Les auteurs tiennent à remercier Nada Naffakh et Marie-Anne Rameix-Welti (Institut Pasteur) pour le virus de RWSN-PB2-Strep.

<html> Un organigramme schématique du protocole est indiqué dans la Fig. 1 et une table de réactifs est présenté ci-dessous. 1. Infection (16 - 24 h) <ol><li> Semences sur des cellules HeLa en 5 150 mm en plastique des boîtes de culture de cellules dans un milieu de Eagle modifié par Dulbecco (DMEM)-moyen-élevé de glucose, de telle sorte qu&#39;ils atteindront environ 80% de confluence le jour suivant (environ 5 x 10 8 cellules). Il est important que les cellules ne parviennent pas à 100% de confluence. </li><li> Le lendemain, diluer le Strep-étiqueté stock de virus de la grippe dans un tampon phosphate salin (PBS) contenant 0,3% de sérumalbumine bovine (BSA) à une multiplicité d&#39;infection de 3. </li><li> Retirez le milieu de croissance à partir des cellules, laver une fois avec du PBS, et ajouter 10 ml de PBS contenant le virus à des cellules. Incuber pendant 1 h à température ambiante. </li><li> Remplacez le milieu d&#39;infection avec du DMEM-hyperglycémie, et poursuivre l&#39;incubation à 37 ° C. </li></ol>&quot;&gt; La durée d&#39;incubation dépend de la phase de l&#39;infection à étudier (voir Discussion). 2. Fractionnement subcellulaire (3 h) <ol><li> Laver les cellules une fois avec du PBS froid et de suspendre les cellules dans du PBS froid avec une raclette en caoutchouc et le transfert d&#39;un tube conique de 50 ml. </li><li> Sédimenter les cellules dans une centrifugeuse de table pendant 5 min à 1000 tours par minute et puis aspirer PBS. </li><li> Resuspendre culot cellulaire dans 10 ml de tampon de saccharose à froid (10 mM HEPES pH 7,9, KCl 10 mM, 2 mg d&#39;acétate de mM (MgOAc), 3 mM CaCl 2, 340 mM de saccharose, 1 mM de dithiothréitol (DTT), 1% mélange inhibiteur de la protéase) . Après la suspension, passer des cellules à travers un embout de pipette 200 pi attaché à une pipette en plastique de 10 ml de sérum de perturber amas de cellules. </li><li> Incuber 10 min sur la glace. Inverser doucement périodiquement pour remettre en suspension les cellules. </li><li> Ajouter de Nonidet P-40 (NP-40) à une concentration finale de 0,5% et à vortex à grande vitesse pendant 15 secondes. Immédiatement centrifuger pendant 10 min à 3500 xg à 4 ° C dans un centrifuge de tablege. </li><li> Enlever le surnageant et conserver à 4 ° C. Il s&#39;agit de la fraction cytoplasmique. Le culot doit être plus petite et plus blanche que la cellule d&#39;origine culot. Toutes les fractions peuvent être conservés pendant au moins 4 h à 4 ° C. </li><li> Laver le culot dans 5 ml de tampon de saccharose à froid, re-centrifugation à l&#39;étape 2.5 et jeter le surnageant. </li><li> Reprendre le culot (contenant des noyaux) dans 1,5 ml tampon d&#39;extraction à froid nucléoplasmique (HEPES 50 mM pH 7,9, 150 mM, 1,5 mM KOAc MgCl 2, 0,1% de NP-40, DTT 1 mM, 1% mélange inhibiteur de la protéase). </li><li> Transférer dans un réfrigérés, tout en verre de 4 ml homogénéisateur Dounce avec un pilon hermétique et homogénéiser les noyaux avec 20 coups. Éviter de faire mousser pendant l&#39;homogénéisation. La résistance devrait augmenter après environ 10 coups. Homogénéisation efficace peut être confirmé sous un microscope à contraste de phase. </li><li> Transfert des noyaux homogénéisé dans un tube de 1,5 ml et incuber 20 min sur une roue tournant à 4 °C. </li><li> Centrifuger à 16000 x g dans une microcentrifugeuse à 4 ° C pendant 10 min. </li><li> Enlever le surnageant et conserver à 4 ° C. Il s&#39;agit de la fraction nucléoplasmique. </li><li> Resuspendre le culot dans 1,5 ml de tampon de digestion (50 mM HEPES pH 7,9, NaCl 10 mM, 1,5 mM MgCl 2, 1 mM DTT, 1% de mélange inhibiteur de la protéase, 100 U / ml Benzonase (pour l&#39;analyse d&#39;ARN, substitut avec 20 U / ml de RNase sans DNase I)) de pré-chauffé à 37 ° C, et incuber 10 min à 37 ° C. </li><li> Ajouter 42 ul de NaCl 5 M (pour une concentration finale de 150 mM de NaCl) et incuber 20 min encore à 4 ° C. </li><li> Centrifuger à 16000 x g dans une microcentrifugeuse à 4 ° C pendant 10 min. </li><li> Enlever le surnageant et conserver à 4 ° C. Il s&#39;agit de la fraction CH150. </li><li> Resuspendre le culot dans 1,5 ml à froid de haute-sel tampon (50 mM HEPES pH 7,9, NaCl 500 mM, 1,5 mM MgCl 2, 0,1% de NP-40, DTT 1 mM, 1% mélange inhibiteur de la protéase) et incuber 20 min sur la glace. </li><li> Centrifuger à 16000 x g &lt;/ Em&gt; dans une microcentrifugeuse à 4 ° C pendant 10 min. </li><li> Enlever le surnageant et conserver à 4 ° C. Il s&#39;agit de la fraction ch500. </li></ol> 3. Strep-tag de purification (2 h) <ol><li> Ajouter 300 pi de 5 M de NaCl à la fraction cytoplasmique (pour une concentration finale de 150 mM de NaCl). </li><li> Aliquote de 100 ul de billes de Sépharose emballés Strep-Tactin par fraction dans un tube de 15 ml conique. </li><li> Ajouter 10 volumes de tampon de lavage (Strep 20 mM HEPES pH 7,9, NaCl 150 mM (500 mM de NaCl pour ch500 échantillons), EDTA 1 mM) à des billes, inverser le tube pour mélanger, et centrifuger 5 min à 1000 x g dans une table centrifuger. </li><li> Jeter le surnageant, répétez l&#39;étape 