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.)

NOTE: Remarques générales: Effectuer toutes les procédures impliquant la manipulation de l&#39;œuf dans des conditions stériles, et la technique stérile doit être utilisé en conséquence. Pré-nettoyer tous les équipements avec 70% d&#39;éthanol avant utilisation. En général, l&#39;inoculation du virus de la grippe et la récolte doivent être effectués dans un laboratoire BSL-2. Toutefois, si ce protocole est utilisé pour propager beaucoup plus pathogène virus de la grippe (par exemple, les souches pandémiques et prépandémiques, souches qui présentent un danger pour la volaille et le bétail, les souches de la grippe aviaire hautement pathogène, le 1918 souche H1N1), les niveaux de biosécurité plus élevés et dans certains cas, des mesures de biosécurité et autorisations sont nécessaires. Le comité institutionnel de biosécurité locale devrait approuver toutes les recherches sur le virus de la grippe. 1. Préparation des oeufs pour l&#39;inoculation <ol><li> Obtenir des œufs aviaires (SPF) exempts d&#39;agents pathogènes fraîchement fécondé sériques d&#39;un fournisseur approprié. Typiquement, des œufs de poule sont utilisés. Les œufs doivent être noused immédiatement à l&#39;arrivée. </li><li> À l&#39;arrivée mettre les œufs dans un incubateur d&#39;œufs humidifié avec un tourneur automatique des oeufs pour faire tourner les oeufs régulièrement. Si un tourneur automatique des oeufs ne est pas disponible, faire pivoter manuellement oeufs au moins 2 à 3 fois par jour (assurez-vous de porter des gants et de garder tout aussi stérile que possible). </li><li> Incuber les œufs à 37 ° C et 55-60% d&#39;humidité pendant 10 à 11 jours. </li></ol> 2. mirage des oeufs <ol><li> Utilisez un candler d&#39;oeuf à examiner l&#39;œuf devant une lumière vive dans une pièce sombre pour vérifier les œufs de l&#39;infertilité par mirage après environ 7 ou 8 jours d&#39;incubation. <ol><li> Essuyez le candler d&#39;œuf avec 70% d&#39;éthanol. Changer de gants souvent et désinfecter avec 70% d&#39;éthanol afin de minimiser le risque de contamination par des agents pathogènes. </li><li> Retirer les oeufs dans l&#39;incubateur et les placer dans des boîtes d&#39;œufs vides. </li><li> Éteignez les lumières dans la salle. </li><li> Maintenez la grande fin de chaque oeuf un à la fois contre le candler. </li><li> Observez l&#39;œuf Détermineze si elle est fertile ou infertile. REMARQUE: les vaisseaux sanguins minces conduisant à un embryon en forme de haricot doit être clairement visible. Les œufs non fécondés apparaissent comme une tache de sang petite avec un jaune d&#39;œuf visible. </li></ol></li><li> Jeter les œufs qui sont fécondés, et retourner les oeufs viables à l&#39;incubateur. Ne laissez pas œufs à l&#39;extérieur de l&#39;incubateur pendant plus de 30 min. Si l&#39;état d&#39;un œuf ne peut être confirmée, marquer, et observer plus tard. </li></ol> 3. Préparation de l&#39;inoculum Virus <ol><li> Diluer virus de la grippe disponibles à environ 10 mars au 10 avril pfu / ml dans du PBS stérile contenant 10 mM HEPES (pH 7,4). Étape critique: Diluer le stock de virus immédiatement avant l&#39;emploi et garder le virus dilué sur la glace tout le temps. </li></ol> 4. Virus de la grippe inoculation par voie allantoïdienne <ol><li> Le jour de l&#39;inoculation du virus (jours 10 ou 11), bougie à nouveau les oeufs et éliminer les embryons morts. Distinguer embryons vivants parla recherche de mouvement à l&#39;intérieur de l&#39;œuf. </li><li> Retirez trois oeufs de l&#39;incubateur, et d&#39;organiser les oeufs dans un support séparé avec le sac d&#39;air vers le haut. </li><li> Marquer l&#39;espace aérien des oeufs avec un marqueur permanent. </li><li> Laver