Diese Arbeit wird teilweise NIH Zuschüsse A1064826 R01-01, U19 AI062627-01, NO1-HHSN26600500032C (SM) unterstützt. Wir danken unseren Labor-Mitglieder zur Diskussion und technische Hilfe, Tina Heil für die kritische Durchsicht des Manuskripts. Wir möchten David Bina, Milwaukee Health Department Virus Labor für Kultur und PCR danken.

<ol>
	<li>Hirst, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+agglutination+of+red+cells+by+allantoic+fluid+of+chick+embryos+infected+with+influenza+virus.">The agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus.</a> Science. 94, 22-23 (1941).</li><li>Jonsson, N., Gullberg, M., Lindberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+polymerase+chain+reaction+as+a+rapid+and+efficient+alternative+to+estimation+of+picornavirus+titers+by+tissue+culture+infectious+dose+50%+or+plaque+forming+units.">Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units.</a> Microbiol. Immunol. 53, 149-154 (2009).</li><li>Kalter, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinating+Behavior+of+Mouse+and+Egg-adapted+Type+A+(PR8)+Influenza+Virus.">Hemagglutinating Behavior of Mouse and Egg-adapted Type A (PR8) Influenza Virus.</a> Science. 110, 184-184 (1949).</li><li>Dulbecco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Plaques+in+Monolayer+Tissue+Cultures+by+Single+Particles+of+an+Animal+Virus.">Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal Virus.</a> Proc. Natl. Acad. Sci. U. S. A. 38, 747-752 (1952).</li><li>Deyde, V. M., Nguyen, T., Bright, R. A., Balish, A., Shu, B., Lindstrom, S., Klimov, A. I., Gubareva, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+molecular+markers+of+antiviral+resistance+in+influenza+A+(H5N1)+viruses+using+a+pyrosequencing+method.">Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using a pyrosequencing method.</a> Antimicrob. Agents Chemother. 53, 1039-1047 (2009).</li><li>Jonsson, N., Gullberg, M., Lindberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+polymerase+chain+reaction+as+a+rapid+and+efficient+alternative+to+estimation+of+picornavirus+titers+by+tissue+culture+infectious+dose+50%+or+plaque+forming+units.">Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units.</a> Microbiol. Immunol. 53, 149-154 (2009).</li><li>Rahman, M., Kieke, B. A., Vandermause, M. F., Mitchell, P. D., Greenlee, R. T., Belongia, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+Directigen+flu+A+B+enzyme+immunoassay+and+direct+fluorescent+assay+for+detection+of+influenza+infection+during+the+2004-2005+season.">Performance of Directigen flu A+B enzyme immunoassay and direct fluorescent assay for detection of influenza infection during the 2004-2005 season.</a> Diagnostic Microbiology and Infectious Disease. 58, 413-418 (2007).</li><li>Landry, M. L., Ferguson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suboptimal+detection+of+influenza+virus+in+adults+by+the+Directigen+Flu+A+B+enzyme+immunoassay+and+correlation+of+results+with+the+number+of+antigen-positive+cells+detected+by+cytospin+immunofluorescence.">Suboptimal detection of influenza virus in adults by the Directigen Flu A+B enzyme immunoassay and correlation of results with the number of antigen-positive cells detected by cytospin immunofluorescence.</a> J. Clin. Microbiol. 41, 3407-3409 (2003).</li><li>Herrmann, B., Larsson, C., Zweygberg, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+and+typing+of+influenza+viruses+A+and+B+by+a+nested+reverse+transcription-PCR:+comparison+to+virus+isolation+and+antigen+detection+by+immunofluorescence+and+optical+immunoassay+(FLU+OIA).">Simultaneous detection and typing of influenza viruses A and B by a nested reverse transcription-PCR: comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay (FLU OIA).</a> J. Clin. Microbiol. 39, 134-138 (2001).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> Am. J. Epidemiol. 27, 493-497 (1938).</li><li>Doerr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins:+into+the+infrared.">Fluorescent proteins: into the infrared.</a> Nat. Meth. 6, 482-483 (2009).</li><li>Shu, B., Wu, K. H., Emery, S., Villanueva, J., Johnson, R., Guthrie, E., Berman, L., Warnes, C., Barnes, N., Klimov, A., Lindstrom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+performance+of+the+CDC+real-time+reverse+transcriptase+PCR+swine+flu+panel+for+detection+of+2009+A+(H1N1)+pandemic+influenza+virus.">Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus.</a> J. Clin. Microbiol. 49, 2614-2619 (2011).</li></ol>

Wir haben nichts zu offenbaren.