3.3 deux fois, puis reprendre le culot perle dans un volume égal de tampon de lavage Strep. </li><li> Ajouter 200 ul de Strep-lavé Tactin suspension talon à chaque fraction (en 1,5 ml microtubes pour nuceloplasmic, des CH150, et ch500 fractions et un tube de 15 ml conique pour la fraction cytoplasmique). </li><li> Tournertubes 1h à 4 ° C sur une roue en rotation. </li><li> Centrifuger les tubes 1 min à 1000 x g à 4 ° C. Rejeter le surnageant. </li><li> Ajouter 1 ml de tampon de lavage à froid Strep dans chaque tube. Transfert perles de tube de 15 ml (fraction cytoplasmique) pour tube de 1,5 ml. Laver tout 1 min tubes à 4 ° C, centrifugation, après que l&#39;étape 3.7. Répétez cette étape de lavage à deux reprises. </li><li> Après la dernière étape de lavage, enlever toutes les traces de tampon de lavage en utilisant un embout de pipette à plat. Ajouter 100 ul de tampon d&#39;élution (tampon de lavage contenant 2,5 Strep desthiobiotine mM) et mélanger délicatement. </li><li> Incuber 15 min sur la glace. Mélanger délicatement de temps en temps des perles en tapant sur le fond des tubes. </li><li> Tubes à centrifuger 1 min à 1000 x g à 4 ° C dans microcentrifugeuse. Recueillir le surnageant contenant éluées Strep-tag vRNPs. </li></ol> 4. Les résultats représentatifs L&#39;efficacité du fractionnement est mieux jugée par analyse par Western blot des lysats de fractionnement à l&#39;aide ANTIBodies spécifiques pour les marqueurs subcellulaires (Fig. 2). En particulier, réussie fractionnement subnucléaire doit afficher peu ou pas de l&#39;ARN polymérase II cellulaire (Pol II) dans le nucléoplasmique, ou soluble fraction nucléaire 4, et le plus doit être extrait avec NaCl 150 mM 5. Une dégradation de Pol II est également reproductible observée dans les cellules infectées par le virus de la grippe par rapport aux cellules non infectées, conformément à la littérature 6. Purification de vRNPs est mieux jugée par l&#39;argent ou la coloration au bleu de Coomassie, comme indiqué pour un éluat cytoplasmique dans la Fig. 3. Dans notre expérience, la plupart Strep-PB2 est purifié dans le cadre d&#39;un vRNP complètement formé, c.-à-polymérase peu soluble est capturé. Cela se reflète dans la grande NP: PA/PB1/PB2 ratio observé par l&#39;argent ou la coloration au bleu de Coomassie. Un ratio plus faible indique une contamination par des ARNases tampon, que plusieurs ARNases ubiquitaires (tels que la RNase A) sont connus pour cliver l&#39;ARN viral, de telle façon que de dissociermangé la polymérase du NP multimères 7. Fait intéressant, le traitement avec Benzonase seul, tandis que suffisante pour digérer l&#39;ARN viral, semble avoir aucun effet sur les ratios stoechiométriques de protéines vRNP. Bien que nous ne pouvons pas expliquer ce phénomène, nous avons observé des perturbations de la vRNPs après digestion avec d&#39;autres RNases (données non présentées), suggérant que Benzonase peut laisser certaines régions structurellement importantes d&#39;ARN intact. Après purification de vRNPs à partir du cytoplasme et nucléoplasme (effectuée sans digestion à la nucléase), 8 bandes faibles, mais discrète à poids moléculaire élevé peut être observé par coloration à l&#39;argent, qui correspondent aux poids moléculaire prédit des 8 segments génomiques de la grippe (voir réf. 3 ). Les quantités relatives de vRNPs purifiés à partir de chaque fraction à 9 heures après l&#39;infection sont présentés dans la figure. 4. La répartition des vRNPs varie au cours de l&#39;infection, avec la plus forte accumulation dans la fraction CH150 à des moments au début, etune accumulation accrue dans le nucléoplasme et le cytoplasme à des moments tardifs. <img src="/files/ftp_upload/4028/4028fig1.jpg" alt="Figure 1"/> Diagramme Figure 1. Du fractionnement subcellulaire. Adapté de la réf. 3. <img src="/files/ftp_upload/4028/4028fig2.jpg" alt="Figure 2"/> Figure 2. Analyse par Western blot des fractions subcellulaires de la grippe les cellules infectées. 10 9 cellules HeLa ont été infectées par la grippe WSN souche du virus pour 9 h avant le fractionnement subcellulaire. Des quantités égales de protéines totales ont été chargés dans chaque couloir et analysées en utilisant les anticorps indiqués. L&#39;ARN polymérase II (Pol II) a été détecté en utilisant clone 8WG16, qui reconnaît toutes les formes du domaine C-terminal (la bande hypophosphorylé est le plus important). Adapté de la réf. 3. <img src="/files/ftp_upload/4028/4028fig3.jpg" alt="Figure 3"/> Figure 3.Argent analyse tache de purifier vRNPs. Des cellules HeLa sont infectées par le RWSN (comme un contrôle négatif) ou RWSN-PB2-Strep pour 9 h, suivie d&#39;une purification de la fraction Strep cytoplasmique. Astérisque indique les bandes qui ne sont pas visibles après digestion par la RNase. Adapté de la réf. 3. <img src="/files/ftp_upload/4028/4028fig4.jpg" alt="Figure 4"/> Figure 4. Argent analyse tache de vRNPs purifiés à partir de différentes fractions cellulaires. 10 9 cellules HeLa ont été infectées par le RWSN-PB2-Strep ou RWSN pour 9 h suivie d&#39;un fractionnement subcellulaire et la purification Strep. Des quantités égales d&#39;éluat de chaque fraction ont été chargés. Adapté de la réf. 3.

