Wir danken SIGA Technologies (Corvallis, OR) für die Bereitstellung von ST-246. DKR wurde durch ein NIH Ausbildungsförderung in der Immunologie an der Universität Boston (5T32AI 7309) unterstützt. Diese Arbeit wurde zum Teil von P41 086180 unterstützt, NIH RO1AI1096159-01 und RO3 (bis JHC).

1. Platte Cells <ol><li> Distanzieren HeLa-Zellen von der Platte und in Wachstumsmedium (DMEM, 2 mM L-glut, 10% FBS) zu verdünnen, um etwa 2,0 x 10 5 Zellen / ml. Pipettieren von 100 ul / Vertiefung in einem schwarzen Wänden, klare Flachboden-96-Well-Platte (20.000 Zellen / Vertiefung). </li><li> Zellen für 24 Stunden inkubieren, bis Konfluenz in einer 37 ° C-Inkubator + 5% CO 2. </li></ol> 2. Zellen infizieren <ol><li> Verdünnen Virus und Zellen infizieren. <ol><li> Tauwetter TRPV (Dreibett-Virus, Früh Venus, Intermediate mCherry, Spät TagBFP) und PLV (Promoter-less Venus) Fluoreszenz-Virus und zerlegen mit Beschallung für 5 Minuten. Alternativ können rohen Virusvorräte mit 0,25 mg / ml Trypsin in Infektionsmedium gemischt 1:1 und bei 37 ° C für 30 min inkubiert. </li><li> Verdünnen rohen Virusvorräte in 37 °-C-Infektion Medien (DMEM, 2 mM L-glut, 2% FBS). Bei hohen MOI-Infektionen (10 PFU / Zelle), verdünnte Virus Lager bis 1,0 x 10 7 PFU / ml unter der Annahme, 5 x 10 4Zellen / Vertiefung und ein Inokulum von 50 ul. Plan zu infizieren 3 Wiederholungsvertiefungen für jede Behandlung mit TRPV und weitere 3 Wiederholungsvertiefungen für die gleiche Behandlung mit PLV auf Hintergrundfluoreszenz spezifisch für jede Behandlung zu berücksichtigen. </li><li> Infizieren Vertiefungen durch Zugabe von 50 &amp;mgr; l / Vertiefung verdünnt Virus in Infektionsmedium. Dies wird als Zeit = 0 h nach Infektion (hpi 0) definiert. </li></ol></li><li> Verdünnen gewünschten Behandlungs Verbindungen in Infektion Medien. Für alle Behandlungen sollte eine einzige Master-Mix 2x Verdünnung hergestellt werden und auf Brunnen (z. B. 350 ul Gesamtvolumen von sechs 96-Brunnen mit jeweils 50 ul) zu replizieren. <ol><li> Verdünnen experimentelle Infektion Verbindungen in Medien. </li><li> Verdünnen Fahrzeug-Steuer Lösungsmittel (zB PBS, DMSO) in Infektions Medien. Hinweis: Ähnliche Endkonzentrationen von Fahrzeug-Steuerlösemittel sollten wie für experimentelle Verbindungen (z. B. angewendet werden, wenn Ihr IBT Lager ist mit DMSO hergestellt und in einem abschließenden concentratio verwendetns von 1 ul / ml, dann mit DMSO allein bei 1 ul / ml auf der Fahrzeugsteuerung). </li><li> Verdünnen Steuer Verbindungen in Infektion Medien. Empfohlene Kontrollverbindungen umfassen: 3,6 um (1 ug / ml) 1-β-D-arabinofuranosylcytosin (AraC), 50 uM (11,7 ug / ml) β-thiosemicarbazon Isatin (IBT), 60 uM (50 ug / ml) Rifampicin, oder 5 &amp;mgr; m (1,9 &amp;mgr; g / ml) ST-246 8,15,16. Siehe Repräsentative Ergebnisse für die erwarteten Effekte. Hinweis: machen diese Verdünnung zu zweimal (2x) der gewünschten Endkonzentration da diese im Verhältnis 1:1 auf das Volumen bereits in die Vertiefung gegeben. </li></ol></li><li> Unmittelbar nach Zugabe des Virus, 50 &amp;mgr; l Medium, das gewünschte Infektion Behandlungen und Kontrollen wie in Schritt 2.2 verdünnt. Hinweis: Um für die Hemmung vor frühzeitig Ausdruck zu testen, Verbindung (en) kann entweder direkt zu der verdünnten Virusinokulum (Schritt 2.1.2) oder hinzugefügt werden, um Zellen vor der Infektion zu hosten (vor