Nous tenons à remercier S. Werner pour fournir les corbeaux utilisées dans cette étude et l&#39;USDA, APHIS, WS, CNRF personnel de soins aux animaux pour les soins et la surveillance des animaux. La mention ou utilisation d&#39;un produit n&#39;implique pas l&#39;approbation de l&#39;USDA. Le financement de cette étude a été fourni par l&#39;USDA, APHIS, les Services vétérinaires.

Notre protocole est adapté de celui que nous avons déjà publié 7. Toutes les procédures impliquant des animaux ont été approuvés par le Comité institutionnel de protection et d&#39;utilisation des animaux de l&#39;United States Department of Agriculture (USDA), Animal et Plant Health Inspection Service (APHIS), Wildlife Services (WS), Centre national de la recherche faunique (CNRF). Une. Corneille gavage <ol><li> Estimer le temps de passage de la «matière cérébrale pseudo &#39;à travers le tube digestif des corneilles d&#39;Amérique. <ol><li> Mélangez 5 ml de cuit et des œufs brouillés ensemble avec un colorant bleu et gavage 1 vol en utilisant un 2 en aiguille de gavage (Figure 1). </li><li> Vérifiez vol toutes les 30 min jusqu&#39;à ce que les matières fécales bleu / vert teinté ne sont plus excrétée. </li></ol></li><li> Obtenir C57BL / 6 cerveaux de souris RML (souche Chandler) infectés non infectées et malades en phase terminale. </li><li> Générer un 20% en poids / volume normal et infecté souris homogénat de cerveau dans un homogénéisateur balle de mélangeur avec 1xtampon phosphate salin (PBS) et des perles de verre, puis centrifuger à 3000 g pendant 1 min pour éliminer plus grand de particules. Retirer le surnageant et diluer avec un volume égal de PBS 1x à générer un poids / volume à 10% dans du PBS stérile. Congeler à -80 ° C jusqu&#39;à ce que nécessaire. </li><li> 17 heures avant de gavage enlever les aliments, mais pas l&#39;eau, des stylos d&#39;oiseau. </li><li> Allouer au hasard corbeaux à des groupes de traitement et de gavage par voie orale chaque vol avec 5 ml d&#39;homogénat de cerveau de souris normales ou infecté en utilisant un 2 en aiguille de gavage. </li><li> Transfert chante dans des cages individuelles et de collecter et mutualiser toutes les matières fécales dans chaque cage pour 4 heures après gavage (Figure 2). </li><li> Homogénéiser les fèces regroupés pour chaque vol dans un homogénéisateur balle de mélangeur avec des perles de verre jusqu&#39;à obtenir une texture uniforme. * Excréments Corneille sont pour la plupart de liquide et peuvent être mélangées facilement. </li><li> Pour chaque vol, diluer 500 pi d&#39;homogénat fécale dans 9,5 ml 1x PBS pour un volume total de 10 ml. </li><li> Centrifuger le homogena fécalete pendant 15 min à 1400 x g et le surnageant extrait. </li><li> Pour réduire au minimum le risque d&#39;une infection microbienne secondaire, traiter surnageant avec 1 ul de 100 unités / ml de pénicilline et 100 pg / ml de streptomycine (Invitrogen, NY) par 100 ul d&#39;homogénat puis placer sous une lumière UV à température ambiante pendant 20 minutes pour réduire risque de contamination virale et bactérienne. Après l&#39;exposition aux UV, les échantillons soniquer dans un appareil à ultrasons Mp 3000 à la mise en 70 pour 30 secondes de perturber la membrane de tous les microbes restants. </li></ol> 2. Souris inoculation <ol><li> Répartir au hasard les souris à des groupes de traitement suivants (Tableau 1): <ol><li> Groupe 1 - souris positifs du traitement par voie intrapéritonéale inoculés (IP) avec 1 ml d&#39;homogénat fécale de corbeaux