Wir möchten S. Werner für die Bereitstellung der in dieser Studie verwendet und USDA Krähen danken, APHIS, WS, NWRC Tierpfleger für Tierpflege und-überwachung. Erwähnen oder die Verwendung eines Produkts nicht USDA Billigung. Die Finanzierung für diese Studie wurde von USDA APHIS, Veterinary Services zur Verfügung gestellt.

Unser Protokoll wird von einem zuvor veröffentlichten wir 7 angepasst. Alle Verfahren, die Tiere wurden von der Institutional Animal Care und Verwenden Ausschuss des United States Department of Agriculture (USDA), Tier-und Pflanzengesundheit Kontrolle Service (APHIS), Wildlife Services (WS), National Wildlife Research Center (NWRC) zugelassen. 1. Crow Gavaging <ol><li> Schätzen Durchlaufzeit &quot;Pseudo Hirnmaterial&quot; durch den Verdauungstrakt der amerikanischen Krähen. <ol><li> Mix 5 ml gekocht und Rührei ganzes Ei mit blauen Farbstoff und eine Magensonde ein Krähe mit einer 2 in der Schlundsonde Nadel (Abbildung 1). </li><li> Überprüfen Krähe alle 30 min bis blau / grün-gefärbten Kot werden nicht mehr ausgeschieden. </li></ol></li><li> Erhalten infizierten und unheilbar krank RML (Chandler Stamm)-infizierten C57BL / 6 Maus Gehirne. </li><li> Generieren Sie einen 20% wt / vol normal und infizierten Maus-Hirn-Homogenat in einer Kugel Mixer Homogenisator mit 1xPhosphat-gepufferte Salzlösung (PBS) und Glasperlen, dann Zentrifugieren bei 3.000 × g für 1 min, um größere Partikel zu entfernen. Überstand entfernen und verdünnt mit einem gleichen Volumen an 1 × PBS, um eine 10% w / v in steriler PBS zu erzeugen. Einfrieren bei -80 ° C, bis sie benötigt. </li><li> 17 Stunden vor der Magensonde zu entfernen Essen, aber nicht Wasser, von Luftfedern. </li><li> Zufällig zuordnen Krähen Behandlungsgruppen und der Schlundsonde oral jede Krähe mit 5 ml entweder normal oder infizierten Mausgehirnhomogenat mit einer 2 in der Schlundsonde Nadel. </li><li> Über kräht auf einzelne Käfige und sammeln und bündeln alle Kot in jedem Käfig für 4 Stunden nach der Magensonde (Abbildung 2). </li><li> Homogenisieren gepoolten Fäkalien für jede Krähe in einem Kugel-Homogenisator Mixer mit Glasperlen, bis eine gleichmäßige Struktur erzielt wird. * Crow Kot sind meist flüssig und kann leicht gemischt werden. </li><li> Für jede Krähe, verdünnte 500 ul der fäkalen Homogenat in 9,5 ml 1x PBS für ein Gesamtvolumen von 10 ml. </li><li> Zentrifugieren Sie die fäkale homogenate für 15 min bei 1.400 xg und extrahieren Sie den Überstand. </li><li> Um der Gefahr eines sekundären mikrobiellen Infektion zu minimieren, behandeln Stand mit 1 ul von 100 Einheiten / ml Penicillin und 100 ug / ml Streptomycin (Invitrogen, NY) pro 100 ul Homogenat dann unter UV-Licht bei Raumtemperatur für 20 min reduzieren Risiko viraler und bakterieller Kontamination. Nach der UV-Belichtung, beschallen Proben in einem Beschallungsgerät 3000 Mp bei Einstellung 70 für 30 Sekunden, um die Membran der verbleibenden Mikroorganismen stören. </li></ol> 2. Maus-Impfung <ol><li> Zufällig Mäuse zuweisen, um die folgenden Behandlungsgruppen (Tabelle 1): <ol><li> Gruppe 1 - Positive Behandlung Mäusen intraperitoneal geimpft (IP) mit 1 ml der fäkalen Homogenat von Krähen oral mit 5ml infizierten Mäusehirnen zwangsernährt. </li><li> Gruppe 2 - Negative Behandlung Mäuse geimpft IP mit 1 ml der fäkalen Homogenat von Krähen oral mit 5ml normalen