Este trabajo fue parcialmente financiado por el Gobierno español &quot;Ministerio de Educación y Ciencia&quot; (proyecto AGL2008-05275-C01/ALI), por el Marco Europeo de Investigación de la Unión siete proyectos financiados VIROBATHE (Contrato N º 513.648), VIROCLIME (Contrato N º 243923 ) y por la Agencia Catalán de agua, Agència Catalana de l&#39;Aigua (ACA), Departamento de Control i Millora dels Ecosistemes acuáticos. Durante el estudio desarrollado Marta Rusiñol era un compañero del catalán Gobierno &quot;AGAUR&quot; (FI-DGR).

1. Concentración de las partículas virales presentes en muestras de agua <ol><li> Recolección y acondicionamiento de muestras de agua <ol><li> Recoger un mínimo de dos réplicas de 10 L por cada muestra en recipientes de plástico con fondo plano y una muestra adicional, como el control del proceso. Esta última muestra se enriquece con una cantidad conocida de partículas virales y se utiliza como control. Nota: Se recomienda contar con un material especial independiente (botellas, tubos, etc) para muestras adicionadas. </li><li> Verificar la calibración del conductímetro y volver a calibrar si es necesario. Preparar un control negativo con agua del grifo previamente ajustado a la conductividad adecuada (ver más abajo 1.6) en un recipiente de plástico adicional L 10. </li><li> Para muestras adicionadas: Añadir el volumen estándar de la lucha contra el virus (aproximadamente 10 5 copias del genoma por cada 10 litros de agua) a la muestra. Mezclar por agitación evitar salpicaduras y aerosoles. Los controles positivos que podría consistir en una cepa poco común de adenovirus sran como HAdV-35 o un bacteriófago como MS2. </li><li> Si la muestra presenta gran cantidad de material en suspensión (arena u otros materiales), hizo que los sedimentos durante 15 minutos. Transferir el agua en un recipiente nuevo. </li><li> Ajustar el pH de la muestra de agua a 3,5 (± 0,1) con la adición de HCl 1 N. Este paso es importante para la concentración de los virus, así que asegúrese de que el pH se ha ajustado correctamente. Mezcle el agua bien por agitación vigorosa, mientras que la adición de HCl. (Nota: Si el pH es inferior a 3,5 añadir NaOH 1 M). </li><li> Ajustar la conductividad. Si la muestra tiene una conductividad de 1500 uS / cm o más, este paso no es necesario. Si la conductividad es inferior a 1.500 uS / cm la formación de material floculado (flóculos) no está garantizada para ajustar la conductividad de 1500 uS / cm por la adición de sales de mar artificial (Sigma). Mezclar enérgicamente con agitación, mientras que la adición de las sales del mar. </li><li> Registre el pH de las muestras antes y después del acondicionamiento, así como uns el volumen de HCl utilizado. La conductividad también deben ser registrados después de ajustar el pH. Siempre desinfectar los electrodos de pH-metro y conductímetro con una nueva solución de HCl, el cloro con una solución de sodio al 10% Tiosulfato y finalmente enjuagar con agua destilada. </li></ol></li><li> Preparación de pre-floculado leche desnatada 1% (PSM) <ol><li> Verificar la calibración del pH-metro y conductímetro y volver a calibrar si es necesario. </li><li> Prepare una solución pre-floculado leche desnatada (1 PSM%, w / v) disolviendo 10 g de polvo de leche descremada (Difco) en un agua de mar artificial L (se disuelven 33,3 g de sal marina artificial en 1 litro de agua del grifo sin cloro y autoclave) y cuidado de ajustar el pH a 3,5 con HCl 1N. Los flóculos deben ser visibles. Prepare la solución justo antes de ser utilizado o se almacenan a 4 ° C durante 24 h. Para decloración uso 1 ml de solución Tiosulfato 