Este trabajo ha sido financiado por la National Medical Research Council, Singapur.

1. Alexa Fluor etiquetado de los virus del dengue <ol><li> Antes de la reacción de marcaje, purificar el virus del dengue, con colchón de sacarosa y preparar los reactivos y equipos necesarios, como se indica en el protocolo. </li><li> Prepare frescos buffer 0,2 M de bicarbonato de sodio, pH 8.5 (tampón etiquetado), y 1,5 millones de hidroxilamina buffer, pH 8,3 (dejar de reactivo), justo antes de que el etiquetado y el filtro de esterilización con filtros de jeringa 0.2μm. </li><li> Diluir aproximadamente 3x10 8 unidades formadoras de placas (UFP) del virus del dengue purificado en 1 ml de buffer de etiquetado en un tubo de 2 ml. Esto puede ser ampliado en proporción para el etiquetado de los lotes de virus. </li><li> Reconstituir el liofilizado Alexa Fluor 594 (AF594) ésteres succinimidilo a 1 mm en el etiquetado tampón inmediatamente antes de la reacción de marcaje. Otros de los fluorocromos Alexa Fluor tinte serie se puede utilizar de acuerdo a las necesidades de uno. Minimizar la exposición a la luz a partir de este paso. </li><li> Agregar 100μl de la 1mM AF594 tinte para el virus diluido mientras se agita suavemente con la punta de la pipeta. </li><li> Incubar la mezcla de reacción de marcaje a temperatura ambiente durante 1 hora en la oscuridad. Mezcla de inversiones suave cada 15 minutos. </li><li> Girar el tubo brevemente en una centrífuga de mesa y añadir 100μl de reactivo de parada a la mezcla de reacción mientras se agita suavemente con la punta de la pipeta. </li><li> Incube a temperatura ambiente durante una hora adicional en la oscuridad. Mezcla de inversiones suave cada 15 minutos. </li></ol> 2. Purificación de Alexa Fluor etiquetados virus del dengue <ol><li> Al mismo tiempo, equilibrar la columna de purificación con el tampón de su elección. En este experimento, una columna PD-10 se equilibró con 25 ml de tampón HNE (Hepes 5 mM, 150 mM NaCl, 0,1 mM EDTA), pH 7,4, antes de su uso. </li><li> Aplicar el virus de la etiqueta en la parte superior de la columna y empezar a recoger el flujo a través de una vez que el virus entra en la etiqueta de la matriz. Llenar la columna con tampón HNE una vez que todos los virus de la etiqueta ha entrado en la matriz. Deseche el primero 2,5 ml de filtrado y recoger el próximo 2 ml de la fracción de virus de la etiqueta. </li><li> Alícuota y almacenar purificada AF594 virus del dengue en la etiqueta -80 ° C, lejos de la fuente de luz. </li></ol><ol><li> Descongelar una alícuota y determinar el título del virus de la etiqueta de la placa de ensayo antes de utilizar el lote de virus de la etiqueta. </li><li> Semillas de 5x10 4 por pocillo de células Vero, que se cultiva en M-199 medio de crecimiento, en una placa de 4 y con un cubreobjetos en la parte inferior de bien un día antes de la infección. </li><li> Quitar el sobrenadante del cultivo de bien y infectar a las células con multiplicidad de infección de un virus de la etiqueta en el volumen de 100μl (diluido en M-199 medio de mantenimiento según sea necesario) durante 10 minutos a 37 ° C. </li><li> Eliminar el inóculo y lavar dos veces en los cubreobjetos 1XPBS. </li><li> Fijar las células en el 3% paraformalydehyde por 30 minutos. </li><li> Lavar los cubreobjetos 3 veces en 1XPBS. </li><li> Permeabilizar las células con una solución de permeabilización que contiene saponina 0,1% y 5% de BSA en 1XPBS por 30 minutos. </li><li> Se incuban las células con diluir centrifugación-aclaró sobrenadante de cultivo monoclonal 3H5 hibridoma anticuerpo durante 1 hora en una cámara húmeda, protegido de la luz. </li><li> Lavar los cubreobjetos 3 veces en tampón de lavado (1XPBS que contienen cloruro de 1 mM de calcio, cloruro de magnesio 1 mM y 0,1% saponina). </li><li> Se incuban las células con AF488 anticuerpo anti-ratón IgG, 1:100 diluida en