Damos las gracias a David Wang, Urisman Anatoly, Amy Kistler, Fischer Kael, Patrick Tang, y Greninger Alexander para el desarrollo inicial y la optimización del protocolo de Virochip. Este trabajo es apoyado por una subvención del NIH K08 y el Premio Descubrimiento Abbott (de CC) y el Instituto Médico Howard Hughes (a JLD).

Los pasos de la prueba Virochip incluyen (1) la extracción de ácidos nucleicos a partir de muestras clínicas, (2) transcripción inversa del ARN extraído y 2 º capítulo de la síntesis de ADNc, (3) amplificación por PCR de cDNA preparada al azar, (3) la incorporación Cy3 tinte fluorescente (4), la hibridación de materiales etiquetados para los microarrays Virochip, y (5) de exploración y el análisis (Figura 1). El protocolo se muestra a continuación demuestran el uso de la prueba Virochip en una muestra de exudado nasal de un niño con una enfermedad similar a la influenza. Secuencias de los cebadores Primer A 5&#39;-GTTTCCCAGTCACGATA-(N 9) -3 &#39; Primer B 5&#39;-GTTTCCCAGTCACGATA-3 &#39; 1. Extracción del ácido nucleico <ol><li> La extracción de ácidos nucleicos a partir de muestras clínicas se deben realizar en el Nivel de Bioseguridad 2 (BSL-2) o más condiciones, ya que estas muestras son potencialmente infecciosas. </li><li> La muestra de exudado nasal se transporta en medio de retención viral. Alícuota de 200 l de la muestra en un tubo de microcentrífuga de 1,5 ml Eppendorf. </li><li> Opcional: pasar de la muestra a través de un filtro de 0,22 micras (para purificar las partículas virales) </li><li> Opcional: muestra pretratamiento con DNasa (que degradan el ADN genómico de acogida, y limpiarnos de protección, encapsulados ácido nucleico viral) utilizando el kit de Turbo DNAsa (Ambion) por las instrucciones del fabricante </li><li> Extracto de la muestra utilizando el Zymo ZR ARN viral kit de extracción o Qiagen Kit Virus Ultrasens según las instrucciones del fabricante. </li></ol> 2. Transcripción reversa y 2 º capítulo de la síntesis de ADNc (&quot;Ronda A&quot;) <ol><li> Añadir un uL 40 pmol / l Primer A 4 uL ARN extraído. Calor a 65 ° C durante 5 minutos en un termociclador (por ejemplo, GeneAmp PCR System 9700) y luego dejar enfriar a temperatura ambiente x 5 min. </li><li> Añadir 5 l de una mezcla maestra que consta de 2 l de 5X buffer RT, 1 l 12,5 mM dNTP, 1 l de agua, 0,5 l 0,1 M TDT, y un 0,5 l RT SSIII (Invitrogen). Se incuba a 42 ° C x 60 minutos. </li><li> En un termociclador programable, el calor a 94 ° C x 2 min (para abortar la reacción de la transcriptasa inversa), se enfría a 10 ° C, se centrifuga el pulso, y luego añadir 5 Sequenase l mezcla que consiste en un búfer de l Sequenase 5X, 3,8 l de agua, y 0,15 l Sequenase (USB Corporation). Para 2 º capítulo de síntesis, la rampa encima de 10 ° C a 37 ° C durante 8 min, mantener 8 minutos, luego caliente a 94 ° C x 2 min (para inactivar Sequenase) y se enfría a 10 ° C (espera). </li><li> Opcional: Rd Una muestra de cDNA se pueden almacenar en este punto a -20 ° C. </li></ol> 3. La amplificación por PCR del ADNc azar Preparado (&quot;Ronda B&quot;) <ol><li> Añadir 5 uL de Rd una muestra de 45 l de una mezcla maestra que consta de 5 l buffer 10X PCR, 1 l 12,5 mM dNTP, 1 l 100 pmol / l B imprimación, 1 l LA KlenTaq (Sigma), y 37 l de agua. </li><li> Ejecutar protocolo de PCR de la siguiente manera: 94 ° C, 2 min → (25 ciclos de 94 ° C, 30 s / 50 ° C, 45 s / 72 ° C, 1 min) → 72 ° C, 5 min → 10 ° C ( espera) </li><li> Compruebe la muestra Rd B en un gel de electroforesis de agarosa al 1,5%. Una prueba de aproximadamente 200 - 1000 pb debe ser visualizado (Figura 2A) </li></ol> 4. Incorporación Cy3 tinte fluorescente <ol><li> Opcional: otra muestra se pueden etiquetar con el colorante fluorescente Cy5 y hibridizada a los microarrays mismo. </li><li> Para incorporar aa-dNTP, añadir 5 uL de la muestra B Rd a 45 l de una mezcla maestra que consta de 5 uL buffer 10X PCR, 1 uL 12,5 mm AA-dNTP (Invitrogen), 1 uL 100 pmol / uL cartilla B, 1, 1 uL KlenTaq LA (Sigma), y 37 uL de agua. Ejecutar protocolo de PCR de la siguiente manera: 94 ° C, 2 min → (15 ciclos de 94 ° C, 30 s / 50 ° C, 45 s / 72 ° C, 1 min) → 72 ° C, 5 min → 10 ° C ( espera) </li><li> Limpiar