Gehirn der Maus zwangsernährt. </li><li> Verdünnen infected und Normal Mausgehirnhomogenat für Gruppen 3 und 4 bis 1:100 w / v in 1x PBS. </li><li> Gruppe 3 - Positive Kontrollmäusen eingeimpft IP mit 1 ml der infizierten Maus-Hirn-Homogenat. </li><li> Gruppe 4 - Negative Kontrollmäusen eingeimpft IP mit 1 ml normalen Maus-Hirn-Homogenat. </li></ol></li></ol><table border="1"><tbody><tr><td></td><td> Treatment Group </td><td> Anzahl der Tiere </td></tr><tr><td> Gruppe 1 </td><td> Scrapie + Crow Kot </td><td> 100 </td></tr><tr><td> Gruppe 2 </td><td> Scrapie - Krähen-Kot </td><td> 25 </td></tr><tr><td> Gruppe 3 </td><td> Scrapie + Maus Gehirn </td><td> 10 </td></tr><tr><td> Gruppe 4 </td><td> Scrapie - Maus Gehirn </td><td> 5 </td></tr></tbody></table> . Tabelle 1 Scrapie Status des Inokulums (positive + negative -) und Anzahl der verwendeten Tiere 7. <ol start="2"><li> Intraperitoneal impfen Maus: <ol><li> Scruff Maus durch dorsalen Nackenfell mit Daumen und Zeigefinger und sanft drehen, um Bauchseite aus. </li><li> Elevate hinteren Ende der Maus, so Kopf ist etwas niedriger. </li><li> Legen Sie 25 G-Nadel 1 cm durch die Haut, 1 cm lateral der Mittellinie, und 1-2 cm vor dem Iliosakralgelenk mit einer Nadel sichernde Spritze. </li><li> Spritzen 1 ml langsam in Maus impfen Körperhöhle. </li></ol></li></ol> 3. Maus-Überwachung <ol><li> Überwachung der Mäuse täglich, bis sie klinische Zeichen der Maus Scrapie auszudrücken. Klinische Symptome können sein: Kyphose, Ataxie, steifen Schwanz, mangelnde Pflege, Abmagerung und Lethargie. </li><li> Ergebnis Mäuse für jede der sechs klinischen Zeichen, wenn sichtbare Anzeichen sind offensichtlich, wobei 0 = keine sichtbar, 1 = mäßig, und 2 = schwere. </li><li> Euthanize Mäuse, wenn die gesamte tägliche Punktzahl für jedes Zeichen zu erreichen ≥ 8 für 1 Tag, ≥ 6 kontinuierlich für 3 Tage, oder bei 365 Tage nach der Inokulation (dpi). </li><li> Ernte Gehirn unmittelbar nach euthanasien und bei -70 ° C </li><li> Scrapie, eine Diagnose zu bestätigen, Digest Gehirnproben mit 3 ul einer 50 ug / ml Proteinase-K-Lösung (PK) wie folgt verdünnt: 3,1. PK ul, 12,5 ul 500 mM EDTA, pH 8, 109,39 ul 1x PBS für 30 min bei 45 ° C, dann inaktivieren PK durch Zugabe von 8 &amp;mgr; l Ladepuffer und Inkubation Proben bei 95 ° C für 5 min. Last Proben auf ein 12% SDS-PAGE-Gel Elektrophorese und Übertragung auf Immobilon-PVDF-Membran und Block mit 5% fettfreier Milch in 0,2% Tween 20 in PBS für 1 h bei Raumtemperatur. Sonde dann mit BAR224 anti-PrP monoklonaler Antikörper, der mit Meerrettich-Peroxidase, verdünnt in Superblock, für 1 Stunde bei Raumtemperatur konjugiert. Spülen Membran für 1 Stunde mit PBS-Tween 20 0,2%. Um zu visualisieren, Inkubation Western-Blot 5 min mit Chemilumineszenzsubstrat und Bild auf einem G-Feld-Gel-Dokumentationssystem. </li></ol>

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Wir zeigen ein Verfahren um den Durchgang von PrP Res Material durch das Verdauungssystem von Krähen zu dokumentieren. Wir verwendeten konventionellen Methoden, um festzustellen, ob Krähen haben die Fähigkeit, PrP Res Material, um die krankheitsfreie geografischen Gebieten transloziert. Andere Widerstand von PrP Res ausgewertet 19-21 und Nagetier 22,23 Verdauungsflüssigkeiten, die beide nicht zu beseitigen Wiederkäuer. Die künftige Anwendung dieser Techniken a...