una solución de permeabilización, de 45 minutos en cámara húmeda, protegido de la luz. </li><li> Lavar los cubreobjetos 3 veces en tampón de lavado y enjuague una vez en agua destilada. </li><li> Frote el borde del cubreobjetos contra una toalla de papel para drenar el exceso de agua y montar a portaobjetos de vidrio con 8μl Mowiol 4-88 Dabco que contiene 2,5%. </li><li> Deje que la solución de montaje para fijar la noche a 4 ° C antes de ver con un microscopio confocal Zeiss. Adquisiciones secuencial se debe realizar un fluoróforo emocionante a la vez y cambiar entre los detectores de forma concomitante. </li><li> Las imágenes son luego analizadas para la co-localización de la tinción de anticuerpos E de proteínas con el virus de la etiqueta con el software LSM Zeiss Zen para estimar el grado de etiquetado. </li></ol> 3. Resultados representante Un ejemplo de la producción del virus del dengue marcado con tinte AF594 se muestra en la Figura 2. Normalmente, menos de 10 veces cae del título inicial debe ser observado tras el etiquetado de éxito. Sin embargo, cabe señalar que todos los buffers tienen que estar preparados para el nuevo etiquetado para tener éxito y Alexa Fluor ésteres succinimidilo debe utilizarse inmediatamente después de la reconstitución, ya que se hidrolizan en ácidos libres no reactivo en soluciones acuosas 8. A continuación, el virus de la etiqueta tiene que ser comprobado por fluorescencia suficiente antes de su uso en los experimentos. Una sencilla prueba de inmunofluorescencia se llevó a cabo en las células Vero y el grado de etiquetado puede estimarse a partir de la co-localización de los virus marcado con anti-E tinción de anticuerpos de proteínas. Cortarcélulas de Al fueron examinados y una imagen confocal típico se muestra en la Figura 3. Co-localización de análisis de las imágenes utilizando el software LSM Zen demostrado superposición coeficientes que van desde 0,65 hasta 0,8, lo que sugiere que aproximadamente el 65 al 80% de los viriones se marcaron con el tinte. <img src="/files/ftp_upload/3168/3168fig1.jpg" alt="Figura 1" /> Cifra que representa el esquema de 1.Overall Alexa Fluor tinte de etiquetado de procedimiento del virus del dengue. En primer lugar, los topes correspondientes y el virus del dengue están preparados purificados. El tinte Alexa Fluor se reconstituye y se añade a la del virus del dengue diluye en el etiquetado de amortiguamiento. La reacción se detuvo una hora más tarde, con la adición de reactivo de parada. Posteriormente, el virus de la etiqueta se purifica a través de una columna de exclusión por tamaño de quitar el tinte libre. Finalmente, el virus de la etiqueta se vuelve a titulados por la placa de ensayo y la prueba de fluorescencia. <img src="/files/ftp_upload/3168/3168fig2.jpg" alt="Figura 2" /> Figura 2. La media de número de viriones viables (ufp / ml) según lo determinado en un ensayo de placa antes y después del AF594 etiquetado. Una alícuota del AF594 etiquetados virus del dengue se descongela y se re-titulado por la placa de ensayo y que por lo general muestran una menor de 10 veces caen de el título de partida. Las barras de error indican la desviación estándar de duplicados. <img src="/files/ftp_upload/3168/3168fig3.jpg" alt="Figura 3" /> Figura 3. Co-localización de las etiquetas AF594 con las proteínas del virus del dengue E en las células Vero. Células cultivadas en cubreobjetos el día anterior estaban infectados con dengue AF594 etiquetados en MOI de 1 por 10 minutos a 37 ° C. Las células fueron fijadas posteriormente y se marcaron con anticuerpos anti-E, y se examina para colocalización de la proteína G (verde) y AF594 etiquetado (rojo). Señales fluorescentes se visualizaron con una lupa 63X utilizando Zeiss LSM 710 microscopio confocal. Barra de escala es 10μm. Amarillos indican las áreas de colocalización, como se muestra en el recuadro.