gavage par voie orale avec 5ml infectés cerveaux de souris. </li><li> Groupe 2 - Négatif souris inoculées de traitement IP avec 1 ml d&#39;homogénat fécale de corbeaux gavage par voie orale avec des cerveaux de souris normales 5ml. </li><li> Diluer infected et normale homogénat de cerveau de souris pour les groupes 3 et 4 à 1:100 p / v dans du PBS 1x. </li><li> Groupe 3 - souris témoins positifs IP inoculé avec 1 ml de la souris infectée-homogénat de cerveau. </li><li> Groupe 4 - Négatif souris témoins inoculées ip avec 1 ml de la normale homogénat de cerveau de souris. </li></ol></li></ol><table border="1"><tbody><tr><td></td><td> Groupe Traitement </td><td> Nombre d&#39;animaux </td></tr><tr><td> Groupe 1 </td><td> Tremblante + Corneille matières fécales </td><td> 100 </td></tr><tr><td> Groupe 2 </td><td> Tremblante - Corneille matières fécales </td><td> 25 </td></tr><tr><td> Groupe 3 </td><td> Cerveau tremblante + souris </td><td> 10 </td></tr><tr><td> Groupe 4 </td><td> Tremblante - cerveau de souris </td><td> 5 </td></tr></tbody></table> . Tableau 1 État de la tremblante inoculum (+ positif, négatif -) et le nombre d&#39;animaux utilisés 7. <ol start="2"><li> Intrapéritonéale inoculer la souris: <ol><li> Scruff souris par dorsale cou fourrure avec le pouce et l&#39;index et faire tourner doucement pour exposer la face ventrale. </li><li> Élever extrémité postérieure de la souris si la tête est légèrement inférieure. </li><li> Insérez l&#39;aiguille 25 G 1 cm à travers la peau, 1 cm de la ligne médiane latérale, et 1-2 cm en avant de l&#39;articulation sacro-iliaque en utilisant une seringue sans aiguille de verrouillage. </li><li> Injecter 1 ml inoculer lentement dans la cavité du corps de la souris. </li></ol></li></ol> 3. Surveillance de souris <ol><li> Surveillance de souris quotidienne jusqu&#39;à ce qu&#39;ils expriment des signes cliniques de la tremblante de la souris. Les signes cliniques peuvent inclure: cyphose, l&#39;ataxie, la queue raide, absence de toilettage, une émaciation, et la léthargie. </li><li> Score des souris pour chacun des signes cliniques 6 lorsque les signes visibles sont évidents, où 0 = aucun visible, 1 = modérée, et 2 = sévère. </li><li> Euthanasier les souris quand scores totaux quotidiens pour chaque signe atteindre ≥ 8 pour 1 jour, ≥ 6 en continu pendant 3 jours, ou à 365 jours après l&#39;inoculation (dpi). </li><li> Cerveaux récolte euthan immédiatement aprèsasie et conserver à -70 ° C. </li><li> Pour confirmer un diagnostic de la tremblante, digérer des échantillons de cerveau avec 3 pi d&#39;un 50 pg / ml de solution de protéinase K (PK) dilué comme suit: 3.1. PK ul, 12,5 ul d&#39;EDTA 500 mM, pH 8, 109,39 ul de 1 x PBS pendant 30 min à 45 ° C, puis inactiver la PK en ajoutant 8 pi de tampon de chargement et l&#39;incubation des échantillons à 95 ° C pendant 5 min. Charger les échantillons sur un gel SDS-PAGE à 12%, électrophorèse et transfert sur membrane PVDF Immobilon bloc et avec 5% de lait écrémé dans 0,2% de Tween 20 dans du PBS pendant 1 heure à température ambiante. Ensuite, la sonde avec un anticorps monoclonal anti-PrP BAR224 conjugué à la peroxydase de raifort, dilué dans Superblock, pendant 1 h à température ambiante. Rincer la membrane pendant 1 h avec du PBS-Tween 20 0,2%. Pour visualiser, incuber Western blot 5 min avec un substrat chimioluminescent et une image sur un système de documentation de gel G-box. </li></ol>

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Aucun conflit d&#39;intérêt déclaré.

