Nos gustaría dar las gracias a S. Werner para la prestación de los cuervos utilizados en este estudio y el USDA, APHIS, WS, NWRC personal de cuidado de los animales para el cuidado de los animales y la vigilancia. Mención o uso de un producto no implica aprobación USDA. Los fondos para este estudio fue proporcionado por la USDA, APHIS, Servicios Veterinarios.

Nuestro protocolo es una adaptación de una publicamos previamente 7. Todos los procedimientos con animales fueron aprobados por el Comité del Departamento de Agricultura de EE.UU. (USDA), Servicio de Inspección de Sanidad Agropecuaria (APHIS), Wildlife Services (WS), Centro Nacional de Investigación de la Vida Silvestre (CNRH) Cuidado de Animales institucional y el uso. 1. Crow gavaging <ol><li> Estimar el tiempo de paso de la &quot;materia cerebral seudo &#39;a través del tracto digestivo de los cuervos americanos. <ol><li> Mezclar 5 ml de cocido y revueltos huevo entero con el tinte azul y sonda nasogástrica 1 cuervo usando un 2 en aguja de sonda (Figura 1). </li><li> Compruebe cuervo cada 30 minutos hasta que las heces de color azul / verde manchado ya no se excreta. </li></ol></li><li> Obtener infectados por RML (cepa Chandler) C57BL / 6 cerebros infectados y enfermos terminales de ratón. </li><li> Generar un 20% en peso / volumen normal y infectada homogeneizado de cerebro de ratón en un homogeneizador mezclador bala con 1xtampón fosfato salino (PBS) y cuentas de vidrio, luego se centrifuga a 3000 × g durante 1 min para eliminar las partículas más grandes. Eliminar el sobrenadante y diluir con un volumen igual de PBS 1x para generar un 10% en peso / vol, en PBS estéril. Congelación a -80 ° C hasta que se necesite. </li><li> 17 horas antes de la alimentación forzada a eliminar la comida, pero no el agua, a partir de plumas de gallo. </li><li> Aleatoriamente asignar cuervos a los grupos de tratamiento y alimentación forzada por vía oral cada cuervo con 5 ml de homogeneizado normal o infectado el cerebro de ratón usando un 2 en aguja de sonda. </li><li> Transferencia cante a jaulas individuales y recopilar y poner en común todas las heces dentro de cada jaula de 4 h después de la alimentación por sonda (Figura 2). </li><li> Homogeneizar heces agrupados para cada cuervo en un homogeneizador mezclador bala con perlas de vidrio hasta que se consigue una textura uniforme. * Heces Crow son en su mayoría líquidos y se pueden mezclar fácilmente. </li><li> Para cada cuervo, diluir 500 l de homogenado fecal en 9,5 ml de 1x PBS para un volumen total de 10 ml. </li><li> Centrifugar la homogena fecalTE durante 15 minutos a 1400 xg y extraer el sobrenadante. </li><li> Para reducir al mínimo la amenaza de una infección microbiana secundaria, el tratamiento de sobrenadante con 1 l de 100 unidades / ml de penicilina y 100 mg / ml de estreptomicina (Invitrogen, NY) por cada 100 l de homogeneizado a continuación, colocar bajo luz UV a temperatura ambiente durante 20 min para reducir riesgo de contaminación viral y bacteriana. Después de la exposición UV, las muestras se somete a ultrasonidos en un baño de ultrasonidos 3000 Mp en el ajuste 70 durante 30 segundos para romper la membrana de los microbios restantes. </li></ol> 2. Inoculación de ratones <ol><li> Aleatoriamente asignar los ratones a los siguientes grupos de tratamiento (Tabla 1): <ol><li> Grupo 1 - ratones de tratamiento positivos