diagnóstico rápido que aquí se presenta facilita la prueba de tiempo-eficiente y preciso de muestras de aves silvestres para la vigilancia de la AIV. Los requisitos de muestra mucho menos estrictas de almacenamiento portátil de RT-PCR son adecuadas para situaciones remotas donde el mantenimiento de la cadena de frío puede ser poco práctico, si los cargadores de nitrógeno líquido o hielo seco no está disponible. Además, encontramos que el análisis de las muestras con los reactivos liofilizados fu...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> RNeasy mini spin columna </td><td> Qiagen </td><td> 74106 </td><td> Incluido en RNeasy Mini Kit </td></tr><tr><td> Tubos de recogida (1,5 y 2 ml) </td><td> Qiagen </td><td> 74106 </td><td> Incluido en RNeasy Mini Kit </td></tr><tr><td> Tampón RLT </td><td> Qiagen </td><td> 74106 </td><td> Incluido en RNeasy Mini Kit </td></tr><tr><td> Buffer RW1 </td><td> Qiagen </td><td> 74106 </td><td> Incluido en RNeasy Mini Kit </td></tr><tr><td> Buffer RPE </td><td> Qiagen </td><td> 74106 </td><td> Incluido en RNeasy Mini Kit </td></tr><tr><td> Agua libre de RNasa </td><td> Qiagen </td><td> 74106 </td><td> Incluido en RNeasy Mini Kit </td></tr><tr><td> 14,3 M β-mercaptoetanol solución </td><td> Fisher Scientific </td><td> BP176100 </td><td></td></tr><tr><td> 100% de etanol </td><td> Fisher Scientific </td><td> NC9602322 </td><td></td></tr><tr><td> Vortex Genie 2, 120 </td><td> Scientific Industries </td><td> SI-0236 </td><td></td></tr><tr><td> TaqMan Influenza A Objetivo 1 (hidrólisis de la sonda) </td><td> Idaho Tecnologías </td><td> Asay-ASY-0109 </td><td></td></tr><tr><td> Tubos capilares LightCycler 20ml </td><td> Roche Applied Science </td><td> 04929292001 </td><td></td></tr><tr><td> Micro-centrífuga con rotor para tubos de 2 ml </td><td> Idaho Tecnologías </td><td></td><td> Incluido en el kit RAPID </td></tr><tr><td> Ruggedized avanzada de dispositivos de identificación de patógenos (RAPID) 7200 </td><td> Idaho Tecnologías </td><td></td><td> Incluido en el kit RAPID </td></tr><tr><td> Pentium portátil basado en Windows XP Professional </td><td> Idaho Tecnologías </td><td></td><td> Incluido en el kit RAPID </td></tr><tr><td> LightCycler software de análisis de datos </td><td> Idaho Tecnologías </td><td></td><td> Incluido en el kit RAPID </td></tr></tbody></table>

rapid diagnosis of avian influenza virus in wild birds: use of a portable rrt-pcr and freeze-dried reagents in the field