Nous démontrons une procédure pour documenter le passage du matériau PrP Res par le système digestif des corneilles. Nous avons utilisé des méthodes conventionnelles pour déterminer si les corbeaux ont la capacité de translocation matériau PrP Res à des zones géographiques indemnes de la maladie. D&#39;autres ont évalué la résistance de la PrP Res de ruminants et de rongeurs 19-21 22,23 sucs digestifs, les deux qui n&#39;ont pas réussi à éliminer. Les futures ...

Les encéphalopathies spongiformes transmissibles (EST) sont des maladies infectieuses mortelles neurodégénératives qui affectent la faune, les animaux domestiques et les humains. L&#39;agent infectieux des EST de maladies semble être mal repliées ou isoformes PrP pathogènes (RES) des protéines prions 1. Les EST animales comprennent la maladie du dépérissement chronique (MDC) dans le cerf mulet (Odocoileus hemionus), le cerf de Virginie (Odocoileus virginianus), le wapiti (Cervus elaphus), et de l&#39;orignal (Alces alces); la tremblante chez les ovins et les caprins; l&#39;encéphalopathie spongiforme bovine ( ESB) chez les bovins domestiques; transmissible encéphalopathie du vison en vison d&#39;élevage, l&#39;encéphalopathie spongiforme féline chez les chats; exotique ongulés encéphalopathie spongiforme dans le zoo exotique rumine de la famille des bovidés et l&#39;encéphalopathie spongiforme bovine chez les primates non humains 8. La maladie EST humaine unique, variant de la maladie de Creutzfeldt-Jakob, est rare et la pensée d&#39;être acquis par la consommation de la PrP Res-contaminalimentaire ATED 9. De même, l&#39;ESB peut infecter les humains si bœuf contaminé est consommé 10. De toutes les maladies de l&#39;EST, tremblante et la MDC sont les deux seuls les épidémies et les sources d&#39;infection autonomes sont présumés être d&#39;un animal à 2,3,11 ainsi que de sources environnementales 4-6. La recherche suggère que la plupart des maladies de l&#39;EST nécessitent des périodes d&#39;incubation prolongées notables d&#39;événements d&#39;exposition naturelles de matières PrP Res à la manifestation de signes cliniques 2-4,6,8 et les barrières d&#39;espèces apparentes minimiser, mais n&#39;éliminent pas le risque de transmission inter-espèces 12-14 . Identification des mécanismes de propagation des prions infectieux (PrP Res) de matériau est extrêmement important pour répondre à des questions sur la façon dont les EST se déplacent à travers le paysage. Des études expérimentales ont suggéré que les insectes 15,16, la volaille et les porcs 17, et la corneille d&#39;Amérique (Corvus brachyrhynchos) 7,18 sont des porteurs passifs ou disperseurs de matériel PrP Res. Passage de matériel PrP Res par le système digestif des corneilles a été récemment documentée, ce qui démontre le rôle qu&#39;ils peuvent jouer dans la dispersion des maladies de l&#39;EST 7. Ces résultats rendent plausible que les corneilles, un trésor, pourraient rencontrer, consomment, et le transport du matériel infectieux par les fèces dépôt, dans les zones exemptes de la maladie. Les procédures que nous démontrons ici ont été utilisés pour documenter le passage du matériau PrP Res par le système de digestion des corbeaux et facilitera grandement l&#39;application de ces méthodes à un autre fixateur et modèles carnivore espèces spécifiques dans la recherche de futurs correspondants. Dans cette étude, les méthodes classiques ont été utilisés pour étudier des moyens non conventionnels de la traite matériau PrP Res, qui pourrait contribuer à la propagation et l&#39;impact global de la matière 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

