The present study was supported by the NIH grants HL120947 (P.C.), HL103868 (P.C.), and the American Heart Association Grant-in-Aid (P.C.)

HINWEIS: Allgemeine Hinweise: Führen Sie alle Verfahren, die Manipulation des Ei unter sterilen Bedingungen und steriler Technik sollte entsprechend verwendet werden. Vorreinigen gesamte Ausrüstung mit 70% Ethanol vor dem Gebrauch. Im allgemeinen sollte Influenza Virusinokulation und Ernte in einem BSL-2 Labor durchgeführt werden. Allerdings, wenn dieses Protokoll wird verwendet, um zu verbreiten viel mehr pathogenen Influenzaviren (zB Pandemie und Pre-Pandemiestämme, Stämme, die eine Gefahr für Geflügel und Vieh, hoch pathogenen aviären Influenza-Stämme, die 1918 H1N1-Stamm darstellen), höhere Biosicherheitsstufen und in einigen Fällen werden Biosicherheitsmaßnahmen und Genehmigungen erforderlich. Der lokale Institutional Biosafety Committee sollte alle Forschung mit Influenza-Virus zu genehmigen. 1. Vorbereitung der Eier für Impfung <ol><li> Erhalten Serum pathogenfreien (SPF) frisch befruchteten Hühnerkeimen aus einem geeigneten Anbieter. Typischerweise sind Hühnereier verwendet. Eier sollten unsed sofort nach Ankunft. </li><li> Nach der Ankunft statt Eier in einem befeuchteten Brutkasten mit einem automatischen Ei Turner, Eier regelmäßig zu drehen. Ist ein automatischer Ei Turner nicht verfügbar ist, von Hand drehen Eier mindestens 2-3 mal pro Tag (stellen Sie sicher, Handschuhe zu tragen und halten alles so steril wie möglich). </li><li> Eiern bei 37 ° C und 55-60% Luftfeuchtigkeit für 10-11 Tage. </li></ol> 2. Eierkerzen <ol><li> Verwenden Sie ein Ei candler, um das Ei vor einem hellen Licht in einem dunklen Raum zu untersuchen, um Eier für Unfruchtbarkeit, die durch Durchleuchten nach ca. 7 oder 8 Tagen Inkubation zu überprüfen. <ol><li> Wischen Sie die Eierdurchleuchter mit 70% Ethanol. Handschuhe häufig wechseln und Desinfektion mit 70% Ethanol, um das Risiko einer Kontamination mit Krankheitserregern zu minimieren. </li><li> Entfernen Sie die Eier aus dem Inkubator und sie in leeren Eierkartons. </li><li> Schalten Sie das Licht in den Raum. </li><li> Halten Sie das große Ende jedes Ei einzeln nacheinander gegen die candler. </li><li> Beobachten Sie das Ei zu determine, wenn es fruchtbar oder unfruchtbar. HINWEIS: Dünne Blutgefäße, die zu einer bohnenförmigen Embryos muss deutlich sichtbar sein. Unbefruchteten Eiern wird als kleiner Blutfleck mit einem sichtbaren Eigelb angezeigt. </li></ol></li><li> Werfen Sie unbefruchtete Eier, die sind, und gibt die lebensfähige Eier in den Inkubator. Lassen Sie keine Eier außerhalb des Inkubators für mehr als 30 min. Wenn der Status eines Eies nicht bestätigt werden kann, markieren Sie ihn und später beobachten sie. </li></ol> 3. Herstellung von Virus Inoculum <ol><li> Verdünne Influenzavirusbestand zu etwa 3. Oktober-4. Oktober pfu / ml in sterilem PBS, enthaltend 10 mM HEPES (pH 7,4). Critical Schritt: Verdünnen Sie die Virusstamm unmittelbar vor der Anwendung und halten die verdünnten Virus auf dem Eis die ganze Zeit. </li></ol> 4. Influenza Virus Impfung über die Allantois Routen <ol><li> Am Tag der Virusinokulation (Tag 10 oder 11), Kerze die Eier wieder und beseitigen tote Embryonen. Unterscheiden Sie Live-EmbryonenSuche nach Bewegung in das Ei. </li><li> Entfernen Sie drei Eier aus dem Inkubator, und ordnen Sie die Eier in einem separaten Halter mit dem Luftsack auf. </li><li> Markieren Sie den Luftraum der Eier mit einem