This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.

Todos los cerdos utilizados en la recogida de los siguientes datos estaban protegidos bajo el Comité de Animales Institucional de Cuidado y Uso Universidad Estatal de Ohio (uso de animales número de protocolo 2009A0134-R1). 1. Preparación de Viral Medio de Transporte y Recogida de muestras Viales <ol><li> Añadir 37 g de Brain Heart Infusion a 900 ml de agua purificada y mezclar bien con una barra de agitación y un agitador magnético mientras se calienta a 70 ° C para disolver completamente el polvo. </li><li> Autoclave el caldo a 121 ° C durante 15 min. Dejar enfriar en la mesa de trabajo hasta que el caldo alcanza la temperatura ambiente. Refrigerar a 4 ° C durante la noche si es necesario. </li><li> Disolver, mediante la mezcla con una barra de agitación magnética, 6,02 g de sal sódica de penicilina G y 10 g de sulfato de estreptomicina en 50 ml de agua purificada a temperatura ambiente. Añadir agua purificada adicional para llevar el volumen total a 100 ml. Filtrar la solución a través de una membrana de polietersulfona 0,22 m en un frasco estéril. </li><li> Agregar asépticamente la solución de penicilina y estreptomicina filtrada en el enfriado caldo de infusión de cerebro y corazón y mezclar bien (barra de agitación o remolino matraz a mano) para hacer que el medio de transporte viral. </li><li> Asépticamente probar el pH del medio de transporte viral; si es necesario, ajustar el pH a 7,4 ± 0,2 utilizando HCl o NaOH. </li><li> Realizar pruebas de control de calidad sobre el medio de transporte viral mediante pruebas de influenza A virus proteína de la matriz por RT-PCR, como se describe por Zhang y Harmon. 22 </li><li> Dispense 5 ml del medio de transporte viral en botellas de polietileno de alta densidad estériles con capacidad de 8 ml. Etiquetar los viales para su uso en el campo. </li><li> Guarde los viales listos para su uso en la crio-cajas estándar a -20 ° C hasta el momento de recogida de muestras. </li></ol> 2. Recolección de Nasal Wipes desde porcina <ol><li> Descongele el número adecuado de viales inmediatamente anteriores a la recogida de muestras; Se necesitará un vial por cerdo muestreado. Deshielo en la sala detemperatura tarda aproximadamente 30-45 min. Mantener los viales descongelados fresco en bolsas de hielo hasta su uso. </li><li> Don equipo de protección personal adecuado como dictados por la situación (por ejemplo, overoles, cubrebotas, respiradores, tapones para los oídos, etc.). </li><li> Entrar en el área de los animales. Si es necesario, limitar el cerdo a un área pequeña pero sin frenar. No despertar descansando animales. </li><li> Póngase un par de guantes de examen desechables. Tenga cuidado de no contaminar los guantes por los cerdos que tocan, personas u objetos inanimados. </li><li> Utilice una mano enguantada para eliminar unas estériles de 5,08 cm x 5,08 cm (2 pulg. X 2 pulg.) De gasa de algodón de su envoltorio. Gasas estériles deben utilizarse siempre que sea posible. Almohadillas de gasa individualmente envueltos son más convenientes de utilizar que aquellos envuelto con dos almohadillas de gasa por paquete. </li><li> Sostenga la almohadilla de gasa de algodón con las yemas de los dedos de una mano la exposición tanto de la almohadilla como sea posible para el muestreo. </li><li> Limpie la almohadilla de gasa a través de hocico de cerdo, teniendoun cuidado especial para entrar en los orificios nasales externos si es posible. Recoger las secreciones nasales visibles (aproximadamente 1 ml) con la misma gasa. </li><li> Utilizando la misma mano, doblar la almohadilla de gasa sobre sí misma para facilitar la colocación en el vial que contiene el medio de transporte viral. </li><li> Utilizando el otro lado, quitar el tapón del vial y colocar la almohadilla de gasa plegada en el vial. Volver a tapar el vial y agitar el vial para asegurar el mezclado de la almohadilla de gasa y medio de transporte viral. </li><li> Quítese los guantes y colóquelos en un recipiente apropiado. Cambie los guantes entre cada cerdo. </li><li> Compruebe el identificador de vial. Número de registro de identificación de cerdos, la edad, el sexo, los signos clínicos, y otras notas que se consideren necesarias por el investigador. </li><li> Enfríe las muestras inmediatamente después de la recolección. Considere hacer alícuotas de la muestra antes de la congelación para evitar múltiples ciclos de congelación-descongelación. Tan pronto como sea posible después de la recolección, colocar las muestras en hielo seco para el transporte al laboratorio. Guarde los viales a -80° C hasta que se inicia la prueba. </li></ol> 3. Detección del Virus de la Influenza Nucleic Acid <ol><li> Descongele las muestras en un baño de grano seco de 37 ° C durante 5 minutos y luego terminar la descongelación de las muestras en la mesa de trabajo a temperatura ambiente durante 20 a 30 min. La adición simultánea de varios viales a las perlas hará que el baño para calentar; se debe tener precaución para no sobrecalentarse los viales. </li><li> Quite 100 l de medio de transporte viral para la extracción de RNA. <ol><li> Use un alto rendimiento (formato de placa de 96) plataforma de perla magnética para la extracción de ARN a partir de un gran número de muestras. 31 </li></ol></li><li> Utilice ensayos de PCR transcriptasa inversa en tiempo real para la detección rápida de la influenza A virus. 31 </li></ol> 4. Aislamiento de Virus de la Influenza de nasales Wipes <ol><li> Descongelar las muestras como se ha descrito anteriormente en la Sección 3.1 </li><li> Tratar muestras descongeladas con sulfato de gentamicina (1000g / ml), anfotericina B (22,5 g / ml), y sulfato de kanamicina (325 mg / ml). </li><li> Inocular en Madin-Darby de riñón canino (MDCK) las células epiteliales como se describió anteriormente. 32 </li></ol>

