This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.

Alle Schweine in der Sammlung der folgenden Daten verwendet werden, wurden unter der Ohio State University Institutional Animal Care und Verwenden Committee geschützt (Tiernutzung Protokollnummer 2009A0134-R1). 1. Herstellung der Virustransportmedium und Probensammlung Vials <ol><li> In 37 g Brain Heart Infusion zu 900 ml gereinigtem Wasser und mischen mit einem Rührstab und einem Magnetrührer unter Erwärmen auf 70 ° C, um das Pulver vollständig aufzulösen. </li><li> Autoklavieren der Brühe bei 121 ° C für 15 min. Kühle auf der Tischplatte, bis die Brühe Raumtemperatur erreicht. Im Kühlschrank bei 4 ° C über Nacht, wenn nötig. </li><li> Aufzulösen, durch Mischen mit einem magnetischen Rührstab, 6,02 g Penicillin G-Natriumsalz und 10 g Streptomycinsulfat in 50 ml gereinigtem Wasser bei Raumtemperatur. Fügen zusätzliches gereinigtes Wasser, um das Gesamtvolumen auf 100 ml zu bringen. Die Lösung wird durch ein 0,22 um-Polyethersulfonmembran in eine sterile Flasche. </li><li> Aseptisch das gefilterte Penicillin und Streptomycin-Lösung hinzufügen, in das gekühlte Brain Heart Infusion Broth und sorgfältig zu vermischen (Rührstab oder Wirbelkolben mit der Hand), um die Virustransportmedium zu machen. </li><li> Aseptisch testen Sie den pH-Wert der Virustransportmedium; Bei Bedarf kann der pH-Wert auf 7,4 ± 0,2 mit HCl oder NaOH. </li><li> Führen Sie Qualitätssicherungstests auf der Virustransportmedium durch die Prüfung für Influenza-A-Virus-Matrixprotein von rRT-PCR, wie von Zhang und Harmon beschrieben. 22 </li><li> Verzichtet werden 5 ml der viralen Transportmediums in sterile hochdichten Polyethylenflaschen mit 8 ml Kapazität. Beschriften Sie die Durchstechflaschen für den Einsatz im Feld. </li><li> Bewahren Sie die ready-to-use Fläschchen in Standard-Kryo-Boxen bei -20 ° C bis zum Probensammlung. </li></ol> 2. Sammlung von Nasal Wipes von Swine <ol><li> Tauen die entsprechende Anzahl von Fläschchen unmittelbar vor der Entnahme zu probieren; ein Fläschchen wird pro Schwein Stichprobe benötigt. Das Auftauen bei RaumTemperatur dauert ca. 30-45 min. Halten Sie die aufgetauten Fläschchen kühl auf Eis Packs bis zur Verwendung. </li><li> Don geeigneter persönlicher Schutzausrüstung, wie diktiert durch die Lage (zB Overalls, Überziehstiefel, Atemschutz, Gehörschutz, etc.). </li><li> Geben Sie den Tierbereich. Bei Bedarf beschränken das Schwein auf einen kleinen Bereich aber nicht zurückhalten. Sie wecken nicht stillstehen Tiere. </li><li> Setzen Sie auf ein Paar Einweguntersuchungshandschuhe. Darauf achten, dass die Handschuhe durch Berühren von Schweinen, Menschen oder leblose Gegenstände kontaminieren. </li><li> Verwenden Sie einen Handschuh in ein steriles 5,08 cm × 5,08 cm zu entfernen (2 in. × 2 in.) Baumwolle Tupfer aus der Verpackung. Steriler Gaze-Pads verwendet werden, wann immer möglich. Einzeln verpackt Gaze-Pads sind bequemer, als die mit zwei Gazekompressen pro Paket verpackt zu verwenden. </li><li> Halten Sie die Baumwolle Gaze mit den Fingerspitzen einer Hand so viel von der Unterlage wie möglich auszusetzen für die Probenahme. </li><li> Wischen Sie die Gaze über den Schweineschnauze, wobeizusätzliche Pflege, um die äußeren Nasenlöcher, wenn möglich in Kraft. Sammeln sichtbaren Nasensekret (etwa 1 ml) mit dem selben Mullbausch. </li><li> Mit der gleichen Seite, falten Sie die Gaze auf sich selbst, um die Platzierung in der Ampulle, die Virustransportmedium zu erleichtern. </li><li> Mit der anderen Hand, entfernen Sie die Kappe von der Durchstechflasche und legen Sie das gefaltete Gaze in das Fläschchen. Verschließen Sie die Röhrchen und schütteln Sie das Fläschchen, um die Durchmischung der Gaze und Virustransportmedium zu gewährleisten. </li><li> Die Handschuhe und in einen geeigneten Behälter zu entfernen. Handschuhe zwischen jedem Schwein ändern. </li><li> Stellen Sie sicher, das Fläschchen Kennung. Nehmen Sie Schwein-Identifikationsnummer, Alter, Geschlecht, klinische Symptome und andere Notizen, die von Prüfer für erforderlich gehalten. </li><li> Kühlen Sie die Proben sofort nach der Entnahme. Überlegen Sie sich Teilmengen der Probe vor dem Einfrieren zu mehreren Auftauen und Einfrieren vermeiden. So bald wie möglich nach der Entnahme, legen Sie die Proben auf Trockeneis für den Transport zum Labor. Lagern Sie die Fläschchen bei -80° C bis zum Test initiiert wird. </li></ol> 3. Der Nachweis von Influenza A Virus-Nukleinsäure <ol><li> Tauen Sie die Proben in einem 37 ° C Trockenperle Bad für 5 Minuten und dann beenden Auftauen der Proben auf dem Labortisch bei Raumtemperatur für 20-30 min. Die gleichzeitige Zugabe von mehreren Ampullen auf die Kügelchen führt dazu, daß das Bad zu erhitzen; Vorsicht ist geboten, um die Durchstechflaschen nicht überhitzen. </li><li> Entfernen 100 ul Virustransportmedium für die RNA-Extraktion. <ol><li> Verwenden ein Hochdurchsatz (96-Well-Platte-Format), Magnetkügelchen Plattform zum Extrahieren von RNA, die aus einer großen Anzahl von Proben. 31 </li></ol></li><li> Verwenden Sie Echtzeit-Reverse-Transkriptase-PCR-Tests zum schnellen Nachweis Influenza A-Virus. 31 </li></ol> 4. Isolierung von Influenza A Virus vom Nasen Wipes <ol><li> Tauen Sie die Proben wie oben in Abschnitt 3.1 beschrieben </li><li> Gönnen aufgetauten Proben mit Gentamicin-Sulfat (1.000ug / ml), Amphotericin B (22,5 &amp; mgr; g / ml) und Kanamycinsulfat (325 ug / ml). </li><li> Inokulieren in Madin-Darby Canine Kidney (MDCK) Epithelzellen wie zuvor beschrieben. 32 </li></ol>