Die jüngste Epidemie Ausbruch der Vogelgrippe in Südostasien und einer globalen Angst vor der Schweinegrippe-Pandemie zu verhängen enorme Bedenken für die öffentliche Gesundheit und Sicherheit. Kritische Schwerpunkt bei der Erzeugung von Impfstoffen für die bestehenden und neuer Stämme von Influenza-Viren gewidmet. Die Verfügbarkeit von schnellen High-Throughput-Technologien für präzise Erkennung und genaue Berechnungen Virustiter wird enorm verbessern Behandlung. Die am häufigsten verwendeten Techniken eingesetzt werden, um Virustiter zu berechnen beinhalten Plaque-bildenden und Influenza Hämagglutinationsassays. Die Plaque-bildenden-Assay wurde zunächst entwickelt, um Bakteriophagen-Titer schätzen und wurde von Renato Dulbecco angenommen zu berechnen Tier virale Titer 4. Um diesen Test durchzuführen, werden 10-fach-Verdünnungen von Influenza lizenzfreie oder tierischen Proben, wie zB BAL-Flüssigkeit hergestellt und 0,1 ml Aliquots werden auf der Monoschicht von MDCK-Zellen inokuliert in 6-Well-Platten. Das Virus wird zur Adsorption auf die MDCK-Zellen von der Ad gefolgt erlaubtdition eines Nährmediums und Agarose. Während der Inkubation, lassen Sie die Original-infizierten Zellen virale Nachkommenschaft, die nur zu den benachbarten Zellen ausbreiten. Die zytopathogene Wirkung des Influenza-Virusinfektion produziert eine kreisförmige Zone von infizierten Zellen genannt Plaque. Jede Plakette wird vermutlich nach Infektion von einer einzigen Zelle mit einem einzigen Viruspartikel von der Replikation des Virus gebildet, gefolgt. Kontrast zwischen den lebenden Zellen und die Plaques können durch Farbstoffe wie Kristallviolett verbessert werden. Ein wesentlicher Nachteil von diesem Assay ist der Mangel an Reproduzierbarkeit und eine enorme Schwankungsbreite innerhalb Experimenten. Ein weiterer Assay verwendet, um die Influenza-Virus-Titer zu bestimmen ist die Hämagglutinationsassay. Die Hämagglutinin-Protein auf der Oberfläche des Influenzavirus effizient bindet an N-Acetylneuraminsäure-enthaltenden Proteine ​​auf Säugern und Vögeln roten Blutkörperchen 1. Diese Wechselwirkung zwischen dem Influenza-Virus und den Erythrozyten führt zu Agglutination in der Form eines Gitters. Der level der Agglutination wird verwendet, um den Titer des Influenza-Virus in Prüfkörper zu schätzen. Erythrozyten-Agglutinations-Assay wird nur für die Titration von einem bekannten Virus zu bestimmen verwendet und kann nicht für Stammidentifizierungsnummer Zwecke verwendet werden. Da dieser Assay nicht zwischen dem Live-und den toten Viren zu unterscheiden, ist es schwierig, eine genaue Berechnung der viralen Titer zu erhalten. Aktuelle klinische Diagnose von Influenza-Infektionen sind auf serologische Tests, Polymerase-Kettenreaktion, direkte Probe Immunfluoreszenz und Zellkultur 1,6 basiert. Die hier beschriebene neue Verfahren hat auch klinische und labordiagnostische Potenzial. Influenza-Nachweis-Kits Directigen Flu-A (BD Biosciences, San Jose, CA) und BinaxNOW (Binax, Inc., Portland, ME) Verwendung Immunchromatographie Assay auf virales Antigen wie Nukleoprotein 7 zu erkennen. Unter Verwendung dieser Kits, Nachweis von