<ol>
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The authors have nothing to disclose.

La recolección de muestras de cerdos utilizando hisopos nasales de poliéster con punta ha demostrado ser útil en la realización de la vigilancia IAV; Sin embargo, el uso del procedimiento de hisopo nasal dificulta los esfuerzos de vigilancia debido al uso requerido de un lazo a la moderación. Toallitas nasales representan un refinamiento de las técnicas de muestreo porcina actuales para minimizar el estrés en las personas y cerdos durante la recogida de muestras. Mientras que el método ha sido desarrollado y val...

<html> Los virus de influenza A (IAV) causan enfermedades respiratorias en muchas especies, incluyendo aves domésticas, cerdos y seres humanos. Debido al reordenamiento del genoma segmentado IAV rápida evolución viral puede ocurrir y nuevas cepas IAV surgir con frecuencia. Cerdos son una especie que puede servir como un recipiente de mezcla para la redistribución de los virus de este tipo a partir de múltiples especies huésped. 1 Actualmente hay tres subtipos principales de IAV comúnmente circulan entre los cerdos de América del Norte (H1N1, H1N2, H3N2), pero múltiples introducciones IAV de los seres humanos tienen llevó a una extensa diversidad IAV dentro de esos subtipos. 2 La rápida evolución de virus de este tipo que infectan cerdos ha sido evidente desde 1998 cuando un triple recombinante IAV que contiene segmentos de genes de virus humanos, aviares y porcinos 3 se generalizó entre los cerdos en los Estados Unidos. 4 El gen interno segmentos de esa IAV triple recombinante siguen siendo muy frecuente entre los virus de este tipo actualmente infectan a los cerdos. 5 &quot;&gt; A nivel mundial, IAV es una causa importante de enfermedad respiratoria en cerdos en la que los signos clínicos típicos incluyen fiebre, anorexia, letargo, tos, dificultad para respirar, estornudos, secreción nasal y poco aumento de peso. IAV puede ser particularmente costosa para sembrar fincas donde reproductiva fracaso debido a la fiebre inducida IAV y lechones débiles nacido se han documentado. 6,7 Dentro de los Estados Unidos, IAV se detecta comúnmente en piaras comerciales y la amplia diversidad antigénica y genómica y la evolución continua entre IAV porcina infectar ha dificultado el control de este virus 8-11. Problemas de salud pública acerca de la aparición de una cepa IAV pandemia resultante de reordenamiento en el cerdo se dio cuenta en 2009 cuando una IAV porcina linaje que contiene segmentos de genes del linaje porcina norteamericana-triple recombinante y el linaje porcina aviar-como Eurasia causó una pandemia mundial en los seres humanos. 12 El virus pandémico (A (H1N1) pdm09) tiene desderedistribución génica con porcina endémica IAV cepas 13,14 y algunas de estas cepas recién reordenadas se han transmitido de nuevo a los seres humanos. 15 La frecuencia de eventos de reordenamiento y la aparición de nuevas cepas IAV con potencial pandémico hace que la vigilancia activa de los virus circulantes IAV en cerdos imprescindible, sobre todo en la interfase porcina humano. La interfaz porcina humana es importante para interespecies bidireccional transmisión de IAV. De humano a los cerdos de transmisión que ocurre en la producción porcina comercial es responsable de una gran cantidad de IAV diversidad actualmente presente en la población porcina. Ferias agrícolas son los parámetros más importantes para el comingling de las personas y los cerdos en los Estados Unidos y son conocidos sitios para la transmisión zoonótica de IAV. 15-21 En 2012, durante el brote de una variante H3N2 IAV, el 93% de los casos reportados asistencia a una feria agrícola en los días antes de la aparición de la enfermedad. 