<ol>
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The authors have nothing to disclose.

Sammeln von Proben von Schweinen unter Verwendung von Polyester-bestückte Nasenabstrichen hat bei der Durchführung von IAV Überwachung bewährt; jedoch die Verwendung des Nasenabstrich Verfahren behindert Überwachungsbemühungen aufgrund der erforderlichen Verwendung einer Schlinge zur Zurückhaltung. Nasal Tücher stellen eine Weiterentwicklung der aktuellen Schweinestichprobentechniken, um Stress auf die Menschen und Schweinen während der Probenahme zu minimieren. Während das Verfahren seit entwickelt und in Aus...

<html> Influenza-A-Viren (IAV) verursachen Atemwegserkrankungen in vielen Arten, darunter Hausgeflügel, Schweinen und Menschen. Durch Neuordnung der segmentierten IAV Genom schnelle virale Evolution kann auftreten, und neue IAV Stämme häufig auftauchen. Schweine sind eine Spezies, die als Mischgefäß für die Neuordnung der iAVS von mehreren Wirtsarten dienen können 1. Derzeit gibt es drei Haupttypen von IAV häufigsten verwendete unter den nordamerikanischen Schweinen (H1N1, H1N2, H3N2) im Umlauf, aber mehrere IAV Einführungen von Menschen haben innerhalb dieser Subtypen führte zu umfangreichen IAV Vielfalt. 2 schnelle Entwicklung der iAVS infizieren Schweine ist seit 1998, als eine dreifache Reassortante IAV enthält Gensegmente aus menschlichen, Vogelgrippe und Schweineviren 3 verbreitete sich unter den Schweinen in den Vereinigten Staaten deutlich. 4 Die interne Gen Segmente von diesem Dreifach Reassortante IAV vor sehr weit verbreitet unter iAVS derzeit infizieren Schweine. 5 &quot;&gt; Weltweit ist die IAV eine wesentliche Ursache für Atemwegserkrankungen bei Schweinen, in der typischen klinischen Symptome sind Fieber, Appetitlosigkeit, Lethargie, Husten, Atemnot, Niesen, Nasenausfluss und schlechte Gewichtszunahme. IAV kann besonders teuer werden, um Betriebe zu säen, wo Fortpflanzungs Versagen aufgrund von IAV induzierte Fieber und schwache geborenen Ferkel wurden dokumentiert 6,7 Innerhalb der Vereinigten Staaten. wird IAV üblicherweise in kommerziellen Schweinebeständen und der umfangreichen Antigens und Genoms Vielfalt und kontinuierlichen Weiterentwicklung unter IAV infizieren Schweine erfasst hat die Kontrolle über diese behindert Virus. 8-11 Der öffentlichen Gesundheit über die Entstehung einer Pandemie IAV-Stamm von Reassortierung was Schweine wurden im Jahr 2009 realisiert werden, wenn ein Schwein-Linie IAV enthält Gensegmente aus dem Triple-Reassortante nordamerikanischen Schweine Abstammung und der eurasischen avian artigen Schweine Linie verursachte eine weltweite Pandemie beim Menschen. 12 Die Pandemie-Virus (A (H1N1) pdm09) verfügt seitmit endemischen Schweine neu angeordneten IAV Stämme 13,14 und einige dieser neu neu angeordneten Stämme zurück auf den Menschen übertragen worden. 