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	<li>Engelhardt, O. G., Smith, M., Fodor, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+influenza+A+virus+RNA-dependent+RNA+polymerase+with+cellular+RNA+polymerase+II.">Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II.</a> J. Virol. 79, 5812-5812 (2005).</li><li>Garcia-Robles, I., Akarsu, H., Müller, C. W., Ruigrok, R., Baudin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+influenza+virus+proteins+with+nucleosomes.">Interaction of influenza virus proteins with nucleosomes.</a> Virology. 332, 329 (2005).</li><li>Chase, G. P., Rameix-Welti, M. A., Zvirbilene, A., Zvirblis, G., Götz, V., Wolff, T., Naffakh, N., Schwemmle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus+ribonucleoprotein+complexes+gain+preferential+access+to+cellular+export+machinery+through+chromatin+targeting.">Influenza virus ribonucleoprotein complexes gain preferential access to cellular export machinery through chromatin targeting.</a> PLoS Pathogens. 7, e1002187 (2011).</li><li>Kimura, H., Tao, Y., Roeder, R. G., Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+RNA+polymerase+II+and+its+transcription+factors+in+an+HeLa+cell:+little+soluble+holoenzyme+but+significant+amounts+of+polymerases+attached+to+the+nuclear+substructure.">Quantitation of RNA polymerase II and its transcription factors in an HeLa cell: little soluble holoenzyme but significant amounts of polymerases attached to the nuclear substructure.</a> Mol. Cell. Biol. 19, 5383-5383 (1999).</li><li>Henikoff, S., Henikoff, J. G., Sakai, A., Loeb, G. B., Ahmad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+profiling+of+salt+fractions+maps+physical+properties+of+chromatin.">Genome-wide profiling of salt fractions maps physical properties of chromatin.</a> Genome Res. 19, 460 (2009).</li><li>Rodriguez, A., Perez-Gonzalez, A., Nieto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus+infection+causes+specific+degradation+of+the+largest+subunit+of+cellular+RNA+polymerase+II.">Influenza virus infection causes specific degradation of the largest subunit of cellular RNA polymerase II.</a> J. Virol. 81, 5315-5315 (2007).</li><li>Ye, Z., Liu, T., Offringa, D. P., McInnis, J., Levandowski, R. 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Pas de conflits d&#39;intérêt déclarés.