10% por cada 100 ml de agua. </li></ol></li><li> Floculación de las partículas virales presentes en las muestras de agua<ol><li> Añadir 100 ml de 1% PSM a la muestra de agua 10-L. </li><li> Agitar las muestras de 80-10 h para permitir que el virus de absorber a los flóculos. Use un temporizador para apagar la agitación después de 8-10 h. </li><li> Detener la agitación y dejar que el sedimento flóculos por la gravedad de 80-10 h. </li></ol></li><li> Recolección y re-disolver los flóculos. Centrifugación <ol><li> Eliminar el sobrenadante mediante una bomba peristáltica y una pipeta de plástico conectada a un tubo de plástico. Para muestras adicionadas el sobrenadante debe ser recogida en una botella y desinfectados de acuerdo a los procedimientos internos. En todos los casos CUIDAR no recoger el sedimento. </li><li> Recoger los sedimentos con los flóculos (500 ml) en una botella de centrífuga. </li><li> Balance de las ollas con la adición de pH 3,5 PSM. </li><li> Centrífuga de las macetas en una centrífuga de alta velocidad a 8.000 xg durante 30 min a 4 ° C. Tan pronto como se detiene la centrífuga, retire con cuidado los botes de centrífuga de la centrifugadora. </li><li> Muy gently verter y desechar el sobrenadante. Siga las medidas apropiadas para material infeccioso. </li><li> 7 ml de solución amortiguadora de fosfato para disolver el precipitado en cada botella de centrífuga. </li><li> Una vez que los copos se han disuelto, medir y añadir tampón fosfato para llegar a un volumen total de 10 ml. </li><li> Homogeneizar el concentrado viral por agitación y distribuir los 10 ml en microtubos limpia que debería estar congelado a -80 ° C hasta que se necesite en su posterior análisis. </li></ol></li></ol> 2. Extracción del ácido nucleico <ol><li> Realizar una extracción de ácidos nucleicos con el QIAamp Viral RNA Mini Kit de las instrucciones del fabricante. Este kit permite la utilización de una plataforma automatizada (como Qiacube, Qiagen). </li></ol> 3. PCR cuantitativa de los adenovirus humanos (HAdV), poliomavirus JC (JCPyV), adenovirus porcino (PADV) y poliomavirus bovina (BPYV) <ol><li> La cuantificación de copias del genoma en las muestras <ol><li> Prepararla mezcla de PCR cuantitativa en un área separada limpia utilizando TaqMan PCR Master Mix Ambiental 2x (Applied Biosystems). La reacción tiene lugar en una placa de reacción de 96 pozos cubierto con óptica cubre adhesivo óptico. Las concentraciones de la mezcla maestra, primers y sondas se describen en la Tabla 1. </li><li> Una vez que la mezcla ha sido preparado, 15μl alícuotas en cada pozo incluyendo los controles (ver 3.2). El volumen total de una reacción después de la adición de la meta se 25μl (mezcla de 15μl de la muestra + 10μl o estándar). </li><li> Añadir el ácido nucleico extracciones de las muestras (10μl) en un área separada. Ejecutar directa y una dilución de diez veces en agua purificada de cada muestra por duplicado. Cubrir los pozos que contienen las muestras con parte de una cubierta adhesiva, tenga la otra parte de la cubierta para el siguiente paso. </li><li> Añadir las diluciones de la suspensión de ADN estándar (10μl) de 10 0 a 10 6 GC/10μl por triplicado y con una micropipeta de uso exclusivo para el Stanestándar de ADN. Es recomendable añadir las normas en una zona equipada con la radiación UV para la destrucción de ADN plásmido, y para limpiar la micropipeta tras cada uso. Cubrir los pozos que contienen las normas de la cubierta adhesiva pre-cortadas. Nota: Para preparar suspensiones estándar para ser utilizado en la cuantificación de copias del genoma, la región del ADN objetivo debe