15 Die Häufigkeit der Reassortierung Veranstaltungen und Entstehung neuer IAV Stämmen mit Pandemie-Potenzial macht die aktive Überwachung der zirkulierenden IAV-Viren bei Schweinen zwingend notwendig, vor allem an der Schweine-Mensch-Schnittstelle. Die Schweine-Mensch-Schnittstelle ist für die bidirektionale Interspezies-Übertragung von IAV. Mensch-zu-Schweine-Übertragung in kommerziellen Schweineproduktion auftritt, ist für eine große Menge von IAV Vielfalt derzeit in der Schweinepopulation vorhanden verantwortlich. Landwirtschaftsmessen sind die größten Einstellungen für die comingling von Menschen und Schweinen in den Vereinigten Staaten und sind dafür bekannt Stellen für den zoonotischen Übertragung von IAV. 15-21 Im Jahr 2012, während des Ausbruchs einer Variante H3N2 IAV, 93% der Fälle berichtet, die Teilnahme an eine Landwirtschaftsmesse in den Tagen vor der Krankheitsbeginn. 15 Genomanalysevon Virusisolaten von Ausstellungs Schweinen gegenüber menschlichen Isolate bestätigte zoonotischen Übertragung. 21 Ausstellung Schweine mit IAV infiziert oft nicht klinischen Anzeichen von Krankheit, 21-23, die die Notwendigkeit einer direkten diagnostischen Tests zu zeigen. Probenahme von sichtlich kranken Schweine allein nicht erfolgreich IAV Prävalenz identifizieren bei Schweinen und kann nicht herangezogen werden, um neue Stämme von IAV Schwellen unter den Schweinen zu identifizieren. Aktive Überwachung ist für die Erkennung von Schwellen Stämme von IAV in Schweinen und ihre Bedrohung sowohl für Schweine und die öffentliche Gesundheit zu beurteilen unbedingt notwendig. Die meisten IAV Überwachungstätigkeiten sind freiwillig und daher minimal störend Verfahren benötigt werden. Die drei großen Schlachttierprobensammelverfahren für IAV infizieren Schweine sind: Nasenabstrichen, Mundflüssigkeiten und Nasentücher. Aktuelle Empfehlungen zur Probenahme einzelnen Schweine zu IAV-Liste das Einfügen von Kunstfaser erkennen Abstrichtupfer in die Nasenlöcher als die bevorzugte Methodeum Nasensekret und Epithelzellen zu sammeln. 24,25 Da Schweine können versuchen, dieses Verfahren, ein Team von Fachpersonal muss Schweinen entweder manuell oder mit einer Schlinge, je nach Größe des Tieres zurückhalten zu vermeiden. 26. Die Zurückhaltung Prozess ist mühsam für die Personal und stressig für die Schweine. Zusätzlich Ausstellung Schweine sind oft in mehreren Wettbewerben auf einer Messe beteiligt, so dass die Wahrnehmung der zusätzlichen Stress auf einem Wettbewerb Tier Besitzer gegenüber den Überwachungs Anstrengungen unternehmen. Mit Wahrscheinlichkeiten der IAV Erkennung im Bereich von 80-100% in IAV infizierten Beständen, haben Mundflüssigkeiten zu einer beliebten Alternative zu Tupfer für den molekularen Nachweis von IAV in Populationen von Schweinenasen. 27,28 Zusätzlich kann Mundflüssigkeiten ein breiteres Fenster der bereitzustellen IAV Erkennung als Nasenabstrichen nach Erstinfektion. Allerdings IAV isoliert von Mundflüssigkeiten problematisch gewesen mit nur 50% der Virusisolierung Versuche was IA29 V Erholung. Verwenden Nasentücher anstelle von Nasenabstrichen bei IAV Überwachung in Schweinen überwindet die oben beschriebenen Einschränkungen. Nasal Tücher nicht die Verwendung eines Rückhalte Snare erfordern und ohne Belastung der Tiere oder Zeugen des Verfahrens durchgeführt werden. Minimale technische Ausbildung ist notwendig, um Nasenwischtücher, die nicht-Tierärzten, einschließlich Schweinebesitzer ermöglicht zu sammeln, um die Überwachungs Proben zu sammeln. Nasen Tücher wurden im Vergleich zu vorher Nasentupfer zum Nachweis und zur Isolierung von Influenza-A-Virus 30 und das detaillierte Protokoll für diese nicht-invasive Methode der Probenahme wird nachstehend beschrieben.