wasserfesten Stift. </li><li> Oberhalb des Luftraums mit 70% Ethanol waschen die Eierschale. </li><li> Die Eier mit dem Luftraum in einem Eierkarton und in einen biologischen Sicherheit Haube (BSL-2). </li><li> Verwenden Sie eine sterile 18G Nadel ein kleines Loch in der Schale auf den Luftsack jedes Ei zu stanzen. Achten Sie darauf, um die Nadel zu tief einfügen, um zu vermeiden, stech den Embryo oder Eigelb. </li><li> Erstellung des verdünnten Influenza-Virus in eine sterile 1 ml-Spritze und fügen Sie eine 22G-Nadel. </li><li> Die Spritze mit Nadel vorsichtig voran in einem Winkel von 45 ° in die Allantoishöhle. </li><li> Injizieren 0,2 ml der verdünnten Influenza-Virus. </li><li> Wiederholen Sie die Injektion mit den anderen zwei Eier, und dann werfen Sie die Spritze und Nadel. </li><li> Dichten Sie das Loch mit einem Tropfen Klebstoff aus einer Klebepistole (oder geschmolzene Paraffinwachs). Be Achten Sie darauf, dass Kleber nicht im Inneren des Eis lecken. </li><li> Legen Sie die Eier in die Brutkasten mit der Luftraum deutete nach oben. </li><li> Wiederholen Sie die Schritte 4,2 bis 4,12, bis alle Eier wurden geimpft. </li></ol> 5. Inkubationszeit <ol><li> Die infizierten Eiern Inkubieren ohne Drehen bei 37 ° C und ca. 60% Luftfeuchtigkeit. </li><li> Wählen optimale Inkubationszeit in Abhängigkeit von der Influenza-Virus-Typ. Inkubieren Influenza A 48 h; inkubieren Influenza B und C für 72 Stunden. </li></ol> 6. Influenza Virus-Ernte <ol><li> Nach Inkubationszeit Chill die Eier bei 4 ° C für mindestens 4 Stunden (oder O / N), um den Embryo zu töten und verengen die Blutgefäße, um das Risiko einer Verunreinigung des infizierten Allantoisflüssigkeit mit Blut zu verringern. Tun dies, um die roten Blutzellen absorbieren das Virus, was die Ausbeute verringert verhindern. Alternativ Chill Eier für 30 min bei -20 ° C; aber dies erhöht die Gefahr einer Kontamination des Blutes. </li><li> Nachdem die Eier ausreichend gekühlt wurde, übertragen Sie die Eier auf einem Bio-Sicherheitshaube. Legen Sie das Ei in eine feste Halterung mit der Luftsack nach oben zeigt. Reinigen Sie das Ei Oberfläche mit 70% Ethanol. </li><li> Verwenden einer sterilen Schere, um die Eierschale über dem Luftsack zu öffnen. Entfernen Sie die Hülle um den Luftsack, während man aufpassen, nicht die Chorioallantoismembran zerstören. </li><li> Öffnen Sie die Chorioallantoismembran mit sterilem stumpfen Pinzette. Vorsichtig zur Seite legen den Embryo und der Dottersack mit einem kleinen Spatel oder Löffel. Darauf achten, dass das Eigelb bersten. </li><li> Mit einer 1 ml-Eppendorf-Pipette vorsichtig sammeln die Allantoisflüssigkeit. Wenn nötig, kippen Sie das Ei ein wenig zur Seite, um so viel Flüssigkeit wie möglich zu sammeln. </li><li> Kombinieren Sie die Allantoisflüssigkeit aller Eier in einen 50-ml-Kunststoff konischen Rohr. Während der Virusernte halten Röhrchen auf Eis zu allen Zeiten. Ein Ei Ausbeuten 5-10 ml eines leicht gelblichen Flüssigkeit. </li><li> Drehe das virushaltige Allantoisflüssigkeit bei 1.000 × g für 10 min bei 46; C bis Trümmer zu pelletieren. Übertragen Sie die klare Flüssigkeit in eine neue 50-ml-Röhrchen auf Eis gehalten. Entsorgen Sie Eier in eine BSL-2 Abfallbehälter. </li></ol> 7. Lagerung <ol><li> Die geklärt Allantoisflüssigkeit (zB 50, 100 oder 500 ul Aliquote) sofort aliquotiert und Snap-freeze in flüssigem Stickstoff. Übertragen gefrorenen Virusstocks bis -80 ° C Gefrierschrank für langfristige Lagerung. </li></ol>