inoculados por vía intraperitoneal (IP) con 1 ml de homogenado fecal de cuervos alimentado a la fuerza con 5ml infectados cerebros de ratón. </li><li> Grupo 2 - Negativo ratones en tratamiento inoculados IP con 1 ml de homogenado fecal de cuervos alimentado a la fuerza con 5ml cerebros normales de ratón. </li><li> Diluir infecteD y normal homogeneizado de cerebro de ratón para los Grupos 3 y 4 a 1:100 w / v en PBS 1x. </li><li> Grupo 3 - ratones de control positivos inoculados IP con 1 ml de infectada homogeneizado de cerebro de ratón. </li><li> Grupo 4 - ratones de control negativo inocularon ip con 1 ml de homogenado normal de cerebro de ratón. </li></ol></li></ol><table border="1"><tbody><tr><td></td><td> Grupo de Tratamiento </td><td> Número de animales </td></tr><tr><td> Grupo 1 </td><td> Tembladera + Crow heces </td><td> 100 </td></tr><tr><td> Grupo 2 </td><td> Tembladera - Crow heces </td><td> 25 </td></tr><tr><td> Grupo 3 </td><td> Scrapie + ratón Cerebro </td><td> 10 </td></tr><tr><td> Grupo 4 </td><td> Scrapie - Ratón Cerebro </td><td> 5 </td></tr></tbody></table> . Cuadro 1 Estado de la tembladera de inóculo (+ positivo, negativo -) y el número de animales utilizados 7. <ol start="2"><li> Intraperitoneal inocular ratón: <ol><li> Ratón Scruff por dorsal piel del cuello con el pulgar y el dedo índice y suavemente gire para exponer la parte ventral. </li><li> Eleve extremo posterior del ratón para que la cabeza es ligeramente inferior. </li><li> Inserte la aguja 25 G 1 cm a través de la piel, 1 cm lateral de la línea media, y 1-2 cm por delante de la articulación sacroilíaca utilizando una jeringa con aguja de bloqueo. </li><li> Inyectar 1 ml inocular lentamente en la cavidad del cuerpo del ratón. </li></ol></li></ol> 3. Monitoreo Ratón <ol><li> Monitorear los ratones diariamente hasta que expresan los signos clínicos de la tembladera ratón. Los signos clínicos pueden incluir: cifosis, ataxia, cola rígida, falta de aseo, emaciación y letargo. </li><li> Puntuación ratones para cada uno de los 6 signos clínicos cuando los signos visibles son evidentes, donde 0 = ninguno visible, 1 = moderado, 2 = y severa. </li><li> La eutanasia a los ratones cuando las puntuaciones diarias totales para cada signo alcanzan ≥ 8 por 1 día, ≥ 6 continua durante 3 días, o 365 días después de la inoculación (dpi). </li><li> Cerebros cosecha inmediatamente después euthanasia y almacenar a -70 ° C. </li><li> Para confirmar un diagnóstico de la tembladera, digerir muestras de cerebro con 3 l de un 50 g / ml de solución de proteinasa K (PK) diluidas de la siguiente manera: 3.1. l de PK, 12,5 l de EDTA 500 mM, pH 8, 109.39 l de 1x PBS durante 30 min a 45 ° C, a continuación, inactivar la PK mediante la adición de 8 l de tampón de carga e incubando las muestras a 95 ° C durante 5 min. Cargar las muestras en un gel de SDS-PAGE al 12%, electroforesis y transferencia a membrana Immobilon PVDF y bloque con 5% de leche sin grasa en 0,2% de Tween 20 en PBS durante 1 hora a temperatura ambiente. A continuación, la sonda con Bar224 anticuerpo monoclonal anti-PrP conjugado con peroxidasa de rábano picante, diluida en superbloque, durante 1 hora a temperatura ambiente. Enjuague la membrana durante 1 hora con PBS-Tween 20 al 0,2%. Para visualizar, incubar Western blot 5 min con sustrato quimioluminiscente y la imagen de un sistema de documentación de gel G-box. </li></ol>