Schritt 2.1.3). </li><li> Inkubieren 24.06 h in einem 37 &amp; #176; + C-Inkubator mit 5% CO 2. </li></ol> 3. Fix Cells <ol><li> Fix Zellen durch Zugabe von 100 ul 8% Para für Infektionen Medien bereits in jedes Well. Inkubation bei Raumtemperatur für 15 min vor Licht geschützt. Hinweis: Es wird empfohlen, 2x PFA (8%), die direkt auf eine Infektion Medien zu Vernebelung von Infektions Vaccinia die auftreten können, zu vermeiden, wenn mit hohem Titer Medien direkt in die Abfallschale vor der Fixierung invertiert. </li><li> Entfernen Fixiermittel durch Umdrehen der Platte in den Abfall Gericht. </li><li> 100 l Raumtemperatur PBS. </li><li> Dichtungsplatte mit optisch klare Klebstoff-Film. Hinweis: Die Platten können bei 4 ° C gelagert werden, wenn nicht sofort gelesen werden. Achten Sie darauf, versiegelten Platten auf Raumtemperatur vor dem Lesen, um Kondensation zu verzerren Spektralphotometer Messungen verhindern zurückzukehren. </li></ol> 4. Quantifizierung Virus Wachstum <ol><li> Messen Endpunkt Fluoreszenz bei 515:530 für Venus, 587:610 für mCherry und 415:457 foder TagBFP (Anregung: Emissionswellenlänge in nm). Vier Messungen sollten pro Vertiefung mit optimierten Verstärkungseinstellungen für jeden Kanal gemittelt werden. Hinweis: Die entsprechenden Emissionswellenlänge kann je nach Modell und Filtereigenschaften des Plattenlesegerät. Dies kann empirisch durch die Durchführung einer Emissionswellenlängen-Scan, um den Punkt, wo es den größten Unterschied zwischen TRPV und PLV für jeden Kanal bestimmen, bestimmt werden. </li><li> Normalisieren Sie die Rohdaten zum Vergleich zwischen den Experimenten zu erleichtern. <ol><li> Für jeden Kanal die mittlere von Wiederholungs TRPV und PLV Brunnen. </li><li> Subtrahieren Sie die Mittel PLV-infizierten Hintergrundmessungen aus den Mittel TRPV-infizierten experimentellen Messungen für jede Behandlung. </li><li> Normalisieren Sie die Daten, indem jede Hintergrund subtrahiert Wert vom Fahrzeug-only-Wert auch. </li><li> Sobald ein Experiment wurde mehrmals, eine Einweg-Varianzanalyse (one-way ANOVA) Test-und Mehrfach c wiederholtERGLEICH Post-Test durchgeführt, um zu bestimmen, ob jede der Behandlungen zeigt einen statistisch signifikanten Unterschied von der Fahrzeug einzige Behandlung für jeden Kanal werden. </li></ol></li></ol> . 5 Alternative Protokoll: Kinetic Plattentests <ol><li> Führen Sie alle Schritte durch Zugabe von Behandlungen (Schritt 2.3). </li><li> Seal Platte mit Haftklebefolie abdecken und in Plattenleseraum auf 37 ° C ins Gleichgewicht Hinweis: Besondere Sorgfalt ist auf Platten bei 37 ° C konstant zu halten, um übermäßige Zellstress und Kondensation zu verhindern. Kinetischen Assays ist es besonders wichtig, einen gepufferten Medium (Natriumbicarbonat und HEPES), um richtigen pH ohne Zusatz von 5% CO 2 erhalten. </li><li> Set-Plattenlesegerät manuell zu gewinnen, um die Sättigung des späteren Zeitpunkten zu verhindern. Anmerkung: Die genaue Verstärkungsoptimierung kann durch Durchführen einer Endpunkt-Assays unter ähnlichen Bedingungen erhalten werden. </li><li> Erwerben Stundenwerte für 8-24 hpi und normalisieren mit ähnlar Verfahren wie in Endpunkt-Assay (Schritt 4.2). </li><li> Individuelle Zeitpunkte für die statistische Signifikanz unter Verwendung ähnlicher Verfahren wie das zuvor beschriebene Endpunkt-Assay analysiert werden. </li></ol>