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			<td height="21" style="height:21px;width:216px;">BBL Brain Heart Infusion</td>
			<td style="width:360px;">Becton, Dickinson and Company</td>
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			<td height="21" style="height:21px;width:216px;">Penicillin G Sodium Salt</td>
			<td style="width:360px;">MP Biomedicals, LLC</td>
			<td style="width:119px;">021194537</td>
			<td>1500 u/mg</td>
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			<td height="21" style="height:21px;width:216px;">Streptomycin Sulfate</td>
			<td style="width:360px;">AMRESCO LLC</td>
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			<td style="width:119px;">30-005-CR</td>
			<td>50 mg/ml</td>
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			<td>5000 ug/ml</td>
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			<td>HDPE with white PP closure</td>
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			<td height="26" style="height:26px;width:216px;">Dermacea Gauze Sponge, 8 ply</td>
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			<td>5.08 cm &#215; 5.08 cm (2 in. &#215; 2 in)&#160;</td>
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nasal wipes for influenza a virus detection and isolation from swine

Überwachung der Influenza-A-Viren bei Schweinen ist entscheidend für die menschliche und tierische Gesundheit, weil Influenza A-Virus schnell entwickelt sich in Schweinepopulationen und neue Stämme werden laufend entstehen. Schweine sind in der Lage, die von verschiedenen Abstammungslinien von Influenza A-Virus so dass sie wichtige Wirte für die Entstehung und Aufrechterhaltung von neuartigen Influenza A-Virus-Stämmen infiziert werden. Sampling Schweine in diverse Einstellungen wie kommerziellen Schweinefarmen, landwirtschaftliche Messen und Märkten für lebende Tiere ist wichtig, einen umfassenden Überblick über die derzeit zirkulierenden IAV-Stämme bereitzustellen. Der aktuelle Goldstandard Schlachttier- Sampling-Technik (dh Sammlung von Nasenabstrichen) ist arbeitsintensiv, weil sie körperliche Zurückhaltung der Schweine erfordert. Nasen Tücher beinhalten Reiben eines Stückes Stoff durch die Schnauze des Schweins mit minimalen oder keiner Beeinträchtigung des Tieres. Die Nasen wischen Verfahren ist einfach durchzuführen und nicht Personal mit professionellen Veterinär- oder Tier Handling Ausbildung. While etwas weniger empfindlich als Nasenabstriche sind Virenerkennung und Isolation gewählten Standort gewährleisten, stellen Nasen wischt eine tragfähige Alternative zum Abtasten einzelner Schweine bei niedriger Belastung Probenahmeverfahren erforderlich sind. Das Verfahren Protokoll beschreibt die notwendigen Schritte, um eine lebensfähige Nasen sammeln wischen von einem einzelnen Schwein.

Erfolgreiche Anwendung dieser Methode ergibt rRT-PCR ergibt sich, dass, begleitet mit der Verwendung eines internen Kontroll während der RNA-Extraktion und rRT-PCR, hat Show Proben enthalten PCR-Inhibitoren in jedem Umwelt Trümmern hob während der Probenahme. Nach der Probenimpfung sollte Virusisolierung Brunnen frei von sichtbaren Umwelt Schutt aus der Probe. Es gibt vernünftige Vereinbarung zwischen rRT-PCR-Ergebnisse und die Virusisolierung Ergebnisse mit dem Verständnis, dass PCR ergibt oft eine höhere IAV pos...

Watch this Scientific Journal Video about Nasal Wipes for Influenza A Virus Detection and Isolation from Swine at JoVE.com

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

nasal-wipes-for-influenza-a-virus-detection-and-isolation-from-swine

Research

JoVE Journal

Immunology and Infection

9.3K Aufrufe.  The Ohio State University. The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. 

Serie: Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

High-throughput Detection Method for 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, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

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, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Affinitätsreinigung des Influenza-Virus Ribonukleoproteinkomplexe aus dem Chromatin der infizierten Zellen

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

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 ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Co-Immunopräzipitation der Maus Mx1 Protein mit dem Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Ein miniaturisiertes Glycan Microarray-Test zur Beurteilung der Avidität und Spezifität von Influenza-A-Virus Hämagglutininarten

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

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 ...

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.