<ol>
	<li>Centers for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+of+deaths+associated+with+seasonal+influenza+---+United+States,+1976-2007.">Estimates of deaths associated with seasonal influenza --- United States, 1976-2007.</a> MMWR. Morbidity and mortality weekly report. 59 (33), 1057-1062 (2010).</li><li>Neumann, G., Noda, T., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+and+pandemic+potential+of+swine-origin+H1N1+influenza+virus.">Emergence and pandemic potential of swine-origin H1N1 influenza virus.</a> Nature. 459 (7249), 931-939 (2009).</li><li>Tumpey, T. M., Garcia-Sastre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenicity+of+Influenza+Viruses+with+Genes+from+the+1918+Pandemic+Virus:+Functional+Roles+of+Alveolar+Macrophages+and+Neutrophils+in+Limiting+Virus+Replication+and+Mortality+in+Mice.">Pathogenicity of Influenza Viruses with Genes from the 1918 Pandemic Virus: Functional Roles of Alveolar Macrophages and Neutrophils in Limiting Virus Replication and Mortality in Mice.</a> J Virol. 79 (23), 14933-14944 (2005).</li><li>Doherty, P. C., Turner, S. J., Webby, R. G., Thomas, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+and+the+challenge+for+immunology.">Influenza and the challenge for immunology.</a> Nat Immu. 7 (5), 449-455 (2006).</li><li>Webster, R. G., Govorkova, 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=Continuing+challenges+in+influenza.">Continuing challenges in influenza.</a> Ann N Y Acad Sci. 1323 (1), 115-139 (2014).</li><li>Szretter, K. J., Balish, A. L., Katz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unit+15G.1+Influenza:+propagation,+quantification,+and+storage.+.">Unit 15G.1 Influenza: propagation, quantification, and storage. .</a> Current protocols in microbiology. , (2006).</li><li>Short, K. R., Kroeze, E. J. B. V., Fouchier, R. A. M., Kuiken, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+influenza-induced+acute+respiratory+distress+syndrome.">Pathogenesis of influenza-induced acute respiratory distress syndrome.</a> Lancet Infect Dis. 14 (4), 57-69 (2014).</li><li>Ito, T., Suzuki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+sialic+acid-galactose+linkages+in+the+chicken+egg+amnion+and+allantois+influence+human+influenza+virus+receptor+specificity+and+variant+selection.">Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.</a> J Virol. 71 (4), 3357-3362 (1997).</li><li>Robertson, J. S., Nicolson, C., Major, D., Robertson, E. W., Wood, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+amniotic+passage+in+the+egg-adaptation+of+human+influenza+virus+is+revealed+by+haemagglutinin+sequence+analyses.">The role of amniotic passage in the egg-adaptation of human influenza virus is revealed by haemagglutinin sequence analyses.</a> J Gen Virol. 74 (Pt 10), 2047-2051 (1993).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells).">Replication and plaque assay of influenza virus in an established line of canine kidney cells).</a> Appl Microbiol. 16 (4), 588-594 (1968).</li></ol>

The authors have nothing to disclose.