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No hay conflictos de interés declarado.

Se demuestra un procedimiento para documentar el paso de material de PrP Res. través del sistema digestivo de cuervos. Se utilizaron los métodos convencionales para determinar si los cuervos tienen la capacidad de trasladar material de PrP Res a las áreas geográficas libres de la enfermedad. Otros han evaluado la resistencia de PrP Res a rumiantes y roedores 19-21 22,23 líquidos digestivos, los cuales no pudieron eliminarlo. Futura aplicación de estas técnicas debe ser ...

Las encefalopatías espongiformes transmisibles (EET) son enfermedades infecciosas mortales neurodegenerativas que afectan a la vida silvestre, los animales domésticos y los seres humanos. El agente infeccioso de las EET parece ser isoformas mal plegadas o patógenos (PrP Res) de proteínas priónicas 1. Las EET animales incluyen la caquexia crónica (CWD) en el venado bura (Odocoileus hemionus), el venado cola blanca (Odocoileus virginianus), el alce (Cervus elaphus), y el alce (Alces alces), la tembladera en ovinos y caprinos; encefalopatía espongiforme bovina ( EEB) en bovinos domésticos; encefalopatía transmisible del visón en el visón de criadero; encefalopatía espongiforme felina en gatos; exótica encefalopatía espongiforme de ungulados exóticos en zoológico rumia de la familia de los bóvidos, y la encefalopatía espongiforme en los primates no humanos 8. La enfermedad TSE humano único, la enfermedad de Creutzfeldt-Jakob variante, es raro y pensado para ser adquirida por el consumo de PrP Res-contamialimentos ATED 9. Del mismo modo, la EEB puede infectar a los seres humanos si carne contaminada se consume 10. De todas las enfermedades EET, la tembladera y la caquexia crónica son los dos únicos con las epidemias y las fuentes de infección autosostenibles se supone que son de un animal a 2,3,11, así como de fuentes ambientales 4-6. La investigación sugiere que la mayoría de las EET requieren períodos de incubación prolongados notables de eventos de exposición naturales de materiales PrP Res a la manifestación de signos clínicos 2-4,6,8 y barreras de las especies aparentes minimizar, pero no eliminan la posibilidad de que, la transmisión entre 12-14 . Identificar mecanismos para la difusión de la (PrP Res) Material prión infeccioso es extremadamente importante para responder a las preguntas acerca de cómo las EET se mueven a través del paisaje. Las investigaciones experimentales han sugerido que los insectos 15,16, aves de corral y cerdos 17, y cuervos americanos (Corvus brachyrhynchos) 7,18 son portadores pasivos o dispersores de material PrP Res. La aprobación de material de PrP Res a través del sistema digestivo de los cuervos se ha documentado recientemente, lo que demuestra el papel que pueden desempeñar en la dispersión de las EET 7. Estos resultados hacen que sea posible que los cuervos, un carroñero, podrían encontrar, consumen, y transportan material infeccioso a través de las heces de depósito, a las zonas libres de la enfermedad. Los procedimientos demostramos aquí se utilizan para documentar el paso de material PrP Res a través del sistema de digestión de los cuervos y facilitará en gran medida la aplicación de estos métodos para otro carroñero y modelos específicos de especies de carnívoros en futuras investigaciones relacionadas. En este estudio se utilizaron métodos convencionales para investigar un medio no convencional de la trata de material PrP Res, que podría contribuir a la difusión y la carga total de material de PrP Res.

<table border="0" cellpadding="0" cellspacing="0" width="545">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RML Chandler strain mouse-adapted scrapie</td>
			<td style="width:112px;">Rocky Mountain Laboratories</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RC57BL/6 mice</td>
			<td style="width:112px;">Hilltop Lab Animals</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">American crows</td>
			<td style="width:112px;">wild captured</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Phosphate buffered Saline</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">70011-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Sonicator</td>
			<td>Misonix</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Proteinase-K solution</td>
			<td style="width:112px;">Roche</td>
			<td align="right" style="width:137px;">3115887001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Loading buffer</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0007 and 0009</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Bis-tris SDS PAGE 12% gel</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0342</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Immobilon PVDF membrane</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">1SEQ00010</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Tween 20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:137px;">P2287</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bullet blender homogenizer</td>
			<td style="width:112px;">Braintree Scientific</td>
			<td style="width:137px;">BBX24B</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">2.3 mm Zirconia/silica beads</td>
			<td style="width:112px;">BioSpec Products</td>
			<td style="width:137px;">11079125Z</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bar224 anti-PrP monoclonal antibody</td>
			<td style="width:112px;">Cayman Chemical</td>
			<td align="right" style="width:137px;">10009035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Superblock</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td align="right" style="width:137px;">37517</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">chemiluminescent substrate</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">WBKLS0500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">G-box gel documentation system</td>
			<td style="width:112px;">Syngene</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

procedures for identifying infectious prions after passage through the digestive system of an avian species