Las aves silvestres han sido implicados en la propagación de la influenza aviar altamente patógena (IAAP) del subtipo H5N1, lo que provocó la vigilancia a lo largo de las rutas migratorias. El muestreo de aves silvestres para el virus de la gripe aviar (AIV) se realiza a menudo en regiones remotas, pero los resultados son a menudo se retrasa debido a la necesidad de transportar las muestras a un laboratorio equipado para las pruebas moleculares. En tiempo real de la transcriptasa inversa reacción de polimerasa en cadena (RT-PCR) es una técnica molecular que ofrece uno de los métodos más precisos y sensibles para el diagnóstico de la AIV. Los protocolos de laboratorio previamente estrictamente necesario para la RT-PCR se están adaptando para el campo. El desarrollo de reactivos liofilizados (liofilizado) que no requieren cadena de frío, con una sensibilidad a nivel de los reactivos húmedos ha llevado a pruebas en el lugar remoto a un objetivo práctico. Aquí presentamos un método para el diagnóstico rápido de la AIV en aves silvestres con una unidad de RT-PCR (Ruggedized avanzada de dispositivos de identificación de patógenos o rápida, las tecnologías de Idaho, Salt Lake City, UT), que cuenta con los reactivos liofilizados (Influenza A Meta 1 Taqman; Asay -ASY-0109, Tecnologías de Idaho). Los reactivos contienen todos los componentes necesarios para las pruebas en las concentraciones adecuadas en un solo tubo: primers, sondas, las enzimas, los tampones y los controles internos positivos, eliminando los errores asociados con el almacenamiento o manejo inadecuado de los reactivos mojado. La unidad portátil realiza una pantalla para la gripe A por objetivo el gen de la matriz y los resultados de los rendimientos en 2-3 horas. Subtipos genéticos también es posible con imprimación H5 y H7, establece que se dirigen al gen de la hemaglutinina. El sistema es adecuado para su uso en muestras cloacales y orofaríngeos procedentes de aves silvestres, como se ha demostrado aquí en el especies de aves playeras migratorias, el playero occidental (Calidrus mauri) capturados en el norte de California. Manejo de los animales siguieron los protocolos aprobados por el Cuidado de Animales y el empleo de los EE.UU. Geological Survey Centro Occidental de Investigación Ecológica y los permisos de las Aves Servicio Geológico de EE.UU. Laboratorio de Anillamiento. La principal ventaja de esta técnica es acelerar el diagnóstico de las aves silvestres, lo que aumenta las posibilidades de contener un brote en una ubicación remota. Sobre el terreno diagnóstico también podrían ser útiles para la identificación y estudio de las personas infectadas en las poblaciones silvestres. La oportunidad de recoger información sobre la biología de acogida (la respuesta inmunológica y fisiológica a la infección) y la ecología espacial (el rendimiento de las aves migratorias infectadas) se proporciona una visión de la medida en que las aves silvestres pueden actuar como vectores para AIV a largas distancias.

Watch this Scientific Journal Video about Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field at JoVE.com

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Este estudio describe el diagnóstico de la gripe aviar en aves silvestres con un portátil de RT-PCR sistema. El método tiene la ventaja de liofilizado reactivos para detectar las aves silvestres en un entorno ajeno al laboratorio, propio de un escenario de brote. Uso de herramientas moleculares ofrece alternativas precisas y sensibles para el diagnóstico rápido.

El diagnóstico rápido del virus de la influenza aviar en aves silvestres: El uso de un portátil de RT-PCR y reactivos liofilizados en el campo

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

rapid-diagnosis-avian-influenza-virus-wild-birds-use-portable-rrt-pcr

Research

JoVE Journal

Immunology and Infection

15.6K vistas.  USGS Western Ecological Research Center. Este estudio describe el diagnóstico de la gripe aviar en aves silvestres con un portátil de RT-PCR sistema. El método tiene la ventaja de liofilizado reactivos para detectar las aves silvestres en un entorno ajeno al laboratorio, propio de un escenario de brote. Uso de herramientas moleculares ofrece alternativas precisas y sensibles para el diagnóstico rápido.

Video: Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Utilizando imágenes bioluminiscentes encargado de investigar la sinergia entre Streptococcus pneumoniae Y el Virus de la Influenza en ratones lactantes

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Investigación

Inmunología e Infección

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

Vigilancia de la Gripe Aviar, con tarjetas FTA: Métodos de Campo, la bioseguridad y los problemas de transporte resueltos

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Asistida por microondas diagnóstico rápido de enfermedades de virus de plantas por microscopía electrónica de transmisión

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Propagación del virus de la influenza en embrionados de pollo Huevos

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Co-inmunoprecipitación de la proteína de ratón Mx1 con la Influenza A nucleoproteína del virus

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Nasal Toallitas para Influenza A Detección de Virus y aislamiento de los cerdos

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

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 ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

In Vitro El desmontaje de virus de influenza A cápsides por centrifugación en gradiente

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.