Influenza verursacht einen großen globalen Krankheitslast, und die Arbeit geht weiter in das Verständnis der Pathogenese der Lungenschädigung 7. Erleichtert die Forschung in dieser tödlichen Krankheit, wurden verschiedene Verfahren entwickelt, um den Influenzavirus 6 ausbreiten. Hier beschreiben wir eine Technik, um Influenza-Virus in Hühnereiern produziert. Der Vorteil dieses Verfahrens ist, dass es in hohem Maße reproduzierbar ist und führt zu großen Mengen von hohem Titer Influenzaviruss...

Influenza A weiterhin eine große Bedrohung für die menschliche Gesundheit sein. Es ist eine potenziell verheerenden Erkrankung der Atemwege mit einem großen globalen Belastung verursacht bis zu 500.000 Todesfälle weltweit pro Jahr 1. Influenza-Viren sind in der Familie Orthomyxoviridae und führen 8 Minus-Sense einzelsträngige RNA in ihrem Genom 1,2. Die hohe Veränderlichkeit (dh antigene &quot;Drift&quot;) des Virusgenoms verhindert Langzeitimmunität. Außerdem ist Influenza zunehmend resistent gegen antivirale Medikamente 3. Das H1N1 2009-Grippe-Pandemie markiert alle herausfordernden Fragen mit dem Influenzaerkrankung (Pandemie-Stamm, anti-virale Resistenz, verzögert Impfstoffproduktion) verbunden. Die Impfstoff-Formulierung wird jährlich von der Weltgesundheitsorganisation für die wahrscheinlichste pathogenen Influenza-Stämme (je eine für H1N1, H3N2 und Influenza B) 4 bestimmt. Da diese Methode beruht auf Annahmen basierte Influenza-Stamms, pathogenen Stämmen werden gelegentlich falsch identifiziert und die Wirksamkeit des Impfstoffes sinkt dramatisch. Darüber hinaus kann das Auftreten von neuen Influenza-Stämme diese präventive Programme verursachen Pandemien 2 zu umgehen. Die Schaffung eines universellen Influenza-Impfstoff ist schwer 5. Daher müssen Erforschung Verständnis der Pathogenese der Lungenschädigung weiter. Um die Anlage der Laborforschung wurden verschiedene Verfahren für die virale Isolation und Vermehrung 6 entwickelt. Menschlichen Influenza-Viren können in einer Vielzahl von Säugetierzellsubstraten verstärkt werden. Jedoch werden die Erzeugung hoher Titer von Viren in großen Mengen am besten in embryonierten Eiern erreicht. Das folgende Protokoll beschreibt eine Technik für die Influenza A-Virusvermehrung und Speicherung von vorhandenen Virusstocks.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Reagent/ Equipment</td>
			<td style="width:225px;">Company</td>
			<td style="width:128px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Phosphate buffered saline (PBS)</td>
			<td style="width:225px;">Cellgro&#160;</td>
			<td style="width:128px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serum pathogen-free (SPF) fertilized chicken eggs&#160;</td>
			<td>VALO BioMedia</td>
			<td style="width:128px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">humidified egg incubator&#160;</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 2100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">automatic egg turner</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 3200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">egg candler</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 3300&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1 ml syringe</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">18G needle</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">20G / 22G needle</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">305176 / 305156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">HEPES</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:128px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ethanol, &#62;99.5%</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:128px;">459844</td>
			<td>diluted to 70% using water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">glue gun &#34;Ad tech Hi Temp Project Pro&#34;</td>
			<td style="width:225px;">Ad tech, Adhesive Technologies, Inc.</td>
			<td>Model 1105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">glue &#34;Ad tech MINI SIZE, Multi Temp&#34;</td>
			<td style="width:225px;">Ad tech, Adhesive Technologies, Inc.</td>
			<td>220-34ZIP30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">MDCK cells</td>
			<td style="width:225px;">ATCC</td>
			<td>CRL-2935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Washed Pooled Turkey Red Blood Cells, 10%</td>
			<td style="width:225px;">Lampire Biological Laboratories</td>
			<td>724908</td>
			<td></td>
		</tr>
	</tbody>
</table>

