Nous remercions David Wang, Anatoly Urisman, Amy Kistler, Kael Fischer, Patrick Tang, et Alexander Greninger pour le développement initial et l&#39;optimisation du protocole Virochip. Ce travail est soutenu par une subvention du NIH K08 et Abbott Discovery Award (pour CC) et le Howard Hughes Medical Institute (au JLD).

Les étapes de l&#39;essai Virochip incluent (1) l&#39;extraction des acides nucléiques à partir d&#39;échantillons cliniques, (2) la transcription inverse de l&#39;ARN extrait et 2 ème brin synthèse de l&#39;ADNc, (3) L&#39;amplification par PCR de l&#39;ADNc amorcés au hasard, (3) l&#39;incorporation colorant fluorescent Cy3 , (4) l&#39;hybridation des matériaux étiquetés pour la puce Virochip, et (5) la numérisation et l&#39;analyse (figure 1). Le protocole présenté ci-dessous montrent l&#39;utilisation de l&#39;essai sur un échantillon Virochip écouvillon nasal d&#39;un enfant avec un syndrome grippal. Séquences des amorces Amorce A 5&#39;-GTTTCCCAGTCACGATA-(N 9) -3 &#39; Primer B 5&#39;-GTTTCCCAGTCACGATA-3 &#39; 1. Extraction des acides nucléiques <ol><li> Extraction des acides nucléiques à partir d&#39;échantillons cliniques doivent être effectuées sous le niveau de biosécurité 2 (BSL-2) ou plus des conditions, comme ces échantillons sont potentiellement infectieux. </li><li> L&#39;échantillon écouvillon nasal est transporté dans un milieu de détention virale. Partie aliquote de 200 ul de l&#39;échantillon dans un tube de 1,5 ml Eppendorf. </li><li> En option: passage de l&#39;échantillon à travers un filtre de 0,22 um (pour purifier des particules virales) </li><li> En option: l&#39;échantillon prétraiter à la DNAse (pour dégrader l&#39;ADN génomique d&#39;hôte et de purifier pour les aires protégées, de l&#39;acide nucléique viral encapsidé) en utilisant le kit turbo DNAse (Ambion) par les instructions du fabricant </li><li> Extraire l&#39;échantillon en utilisant l&#39;ARN viral Zymo ZR Extraction Kit ou Kit Qiagen Virus Ultrasens selon les instructions du fabricant. </li></ol> 2. Transcription inverse et 2 ème brin synthèse de l&#39;ADNc («Round A») <ol><li> Ajouter 1 UL 40 pmol / uL Primer A à 4 pl extrait l&#39;ARN. Chauffer à 65 ° C pendant 5 min dans un thermocycleur (par exemple GeneAmp PCR System 9700), puis laisser refroidir à température ambiante température x 5 min. </li><li> Ajouter 5 ul d&#39;un mélange maître comprenant 2 uL de tampon RT 5X, 1 pi 12,5 mM dNTP, 1 uL d&#39;eau, 0,5 pi DTT 0,1 M et 0,5 uL SSIII RT (Invitrogen). Incuber à 42 ° C x 60 minutes. </li><li> Dans un thermocycleur programmable, de la chaleur à 94 ° C x 2 minutes (pour annuler la réaction de la transcriptase inverse), laisser refroidir à 10 ° C, centrifuger le pouls, puis ajouter 5 mélangez Sequenase uL composé d&#39;une mémoire tampon Sequenase uL 5X, 3,8 pi d&#39;eau, et 0,15 uL Sequenase (USB Corporation). Pour 2 ème synthèse des brins, montée en puissance de 10 ° C à 37 ° C en 8 min, tenir 8 min, puis chauffer à 94 ° C x 2 minutes (pour inactiver Sequenase) et laisser refroidir à 10 ° C (en attente). </li><li> En option: Rd Un échantillon d&#39;ADNc peut être conservé à ce point à -20 ° C. </li></ol> 3. Amplification par PCR de hasard apprêté ADNc («Ronde B&quot;) <ol><li> Ajouter 5 uL de Rd Un échantillon de 45 ul d&#39;un mélange maître constitué de 5 tampon PCR 10X uL, 1 pi 12,5 mM dNTP, 1 ul amorce B 100 pmol / ul, 1 ul Klentaq LA (Sigma), et 37 pi d&#39;eau. </li><li> Exécuter protocole de PCR comme suit: 94 ° C, 2 min → (25 cycles de 94 ° C, 30 s / 50 ° C, 45 s / 72 ° C, 1 mn) → 72 ° C, 5 min → 10 ° C ( attente) </li><li> Vérifier l&#39;échantillon B Rd sur un gel d&#39;électrophorèse d&#39;agarose 1,5%. Un frottis d&#39;environ 200 - 1000 pb devrait être visualisées (figure 2A) </li></ol> 4. Cy3 Incorporation colorant fluorescent <ol><li> En option: un autre échantillon peut être étiqueté avec le colorant fluorescent et Cy5 hybride à la puce même. </li><li> Pour intégrer aa-dNTP, ajouter 5 uL de Rd échantillon B à 45 uL d&#39;un master mix composé de 5 tampon PCR 10X uL, 1 uL 12,5 mm AA-dNTP (Invitrogen), 1 uL 100 apprêt pmol / ul B, 1, 1 uL Klentaq LA (Sigma), et 37 d&#39;eau ul. Exécuter