<ol>
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Die Autoren haben nichts zu offenbaren und keine Patente auf Viren oder Screening-Verfahren anhängig.

Hier haben wir den praktischen Einsatz eines mehrstufigen Vaccinia-Reportervirus (TRPV), die reproduzierbar ist, Echtzeit-Feedback Maßnahmen der Virusreplikation liefert beschrieben. Durch die Verwendung von gut definierten Pockenvirus-Inhibitoren, konnten wir zeigen, dass TRPV reagiert in einer Weise, die mit dem Wirkmechanismus verstanden, für jeden Inhibitor. Während die TRPV Virus bietet die umfassendsten Informationen über Viren Bühne Progression, zweistufig (IREV, LREV) und einstufigen (EV, IV, LV)-Viren kön...

Traditionell wird Virus-Expressions mit molekularbiologischen Techniken (zB Northern Blot, Western Blot, Microarray-Hybridisierung, etc.) 1 untersucht. Diese Verfahren sind in der Lage, detaillierte Informationen in Bezug auf die Kategorisierung Expressionsänderungen der einzelnen mRNAs bzw. Proteine, sind sie typischerweise nicht zugänglich Echtzeit-und Hochdurchsatzverfahren. Alternative Ansätze mit Fluoreszenz-basierten Reporter haben bisher bei der Arbeit mit Pockenviren angewandt worden; hat jedoch ihre Entwicklung und Nutzung durch abwechslungsreiche Ziele motiviert. Mehrere solche Verfahren wurden zur Selektion von rekombinanten Viren 2,3 entwickelt. Diese Techniken auszudrücken EGFP auf ordnungsgemäße Einbau und die Expression von exogener DNA aus rekombinanten Vaccinia-Klonen. Ebenso mehrere Vacciniastämme stabil exprimieren EGFP löslich oder GFP-markierten Proteine ​​unter einer nativen Vaccinia-Promotor exprimiert sind weit verbreitet. Diese sind typ.tisch verwendet werden, um das Eindringen von Viren und die Replikation während Antikörper-basierten Neutralisationstests, chemische Hemmung oder Vergleich der antiviralen Wirksamkeit 4-6 quantifizieren. Obwohl diese Viren nützlich erwiesen haben, sind sie in ihrer Fähigkeit, Informationen über den Punkt der Inhibition durch ihre Nutzung eines einzelnen mehrdeutigen frühen / späten viralen Promotor bereitzustellen begrenzt. Bisherige Verfahren haben auch die Verwendung von EGFP Protein gemacht mit suboptimalen Signal-zu-Rauscheigenschaften. Aufgrund der Komplexität des Pockenvirus-Replikation und der Mangel an vorhandenen Werkzeugen zur Verfügung, um Echtzeit-Änderungen in der viralen Genexpression zu untersuchen, haben wir eine Reihe von Einzel-und Mehrstufen-Reporter Viren 7,8 entwickelt. Wie bereits in früheren Veröffentlichungen beschrieben, diese Viren Ausdruck ein, zwei, oder drei spektral unterschiedlichen fluoreszierenden Proteinen aus nativen Vaccinia-Promotoren in der frühen, mittleren oder späten Stadien der Infektion. Diese Viren können unsed als Indikatoren für die Virusreplikation Fortschritte mit einem Fluoreszenzmikroskop und die sie für Hochdurchsatz-Platte-basierte Assays Leser gleich gut geeignet sind. Diese Viren sind einfach anstelle des Wildtyp-Virus verwenden, wachsen mit ähnlichen Kinetik gleichwertige Titer 7 zu erreichen. Bei der Planung und Erstellung dieser Viren wurde viel Sorgfalt bei der Auswahl der Fluorophore, die hohen Signal-zu-Hintergrund-Merkmale mit überlegener Effizienz Klapp müssen zuverlässige Quantifizierung mit schneller Rückmeldung an Veränderungen in der Expression (Venus, mCherry und TagBFP) Erleichterung aufgenommen. Zusätzlich Kombinationen von viralen Promotoren wurden ausgewählt, die eine hohe Wiedergabetreue, eindeutige Phase-spezifische Expression, die Bereitstellung von Informationen über das gesamte Spektrum der früh (C11R-Promotor), Mittelstufe (G8R Promotor) und späten (F17R Promotor) Genexpression zu produzieren, im Gegensatz zu zweideutig frühen / späten Veranstalter. Diese Viren können als ein Werkzeug verwendet werden, für investigating den Lebenszyklus des prototypischen Pockenvirus, Vaccinia-Virus. Vieles ist noch nicht bekannt über die Wirt-Virus-Interaktion trotz seiner langen Geschichte der Studie. Vaccinia ist komplex, produziert mehr als 200 einzigartige Proteine, von denen viele immunmodulatorische