la muestra Rd C con el ADN limpio y Concentrador-5 Kit (Zymo de Investigación) por las instrucciones del fabricante. Eluyen en 10 mL. </li><li> Añadir 1 l de bicarbonato 1M (Sigma) y 1 uL Cy5 (GE Healthcare) a la muestra C Rd. </li><li> Incubar x 1 hora en la oscuridad. </li><li> Limpiar la muestra Cy3 marcado con el ADN limpio y Concentrador-5 Kit (Zymo de Investigación) por las instrucciones del fabricante. Eluyen en 12 mL. </li></ol> 5. La hibridación de microarrays Virochip <ol><li> Opcional: use 1.5 l para comprobar la concentración de cDNA y la cantidad de colorante incorporado en un espectrofotómetro Nanodrop (para la normalización de las muestras) </li><li> En un tubo de strip PCR añadir un buffer l fragmentación 25X, 5 l 5X buffer de bloqueo, 9,5 l (o la cantidad de normalizar) de Cy3 marcado de la muestra, y el agua hasta un volumen total de 25 l (Agilent) </li><li> Añadir 25 l de tampón de hibridación 2X Gex (Agilent). Centrifugar brevemente para eliminar las burbujas. </li><li> Carga de 40 l de la muestra de diapositivas en la junta. </li><li> Lugar Agilent impreso Virochip array (Universidad de Californiuna, San Francisco / Agilent) (lado marcado &quot;Agilent&quot; hacia abajo) en la parte superior de la diapositiva junta y apretar el tornillo con firmeza. </li><li> Hibridación noche a la mañana en el horno 65 ° C, ajuste de velocidad a 10 rpm. </li><li> Para lavar las matrices, precalentar el buffer 2 (Agilent) a 37 ° C. Desmontar la junta de diapositivas / array submarino sándwich en un buffer (Agilent). Lave en tampón 1 x 1 min. Lave en tampón precalentado 2 x 1 min. Poco a poco eliminar diapositivas de tampón 2, teniendo cuidado de evitar las burbujas (uso Kimwipe para absorber la humedad adicional, teniendo cuidado de evitar el contacto directo con el lado activo de la matriz) </li><li> Lugar de diapositivas en el soporte de diapositivas e insertar en el escáner matriz. </li></ol> 6. Virochip exploración y análisis <ol><li> Exploración Cy3 marcado matriz en 5 micras o 2 micras según el protocolo del instrumento de exploración estándar (Figura 2B). </li><li> Analizar microarrays Virochip por inspección visual, análisis de cluster, o el programa automatizado Virochip análisis, E-Predecir 11. Por cada uno de estos tres métodos, el virus de la influenza se detecta fácilmente en la muestra de exudado nasal (de un niño con enfermedad tipo influenza) (Figura 2 C) </li></ol> 7. Los resultados representativos: Cuando el protocolo se hace correctamente, una prueba debe ser visto en la electroforesis en gel de agarosa después del paso de Rd B (Figura 2). Los microarrays Virochip en la plataforma de Agilent será difícil de analizar visualmente (Figura 2B), pero si hay un virus que debe ser fácilmente identificados por inspección visual, análisis de conglomerados, y / o E-Predecir (fig. 1C). Las muestras pueden ser enriquecida con las cantidades medidas de ácido nucleico de un virus (virus del mosaico del tabaco; bacteriófago MS2) para servir como un control positivo para el ensayo. <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="Figura 1" /> Figura 1. Esquemática de Virochip procesamiento y análisis de microarrays. Extraído de ácidos nucleicos a partir de muestras clínicas son amplificado al azar, etiquetados con tintes fluorescentes, y hibridizada a los microarrays Virochip. Microarrays se escanean a una resolución de 2 micras y se analizaron mediante una variedad de herramientas computacionales, incluyendo E-Predecir. <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="Figura 2" /> Figura 2. Pasos en el ensayo de Virochip Después de la amplificación por PCR al azar, una mancha de 200 -. 1.000 pares de bases puede ser visualizado por electroforesis en gel (A) (B) Tres microarrays Virochip de los 8 conjuntos / portaobjetos de vidrio se muestran, con una pequeña región. de un microarray de soplado en el recuadro en la esquina inferior derecha. (C) automatizado de análisis de microarrays viral mediante E-Predecir revelando la presencia de virus de influenza A en la muestra clínica. La puntuación alta similitud (s = 0,55) corresponde a un p-valor que se aproxima a cero, lo que hace esta una predicción muy importante.