la coquille dessus de l&#39;espace aérien avec 70% d&#39;éthanol. </li><li> Mettez les œufs avec l&#39;air l&#39;espace dans une boîte d&#39;œufs et dans une hotte de biosécurité (BSL-2). </li><li> Utiliser une aiguille de 18G stérile pour percer un petit trou dans la coquille sur le sac d&#39;air de chaque œuf. Veillez à ne pas insérer l&#39;aiguille trop profondément pour éviter poignarder l&#39;embryon ou le jaune. </li><li> Dessinez le virus de la grippe diluée dans une seringue de 1 ml stérile et fixer une aiguille de 22G. </li><li> Avancer avec précaution la seringue avec une aiguille à un angle dans la cavité allantoïdienne 45 °. </li><li> Injecter 0,2 ml du virus dilué de la grippe. </li><li> Répéter l&#39;injection avec les deux autres oeufs, puis jeter la seringue et l&#39;aiguille. </li><li> Sceller le trou avec une goutte de colle à partir d&#39;un pistolet à colle (ou de la cire de paraffine fondue). Be Veillez à ce que la colle ne fuit pas à l&#39;intérieur de l&#39;œuf. </li><li> Placez les oeufs de nouveau dans l&#39;incubateur d&#39;œufs avec l&#39;espace d&#39;air pointé vers le haut. </li><li> Répétez les étapes 4.2 à 4.12 jusqu&#39;à ce que tous les oeufs ont été inoculés. </li></ol> 5. Temps d&#39;incubation <ol><li> Incuber les œufs infectés sans tourner à 37 ° C et ~ 60% d&#39;humidité. </li><li> Choisir le temps d&#39;incubation optimal en fonction du type de virus de la grippe. Incuber la grippe A pendant 48 h; incuber la grippe B et C pendant 72 heures. </li></ol> 6. Virus de la grippe récolte <ol><li> Après la période d&#39;incubation, les oeufs refroidir à 4 ° C pendant un minimum de 4 heures (ou O / N) pour tuer l&#39;embryon et la constriction des vaisseaux sanguins pour réduire le risque de contamination du fluide allantoïque infecté avec le sang. Pour ce faire, pour empêcher les globules rouges de l&#39;absorption du virus, ce qui réduit le rendement. Alternativement, les œufs de refroidissement pendant 30 min à -20 ° C; Mais cela augmente le risque de contamination par le sang. </li><li> Après les oeufs ont été suffisamment refroidi, transférer les oeufs à une hotte de biosécurité. Placez l&#39;oeuf dans un support solide avec le sac d&#39;air vers le haut. Nettoyer la surface de l&#39;œuf avec 70% d&#39;éthanol. </li><li> Utilisez des ciseaux stériles pour ouvrir la coquille dessus de la poche d&#39;air. Retirer la coquille autour de la poche d&#39;air tout en faisant attention de ne pas détruire la membrane chorioallantoïque. </li><li> Ouvrez la membrane chorioallantoïque avec des pinces stériles contondants. Déplacez doucement côté l&#39;embryon et le sac jaune avec une petite spatule ou une cuillère. Prenez soin de ne pas rompre le jaune. </li><li> L&#39;utilisation d&#39;un 1 ml pipette Eppendorf, recueillir soigneusement le fluide allantoïque. Si nécessaire, inclinez l&#39;œuf un peu sur le côté pour recueillir autant de liquide que possible. </li><li> Mélanger le liquide allantoïdien de tous les oeufs dans un tube en plastique conique de 50 ml. Garder les tubes sur la glace en tout temps pendant la récolte de virus. Un rendement d&#39;oeufs 5-10 ml d&#39;un fluide légèrement jaunâtre. </li><li> Spin le fluide allantoïque contenant le virus à 1000 g pendant 10 min à 46; C pour sédimenter les débris. Transférer le liquide clair dans un nouveau tube de 50 ml conservé dans la glace. Éliminer œufs dans un conteneur à déchets BSL-2. </li></ol> 7. Stockage <ol><li> Aliquoter immédiatement le fluide clarifié allantoïdien (par exemple, 50, 100 ou 500 ul aliquotes) et enclenchez-gel dans l&#39;azote liquide. Transfert stocks de virus congelés à -80 ° C congélateur pour le stockage à long terme. </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.