und hosten antagonistisch. Bei Infektion beginnt Vaccinia-Virus sofort frühen mRNA-Transkription. Dies wird durch eine RNA-Polymerase und Transkriptionsfaktoren, die auf der viralen Genoms während der Virion Verpackungs geladen und in einem angehaltenen Zustand, bis nachfolgende Infektion gehalten wurden, erleichtert. Diese frühe Ausdruck produziert vor allem Proteine ​​für die Unterdrückung der das Immunsystem des Wirts (mRNA Decapping, dsRNA-Sequestrierung und Köder Rezeptorproteine ​​sowie Inhibitoren der Apoptose, Stressantwort und Toll, IL, und NF-kB-Signalisierung) und Genom-Replikation erforderlich. Frühe Ausdruck produziert auch Transkriptionsfaktoren für Zwischenausdruck erforderlich. Zwischen Ausdruck schließt die Expression von Transkriptionsfaktoren spät. DiesAusdruck Kaskade führt zur Produktion von strukturellen und enzymatischen Virusproteine ​​während der späten Stadien der Infektion, die für die komplette Montage des reifen Virions Vaccinia notwendig sind. Unsere Gruppe von fluoreszierenden Reporter-Viren ermöglicht rasche Fortschritte im Verständnis der Biologie Pockenvirus hergestellt werden. Eine der häufigsten und zeitraubenden Methoden auf dem Gebiet der Virologie ist die Wachstumstest. Dies beinhaltet typischerweise Infizieren von Zellen, Festlegung einer Reihe von Behandlungen, Virus-Ernte durch Lyse von infizierten Zellen und der Quantifizierung des erhaltenen Virustiter durch Plaque-Assay. Mit den hier beschriebenen Reporter Viren ermöglicht Echtzeit-Messung der Viruswachstum, die leicht untersucht und verglichen zwischen zahlreichen Behandlungen, die parallel ausgeführt werden können. Wir sehen dieses Set von Reagenzien werden in verschiedenen Protokollen für die Identifizierung Veränderungen im viralen Genexpression in Reaktion auf Medikamente, RNAi-k verwendet werdennockdown oder Wirtsspektrum Einschränkung. Diese Methode ermöglicht auch High-Content-Analyse in einem größeren Maßstab als bisher zur Verfügung, so dass für Hochdurchsatz-Anti-Virus-Drogen-Bildschirme für speziell definierte Zielstufen von Interesse. Während zahlreiche mögliche Behandlungen zur Bekämpfung von Pockenvirus-Infektion identifiziert worden sind, die einzige von der FDA zugelassenen Therapien wirksam zur Behandlung von Pockenvirusinfektion sind die acyclischen Nucleosid-Phosphonats, Cidofovir, und Behandlung mit Vaccinia-Immunglobulin 9,10. Trotz der Ausrottung der Pocken im Jahr 1977 11, bleiben Pockenviren eine erhebliche Bedrohung für die menschliche Gesundheit 12. Einstellung der weit verbreiteten Impfung gegen Variola-Virus hat zu einer erhöhten Anfälligkeit für andere Pockenviren 13 geführt. Zum Beispiel werden die Regionen Zentralafrikas zuvor durch Impfung geschützt erleben einen Anstieg der Affenpocken-Virus-Infektionen 14. Erhebliche Bedenken hat auchhob in Bezug auf die latente Anfälligkeit für vorsätzliche Freisetzung von Variola-Virus. Aufgrund der begrenzten Therapien derzeit zur Verfügung, es besteht ein dringender Bedarf für die Entwicklung von neuartigen Behandlungen. Diese Reporterviren erlauben eine schnelle und Hochdurchsatz-Screening niedermolekularer Inhibitor zur Hemmung einer spezifischen Phase der Virusreplikation. Identifizierung von Inhibitoren, die viralen Expressions Stufen Ziel derzeit nicht das Ziel der Hemmung wird die Entwicklung von Kombinationstherapien mit erhöhter Potenz zu erleichtern. Man kann viel über Vaccinia Zellbiologie durch Beobachtung von Veränderungen in jeder Stufe der Genexpression zu entnehmen. Dämpfung durch chemische oder genetische Manipulation einer infizierten Wirtszelle wird in der Regel in Form von geringeren Virustiter ausgedrückt. Doch beim Vergleich der Entwicklung in den einzelnen Phasen des viralen Expressionskaskade, kann man ein vollständigeres Verständnis der Virus-Fitness ist Auswirkungen zu erhaltendurch eine besondere Behandlung ed. Diese Daten haben gezeigt, dass auch mit der traditionellen Virustiter Ausgangs korrelieren, aber detailliertere mechanistische Informationen sowie hohe Durchsatzkapazitäten 7.

