Este trabajo es apoyado en parte, las subvenciones del NIH R01 A1064826-01, Sub-19 AI062627-01, no1-HHSN26600500032C (a SM). Agradecemos a nuestros miembros del laboratorio en busca de ayuda la discusión y el técnico, Tina Heil para la revisión crítica del manuscrito. Nos gustaría dar las gracias a David Bina, Milwaukee Departamento de Salud del Laboratorio de cultivo de virus y PCR.

<ol>
	<li>Hirst, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+agglutination+of+red+cells+by+allantoic+fluid+of+chick+embryos+infected+with+influenza+virus.">The agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus.</a> Science. 94, 22-23 (1941).</li><li>Jonsson, N., Gullberg, M., Lindberg, 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=Real-time+polymerase+chain+reaction+as+a+rapid+and+efficient+alternative+to+estimation+of+picornavirus+titers+by+tissue+culture+infectious+dose+50%+or+plaque+forming+units.">Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units.</a> Microbiol. Immunol. 53, 149-154 (2009).</li><li>Kalter, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinating+Behavior+of+Mouse+and+Egg-adapted+Type+A+(PR8)+Influenza+Virus.">Hemagglutinating Behavior of Mouse and Egg-adapted Type A (PR8) Influenza Virus.</a> Science. 110, 184-184 (1949).</li><li>Dulbecco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Plaques+in+Monolayer+Tissue+Cultures+by+Single+Particles+of+an+Animal+Virus.">Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal Virus.</a> Proc. Natl. Acad. Sci. U. S. A. 38, 747-752 (1952).</li><li>Deyde, V. M., Nguyen, T., Bright, R. A., Balish, A., Shu, B., Lindstrom, S., Klimov, A. I., Gubareva, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+molecular+markers+of+antiviral+resistance+in+influenza+A+(H5N1)+viruses+using+a+pyrosequencing+method.">Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using a pyrosequencing method.</a> Antimicrob. Agents Chemother. 53, 1039-1047 (2009).</li><li>Jonsson, N., Gullberg, M., Lindberg, 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=Real-time+polymerase+chain+reaction+as+a+rapid+and+efficient+alternative+to+estimation+of+picornavirus+titers+by+tissue+culture+infectious+dose+50%+or+plaque+forming+units.">Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units.</a> Microbiol. Immunol. 53, 149-154 (2009).</li><li>Rahman, M., Kieke, B. A., Vandermause, M. F., Mitchell, P. D., Greenlee, R. T., Belongia, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+Directigen+flu+A+B+enzyme+immunoassay+and+direct+fluorescent+assay+for+detection+of+influenza+infection+during+the+2004-2005+season.">Performance of Directigen flu A+B enzyme immunoassay and direct fluorescent assay for detection of influenza infection during the 2004-2005 season.</a> Diagnostic Microbiology and Infectious Disease. 58, 413-418 (2007).</li><li>Landry, M. L., Ferguson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suboptimal+detection+of+influenza+virus+in+adults+by+the+Directigen+Flu+A+B+enzyme+immunoassay+and+correlation+of+results+with+the+number+of+antigen-positive+cells+detected+by+cytospin+immunofluorescence.">Suboptimal detection of influenza virus in adults by the Directigen Flu A+B enzyme immunoassay and correlation of results with the number of antigen-positive cells detected by cytospin immunofluorescence.</a> J. Clin. Microbiol. 41, 3407-3409 (2003).</li><li>Herrmann, B., Larsson, C., Zweygberg, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+and+typing+of+influenza+viruses+A+and+B+by+a+nested+reverse+transcription-PCR:+comparison+to+virus+isolation+and+antigen+detection+by+immunofluorescence+and+optical+immunoassay+(FLU+OIA).">Simultaneous detection and typing of influenza viruses A and B by a nested reverse transcription-PCR: comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay (FLU OIA).</a> J. Clin. Microbiol. 39, 134-138 (2001).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> Am. J. Epidemiol. 27, 493-497 (1938).</li><li>Doerr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins:+into+the+infrared.">Fluorescent proteins: into the infrared.</a> Nat. Meth. 6, 482-483 (2009).</li><li>Shu, B., Wu, K. H., Emery, S., Villanueva, J., Johnson, R., Guthrie, E., Berman, L., Warnes, C., Barnes, N., Klimov, A., Lindstrom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+performance+of+the+CDC+real-time+reverse+transcriptase+PCR+swine+flu+panel+for+detection+of+2009+A+(H1N1)+pandemic+influenza+virus.">Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus.</a> J. Clin. Microbiol. 49, 2614-2619 (2011).</li></ol>

No tenemos nada que revelar.