L&#39;influenza cause une grande charge mondiale de morbidité, et le travail se poursuit dans la compréhension de la pathogenèse des lésions pulmonaires 7. Pour la recherche facilitée dans cette maladie mortelle, diverses méthodes ont été développées pour propager le virus de la grippe 6. Ici, nous décrivons une technique pour produire des virus de la grippe dans les oeufs de poulet. L&#39;avantage de cette méthode est qu&#39;il est hautement reproductible et se traduit par de grandes q...

La grippe A continue d&#39;être une menace majeure pour la santé humaine. Ce est une maladie respiratoire potentiellement dévastatrice avec une grande charge mondiale causant jusqu&#39;à 500 000 décès dans le monde chaque année 1. Les virus grippaux sont dans la famille des Orthomyxoviridae et portent huit sens négatif ARN simple brin dans leur génome 1,2. La haute mutabilité (ce est à dire, &quot;dérive&quot; antigénique) du génome viral empêche immunité à long terme. En outre, la grippe est de plus en plus résistantes aux médicaments anti-viraux 3. La pandémie de grippe H1N1 2009 a mis en évidence toutes les questions difficiles associés à la maladie de la grippe (souche pandémique, la résistance anti-virale, la production de vaccins retardé). La formulation de vaccin est déterminé annuellement par l&#39;Organisation mondiale de la santé pour les souches de la grippe les plus susceptibles pathogènes (un pour chaque H1N1, H3N2 et la grippe B) 4. Parce que cette méthode est basée sur la souche de grippe annoncées, les souches pathogènes sont parfois mal identifiés et l&#39;efficacité du vaccin diminue considérablement. En outre, l&#39;apparition de nouvelles souches de la grippe peut contourner ces programmes de prévention en cas de pandémie provoquant la maladie 2. La création d&#39;un vaccin contre la grippe universelle a été difficile à atteindre 5. Par conséquent, la recherche doit se poursuivre dans la compréhension de la pathogenèse des lésions pulmonaires. Pour la recherche en laboratoire de l&#39;installation, plusieurs méthodes ont été développées pour l&#39;isolement du virus et la propagation 6. Virus de la grippe humaine peuvent être amplifiés dans une variété de substrats de cellules de mammifères. Cependant, générant virus de titre élevé en grandes quantités sont mieux atteints dans des oeufs embryonnés. Le protocole suivant décrit une technique pour la grippe A propagation du virus et le stockage de stocks de virus existants.

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	<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

L&#39;infection grippale est associée à environ 36 000 morts et plus de 200 000 hospitalisations chaque année aux États-Unis. L&#39;émergence continue de nouvelles souches de virus de la grippe en raison de la mutation et de réassortiment complique le contrôle du virus et nécessite le développement permanent de nouveaux médicaments et vaccins. L&#39;étude de la grippe en laboratoire nécessite une méthode fiable et rentable pour la propagation du virus. Ici, un protocole complet est fourni pour la grippe A propagation des virus dans des œufs de poule fertiles, ce qui donne toujours des stocks viraux à titre élevé. En bref, le sérum (SPF) des œufs de poule fécondés exempts d&#39;agents pathogènes sont incubées à 37 ° C et 55-60% d&#39;humidité pendant 10 à 11 jours. Sur cette période, le développement de l&#39;embryon peut être facilement contrôlé en utilisant un candler d&#39;oeuf. Virus inoculation est effectuée par injection de stock de virus dans la cavité allantoïque en utilisant une aiguille. Après 2 jours d&#39;incubation à 37 ° C, les oeufs sontréfrigérés pendant au moins 4 heures à 4 ° C. La coquille d&#39;oeuf au-dessus de la poche d&#39;air et la membrane chorio-allantoïde sont ensuite ouvert avec précaution, et le fluide allantoïque contenant le virus est récolté. Le liquide est éliminé de débris par centrifugation, aliquoté et transféré à -80 ° C pour stockage à long terme. La grande quantité (5-10 ml de liquide par oeuf contenant virus) titre et de haute virus qui est habituellement obtenue avec ce protocole a fait l&#39;utilisation d&#39;œufs pour la préparation de virus notre méthode favorable, en particulier pour les études in vitro qui nécessitent de grandes quantités de virus où des dosages élevés de la même stock de virus sont nécessaires.

Plusieurs méthodes ont été développées pour titrer le virus de la grippe. Une considération lors du choix d&#39;une méthode appropriée est que certains déterminer particules totales, indépendamment de la viabilité (par exemple, le dosage de l&#39;hémagglutinine) alors que d&#39;autres sont basés sur l&#39;infectiosité, qui permettra d&#39;évaluer virions viables (par exemple, essai de plaque) 6. Ici, le titre viral de liquide allantoïdien recueilli à partir d&#39;œufs de po...

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.

Virus de la grippe Propagation dans embryonnés de poulet Oeufs

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

42.7K vues.  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.

Vidéo: 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.

Génération de virus recombinants de la grippe des ADN plasmidique

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

Recherche

Immunologie et infection

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.

Méthode de détection à haut débit pour les virus de la grippe

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.

Purification par affinité des complexes virus de la grippe ribonucléoprotéiques de la chromatine des cellules infectées

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.

Propagation d&#39;<em&gt; Virus-01 Homalodisca coagulata</em&gt; Par<em&gt; Homalodisca vitripennis</em&gt; Culture Cellulaire

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

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.

Lingettes nasal pour la grippe détection d&#39;un virus et l&#39;isolement des porcs

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.

Mesurer l&#39;attachement et l&#39;internalisation du virus grippal A dans les cellules A549 par cytométrie en flux

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.

Neutrophiles Isolement et analyse pour déterminer leur rôle dans le lymphome cellulaire Sensibilité aux agents thérapeutiques

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 désassemblage du virus grippal A capsides par Centrifugation Gradient

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.

Études de Virus influenza de type A dans un modèle murin d’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 ...

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.

Génération des variantes d’évasion de neutraliser les anticorps monoclonaux Influenza Virus

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 ...

Methods Article

Virus de la grippe Propagation dans embryonnés de poulet Oeufs