protocole de PCR comme suit: 94 ° C, 2 min → (15 cycles de 94 ° C, 30 s / 50 ° C, 45 s / 72 ° C, 1 mn) → 72 ° C, 5 min → 10 ° C ( attente) </li><li> Nettoyer l&#39;échantillon Rd C avec l&#39;ADN propre et Concentrateur-5 Kit (Zymo Research) par les instructions du fabricant. Eluer dans 10 ul. </li><li> Ajouter 1 uL de bicarbonate de 1M (Sigma) et 1 ul Cy5 (GE Healthcare) à l&#39;échantillon Rd C. </li><li> Incuber x 1 heure dans l&#39;obscurité. </li><li> Nettoyer l&#39;échantillon Cy3-étiquetés avec l&#39;ADN propre et Concentrateur-5 Kit (Zymo Research) par les instructions du fabricant. Eluer dans 12 uL. </li></ol> 5. Hybridation d&#39;Virochip Microarray <ol><li> En option: utiliser 1,5 uL de vérifier la concentration d&#39;ADNc et la quantité de colorant incorporé sur un spectrophotomètre Nanodrop (pour la normalisation des échantillons) </li><li> Dans un tube de la bande de PCR ajouter 1 pi de tampon de fragmentation 25X, 5 pl de tampon 5X blocage, 9,5 ul (ou le montant de normaliser) de Cy3-étiquetés de l&#39;échantillon, et l&#39;eau pour un volume total de 25 uL (Agilent) </li><li> Ajouter 25 uL de tampon d&#39;hybridation 2X Gex (Agilent). Centrifuger brièvement pour éliminer les bulles. </li><li> Charge à 40 uL de l&#39;échantillon sur la glissière du joint. </li><li> Placez-imprimés Agilent array Virochip (Université de Californiune, San Francisco / Agilent) (côté marqué &quot;Agilent&quot; vers le bas) sur le dessus de la glissière joint et fixez la vis serrée. </li><li> Hybrider nuit dans le four à 65 ° C, réglage de la vitesse à 10 tpm. </li><li> Pour laver les tableaux, préchauffer le tampon de 2 (Agilent) à 37 ° C. Prenez part sous joint sandwichs glisser / tableau dans un tampon de 1 (Agilent). Laver dans le tampon de 1 x 1 min. Laver dans le tampon préchauffé 2 x 1 min. Retirer lentement glisser du tampon 2, en prenant soin d&#39;éviter les bulles (utilisation Kimwipe pour évacuer l&#39;humidité supplémentaire en prenant soin d&#39;éviter de toucher directement l&#39;aspect actif de la matrice) </li><li> Placez diapositives dans le support de diapositives et insérer dans scanneur tableau. </li></ol> 6. Virochip balayage et d&#39;analyse <ol><li> Balayage Cy3 étiquetés tableau à 5 um um ou 2 selon le protocole de l&#39;instrument de balayage standard (figure 2B). </li><li> Analyser microarrays Virochip par inspection visuelle, l&#39;analyse typologique, ou le programme d&#39;analyse automatisée Virochip, E-Predict 11. En chacun de ces trois méthodes, le virus de l&#39;influenza est facilement détectée dans l&#39;échantillon écouvillon nasal (à partir d&#39;un enfant avec syndrome grippal) (figure 2C) </li></ol> 7. Les résultats représentatifs: Lorsque le protocole est fait correctement, un frottis devraient être visibles sur l&#39;électrophorèse sur gel d&#39;agarose après l&#39;étape Rd B (figure 2A). Les biopuces Virochip sur la plateforme Agilent sera difficile d&#39;analyser visuellement (figure 2B), mais si un virus est présent, il devrait être facilement identifiés par l&#39;inspection visuelle, l&#39;analyse typologique, et / ou E-Predict (figure 1C). Les échantillons peuvent être dopés avec des quantités mesurées de l&#39;acide nucléique d&#39;un virus connu (par exemple le virus de la mosaïque du tabac; bactériophage MS2) pour servir de contrôle positif pour le dosage. <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="Figure 1" /> Figure 1. Schéma des biopuces Virochip traitement et d&#39;analyse. Extraite d&#39;acides nucléiques à partir d&#39;échantillons cliniques sont amplifiés au hasard, marqués avec des colorants fluorescents, et hybridées à la puce Virochip. Microarrays sont numérisées à une résolution de 2 um et analysées en utilisant une variété d&#39;outils informatiques, y compris E-Predict. <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="Figure 2" /> Figure 2. Étapes dans le dosage de Virochip Après amplification par PCR aléatoire, un frottis de 200 -. 1000 pb peut être visualisé par électrophorèse sur gel (A) (B) Trois puces Virochip sur les 8 tableaux / lame de verre sont présentés, avec une petite région. d&#39;une biopuce soufflé dans l&#39;encart dans le coin inférieur droit. (C) automatisé biopuces d&#39;analyse virale en utilisant E-Predict révélant la présence de virus influenza A dans l&#39;échantillon clinique. Le score de similarité élevé (s = 0,55) correspond à une p-valeur qui est proche de zéro, ce qui rend cette une prédiction extrêmement important.