El brote de la epidemia reciente de gripe aviar en el sudeste asiático y el temor global de la pandemia de gripe porcina imponen enormes problemas para la salud pública y la seguridad. Enfoque crítico se ha dedicado en la generación de vacunas para las cepas existentes y nuevas de virus de la influenza. La disponibilidad de los rápidos tecnologías de alto rendimiento para la detección precisa y exacta los cálculos título viral tremendamente a mejorar el tratamiento. Las técnicas más comúnmente utilizadas empleadas para calcular título del virus incluyen la placa ensayos de hemaglutinación formadoras y la influenza. El ensayo de formación de placas fue desarrollado para estimar los títulos de bacteriófagos y fue adoptado por Renato Dulbecco para el cálculo de los títulos virales de los animales 4. Para realizar este ensayo, 10-diluciones de stock de la influenza o especímenes de animales tales como líquido de BAL se preparan y alícuotas de 0,1 ml se inoculan sobre la monocapa de células MDCK a 6-y placas. El virus se permite para adsorberse sobre las células MDCK seguidos por el anunciocondición de un medio nutritivo y agarosa. Durante la incubación, las células infectadas originales liberar progenie viral que se extendió sólo a las células vecinas. El efecto citopatogénico de la infección viral de la influenza produce una zona circular de las células infectadas llamado una placa. Cada placa está presumiblemente formado después de la infección de una sola célula con una partícula de virus único seguido por la replicación del virus. Contraste entre las células vivas y placas las puede ser mejorada por los tintes tales como cristal violeta. Un inconveniente importante de este ensayo es la falta de reproducibilidad y enormes variaciones dentro de experimentos. Otro ensayo utilizado para determinar el título viral de la influenza es el ensayo de hemaglutinación. El presente proteína hemaglutinina en la superficie del virus de influenza de manera eficiente se une a la N-acetilneuramínico que contienen ácido-proteínas en células de la sangre de mamíferos y aves rojas 1. Esta interacción entre el virus de la influenza y los eritrocitos los conduce a la aglutinación en la forma de una celosía. El lEvel de aglutinación se utiliza para estimar el tıtulo del virus de la gripe en muestras de prueba. Ensayo de aglutinación eritrocitaria sólo se utiliza para determinar la titulación de un virus conocido y no puede ser usado para los propósitos identificación de la cepa. Puesto que este ensayo no hace diferencia entre el vivo y los virus muertos, es difícil para obtener un cálculo preciso de los títulos virales. Actuales diagnósticos clínicos de las infecciones de gripe se basan en pruebas serológicas, la reacción en cadena de la polimerasa, inmunofluorescencia directa de muestras y el cultivo de células de 1,6. El nuevo método descrito aquí también tiene el potencial de diagnóstico clínico y de laboratorio. Detección de la influenza kits Directigen FLU-A. (BD Biosciences, San Jose, CA) y BinaxNOW (Binax, Inc., Portland, ME) ensayo de uso inmunocromatografía para detectar el antígeno viral, como la nucleoproteína 7 El uso de estos equipos, la detección del virus se puede lograr en los 15 minutos, sin embargo, todas las muestras negativas tienen que ser más screened por el método de cultivo celular. Diversos estudios también han informado de una baja sensibilidad de estos kits, en particular, cuando la infección fue leve o baja 8. En nuestro método, hemos consistentemente detectar un precio tan bajo como 100 UFP de virus. Esto sugiere que nuestra técnica es útil en la detección de virus H1N1 en muestras clínicas, incluso cuando la infección es leve. Un método basado en la fluorescencia para detectar antígenos virales se ha informado, donde ya sea especímenes aspirado nasofaríngeo se analizaron directamente o se inocularon en una monocapa de células susceptibles a la multiplicación del virus y los análisis posteriores 9. Sin embargo, estos métodos están limitados para propósitos de diagnóstico y no son adecuadas para los cálculos de título del virus. Q-PCR ayuda a identificar la presencia de ARN viral y no estimar la infectividad de virus vivos. En comparación con estos ensayos, IR con base de tinte sistema de análisis ayudará en la detección y la enumeración de título viral en muestras clínicas y de laboratorio. En caso de brote de influenza, escrítico para diagnosticar miles de muestras en un período de tiempo corto. Nuestro método basado en una placa de 384 pocillos y un escáner de infrarrojos puede escanear 6 placas a la vez (más de 2.000 muestras), por lo tanto ofrece una mayor ventaja sobre otros métodos de inmunofluorescencia directa. Viabilidad de la tramitación rápida de las miles de muestras clínicas podría