influenza virus propagation in embryonated chicken eggs

Influenza-Infektion ist mit rund 36.000 Tote und mehr als 200.000 Krankenhauseinweisungen pro Jahr in den Vereinigten Staaten verbunden. Die kontinuierliche Entstehung neuer Influenza-Virus-Stämme aufgrund von Mutation und Wiederzusammenstellung erschwert die Kontrolle des Virus und die ständige Entwicklung neuer Arzneimittel und Impfstoffe. Die Laboruntersuchung von Influenza erfordert ein zuverlässiges und kostengünstiges Verfahren für die Vermehrung des Virus. Hier wird ein umfassendes Protokoll für Influenza-A-Virusvermehrung in fruchtbare Hühnereiern, die konsequent liefert hohen Titer Virusbeständen. 11 Tage - Kurz gesagt, werden Serum pathogenfreien (SPF) befruchtete Hühnereier bei 37 ° C und 55-60% Luftfeuchtigkeit für 10 inkubiert. In diesem Zeitraum können die Entwicklung des Embryos einfach mit ein Ei candler überwacht werden. Virusinokulation erfolgt durch Injektion von Virusstocks in die Allantois-Höhle unter Verwendung einer Nadel durchgeführt. Nach 2 Tagen Inkubation bei 37 ° C, sind die Eiergekühlt für mindestens 4 Stunden bei 4 ° C. Die Eischale oberhalb der Luftsack und die Chorioallantoismembran werden dann vorsichtig geöffnet und die Allantoisflüssigkeit, die das Virus geerntet. Das Fluid wird von Bruchstücken durch Zentrifugation geklärt, in Aliquote aufgeteilt und bis -80 ° C zur Langzeitlagerung transferiert. Die große Menge (5-10 ml des virushaltigen Fluids pro Ei) und hoher Virustiter in der Regel mit diesem Protokoll erreicht hat die Verwendung von Eiern für die Viruspräparation unserer günstiges Verfahren zur in vitro-Studien, die größere Mengen, insbesondere des Virus in denen hohe Dosierungen des gleichen Virusstock erforderlich.

Mehrere Verfahren sind entwickelt worden, um Influenza-Virus zu titrieren. Eine Überlegung bei der Auswahl eines geeigneten Verfahrens ist, dass einige zu bestimmen Gesamtpartikel unabhängig von Lebensfähigkeit (zB Hämagglutinin-Assay), während andere auf die Infektiosität der Basis, die lebensfähigen Virionen bewertet wird (zB Plaque-Test) 6. Hier wurde der Virustiter von Allantoisflüssigkeit aus Hühnereiern mit einer Maus-adaptierten Influenzavirus (A / PR / 8/34) beimpft gesammel...

Watch this Scientific Journal Video about Influenza Virus Propagation in Embryonated Chicken Eggs at JoVE.com

Influenza Virus Propagation in Embryonated Chicken Eggs

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 ...

influenza-virus-propagation-in-embryonated-chicken-eggs

Research

JoVE Journal

Immunology and Infection

42.6K Aufrufe.  Cedars-Sinai Medical Center. 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.

Serie: Influenza Virus Propagation in Embryonated Chicken Eggs

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

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 ...

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce&#39;s disease (PD) of grapevine. Pierce&#8217;s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

Ausbreitung von<em&gt; Homalodisca coagulata Virus-01</em&gt; Über<em&gt; Homalodisca vitripennis</em&gt; Zellkultur

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

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 ...

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 ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Mess Bindung und Internalisierung von Influenza A-Virus in A549-Zellen mittels Durchflusszytometrie

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophilen-Isolierung und Analyse zur Bestimmung ihrer Rolle in der Lymphom-Zellempfindlichkeit therapeutische Mittel

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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.