<ol>
	<li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+and+genotyping+of+viral+pathogens.">Microarray-based detection and genotyping of viral pathogens.</a> Proc Natl Acad Sci U S A. 99, 15687-15692 (2002).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+a+critical+respiratory+illness+caused+by+human+metapneumovirus+by+use+of+a+pan-virus+microarray.">Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.</a> J Clin Microbiol. 45, 2340-2343 (2007).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+detection+of+human+parainfluenzavirus+4+infection+associated+with+respiratory+failure+in+an+immunocompetent+adult.">Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.</a> Clin Infect Dis. 43, e71-e76 (2006).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+DNA+microarrays+for+detection+of+viruses+in+acute+respiratory+tract+infections+in+children.">Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.</a> J Pediatr. 153, 76-83 (2008).</li><li>Kistler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-viral+screening+of+respiratory+tract+infections+in+adults+with+and+without+asthma+reveals+unexpected+human+coronavirus+and+human+rhinovirus+diversity.">Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity.</a> J Infect Dis. 196, 817-825 (2007).</li><li>Rota, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+coronavirus+associated+with+severe+acute+respiratory+syndrome.">Characterization of a novel coronavirus associated with severe acute respiratory syndrome.</a> Science. 300, 1394-1399 (2003).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+discovery+and+sequence+recovery+using+DNA+microarrays.">Viral discovery and sequence recovery using DNA microarrays.</a> PLoS Biol. 1, e2-e2 (2003).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+Gammaretrovirus+in+prostate+tumors+of+patients+homozygous+for+R462Q+RNASEL+variant.">Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.</a> PLoS Pathog. 2, 25-25 (2006).</li><li>Kistler, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+divergent+avian+bornaviruses+from+cases+of+proventricular+dilatation+disease:+identification+of+a+candidate+etiologic+agent.">Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.</a> Virol J. 5, e25-e25 (2008).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardioviruses+related+to+Theiler's+murine+encephalomyelitis+virus+in+human+infections.">Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections.</a> Proc Natl Acad Sci U S A. 105, 14124-14129 (2008).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Predict:+a+computational+strategy+for+species+identification+based+on+observed+DNA+microarray+hybridization+patterns.">E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.</a> Genome Biol. 6, R78-R78 (2005).</li></ol>

El protocolo Virochip como se describe aquí es complejo y requiere un técnico de investigación meticulosa y especializada. La concentración correcta de los reactivos y las condiciones de PCR, el etiquetado y la hibridación son críticos. El protocolo Virochip se puede modificar fácilmente para adaptarse a un análisis de los tejidos enfermos mediante el uso de un método de extracción de tejidos, tales como TRIzol (Invitrogen) para la extracción de ácidos nucleicos. Las sondas se pueden añadir o eliminar si es necesario para obtener el espectro deseado y amplitud de la cobertura, por ejemplo, las sondas de detección de blancos no virales tales como bacterias y hongos pueden ser diseñados y agregó que &quot;en la marcha&quot;. Algunas aplicaciones de la prueba Virochip actualmente en desarrollo incluyen un amplio espectro de diagnóstico viral en el laboratorio clínico, investigación de brotes, detección pureza de las drogas y vacunas, y el descubrimiento de nuevos patógenos virales.

<table border="1"><tbody><tr><th> Nombre del reactivo / material </th><th> Nombre del reactivo / material </th></tr><tr><td> Primera </td><td> 5&#39;-GTTTCCCAGTCACGATA (N9) -3 &#39; </td></tr><tr><td> Primer B </td><td> 5&#39;-GTTTCCCAGTCACGATA-3 &#39; </td></tr><tr><td rowspan="5"> RT Master Mix (5 l) </td><td> 2 l de 5X buffer RT </td></tr><tr><td> 1 l 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 l de agua </td></tr><tr><td> 0,5 l 0,1 M TDT </td></tr><tr><td> 0,5 l RT SSIII (Invitrogen) </td></tr><tr><td rowspan="3"> Sequenase Mix (5 l) </td><td> Un buffer l Sequenase 5X </td></tr><tr><td> 3,8 l de agua </td></tr><tr><td> 0,15 l Sequenase (USB Corporation) </td></tr><tr><td rowspan="5"> ª B PCR Master Mix (45 l) </td><td> 5 l buffer 10X PCR </td></tr><tr><td> 1 l 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 l 100 pmol / l cartilla B </td></tr><tr><td> 1 l LA KlenTaq (Sigma) </td></tr><tr><td> 37 l de agua </td></tr><tr><td rowspan="5"> º C PCR Master Mix (45 l) </td><td> Buffer 5 uL 10X PCR </td></tr><tr><td> 1 l 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 l 100 pmol / l cartilla B </td></tr><tr><td> 1 l LA KlenTaq (Sigma) </td></tr><tr><td> 37 l de agua </td></tr><tr><td rowspan="4"> La hibridación de amortiguación (25 l) </td><td> Un buffer l fragmentación 25X (Agilent) </td></tr><tr><td> 5 l 5X buffer de bloqueo (Agilent) </td></tr><tr><td> 9,5 l (o la cantidad de normalizar) de Cy3 marcado de la muestra </td></tr><tr><td> Agregar agua a volumen total de 25 l </td></tr><tr><td> Agilent Virochip microarrays </td><td> permiso en espera, disponible a partir de Chiu laboratorio, UCSF </td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