Übertragbare spongiforme Enzephalopathien (TSE) sind tödliche Infektions neurodegenerative Erkrankungen, die Tierwelt auswirken, Haustieren und Menschen. Der Erreger der TSE-Erkrankungen scheint falsch gefaltete oder pathogenen Isoformen (PrP Res) von Prion-Proteinen 1 sein. Tier TSE-Erkrankungen gehören Chronic Wasting Disease (CWD) in Maultierhirsch (Odocoileus hemionus), Weißwedelhirsche (Odocoileus virginianus), Elch (Cervus elaphus) und Elch (Alces alces), Scrapie bei Schafen und Ziegen, die bovine spongiforme Enzephalopathie ( BSE) in Hausrinder; tragbare Nerz Enzephalopathie bei Zucht Nerz, Feline Spongiforme Enzephalopathie bei Katzen, exotische Huftiere spongiforme Enzephalopathie in exotischen Zoo sinniert der Familie Hornträger und spongiforme Enzephalopathie in nicht-menschlichen Primaten 8. Der einzelne Mensch TSE-Erkrankung, Variante der Creutzfeldt-Jakob-Krankheit ist selten und vermutlich durch den Verzehr von PrP Res-Contamin erworben werdenATED Essen 9. Ebenso BSE Menschen infizieren können, wenn verseuchtes Rindfleisch verbraucht 10. Von all den TSE-Erkrankungen, Scrapie und CWD sind nur zwei mit sich selbst erhaltende Epidemien und Quellen für die Infektion sind vermutlich von Tier zu Tier 2,3,11 sowie aus Umweltquellen 4-6 sein. Forschung schlägt vor, dass die meisten TSE-Erkrankungen erfordern bemerkenswerte längere Inkubationszeiten aus natürlichen Belichtung Ereignisse des PrP Res Material zur Manifestation klinischer Symptome 2-4,6,8 und Scheinspeziesbarrieren zu minimieren, aber nicht das Potenzial für die, Übertragung zwischen 12-14 beseitigen . Identifizieren Mechanismen für die Ausbreitung von Infektions Prionen (PrP Res) Material ist für die Beantwortung von Fragen, wie TSE-Erkrankungen durch die Landschaft bewegen extrem wichtig. Experimentelle Untersuchungen haben ergeben, dass 15,16, Geflügel und Schweine 17, und amerikanische Krähen (Corvus brac Insektenhyrhynchos) 7,18 passive Träger oder Dispergatoren von PrP Res Material. Passage von PrP Res Material durch das Verdauungssystem von Krähen hat kürzlich dokumentiert, was die Rolle, die sie in Ausbreitung von TSE-Erkrankungen 7 zu spielen. Diese Ergebnisse machen es plausibel, dass Krähen, ein Aasfresser, auftreten können, zu konsumieren und zu transportieren infektiösem Material über Kot Abscheidung, um seuchenfreie Gebiete. Die Verfahren, die wir hier zeigen, wurden verwendet, um den Durchgang von PrP Res Material durch das Verdauungssystem von Krähen zu dokumentieren und die Anwendung dieser Methoden auf andere Fänger und Raubtierarten spezifische Modelle in verwandten zukünftige Forschung erheblich erleichtern. In dieser Studie wurden konventionelle Methoden verwendet werden, um eine unkonventionelle Mittel des Menschenhandels PrP Res Material, das für die Verbreitung und der Gesamtbelastung von PrP Res Material beitragen könnten, zu untersuchen.