ser clonado en un plásmido y lineal. En la siguiente dirección se encuentra un procedimiento detallado de cómo crear curvas de nivel con las plantillas de ADN plásmido para ser utilizados en qPCR: <a href="http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf">http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf</a> </li><li> Realizar la qPCR en un sistema adecuado de seleccionar los parámetros adecuados (teniendo en cuenta el uso de la cubierta adhesiva y el volumen total en cada pozo, etc.) Tras la activación del AmpliTaq Gold durante 10 min a 95 ° C, 40 ciclos de amplificación se realizan de la siguiente manera: 15 s a 95 ° Cy 1 min a 60 ° C durante HAdV, JCPyV y BPYV, y 15 s a 95 ° C, 20 a 55 ° C y 20 a 60 ° C para PADV. </li><li> Una vez que las reacciones se han completado, los datos almacenados y los resultados como se describe en el manual del usuario de los equipos utilizados. La cantidad de ADN se define como la mediana de los datos obtenidos después de corregir el factor de dilución cuando sea necesario. </li></ol></li><li> Controles <ol><li> Utilice los controles positivos y negativos. El ensayo debe incluir más de un no-modelo de control (NTC) para probar la mezcla no produce fluorescencia. Es aconsejable que ejecute el control de procesos positivos con el fin de evaluar la potencial inhibición enzimática debido a los inhibidores presentes en las muestras estudiadas. </li><li> Registrar los resultados de los ensayos de qPCR de dos diferentes diluciones del ADN estándar y de control de procesos. Usar los resultados para elaborar gráficos de control para el control de calidad (QC) los programas relacionados con la sensibilidad y la eficiencia de los ensayos. </li></ol></li><li> La confirmación deresultados <ol><li> Los resultados positivos pueden ser confirmado mediante secuenciación PCR anidada y de nucleótidos de los amplicones, la producción de datos sobre las secuencias de nucleótidos de las cepas detectadas 1,5,6,7,9,12. </li></ol></li></ol> Siguiendo el procedimiento descrito, los virus humanos y animales han sido detectados y cuantificados en las aguas de baño, agua de mar y agua de los ríos 2,3. Como ejemplo representativo, las muestras de agua subterránea de las áreas que presentan altos niveles de nitratos fueron evaluados para definir las fuentes de la contaminación. Diez litros de muestras de agua fueron recolectadas en cuatro pozos diferentes en las zonas rurales de una región del noreste de España. Cinco réplicas se obtuvieron en cada una repetición bienestar sembrado con adenovirus humanos 2 utilizadas como control de procesos. Las muestras fueron procesadas de acuerdo al protocolo representadas en la figura 1. Las cuatro réplicas analizadas en uno de los cuatro sitios estudiados mostraron resultados positivos para PADV (valor medio 7.74x10 2 GC / L), que se relaciona con la presencia de purines de cerdo en los alrededores del sitio de muestreo y apoyaría la contaminación fecal porcina como la fuente de nitratos en el agua subterránea (Tabla 2). 4. Los resultados representativos: <img src="/files/ftp_upload/2820/2820fig1.jpg" alt="Figura 1" /> Figura 1. Procedimiento para la detección y cuantificación de virus en el agua. <img src="/files/ftp_upload/2820/2820table1.jpg" alt="Tabla 1" /> Tabla 1. Concentración de los cebadores y sondas para ensayos de PCR cuantitativa. <img src="/files/ftp_upload/2820/2820table3.jpg" alt="Tabla 3" /> Tabla 2. Detección y cuantificación de adenovirus humanos y animales y poliomavirus en muestras de agua subterránea. n número de réplicas analizadas Porcentaje% de los positivos se replica (-) No detectados