<table border="0" cellpadding="0" cellspacing="0" width="1249">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:747px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:104px;">11995-065</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fetal Bovine Serum - Optima, Heat Inactivated (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">200 mM L-Glutamine, (L-glut)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3712</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Trypsin, 2X, Sterile, Irradiated</td>
			<td>Worthington Biochemical Corp.</td>
			<td>TRLVMF</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl Sulfoxide, Cell Culture (DMSO)</td>
			<td>American Bioanalytical</td>
			<td>AB03091</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1-&#946;-D-Arabinofuranosylcytosine&#160; (AraC), MW= 280 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>C6645</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">isatin &#946; -thiosemicarbazone (IBT), MW= 234 g/mol</td>
			<td>Fisher Scientific</td>
			<td>NC9075202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Rifampicin, MW= 823 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">ST-246, MW= 376 g/mol</td>
			<td>SIGA Labs, Corvallis, OR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">8% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TempPlate RT optically clear film</td>
			<td>USA Scientific</td>
			<td>2978-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

vaccinia reporter viruses for quantifying viral function at all stages of gene expression

Pockenviren sind eine Familie von Doppelstrang-DNA-Viren, die aktive menschliche Krankheitserreger wie Affenpocken, Dell contagiousum und Contagalo Virus enthalten. Die Familie umfasst auch die Pocken-Virus, Variola. Aufgrund der Komplexität des Pockenvirus-Replikation, bleiben noch viele Fragen hinsichtlich ihrer Genexpression Strategie. In diesem Artikel beschreiben wir die Konzeption und die Verwendung von rekombinanten Vaccinia-Viren, die Echtzeitmessung von einzelnen und mehrfachen Stufen der viralen Genexpression in einem Format mit hohem Durchsatz zu ermöglichen. Dies wird durch die Verwendung von spektral unterschiedlichen fluoreszierenden Proteine ​​als Reporter für jede der drei Phasen der Virusreplikation ermöglicht. Diese Viren einen hohen Signal-zu-Rausch-Verhältnis unter Beibehaltung Stufe spezifische Expressionsmuster, wodurch plattenbasierten Assays und mikroskopische Beobachtungen der Virus Vermehrung und Replikation. Diese Tools verwendet haben für antivirale Entdeckung, Studien der Virus-Wirt-Interaktion, und evolutionäre biologie.

Als ein Beispiel für die typische Verwendung dieser Viren wurde das dreifache Fluoreszenz-Reportervirus verwendet, um den Punkt der Hemmung von mehreren wohldefinierten Pockenvirus-Inhibitoren zu vergleichen. HeLa-Zellen wurden in Gewebekultur behandelt vergoldet schwarz-mauert klar Flachboden-96-Well-Platten und über Nacht inkubiert. Konfluente Monolayer wurden bei einer Multiplizität der Infektion (MOI) von 10 unter Verwendung von entweder dreifache Reporter Virus (TRPV, Tabelle...

Watch this Scientific Journal Video about Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression at JoVE.com