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Aunque AF594 colorante se utilizó en este informe, una amplia gama de fluoróforos en la serie Alexa Fluor ésteres succinimidilo está disponible con la química de etiquetado similar. Esto podría extender la aplicación de etiquetado más allá de la imagen. La citometría de flujo se puede utilizar como una alternativa a la microscopía confocal para estimar el grado de etiquetado para los fluoróforos que puede ser excitado y detectado por la máquina de FACS. Alexa Fluor colorantes so...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> De bicarbonato de sodio </td><td> Sigma-Aldrich </td><td> S6297 </td><td></td></tr><tr><td> Hidroxilamina </td><td> Sigma-Aldrich </td><td> 159417 </td><td></td></tr><tr><td> De hidróxido de sodio </td><td> Merck </td><td> 106498 </td><td></td></tr><tr><td> AF594 ésteres succinimidilo </td><td> Molecular Probes, Invitrogen </td><td> A20004 </td><td></td></tr><tr><td> PD-10 columna </td><td> GE Healthcare </td><td> 17-0851-01 </td><td></td></tr><tr><td> Hepes </td><td> Sigma-Aldrich </td><td> H6147 </td><td></td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> S3014 </td><td></td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E9884 </td><td></td></tr><tr><td> M-199 </td><td> Invitrogen </td><td> 11150 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070.03 </td><td></td></tr><tr><td> 4 y placa </td><td> Nunc </td><td> 176740 </td><td></td></tr><tr><td> Cubreobjetos </td><td> Einst </td><td> 0111520 </td><td></td></tr><tr><td> Portaobjetos del microscopio </td><td> Vela marca </td><td> 7105 </td><td></td></tr><tr><td> 3H5 hibridoma </td><td> ATCC </td><td> HB46 </td><td></td></tr><tr><td> 10x PBS </td><td> Primera base </td><td> BUF-2040-10X1L </td><td></td></tr><tr><td> Saponina </td><td> Sigma-Aldrich </td><td> S4521 </td><td></td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A7906 </td><td></td></tr><tr><td> Cloruro de magnesio </td><td> Sigma-Aldrich </td><td> M2670 </td><td></td></tr><tr><td> Cloruro de calcio </td><td> Sigma-Aldrich </td><td> C3306 </td><td></td></tr><tr><td> Paraformaldehído </td><td> Sigma-Aldrich </td><td> 15,812-7 </td><td></td></tr><tr><td> Mowiol 4-88 </td><td> Calbiochem </td><td> 475904 </td><td></td></tr><tr><td> Dabco </td><td> Sigma-Aldrich </td><td> D27802 </td><td></td></tr><tr><td> Centrífuga de mesa </td><td> Eppendorf </td><td> 5424 </td><td></td></tr><tr><td> Microscopio confocal </td><td> Zeiss </td><td> LSM 710 </td><td></td></tr></tbody></table> Para preparar la M-199 medio de crecimiento, añadir 50 ml de FBS, de 5 ml de piruvato de sodio y 5 ml de la no-aminoácidos esenciales para 500 ml de M-199, filtro estéril. Para preparar la M-199 medio de mantenimiento, agregar 15 ml de FBS, de 5 ml de piruvato de sodio y 5 ml de la no-aminoácidos esenciales para 500 ml de M-199, filtro estéril.