<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

Infektiöse Prionen (PrP Res) Material ist wahrscheinlich die Ursache der tödlichen, neurodegenerative übertragbare spongiforme Enzephalopathie (TSE) Krankheiten ein. Übertragung von TSE-Erkrankungen wie Chronic Wasting Disease (CWD), wird vermutet, von Tier zu Tier 2,3 sowie aus Umweltquellen 4-6 sein. Reiniger und Fleischfresser haben Potenzial, PrP Res Material durch Konsum und die Ausscheidung von CWD-kontaminierten Aas transloziert. Neuere Arbeiten haben Gang von PrP Res Material durch das Verdauungssystem der amerikanischen Krähen (Corvus brachyrhynchos), einer gemeinsamen nordamerikanischen Schnitzel 7 dokumentiert. Wir beschreiben Verfahren verwendet werden, um Durchgang von PrP Res Material durch amerikanische Krähen zu dokumentieren. Krähen wurden mit RML-Dehnungs-Maus-adaptierten Scrapie zwangsernährt und ihren Kot gesammelt wurden 4 Stunden nach der Magensonde. Crow Kot wurden dann gesammelt und in intraperitonealC57BL / 6 Mäusen. Die Mäuse wurden täglich überwacht, bis sie ausgedrückt klinischen Zeichen der Maus Scrapie und wurden danach getötet. Asymptomatische Mäuse wurden bis zu 365 Tage nach der Impfung überwacht. Western-Blot-Analyse wurde durchgeführt, um Krankheitsstatus zu bestätigen. Ergebnisse zeigten, dass Prionen infektiös bleiben nach einer Reise durch das Verdauungssystem von Krähen und im Kot vorhanden sind, was zu Krankheit in Versuchsmäusen.

Die angewendeten Verfahren zeigen, dass das Verdauungssystem von Krähen nicht PrP Res Infektiosität 4 Stunden nach orale Gabe von Scrapie Hirnhomogenat 7 zu beseitigen. Alle zwanzig Krähen, die mit PrP Res Material zwangsernährt wurden anschließend PrP Res Material, um Mäuse übertragen über Kot. Kranke Mäuse wurden durch klinische Manifestation der Maus-Scrapie-Krankheit Anzeichen und Bestätigung wurde durch Western-Blot-Analyse abgeschlossen identifiziert. <p cl...

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

Aasfresser haben Potenzial, infektiöse Prionen übertragbaren spongiformen Enzephalopathie in ihren Kot zu krankheitsfreien Gebieten transloziert. Wir detailliert Methoden verwendet werden, um festzustellen, ob der Maus-adaptierten Scrapie-Prionen bleiben nach dem Durchgang aber den Verdauungstrakt der amerikanischen Krähen (Corvus brachyrhynchos), einer gemeinsamen Verbraucher toter Tiere ansteckend.

Verfahren zur Identifizierung von Infektions Prionen Nach Passage durch das Verdauungssystem von eine Vogelart

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 Aufrufe.  USDA.  Aasfresser haben Potenzial, infektiöse Prionen übertragbaren spongiformen Enzephalopathie in ihren Kot zu krankheitsfreien Gebieten transloziert. Wir detailliert Methoden verwendet werden, um festzustellen, ob der Maus-adaptierten Scrapie-Prionen bleiben nach dem Durchgang aber den Verdauungstrakt der amerikanischen Krähen (Corvus brachyrhynchos), einer gemeinsamen Verbraucher toter Tiere ansteckend. 

Serie: 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.

Entwicklung eines Negativ Selektionsmarker für 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 ...

Forschung

Immunologie und Infektion

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.

Propagierung und Erkennen einer ansteckenden Molecular Clone von Maedi-Visna Virus, das Green Fluorescent Protein exprimiert

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.

Proteinfehlfaltungen Cyclic Amplification von Prionen

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 Entschlossenheit Konzentration Durch Plaque-Assays: Mit Tradition und neuen Überzugssysteme

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.

Sicherheitsvorkehrungen und Betriebsverfahren in einem (A) BSL-4-Labor: 1. Biosafety Level 4 Anzug Labor Suite Einreise- und Ausreiseverfahren

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.

Sicherheitsvorkehrungen und Betriebsverfahren in einem (A) BSL-4-Labor: 2. Allgemeine 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 ...

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.

Sicherheitsvorkehrungen und Betriebsverfahren in einem (A) BSL-4-Labor: 4. Medizinische Bildgebende Verfahren

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.

Sicherheitsvorkehrungen und Betriebsverfahren in einem (A) BSL-4-Labor: 3. Aerobiologie

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 Infectious Zellkultursystem und die<em&gt; In Vitro</em&gt; Prophylaktische Wirkung von Interferonen

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.

Isolieren von Lymphozyten aus der Maus kleinen intestinalen Immunsystems

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