El diagnóstico de la causa viral de muchas enfermedades infecciosas es difícil debido a la secuencia de la diversidad inherente a los virus, así como la aparición continua de nuevos patógenos virales, como el coronavirus del SRAS y 2009, la pandemia del virus H1N1 de la gripe, que no son detectables por los métodos tradicionales. Para enfrentar estos desafíos, ya hemos desarrollado y validado una plataforma de microarrays pan-viral llamada Virochip con la capacidad de detectar todos los virus conocidos, así como nuevas variantes sobre la base de la homología de secuencia conservada 1. Utilizando el Virochip, hemos identificado la gama completa de los virus asociados con las infecciones respiratorias, incluyendo los casos de enfermedades inexplicables crítico en los pacientes hospitalizados, con una sensibilidad igual o superior a 5.2 convencionales de ensayos clínicos. El Virochip también se ha utilizado para identificar nuevos virus, incluyendo el coronavirus del SRAS 6,7, un clado rinovirus novela 5, XMRV (un retrovirus relacionados con el cáncer de próstata) 8, bornavirus aviar (la causa de una enfermedad degenerativa en los loros) 9, y una novela Cardiovirus en niños con enfermedades respiratorias y diarreicas 10. La versión actual de la Virochip ha sido portado a una plataforma de microarrays de Agilent y consta de ~ 36.000 sondas derivadas de más de ~ 1.500 virus en el GenBank de diciembre de 2009. Aquí nos demuestran los pasos a seguir en el procesamiento de un ensayo de Virochip de principio a fin (~ 24 horas el tiempo de entrega), incluyendo la extracción de ácidos nucleicos de muestras, amplificación por PCR utilizando cebadores aleatorios, la incorporación de un colorante fluorescente, y la hibridación de microarrays, la exploración y el análisis.

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

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

El Virochip es un microarray de pan-viral diseñada para detectar de forma simultánea todos los virus conocidos, así como nuevos virus sobre la base de la homología de secuencia conservada. Aquí se demuestra cómo ejecutar un ensayo de Virochip para analizar las muestras clínicas para determinar la presencia de virus conocidos y desconocidos.

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

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

using-pan-viral-microarray-assay-virochip-to-screen-clinical-samples

Research

JoVE Journal

Immunology and Infection

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

18.7K vistas.  University of California, San Francisco. El Virochip es un microarray de pan-viral diseñada para detectar de forma simultánea todos los virus conocidos, así como nuevos virus sobre la base de la homología de secuencia conservada. Aquí se demuestra cómo ejecutar un ensayo de Virochip para analizar las muestras clínicas para determinar la presencia de virus conocidos y desconocidos.

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

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

El establecimiento de un cultivo líquido cubierto de las vías respiratorias humanas epiteliales polarizadas Calu-3 células para estudiar la respuesta del huésped celular a patógenos respiratorios<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Investigación

Inmunología e Infección

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Usando Click Química para medir el efecto de la infección viral en el Host-Cell ARN Síntesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

El uso de proteínas fluorescentes de Seguimiento glicosoma Dinámica en el tripanosoma africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

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 ...

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

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

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

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

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

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Utilización de cápsulas para la tinción negativa de muestras víricas en biocontención

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonofagocítico asesinato ensayo para evaluar las respuestas inmunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Determinación de la eficiencia de los inhibidores químicos contra el crecimiento de toxoplasma intracelular Gondii mediante un ensayo de crecimiento basado en Luciferase

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Medición de anticuerpos que inducen la fagocitosis adquirida naturalmente a los parásitos plasmodium falciparum mediante un ensayo basado en citometría de flujo

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Diagnóstico de la infección bacteriana respiratoria humana mediante amplificación isotérmica

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Autor destacado: Avance en la detección rápida de patógenos respiratorios mediante chip microfluídico 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Detección rápida de patógenos bacterianos que causan infecciones del tracto respiratorio inferior a través de la amplificación isotérmica mediada por bucle basada en chips microfluídicos

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...