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Wir beschreiben die Verwendung eines fluoreszierenden Reporter Vaccinia-Virus, das Echtzeit-Messung der viralen Infektiosität und Genexpression ermöglicht durch die Stufe-spezifische Expression von spektral unterschiedlichen Fluorophoren Reporter. Wir detailliert ein plattenbasierte Verfahren zum genauen Identifizieren der Phase, in der die Virusreplikation in Reaktion auf kleine Molekül Inhibierung betroffen.

Vaccinia-Viren Reporter zur Quantifizierung von Viral-Funktion auf allen Stufen der Genexpression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

vaccinia-reporter-viruses-for-quantifying-viral-function-at-all

Research

JoVE Journal

Immunology and Infection

11.2K Aufrufe.  Boston University School of Medicine.  Wir beschreiben die Verwendung eines fluoreszierenden Reporter Vaccinia-Virus, das Echtzeit-Messung der viralen Infektiosität und Genexpression ermöglicht durch die Stufe-spezifische Expression von spektral unterschiedlichen Fluorophoren Reporter. Wir detailliert ein plattenbasierte Verfahren zum genauen Identifizieren der Phase, in der die Virusreplikation in Reaktion auf kleine Molekül Inhibierung betroffen. 

Serie: Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Generierung von Multivirus-spezifischen T-Zellen zu verhindern / zu behandeln virale Infektionen nach allogenen hämatopoetischen Stammzelltransplantation

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Forschung

Immunologie und Infektion

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&quot;Bioluminescent &#39;Reporter Phage für den Nachweis von Kategorie A bakteriellen Erregern

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Quantitative Analysen aller Influenza Typ A Viral Hämagglutininen und Neuraminidasen mit Universal Antikörper in Einfache Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

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

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Kostengünstige Methode für Mikrobielle Source Tracking mit spezifischen menschlichen und tierischen Viren

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

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Isolierung von Fidelity Varianten von RNA-Viren und Charakterisierung von Virus-Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Aufbau einer Liquid-bedeckten Kultur Polarized Menschliche Airway Epithelial Calu-3-Zellen, um Host-Zell-Antwort, um Atemwegspathogene Studie<em&gt; In-vitro-</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Maus fötalen Leberkultursystem zur Ziel Gen-Funktionen in der frühen und späten Stadien der Klemme Erythropoese Dissect

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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

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

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

Die intranasale Verabreichung von rekombinantem Influenza Vaccines in chimären Maus-Modelle zu Mucosale Immunität Studieren

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

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Eine einfache und effiziente Methode zu konstruieren Mutant Vacciniavirusvektoren

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Entwicklung eines Systems Hepatitis-B-Virus Reporter den frühen Stadien des Replikationszyklus zu überwachen

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...