15 Análisis genómicode los aislamientos virales de cerdos de exhibición frente a las cepas humanas confirmado la transmisión zoonótica. porcina 21 Exposición infectado con IAV menudo no muestran signos clínicos de la enfermedad, 21-23 que indican la necesidad de pruebas diagnósticas directas. El muestreo de los cerdos visiblemente enfermos por sí sola no identificar con éxito prevalencia IAV en los cerdos y no puede ser invocada para identificar nuevas cepas de IAV emergentes entre los cerdos. La vigilancia activa es absolutamente necesario para la detección de cepas emergentes de IAV en cerdos y para evaluar su amenaza tanto para los cerdos y la salud pública. La mayoría de las actividades de vigilancia IAV son voluntarios y por lo tanto se necesitan métodos mínimamente perturbadores. Los tres principales procedimientos de recogida de muestras ante mortem para IAV infectar cerdos son: hisopos nasales, fluidos orales, nasales y toallitas. Las recomendaciones actuales para el muestreo de los cerdos individuales para detectar lista IAV la inserción de fibra sintética con punta de hisopos en las fosas nasales como el método preferidopara recoger las secreciones nasales y células epiteliales. 24,25 Debido a que los cerdos pueden tratar de evitar este procedimiento, un equipo de personal capacitado debe contener cerdos ya sea manualmente o con una trampa en función del tamaño del animal. 26 El proceso de retención es laboriosa para el personal y estresante para los cerdos. Además, porcina exposiciones suelen participar en varias competiciones en una feria por lo que la percepción de estrés añadido en un animal de la competencia puede hacer que los propietarios resistente a los esfuerzos de vigilancia. Con probabilidades de detección IAV que van desde 80-100% en IAV rebaños infectados, fluidos orales se han convertido en una alternativa popular a hisopos nasales para la detección molecular de IAV en las poblaciones de la especie porcina. 27,28 Además, los fluidos orales pueden proporcionar una ventana más amplia de detección IAV de hisopos nasales después de la infección inicial. Sin embargo, el aislamiento a partir de fluidos orales IAV ha sido problemático con sólo el 50% de los intentos de aislamiento del virus resultante en IAV recuperación. 29 El uso de toallitas nasales en lugar de hisopos nasales durante la vigilancia IAV en cerdos supera las limitaciones descritas anteriormente. Toallitas nasales no requieren el uso de una trampa de restricción y se puede realizar sin hacer hincapié en los animales o los testigos del procedimiento. Es necesaria una formación técnica mínima para recoger trapos nasales, lo que permite a los profesionales no veterinarios, incluidos los propietarios de cerdos, para recoger las muestras de vigilancia. Toallitas nasales fueron previamente en comparación con hisopos nasales para la detección y el aislamiento de virus de influenza A 30 y el protocolo detallado para este método no invasivo de muestreo se describe a continuación.