Virus kann innerhalb von 15 min erreicht werden, doch, haben alle negativen Proben zur weiteren s betragencreened durch die Zellkultur-Verfahren. Verschiedene Studien haben auch geringe Empfindlichkeit dieser Kits, vor allem, wenn die Infektion mild oder niedrigen 8 berichtet wurde. Bei unserer Methode erkennen wir konsequent so niedrig wie 100 PFU des Virus. Dies legt nahe, unsere Technik ist hilfreich bei der Erkennung H1N1-Virus in klinischen Proben, selbst wenn die Infektion mild ist. Ein Fluoreszenz-basierte Methode zur viralen Antigene zu detektieren wurde berichtet, aus denen entweder Nasen-Rachen-Aspirat Proben direkt analysiert wurden oder wurden auf eine Monoschicht von empfänglichen Zellen zur Virusvermehrung und die anschließenden Auswertungen 9 inokuliert. Allerdings sind diese Methoden für diagnostische Zwecke beschränkt und eignen sich nicht für Virustiter Berechnungen. Q-PCR hilft, um das Vorhandensein von viraler RNA zu identifizieren und schätzt nicht die die Infektiosität von Lebend-Viren. Im Vergleich zu diesen Assays, wird IR-Farbstoff-basierten Assay-System in der Detektion und virale Titer-Enumeration in der klinischen und Laborproben zu helfen. Im Falle einer Grippewelle, ist esentscheidend für Tausende von Proben in einer kurzen Zeitperiode zu diagnostizieren. Unsere Methode auf einer 384-Well-Platte und IR-Scanner basieren, können 6-Platten in einer Zeit (&gt; 2.000 Proben) zu scannen, und bietet daher einen größeren Vorteil gegenüber anderen direkten Fluoreszenz-Methoden. Machbarkeit der schnellen Verarbeitung von Tausenden von klinischen Proben könnten reduzieren Test Variabilität, Länge der Assay-Zeit und Kosten. Die Verwendung von einem M2-spezifischen Antikörper bietet den Vorteil, dass unabhängig von Belastung Unterschiede in den H und N-Proteine. Das M2-Protein ist relativ in den meisten Influenza-Stämme infizieren Menschen konserviert. Wenn Antikörper, die spezifisch an zirkulierenden Flecken mit unserem System kombiniert werden kann man messen sowohl den Titer des Virus zu identifizieren und die Belastung. Da jedoch M2 ist das Ziel für den Amantadin-ähnliche Medikamente, können Mutanten auf, die das Bindungsstelle für den hier verwendeten mAb ändern. Dies stellt eine potentielle Beschränkung für dieses System in Individuen, die durch diese Medikamente behandelt werden. MehrereTests werden verwendet, um Influenza-Titer in Testproben aufzuzählen. Um Virustiter, 50% Gewebekultur infektiöser Dosis (TCID 50) und Eier Allantois-Flüssigkeit-basierte Assays zu berechnen sind weit verbreitet 1,10 verwendet. Stellen Sie jedoch Bedarf von mehreren Tagen diese Methoden weniger zuverlässig. Ferner wird in dieser Methoden Berechnungen auf 50% Veränderung in der Anzahl der Plaques Basis stellen theoretische, aber nicht genau viralen Titrationen. Plaque-bildenden Tests können innerhalb von 96 Stunden durchgeführt werden, jedoch ist mangelnde Reproduzierbarkeit ein wichtiges Anliegen der virale Titer Berechnungen. Die vorliegende Technik hier beschriebene Vorgehen hat mehrere deutliche Vorteile. Erstens ist es ein hoch reproduzierbare Technik mit konsistenten Ergebnissen