<ol>
	<li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+and+genotyping+of+viral+pathogens.">Microarray-based detection and genotyping of viral pathogens.</a> Proc Natl Acad Sci U S A. 99, 15687-15692 (2002).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+a+critical+respiratory+illness+caused+by+human+metapneumovirus+by+use+of+a+pan-virus+microarray.">Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.</a> J Clin Microbiol. 45, 2340-2343 (2007).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+detection+of+human+parainfluenzavirus+4+infection+associated+with+respiratory+failure+in+an+immunocompetent+adult.">Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.</a> Clin Infect Dis. 43, e71-e76 (2006).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+DNA+microarrays+for+detection+of+viruses+in+acute+respiratory+tract+infections+in+children.">Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.</a> J Pediatr. 153, 76-83 (2008).</li><li>Kistler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-viral+screening+of+respiratory+tract+infections+in+adults+with+and+without+asthma+reveals+unexpected+human+coronavirus+and+human+rhinovirus+diversity.">Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity.</a> J Infect Dis. 196, 817-825 (2007).</li><li>Rota, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+coronavirus+associated+with+severe+acute+respiratory+syndrome.">Characterization of a novel coronavirus associated with severe acute respiratory syndrome.</a> Science. 300, 1394-1399 (2003).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+discovery+and+sequence+recovery+using+DNA+microarrays.">Viral discovery and sequence recovery using DNA microarrays.</a> PLoS Biol. 1, e2-e2 (2003).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+Gammaretrovirus+in+prostate+tumors+of+patients+homozygous+for+R462Q+RNASEL+variant.">Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.</a> PLoS Pathog. 2, 25-25 (2006).</li><li>Kistler, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+divergent+avian+bornaviruses+from+cases+of+proventricular+dilatation+disease:+identification+of+a+candidate+etiologic+agent.">Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.</a> Virol J. 5, e25-e25 (2008).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardioviruses+related+to+Theiler's+murine+encephalomyelitis+virus+in+human+infections.">Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections.</a> Proc Natl Acad Sci U S A. 105, 14124-14129 (2008).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Predict:+a+computational+strategy+for+species+identification+based+on+observed+DNA+microarray+hybridization+patterns.">E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.</a> Genome Biol. 6, R78-R78 (2005).</li></ol>

Le protocole Virochip comme décrit ici est complexe et nécessite un technicien de recherche minutieux et compétent. Concentrations de réactifs et des conditions correctes pour la PCR, l&#39;étiquetage, et l&#39;hybridation sont critiques. Le protocole Virochip peut être facilement modifié pour accueillir l&#39;analyse des tissus malades par l&#39;utilisation d&#39;une méthode d&#39;extraction des tissus tels que Trizol (Invitrogen) pour l&#39;extraction des acides nucléiques. Les sondes peuvent être ajoutées ou supprimées au besoin d&#39;obtenir le spectre désiré et l&#39;étendue de la couverture, par exemple, des sondes pour la détection de cibles non viraux tels que les bactéries et les champignons peuvent être conçus et ajouté «à la volée&quot;. Certaines applications de l&#39;analyse Virochip actuellement en développement comprennent large spectre diagnostic des maladies virales dans les laboratoires cliniques, l&#39;investigation des flambées, le dépistage pureté des médicaments et des vaccins, et la découverte de nouveaux agents pathogènes viraux.