Prion infectieux (PrP Res) matériau est probablement la cause d&#39;accidents mortels, neurodégénérative encéphalopathie spongiforme transmissible (EST) maladies 1. Transmission de maladies de l&#39;EST, telles que la maladie du dépérissement chronique (MDC), est présumé être d&#39;un animal à 2,3 ainsi que de sources environnementales 4-6. Charognards et carnivores ont un potentiel de translocation matériau PrP Res par la consommation et l&#39;excrétion de charogne CWD contaminés. Des travaux récents ont documenté le passage du matériau PrP Res par le système digestif des corneilles d&#39;Amérique (Corvus brachyrhynchos), un trésor commun nord-américain 7. Nous décrivons les procédures utilisées pour documenter le passage du matériau PrP Res par des corneilles d&#39;Amérique. Crows ont été gavés avec RML-souche de tremblante adaptée à la souris et leurs excréments ont été recueillis 4 h après gavage. excréments de corneille ont ensuite été regroupées et injectées par voie intrapéritonéale àSouris C57BL / 6. Les souris ont été suivies quotidiennement jusqu&#39;à ce qu&#39;ils ont exprimé des signes cliniques de la tremblante de la souris et ont ensuite été euthanasiés. Souris asymptomatiques ont été suivis pendant 365 jours après inoculation. Western blot a été effectuée pour confirmer le statut de la maladie. Les résultats ont révélé que les prions infectieux restent après avoir traversé le système digestif des corbeaux et sont présents dans les fèces, ce qui provoque la maladie chez des souris d&#39;essai.

Les procédures utilisées démontrent que le système digestif des corbeaux n&#39;élimine pas PrP Res infectivité 4 h après gavage oral de la tremblante homogénat de cerveau 7. Tous les vingt corbeaux qui ont été gavés avec du matériel PrP Res été transmises matériau PrP Res par les fèces de souris. Souris malades ont été identifiés par la manifestation de signes cliniques de la tremblante à la souris et la confirmation de la maladie a été complétée par un...

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

Les charognards ont le potentiel de translocation infectieuses transmissibles prions de l&#39;encéphalopathie spongiforme dans leurs excréments dans les zones exemptes de la maladie. Nous détail les méthodes utilisées pour déterminer si prions de la tremblante adaptée à la souris restent infectieux après le passage à travers le tractus digestif des corneilles d&#39;Amérique (Corvus brachyrhynchos), une consommation courante des animaux morts.

Procédures d&#39;identification des prions infectieux Après passage dans le système digestif d&#39;une espèce aviaire

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 vues.  USDA.  Les charognards ont le potentiel de translocation infectieuses transmissibles prions de l'encéphalopathie spongiforme dans leurs excréments dans les zones exemptes de la maladie. Nous détail les méthodes utilisées pour déterminer si prions de la tremblante adaptée à la souris restent infectieux après le passage à travers le tractus digestif des corneilles d'Amérique (Corvus brachyrhynchos), une consommation courante des animaux morts. 

Vidéo: 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.

Développement d&#39;un marqueur sélectionnable négatif pour les 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 ...

Recherche

Immunologie et infection

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.

Détection de multiplication et un clone moléculaire infectieux du virus Maedi-visna qui exprime 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 ...

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.

Protein Misfolding Cyclic Amplification des prions

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.

Détermination de la concentration virale par Plaque Essais: Systèmes calque à l&#39;aide traditionnels et nouveaux

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.

Mesures de sécurité et procédures d&#39;exploitation dans un (A) 4-BSL laboratoire: Procédures 1. biosécurité de niveau 4 Suit Laboratoire Suite entrée et de sortie

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.

Mesures de sécurité et procédures d&#39;exploitation dans un (A) de laboratoire BSL-4: 2. Pratiques générales

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.

Mesures de sécurité et procédures d&#39;exploitation dans un (A) 4-BSL laboratoire: 4. Procédures d&#39;imagerie médicale

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.

Mesures de sécurité et procédures d&#39;exploitation dans un (A) BSL-4 laboratoire: 3. Aerobiology

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.

Zika Virus système infectieuse Culture cellulaire et la<em&gt; In vitro</em&gt; Effet prophylactique des interférons

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.

Isoler des Lymphocytes du système immunitaire Intestinal souris petit

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