Priónica infecciosa (PrP Res) material es probablemente la causa de la fatal neurodegenerativa encefalopatía espongiforme transmisible (EET) enfermedades 1. La transmisión de las EET, como la caquexia crónica (CWD), se presume que es de un animal a 2,3, así como de fuentes ambientales 4-6. Los carroñeros y carnívoros tienen potencial para trasladar material de PrP Res a través del consumo y la excreción de carroña CWD-contaminada. Trabajos recientes han documentado el paso de material PrP Res a través del sistema digestivo de los cuervos americanos (Corvus brachyrhynchos), un carroñero norteamericano común 7. Se describen los procedimientos utilizados para documentar el paso de material PrP Res través de cuervos americanos. Cuervos fueron alimentadas por sonda con RML-tembladera cepa adaptada a ratón y sus heces se recogieron 4 h después de alimentación forzada. Crow heces fueron reunidas después y se inyectaron por vía intraperitoneal enC57BL / 6 ratones. Los ratones fueron controlados diariamente hasta que expresaron los signos clínicos de la tembladera de ratón y se sacrificaron a partir de entonces. Ratones asintomáticos fueron monitoreados hasta 365 días después de la inoculación. Se realizó un análisis de Western blot para confirmar el estado de la enfermedad. Los resultados revelaron que los priones infecciosos permanecen después de viajar a través del sistema digestivo de cuervos y están presentes en las heces, causante de la enfermedad en los ratones de prueba.

Los procedimientos utilizados demuestran que el sistema digestivo de cuervos no elimina la PrP Res. infectividad 4 horas después de la alimentación forzada oral de homogeneizado de cerebro tembladera 7. Los veinte cuervos que fueron alimentadas por sonda con material de PrP Res transmiten posteriormente el material PrP Res vía heces a los ratones. Ratones enfermos se identificaron por la manifestación de signos-ratón de tembladera clínicos y la confirmación de la enferm...

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Los carroñeros tienen potencial para trasladar infecciosas priones de encefalopatía espongiforme transmisible en sus heces a las zonas libres de la enfermedad. Nos detalle los métodos utilizados para determinar si priones de scrapie adaptados-ratón permanecen infecciosa después del paso a través del tracto digestivo de cuervos americanos (Corvus brachyrhynchos), un consumidor común de animales muertos.

Procedimientos para la Identificación de priones infecciosos después de pasar por el sistema digestivo de una especie de aves

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

procedures-for-identifying-infectious-prions-after-passage-through

Research

JoVE Journal

Immunology and Infection

11.0K vistas.  USDA.  Los carroñeros tienen potencial para trasladar infecciosas priones de encefalopatía espongiforme transmisible en sus heces a las zonas libres de la enfermedad. Nos detalle los métodos utilizados para determinar si priones de scrapie adaptados-ratón permanecen infecciosa después del paso a través del tracto digestivo de cuervos americanos (Corvus brachyrhynchos), un consumidor común de animales muertos. 

Video: Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Desarrollo de un marcador negativo seleccionable para Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Investigación

Inmunología e Infección

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

Propagación y detección de un clon infeccioso molecular de Maedi-Visna virus que expresa la proteína fluorescente verde

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Mal plegamiento de proteínas priones de amplificación cíclica

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Viral Concentración Determinación A través de placa Ensayos: El uso de tradicionales y novedosos sistemas de superposición

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

Medidas de seguridad y procedimientos de operación en una (A) BSL-4 de laboratorio: 1. Nivel de Bioseguridad 4 Traje Laboratorio de Entrada y Salida Procedimientos suite

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

Medidas de seguridad y procedimientos de operación en una (A) de laboratorio BSL-4: 2. Prácticas generales

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

Medidas de seguridad y procedimientos de operación en una (A) BSL-4 de laboratorio: 4. Procedimientos de imágenes médicas

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

Medidas de seguridad y procedimientos de operación en una (A) BSL-4 de laboratorio: 3. Aerobiología

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Virus Zika Sistema infecciosa cultivo celular y la<em&gt; In Vitro</em&gt; Profiláctica Efecto de los interferones

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Aislar los linfocitos desde el sistema de inmune Intestinal pequeño ratón

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...