Alors que de nombreuses études ont récemment identifié des protéines individuelles ou des réseaux cellulaires impliqués dans l&#39;infection du virus de la grippe 8, la signification fonctionnelle de la majorité de ces interactions demeure incertaine. Compte tenu de la dépendance absolue de la chromatine à base de fonctions pour la grippe virus à ARN de synthèse et de la nature complexe biophysique et biochimique du noyau 9, de nouvelles techniques seront nécessaires pour élucider ces ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> DMEM-hyperglycémie </td><td> Gibco </td><td> 11965-092 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A9418 </td><td></td></tr><tr><td> Inhibiteur de la protéase Mix G </td><td> Serva </td><td> 39101 </td><td></td></tr><tr><td> Benzonase nucléase </td><td> Novagen </td><td> 71206 </td><td> 25 U / pl </td></tr><tr><td> DNase I, sans RNase </td><td> ThermoScientific </td><td> EN0523 </td><td> 50 U / pl </td></tr><tr><td> Dounce homogénéisateur </td><td> Wheaton </td><td> 432-1271 </td><td> Usage de type &quot;B&quot; pilon </td></tr><tr><td> Strep-Tactin Sepharose </td><td> IBA GmbH </td><td> 2-1201-025 </td><td> 50% en format de colonne de suspension peut également être utilisé </td></tr><tr><td> Desthiobiotine </td><td> IBAGmbH </td><td> 2-1000-002 </td><td></td></tr></tbody></table>

affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells

Comme tous les virus à ARN à brin négatif, le génome de virus de la grippe est conditionnée sous forme de complexes ribonucléoprotéiques virales (vRNP), dans lequel le génome simple brin est encapsidé par la nucléoprotéine (NP), et associé avec le complexe polymérase trimère constitué de la PA, PB1, PB2 et sous-unités. Cependant, contrairement à la plupart des virus à ARN, virus de la grippe virale effectuer la synthèse d&#39;ARN dans le noyau des cellules infectées. Fait intéressant, la synthèse d&#39;ARNm viral utilise cellulaires pré-ARNm comme amorces, et il a été proposé que ce processus se déroule sur la chromatine 1. Interactions entre la polymérase virale et l&#39;hôte l&#39;ARN polymérase II, ainsi qu&#39;entre le NP et les nucléosomes d&#39;accueil ont également été caractérisées 1,2. Récemment, la génération de virus grippaux recombinants codant pour une seule Strep-Tag génétiquement fusionnée à l&#39;extrémité C-terminale de la sous-unité PB2 de la polymérase virale (RWSN-PB2-streptococcique 3) a êtrefr décrit. Ces virus recombinants permettre la purification de complexes contenant PB2, y compris, à partir de vRNPs cellules infectées. Pour obtenir purifié vRNPs, des cultures de cellules sont infectées, et vRNPs sont purifiés par affinité à partir de lysats dérivés de ces cellules. Toutefois, les procédures de lyse utilisées à ce jour ont été basées sur une seule étape de lyse détergent, qui, malgré la présence d&#39;une nucléase générale, souvent d&#39;extraire la chromatine liée seul matériau inefficace. Notre travail préliminaire suggère qu&#39;une grande partie de vRNPs nucléaires n&#39;ont pas été extraites lors de la lyse cellulaire traditionnel, et ne pouvait donc pas être purifié par affinité. Pour augmenter cette efficacité de l&#39;extraction, et de séparer la chromatine liée de la non-chromatine assortis vRNPs nucléaires, nous avons adapté un protocole étape par étape d&#39;extraction subcellulaire à la grippe les cellules infectées. En bref, cette procédure sépare tout d&#39;abord les noyaux de la cellule, puis extraits solubles des protéines nucléaires (appelée ici &quot;nucléoplasmique&quot; fraction).Le reste insoluble nucléaire est ensuite digéré par Benzonase, un ADN non spécifique / ARN nucléase, suivie de deux étapes d&#39;extraction de sel: la première utilisation de NaCl 150 mM (appelé &quot;CH150&quot;), puis 500 mM de NaCl (&quot;ch500&quot;) (Fig. 1 ). Ces étapes d&#39;extraction de sel ont été choisis en fonction de notre observation selon laquelle 500 mM de NaCl était suffisante pour solubiliser plus de 85% de vRNPs nucléaires tout en permettant la liaison de vRNPs marqués à la matrice d&#39;affinité. Après fractionnement subcellulaire des cellules infectées, il est possible de purifier par affinité vRNPs PB2-étiqueté à partir de chaque fraction individuelle et d&#39;analyser leurs protéines et d&#39;ARN en utilisant des composants Western Blot et par extension d&#39;amorce, respectivement. Récemment, nous avons utilisé cette méthode pour découvrir que l&#39;exportation vRNP forme des complexes au cours de points de retard après l&#39;infection sur la fraction la chromatine extraite avec 500 mM de NaCl (ch500) 3.

Watch this Scientific Journal Video about Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells at JoVE.com

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

Les virus grippaux répliquer leur génome à ARN en association avec la cellule hôte chromatine. Ici, nous présentons une méthode pour purifier intactes complexes ribonucléoprotéiques virales de la chromatine des cellules infectées. Purifiée des complexes viraux peuvent être analysés à la fois par Western blot et par extension d&#39;amorce de la teneur en protéines et d&#39;ARN, respectivement.

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 ...

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Immunology and Infection

13.0K vues.  Universitätsklinikum Freiburg. Les virus grippaux répliquer leur génome à ARN en association avec la cellule hôte chromatine. Ici, nous présentons une méthode pour purifier intactes complexes ribonucléoprotéiques virales de la chromatine des cellules infectées. Purifiée des complexes viraux peuvent être analysés à la fois par Western blot et par extension d'amorce de la teneur en protéines et d'ARN, respectivement.

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

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 ...

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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, ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

Purification de vacuoles agent pathogène à partir<em&gt; Legionella</em&gt; Infectées par les phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

L&#39;expression de l&#39;hémagglutinine recombinante fonctionnelle et la neuraminidase du virus de la nouvelle grippe H7N9 à l&#39;aide du système d&#39;expression baculovirus

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

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.

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 ...

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-immunoprécipitation de la protéine souris Mx1 avec le virus Influenza A nucléoprotéine

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.

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 ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Analyse phénotypique et fonctionnelle des cellules T activées Regulatory Isolés de chorioméningite lymphocytaire Souris infectées par le virus chroniques

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

Détermination de biofilm Initiation sur les cellules infectées par le virus par les bactéries et les champignons

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

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 ...