reducir la variabilidad de ensayo, la duración del tiempo de ensayo y los costes. El uso de un anticuerpo M2-específico ofrece la ventaja de ser independiente de la cepa diferencias en las proteínas H y N. La proteína M2-está relativamente conservada en la mayoría de las cepas de influenza que infectan a los seres humanos. Si los anticuerpos específicos a las manchas circulantes se combinan con nuestro sistema de una puede medir tanto el título de virus e identificar la cepa. Sin embargo, desde M2 ​​es el objetivo para los fármacos amantadina-como, los mutantes pueden surgir que podrían cambiar el sitio de unión para el mAb utilizado aquí. Esto representa una limitación potencial de este sistema en los individuos que están siendo tratados por estas drogas. MúltipleLos ensayos se utilizan para enumerar los títulos de la gripe en muestras de ensayo. Para calcular el virus de la titulación, el 50% de la dosis infecciosa de cultivo de tejidos (TCID 50) y de huevo líquido alantoideo ensayos basados ​​son ampliamente utilizados 1,10. Sin embargo, los requisitos de días múltiples hacer estos métodos menos fiables. Además, en estos métodos de cálculos basados ​​en el cambio de 50% en el número de placas proporcionar titulaciones virales teóricas pero no exacta. La placa que forma los ensayos se pueden realizar dentro de las 96 horas, sin embargo, la falta de reproducibilidad es una preocupación importante en los cálculos del título viral. La presente técnica descrita aquí tiene varias ventajas distintivas. En primer lugar, es una técnica altamente reproducible con resultados consistentes. En segundo lugar, los cálculos de título viral se basan en la simple cuantificación de la intensidad de fluorescencia que se comparan contra los definidos por las curvas de calibración. En tercer lugar, dos o más anticuerpos primarios se puede utilizar para diagnosticar múltiples serotipos en una muestra dada. Además, IR tinte conjugado con anticuerpos tienen minimAl autofluorescencia y se han utilizado para la celda de imagen 11. UV o visual de radiación (300-600 nM) resulta en autofluorescencia alta celular debido a la presencia de altos cuánticos endógenos fluoróforos rendimiento. Estos incluyen los aminoácidos aromáticos, los pigmentos de lipo, nucleótidos piridínico (NADPH), la elastina, colágeno y coenzimas Flavin. Esta propiedad intrínseca de las células hace que sea difícil de utilizar estas fuentes de radiación como sondas para cuantificar la expresión de proteínas específicas. En contraste, cerca-IR tintes excitar y emiten entre 670-1000 nm. Muchas moléculas celulares de acogida e hidrocarburos aromáticos policíclicos no excitan en esta longitud de onda y debido a la dispersión de la luz reducida, emiten fluorescencia de fondo extremadamente bajo. Además, la ventana entre el fondo y detección específica se mejoró enormemente debido a un alto coeficiente de extinción de los fluoróforos cerca-IR. Fotoestabilidad, superior señal-a-ruido, es extremadamente relevante en la cuantificación de los títulos virales. El uso de microflcámaras uidic automatizará el proceso de selección a través de controles robóticos, y podría permitir poner a prueba las muestras clínicas altamente contagiosas con facilidad. Así, la utilización de los cerca-IR colorantes en estos métodos es ideal y muy fiable para enumerar los títulos virales en muestras de tejido.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Albúmina de suero bovino </td><td> Atlanta Productos Biológicos </td><td> S11750 </td><td></td></tr><tr><td> PR8 virus de la </td><td></td><td></td><td> Desde el Dr. Thomas M. Moran </td></tr><tr><td> Cabra anti-ratón IRDye @ 800 </td><td> LI-COR biociencias </td><td> 926-32210 </td><td> Dilución 1:200 </td></tr><tr><td> LI-COR IR escáner Odyssey </td><td> LI-COR-biociencias </td><td> 9201-01 </td><td></td></tr><tr><td> 384 pozos de la placa de fondo plano </td><td> Nalge Nunc internacional </td><td> 164730 </td><td></td></tr><tr><td> 96 pozos de la placa de fondo plano </td><td> Nalge Nunc internacional </td><td> 165305 </td><td></td></tr><tr><td> Medio DMEM </td><td> Invitrogen </td><td> 11965126 </td><td></td></tr><tr><td> Medio RPMI </td><td> Invitrogen </td><td> 11875135 </td><td></td></tr><tr><td> De bicarbonato de sodio </td><td> Invitrogen </td><td> 25080 </td><td></td></tr><tr><td> L-glutamina 100 veces </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Trypson-EDTA </td><td> Invitrogen </td><td> R001100 </td><td></td></tr><tr><td> Sodio Piruvato </td><td> Invitrogen </td><td> 11360070 </td><td></td></tr><tr><td> Anti-M-2 de anticuerpos </td><td></td><td></td><td> Desde el Dr. Thomas M. Moran </td></tr><tr><td> Anti-NP MAB </td><td> CDC </td><td> V2S2208 </td><td> Se obtiene por un MTA </td></tr></tbody></table>