visualizing dengue virus through alexa fluor labeling

Los primeros eventos en la interacción entre el virus y la célula puede tener gran influencia en el resultado de la infección. Determinación de los factores que influyen en esta interacción podría conducir a una mejor comprensión de la patogénesis de la enfermedad y así influir en el diseño de vacunas o terapéuticos. Por lo tanto, el desarrollo de métodos para investigar esta interacción sería útil. Los recientes avances en el desarrollo de fluoróforos 1-3 y tecnología de imagen 4 puede ser aprovechado para mejorar nuestros conocimientos actuales sobre la patogénesis del dengue y así allanar el camino para reducir los millones de infecciones de dengue ocurren anualmente. El virus del dengue tiene una envoltura externa andamio que consta de 90 sobre glicoproteína (E) dímeros que protejan el depósito nucleocápside, que contiene una sola hebra de ARN del genoma positivo 5. Las subunidades proteicas idénticas en la superficie del virus por lo tanto pueden ser etiquetados con un colorante reactivo de amina y se visualizan a través de microscopía de inmunofluorescencia. A continuación, presentamos un método simple de etiquetado de los virus del dengue con Alexa Fluor succinimidil éster tinte disuelve directamente en un buffer de bicarbonato de sodio que se produjo el virus altamente viable después del marcaje. No existe un procedimiento normalizado para el etiquetado de los virus vivos y el protocolo existente de un fabricante para el etiquetado de proteínas por lo general requiere la reconstitución de un colorante en dimetilsulfóxido. La presencia de dimetilsulfóxido, incluso en cantidades mínimas, puede bloquear la infección del virus de la producción y también inducir la citotoxicidad celular 6. La exclusión del uso de dimetilsulfóxido en este protocolo lo que reduce esta posibilidad. Alexa Fluor tiene tintes fotoestabilidad superior y son menos sensibles al pH que los tintes comunes, como la fluoresceína y rodamina 2, lo que es ideal para el estudio de la captación celular y el transporte endosomal del virus. La conjugación de Alexa Fluor tinte no afecta el reconocimiento de virus del dengue etiquetados por el virus de anticuerpos específicos y sus supuestos receptores de siete células del huésped. Este método podría tener aplicaciones útiles en los estudios virológicos.

Watch this Scientific Journal Video about Visualizing Dengue Virus through Alexa Fluor Labeling at JoVE.com

Visualizing Dengue Virus through Alexa Fluor Labeling

Aprovechando los avances en el desarrollo de fluoróforo y la tecnología de imágenes, un método sencillo de Alexa Fluor etiquetado de los virus del dengue se ideó para visualizar las primeras interacciones entre el virus y la célula.

Visualización de virus del dengue a través de Alexa Fluor etiquetado

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

visualizing-dengue-virus-through-alexa-fluor-labeling

Research

JoVE Journal

Immunology and Infection

13.7K vistas.  DSO National Laboratories. Aprovechando los avances en el desarrollo de fluoróforo y la tecnología de imágenes, un método sencillo de Alexa Fluor etiquetado de los virus del dengue se ideó para visualizar las primeras interacciones entre el virus y la célula.

Video: Visualizing Dengue Virus through Alexa Fluor Labeling

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 ...

Investigación

Inmunología e Infección

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Evaluación de la eficacia y la toxicidad de los ARN del VIH-1 Orientación de la producción para uso en terapia génica o de drogas

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Rescate y Caracterización del Virus Recombinante de un Nuevo Mundo Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualización de macrófagos trampas extracelular mediante Microscopía Confocal

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Una citometría de flujo Simple basado en análisis para determinar In Vitro anticuerpo dependiente del realce del Virus del Dengue usando suero convaleciente Zika Virus

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

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 ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

Medición de ARN del Virus del Dengue en el sobrenadante de cultivo de las células infectadas por reacción en cadena de la polimerasa cuantitativo en tiempo real

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identificación de la codificación y las clases de ARN no codificante expresado en cerdos sangre entera

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

Un modelo murino de enfermedad similar a la encefalitis vírica aguda inducida por el virus del dengue

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Prueba IN Vitro ELISA para evaluar la potencia de la vacuna contra la rabia

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...