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Dos solicitudes de patente presentadas en 2009 para proteger la propiedad intelectual de los protocolos para la cuantificación de PADV y BPYV.

<html> El procedimiento descrito se cumplen las condiciones para un método de ajuste de rutina de los laboratorios de salud ambiental y pública: reproducible, fiable, sencillo y rentable. El protocolo es sencillo, sin embargo, deben seguirse estrictamente. Baja conductividad en las muestras sin necesidad de añadir la concentración de sales pidió agua de mar artificial reduciría dramáticamente la recuperación de los virus, como sería el caso si el tiempo de agitación para la floculación es significativamente re...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Centrífuga de alta velocidad (8.000 xg) </td><td> Berckman Coulter </td><td> Avanti J-20XP </td><td></td></tr><tr><td> pH-metro, termómetro y conductímetro </td><td> Afora </td><td> LPPC3003 </td><td></td></tr><tr><td> Tubos de plástico 100-200 cm de longitud </td><td> Deltalab </td><td> 350059 </td><td></td></tr><tr><td> Se graduó pipetas desechables estériles </td><td> Labclinics </td><td> PN10E1 </td><td></td></tr><tr><td> Tubos estériles de plástico de 1,5 y 10-15 ml (Eppendorf, halcones, etc) </td><td> Afora </td><td> KA298/00 </td><td></td></tr><tr><td> Ollas centrifugadora (500 mL) </td><td> Fisher Scientific </td><td> SE5753512 </td><td></td></tr><tr><td> Agitadores magnéticos e imanes (uno porde la muestra) </td><td> Fisher Scientific </td><td> 10510 </td><td></td></tr><tr><td> De vidrio o recipientes de plástico con fondo plano para permitir el uso de agitadores magnéticos </td><td> Deltalab </td><td> 191642 </td><td></td></tr><tr><td> Una bomba peristáltica para retirar el sobrenadante (o una bomba de vacío por chorro de agua) </td><td> Watson-Marlow </td><td> 323E / D </td><td></td></tr><tr><td> Temporizador para apagar la agitación después de 8-10 horas </td><td> Deltalab </td><td> 900400 </td><td></td></tr><tr><td> El ácido clorhídrico (1 N y 0,1 N) </td><td> Panreac </td><td> 141020.1611 </td><td></td></tr><tr><td> Hidróxido de sodio (1N) </td><td> Panreac </td><td> 131687.1211 </td><td></td></tr><tr><td> Sales del agua de mar artificial mar </td><td> Sigma </td><td> S9883 </td><td></td></tr><tr><td> La leche desnatada (SM) </td><td> Difco </td><td> 232100 </td><td></td></tr><tr><td> Fosfato de pH 7, 5 </td><td></td><td></td><td> 1:2 v / v de estériles Na 2 HPO 4 0,2 M y NaH 2 PO 4 0,2 M a pH 7,5 </td></tr><tr><td> Tiosulfato </td><td> Panreac </td><td> 121879.1209 </td><td> Prepare una solución de 10% en agua </td></tr><tr><td> QIAamp Viral RNA Mini Kit </td><td> Qiagen </td><td> 52904 </td><td></td></tr><tr><td> De 96 pocillos placa de reacción óptica (500 unidades) </td><td> Applied Biosystems </td><td> 43426659 </td><td></td></tr><tr><td> Óptica cubre adhesivo (100 unidades) </td><td> Applied Biosystems </td><td> 4311971 </td><td></td></tr><tr><td> TaqMan PCR Master Mix Ambiental 2x </td><td> Applied Biosystems </td><td> 4396838 </td><td></td></tr></tbody></table>

cost-effective method for microbial source tracking using specific human and animal viruses