high-throughput detection method for influenza virus

Virus de la gripe es un patógeno respiratorio que causa un alto grado de cada año, la morbilidad y la mortalidad en varias partes del mundo. Por lo tanto, el diagnóstico preciso de la cepa infectante y la detección rápida de alto rendimiento de un gran número de muestras clínicas es fundamental para controlar la propagación de las infecciones de la pandemia. Actuales diagnósticos clínicos de las infecciones de gripe se basan en pruebas serológicas, la reacción en cadena de la polimerasa, inmunofluorescencia directa de muestras y 1,2 de cultivo celular. Aquí, se presenta el desarrollo de una nueva técnica de diagnóstico utilizada para detectar virus vivos de influenza. Hemos utilizado la humana adaptada a ratón A/PR/8/34 (PR8, el H1N1) virus de la 3 para probar la eficacia de esta técnica utilizando células MDCK 4. Células MDCK (10 4 o 5 x 10 3 por pocillo) se cultivaron en 96 - o placas 384-así, infectados con PR8 y proteínas virales se detectaron utilizando anti-M2 seguido por un IR tinte-conjugado seganticuerpo secundario. M2 5 y la hemaglutinina 1 son dos proteínas marcadoras principales que se utilizan en muchas pruebas de diagnóstico diferentes. El empleo de IR-dye-conjugado con anticuerpos secundarios minimizó la autofluorescencia asociado con otros tintes fluorescentes. El uso de anticuerpos anti-M2 nos permitió utilizar la intensidad de fluorescencia antígeno-específica como un indicador directo de la cantidad de virus. Para enumerar la intensidad de fluorescencia, se utilizó el LI-COR Odyssey basado en escáner de infrarrojos. Este sistema utiliza dos canales de detección basados ​​en láser de infrarrojos para identificar fluoróforos y diferenciarlos del ruido de fondo. El primer canal excita a 680 nm y emite a 700 nm para ayudar a cuantificar el fondo. El segundo canal detecta fluoróforos que excitan a 780 nm y emisión a 800 nm. Escaneo de PR8-las células infectadas MDCK en el escáner de infrarrojos indicó un título viral dependiente de fluorescencia brillante. Una correlación positiva de intensidad de fluorescencia a título del virus a partir de 10 2 -10 5 UFP podría ser de Conconsistentemente observado. Positividad mínima detectable, pero siempre visto con 10 2 -10 3 UFP PR8 títulos virales demostrado la alta sensibilidad de los tintes de infrarrojo cercano. La relación señal-ruido se determinó mediante la comparación de los mock-infectados o isotipo tratados con anticuerpo-células MDCK. Uso de las intensidades de fluorescencia de 96 - o formatos de placas de 384 pocillos, se construyeron las curvas estándar de valoración. En estos cálculos, la primera variable es el título viral, mientras que la segunda variable es la intensidad de la fluorescencia. Por lo tanto, hemos utilizado la distribución exponencial para generar una curva de ajuste para determinar la relación polinomio entre los títulos virales e intensidades de fluorescencia. En conjunto, llegamos a la conclusión de que IR con base de tinte sistema de detección de la proteína puede ayudar a diagnosticar las cepas de virus infectante y precisa enumerar el título de los patógenos que infectan.

Watch this Scientific Journal Video about High-throughput Detection Method for Influenza Virus at JoVE.com

High-throughput Detection Method for Influenza Virus

Este método describe el uso de infrarrojos sistema colorante formación de imágenes basada para la detección de H1N1 en bronquioalveolar lavado (BAL) de líquidos de los ratones infectados en una alta sensibilidad. Esta metodología se puede realizar en un 96 - o 384-así placa, requiere &lt;10 l de volumen material de prueba y tiene el potencial para el cribado simultáneo de múltiples patógenos.

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, ...

high-throughput-detection-method-for-influenza-virus

Research

JoVE Journal

Immunology and Infection

26.0K vistas.  Blood Research Institute. Este método describe el uso de infrarrojos sistema colorante formación de imágenes basada para la detección de H1N1 en bronquioalveolar lavado (BAL) de líquidos de los ratones infectados en una alta sensibilidad. Esta metodología se puede realizar en un 96 - o 384-así placa, requiere 

Video: High-throughput Detection Method for Influenza Virus

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 ...

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 ...

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 ...

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 ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

La detección de alto rendimiento de los patógenos respiratorios de especímenes animales por PCR nanoescala

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

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.

Estudios de Virus de influenza A en un modelo murino de infección

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 ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Generación de variantes de Escape de neutralizar anticuerpos Monoclonal del Virus de la Influenza

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

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

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

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

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

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