führen. Zweitens Virustiter Berechnungen basieren auf einfachen Quantifizierung der Fluoreszenzintensitäten, die gegen definierte Standard-Kurven im Vergleich beruhen. Drittens kann zwei oder mehr primären Antikörper verwendet, um mehrere Serotypen in einer gegebenen Probe zu diagnostizieren. Außerdem haben IR-Farbstoff-konjugierte Antikörper minimal Autofluoreszenz und haben für Cell Imaging 11 verwendet worden. UV-oder Stoss mit niedriger Energie (300-600 nM) führt zu hohen zelluläre Autofluoreszenz aufgrund der Gegenwart von hohen Quantenausbeuten nachgebenden endogener Fluorophore. Dazu gehören aromatische Aminosäuren, Lipo-Pigmente, pyridinischer Nukleotide (NADPH), Elastin, Kollagen und Flavin-Coenzymen. Diese intrinsische Eigenschaft von Zellen macht es schwierig, diese Quellen von Strahlung zu nutzen als Sonden die Expression spezifischer Proteine ​​zu quantifizieren. Im Gegensatz dazu anregen NIR-Farbstoffe und emittieren zwischen 670-1000 nm. Viele Gastgeber zellulären Molekülen und polycyclische aromatische Kohlenwasserstoffe nicht bei dieser Wellenlänge anregen und aufgrund der geringeren Lichtstreuung, geben extrem geringe Hintergrundfluoreszenz. Ferner wird das Fenster zwischen dem Hintergrund und spezifischen Nachweis erheblich verbessert aufgrund einer hohen Extinktionskoeffizienten der Nah-IR-Fluorophore. Photostabilität, Hervorragendes Signal-Rausch-Verhältnis, ist äußerst relevant bei der Quantifizierung der viralen Titer. Verwendung von Microfluidic Kammern wird das Screening-Verfahren durch Roboter-Steuerungen zu automatisieren, und könnten es uns ermöglichen, hoch ansteckende klinischen Proben mit Leichtigkeit zu testen. Somit ist die Nutzung der NIR-Farbstoffe in diesen Verfahren ideale und höchst zuverlässige, um virale Titer in Gewebeproben aufzuzählen.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Rinderserumalbumin </td><td> Atlanta Biologicals </td><td> S11750 </td><td></td></tr><tr><td> PR8-Virus </td><td></td><td></td><td> Von Dr. Thomas Moran M </td></tr><tr><td> Ziege anti-Maus-IRDye @ 800 </td><td> LI-COR Biosciences </td><td> 926-32210 </td><td> 1:200-Verdünnung </td></tr><tr><td> LI-COR Odyssey IR-Scanner </td><td> LI-COR Biosciences- </td><td> 9201-01 </td><td></td></tr><tr><td> 384-Brunnen flache Bodenplatte </td><td> Nalge Nunc internationalen </td><td> 164730 </td><td></td></tr><tr><td> 96-Brunnen flache Bodenplatte </td><td> Nalge Nunc internationalen </td><td> 165305 </td><td></td></tr><tr><td> DMEM-Medium </td><td> Invitrogen </td><td> 11965126 </td><td></td></tr><tr><td> RPMI-Medium </td><td> Invitrogen </td><td> 11875135 </td><td></td></tr><tr><td> Natriumbicarbonat </td><td> Invitrogen </td><td> 25080 </td><td></td></tr><tr><td> L-Glutamin 100x </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Trypson-EDTA </td><td> Invitrogen </td><td> R001100 </td><td></td></tr><tr><td> Natriumpyruvat </td><td> Invitrogen </td><td> 11360070 </td><td></td></tr><tr><td> Anti-M-2-Antikörper </td><td></td><td></td><td> Von Dr. Thomas Moran M </td></tr><tr><td> Anti-NP-mAb </td><td> CDC </td><td> V2S2208 </td><td> Erhalten mit einem MTA </td></tr></tbody></table>