La contaminación microbiana del medio ambiente representa un riesgo significativo para la salud. Clásicos indicadores fecales bacterianos han demostrado que tienen limitaciones significativas, los virus son más resistentes a procesos de inactivación y muchos indicadores estándar fecal no informan sobre la fuente de contaminación. El desarrollo de métodos eficientes para la concentración de virus a partir de agua y ensayos moleculares facilita la aplicación de los virus como indicadores de contaminación fecal y herramientas de software como el seguimiento microbiano (MST). Adenovirus y poliomavirus son virus ADN infectar a especies específicas de vertebrados incluyendo a los humanos y son persistentemente excreta en las heces y / o de orina en todas las áreas geográficas estudiadas. En estudios anteriores, hemos sugerido la cuantificación de los adenovirus humanos (HAdV) y poliomavirus JC (JCPyV) por PCR cuantitativa (qPCR) como un índice de contaminación fecal humana. Recientemente, se han desarrollado ensayos de PCR cuantitativa para la cuantificación específica del porcine adenovirus (PADV) y poliomavirus bovina (BPYV) como animales marcadores de contaminación fecal con la sensibilidad de copias del genoma 1.10 por tubo de ensayo. En este estudio, se presenta el procedimiento a seguir para identificar la fuente de contaminación en muestras de agua usando estas herramientas. Como ejemplo de resultados representativos, el análisis de virus en el agua subterránea presenta altos niveles de nitratos se muestra. Detección de virus en las aguas contaminadas de baja o moderada requiere la concentración de los virus por lo menos de varios litros de agua en un volumen mucho menor, un procedimiento que generalmente consta de dos etapas de concentración en serie. Este procedimiento un tanto engorroso y la variabilidad observada en la recuperación viral significativamente interferir con el procesamiento simultáneo de un gran número de muestras de agua. Con el fin de eliminar los cuellos de botella causados ​​por los procedimientos de dos pasos que hemos aplicado un protocolo de un solo paso desarrollado en Studie anteriors, y aplicable a una diversidad de matrices de agua. El procedimiento incluye: la acidificación de las muestras de agua de diez litros, floculación de la leche desnatada en polvo, sedimentación por gravedad de los materiales floculada, la recogida del precipitado y centrifugación, resuspensión del precipitado en tampón fosfato 10 ml. El concentrado viral se utiliza para la extracción de ácidos nucleicos virales y los adenovirus y poliomavirus específicos de interés se han cuantificado mediante qPCR. Alto número de muestras se podrá analizar simultáneamente con este método de concentración de bajo costo. El procedimiento se ha aplicado al análisis de las aguas de baño, agua de mar y agua de los ríos y en este estudio, se presentan los resultados de analizar muestras de agua subterránea. Este método cuantitativo de alto rendimiento es fiable, sencilla y rentable.

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Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

El estudio describe un método costo-efectivo para la identificación de la fuente de contaminación fecal / orina o la contaminación por nitratos en el agua utilizando qPCR para la cuantificación específica de los virus de ADN humano / porcino / bovino, adenovirus y poliomavirus, propuesto como herramientas de MST.

Método rentable para el Rastreo de uso microbiana específica virus humanos y animales

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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12.1K vistas.  University of Barcelona. El estudio describe un método costo-efectivo para la identificación de la fuente de contaminación fecal / orina o la contaminación por nitratos en el agua utilizando qPCR para la cuantificación específica de los virus de ADN humano / porcino / bovino, adenovirus y poliomavirus, propuesto como herramientas de MST.

Video: Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Investigación

Inmunología e Infección

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

Supervisión efectiva de laboratorio integral y costo del VIH / SIDA: un modelo a seguir de África

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus Crecimiento con hemoglobina humana como una fuente de hierro

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

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 ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Crecimiento por parejas Ensayo de competición para determinar la aptitud de replicación de virus de inmunodeficiencia Humanos

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Administración intranasal de vacunas contra la influenza recombinante en modelos de ratón quimérico para el Estudio de la mucosa Inmunidad

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Cromosomas artificiales bacterianos: una herramienta de Genómica Funcional para el Estudio de cadena positiva virus de ARN

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Medición de Influenza neutralizando las respuestas del anticuerpo al virus a (H3N2) en suero humano por microneutralización ensayos utilizando células de MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

VirWaTest, un método de punto de uso para la detección de virus en muestras de agua

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Cuantificación de células CD4+ T específicas de antígenos humanos que utilizan éster de succinidilo de carboxifluoresceína

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...