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			<td height="21" style="height:21px;width:216px;">BBL Brain Heart Infusion</td>
			<td style="width:360px;">Becton, Dickinson and Company</td>
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			<td height="21" style="height:21px;width:216px;">Penicillin G Sodium Salt</td>
			<td style="width:360px;">MP Biomedicals, LLC</td>
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			<td>1500 u/mg</td>
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			<td height="21" style="height:21px;width:216px;">Streptomycin Sulfate</td>
			<td style="width:360px;">AMRESCO LLC</td>
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			<td height="21" style="height:21px;width:216px;">Gentamicin Solution</td>
			<td style="width:360px;">Mediatech, Inc.</td>
			<td style="width:119px;">30-005-CR</td>
			<td>50 mg/ml</td>
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			<td height="26" style="height:26px;width:216px;">Dermacea Gauze Sponge, 8 ply</td>
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			<td>5.08 cm &#215; 5.08 cm (2 in. &#215; 2 in)&#160;</td>
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nasal wipes for influenza a virus detection and isolation from swine

La vigilancia de los virus de la gripe A en cerdos es fundamental para la salud humana y animal debido a la influenza A virus evoluciona rápidamente en poblaciones porcinas y las nuevas cepas están surgiendo continuamente. Los cerdos son capaces de ser infectados por diversos linajes del virus de la gripe A haciéndolos anfitriones importantes para la aparición y el mantenimiento de la nueva influenza A cepas del virus. Muestreo cerdos en diversos entornos, como las granjas comerciales de cerdos, ferias agrícolas y mercados de animales vivos es importante proporcionar una visión completa de las cepas circulantes de IAV. La técnica actual estándar de oro ante mortem de muestreo (es decir, colección de hisopos nasales) es mano de obra intensiva, ya que requiere la restricción física de los cerdos. Toallitas nasales implican frotar un trozo de tela a través del hocico del cerdo con mínima o ninguna restricción del animal. La nasal limpie procedimiento es sencillo de realizar y no requiere personal con veterinaria profesional o capacitación manejo de animales. While ligeramente menos sensible que hisopos nasales, las tasas de detección de virus y de aislamiento son adecuados para hacer nasal limpia una alternativa viable para el muestreo de los cerdos individuales cuando se requieren métodos de muestreo de baja tensión. El protocolo de procedimiento describe los pasos necesarios para cobrar una nasal viable limpie de un cerdo individual.

El uso exitoso de este método produce RT-PCR resulta que, acompañado con el uso de un control interno durante la extracción de RNA y RT-PCR, se muestran muestras no contenían inhibidores de PCR de cualquier residuo ambiental recogió durante el muestreo. Después de la inoculación de la muestra, los pozos de aislamiento del virus deben estar libres de escombros ambiental visible de la muestra. Existe un acuerdo razonable entre los resultados de RT-PCR y los resultados de aislamiento de virus con el entendimiento de...

Watch this Scientific Journal Video about Nasal Wipes for Influenza A Virus Detection and Isolation from Swine at JoVE.com

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

nasal-wipes-for-influenza-a-virus-detection-and-isolation-from-swine

Research

JoVE Journal

Immunology and Infection

9.3K vistas.  The Ohio State University. The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. 

Video: Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

Investigación

Inmunología e Infección

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 ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX colorimétrico de detección de un microarray de ADN para la influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

De alto rendimiento Método de detección de virus de la influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Purificación por afinidad de los complejos de ribonucleoproteína Influenza Virus de la cromatina de las células infectadas

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

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 ...

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 ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Un ensayo de microarrays miniaturizada glicanos para la evaluación de avidez y especificidad del virus de la gripe A Hemaglutininas

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

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 ...