high-throughput detection method for influenza virus

Influenza-Virus ist ein Krankheitserreger, der Atemwege ein hohes Maß an Morbidität und Mortalität in jedem Jahr in verschiedenen Teilen der Welt verursacht. Daher ist eine genaue Diagnose des infizierenden Stammes und schnelle Hochdurchsatz-Screening einer großen Zahl von klinischen Proben ausschlaggebend für die Ausbreitung der Pandemie-Infektionen zu kontrollieren. Aktuelle klinische Diagnose von Influenza-Infektionen sind auf serologische Tests, Polymerase-Kettenreaktion, direkte Probe Immunfluoreszenz und Zellkultur 1,2 basiert. Hier berichten wir über die Entwicklung eines neuartigen Diagnose-Technik verwendet, um Live-Influenza-Viren zu erkennen. Wir nutzten die Maus-adaptierten menschlichen A/PR/8/34 (PR8, H1N1)-Virus 3, um die Wirksamkeit dieser Technik unter Verwendung MDCK-Zellen 4 zu testen. MDCK-Zellen (10 4 oder 5 x 10 3 pro Vertiefung) wurden in 96 kultiviert - oder 384-Well-Platten, mit PR8 und viralen Proteinen infiziert wurden unter Verwendung von anti-M2 gefolgt von einer IR-Farbstoff-konjugierten Sek.ondary antibody. M2 5 und Hämagglutinin 1 sind zwei bedeutende Marke Proteine ​​in vielen verschiedenen diagnostischen Assays verwendet. Der Einsatz IR-Farbstoff-konjugierten sekundären Antikörpern minimierte die Autofluoreszenz mit anderen fluoreszierenden Farbstoffen zugeordnet. Die Verwendung von Anti-M2-Antikörper erlaubte uns, die Antigen-spezifischen Fluoreszenzintensität als direkte Metrik von viralen Menge die verwenden. Um die Intensität der Fluoreszenz aufzählen, nutzten wir die LI-COR Odyssey IR-basierten Scanner. Dieses System verwendet zwei Kanal Laser-basierter IR-Erkennungen zu Fluorophoren identifizieren und unterscheiden sie von Hintergrundrauschen. Der erste Kanal Anregung bei 680 nm und emittiert bei 700 nm zu helfen, den Hintergrund zu quantifizieren. Der zweite Kanal erkennt Fluorophore anzuregen, dass bei 780 nm und 800 nm emittieren. Scannen von PR8-infizierten MDCK-Zellen in der IR-Scanner zeigten eine virale Titer-abhängige helle Fluoreszenz. Eine positive Korrelation der Fluoreszenzintensität zu Virustiter ab 10 2 -10 5 PFU könnte conkonsequent beobachtet. Minimal, aber nachweisbare Positivität konsequent gesehen mit 10 2 -10 3 PFU PR8 virale Titer zeigte die hohe Empfindlichkeit der Nah-IR-Farbstoffe. Das Signal-zu-Rausch-Verhältnis wurde durch Vergleich der Mock-infizierten oder Isotyp-Antikörper behandelten MDCK-Zellen bestimmt. Mit Hilfe der Fluoreszenz-Intensitäten von 96 - oder 384-Well-Platte-Formate, konstruierten wir Standard-Titrationskurven. In diesen Rechnungen ist die erste Variable der virale Titer, während die zweite Variable ist die Fluoreszenzintensität. Deshalb nutzten wir die exponentielle Verteilung, um eine Kurve anpassbare, um das Polynom Beziehung zwischen den viralen Titer und Fluoreszenz-Intensitäten bestimmen, zu erzeugen. Zusammen schließen wir, dass IR-Farbstoff-basierten Protein Detection System kann bei der Diagnose von viralen Stämmen infiziert und genau aufzuzählen den Titer der infizierenden Erreger.

Watch this Scientific Journal Video about High-throughput Detection Method for Influenza Virus at JoVE.com

High-throughput Detection Method for Influenza Virus

Diese Methode beschreibt die Verwendung von Infrarot-Farbstoff basierende Imaging-System für den Nachweis von H1N1 in bronchioalveolar Lavage (BAL) Flüssigkeit von infizierten Mäuse mit einer hohen Empfindlichkeit. Diese Methodik in einem 96 durchgeführt werden kann - oder 384-Well-Platte, erfordert &lt;10 ul Volumen von Testmaterial und hat das Potenzial für die gleichzeitige Screening von mehreren Erregern.

High-Throughput Nachweisverfahren für Influenza-Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

high-throughput-detection-method-for-influenza-virus

Research

JoVE Journal

Immunology and Infection

26.0K Aufrufe.  Blood Research Institute. Diese Methode beschreibt die Verwendung von Infrarot-Farbstoff basierende Imaging-System für den Nachweis von H1N1 in bronchioalveolar Lavage (BAL) Flüssigkeit von infizierten Mäuse mit einer hohen Empfindlichkeit. Diese Methodik in einem 96 durchgeführt werden kann - oder 384-Well-Platte, erfordert 

Serie: High-throughput Detection Method for Influenza Virus

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Erzeugung von rekombinanten Influenza Virus von Plasmid-DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Forschung

Immunologie und Infektion

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Mit Bioluminescent Imaging, um Synergie von Untersuchen Streptococcus pneumoniae Und Influenza A Virus in Infant Mäuse

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX kolorimetrischen Nachweis einer DNA Microarray für Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza Virus Vermehrung in embryonierten Hühnereiern

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Nasal Tücher für Influenza-A-Virus-Erkennung und Isolierung von Schweine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

In - vitro - Zerlegung von Influenza - A - Virus Kapside durch Gradientenzentrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Hochdurchsatz-Nachweis von Respiratory Pathogens in Tier Proben durch nanoskalige PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza-A-Virus-Studien in einem Mausmodell der Infektion

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Generation von Escape Varianten des Influenza-Virus Monoclonal Antikörper neutralisieren

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Schnelle, sichere und einfache manuelle am Krankenbett Nukleinsäure-Extraktion für den Nachweis des Virus in Vollblut-Proben

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...