<table border="1"><tbody><tr><th> Nom de réactif / matériel </th><th> Nom de réactif / matériel </th></tr><tr><td> Primera </td><td> 5&#39;-GTTTCCCAGTCACGATA-(N9) -3 &#39; </td></tr><tr><td> Primer B </td><td> 5&#39;-GTTTCCCAGTCACGATA-3 &#39; </td></tr><tr><td rowspan="5"> RT Mix Master (5 uL) </td><td> 2 ul de tampon 5X RT </td></tr><tr><td> 1 ul 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 uL d&#39;eau </td></tr><tr><td> 0,5 ul DTT 0,1 M </td></tr><tr><td> 0,5 ul SSIII RT (Invitrogen) </td></tr><tr><td rowspan="3"> Sequenase Mix (5 uL) </td><td> 1 pl de tampon 5X Sequenase </td></tr><tr><td> 3,8 uL d&#39;eau </td></tr><tr><td> 0,15 uL Sequenase (USB Corporation) </td></tr><tr><td rowspan="5"> Rd B PCR Master Mix (45 ul) </td><td> 5 ul de tampon 10X PCR </td></tr><tr><td> 1 ul 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 ul 100 amorce B pmol / uL </td></tr><tr><td> 1 ul Klentaq LA (Sigma) </td></tr><tr><td> 37 uL d&#39;eau </td></tr><tr><td rowspan="5"> Rd C PCR Master Mix (45 ul) </td><td> 5 uL de tampon 10X PCR </td></tr><tr><td> 1 ul 12,5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 ul 100 amorce B pmol / uL </td></tr><tr><td> 1 ul Klentaq LA (Sigma) </td></tr><tr><td> 37 uL d&#39;eau </td></tr><tr><td rowspan="4"> Tampon d&#39;hybridation (25 ul) </td><td> 1 pi de tampon de fragmentation 25X (Agilent) </td></tr><tr><td> 5 ul de tampon 5X blocage (Agilent) </td></tr><tr><td> 9,5 ul (ou le montant de normaliser) de Cy3-étiquetés de l&#39;échantillon </td></tr><tr><td> Ajouter de l&#39;eau au volume total de 25 uL </td></tr><tr><td> Agilent Virochip biopuces </td><td> licence en attendant, disponible à Chiu laboratoire, UCSF </td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

Le diagnostic des causes virales de nombreuses maladies infectieuses est difficile en raison de la diversité des séquences inhérentes de virus ainsi que l&#39;émergence continue de nouveaux agents pathogènes viraux, tels que le coronavirus du SRAS et de grippe pandémique H1N1 2009 virus, qui ne sont pas détectables par les méthodes traditionnelles. Pour relever ces défis, nous avons précédemment développé et validé une plateforme biopuces pan-virale appelée Virochip avec la capacité de détecter tous les virus connus ainsi que de nouveaux variants sur la base d&#39;une homologie de séquence conservée 1. Utilisation de la Virochip, nous avons identifié le spectre complet des virus associés à des infections respiratoires, y compris des cas de maladie grave inexpliquée chez les patients hospitalisés, avec une sensibilité équivalente ou supérieure à 2-5 tests cliniques classiques. Le Virochip a également été utilisé pour identifier de nouveaux virus, y compris le coronavirus du SRAS 6,7, un clade rhinovirus roman 5, XMRV (un rétrovirus liés à la prostate) 8, bornavirus aviaire (la cause d&#39;une maladie débilitante chez les perroquets) 9, et un roman dans cardiovirus enfants souffrant de maladies respiratoires et diarrhéiques 10. La version actuelle de la Virochip a été porté à une plateforme Agilent microarray et se compose de 36 000 ~ sondes issues de plus de 1500 ~ virus dans GenBank à partir de Décembre 2009. Nous montrerons les étapes impliquées dans le traitement d&#39;un test Virochip du début à la fin (temps ~ retournement 24 heures), y compris l&#39;extraction des acides nucléiques de l&#39;échantillon, l&#39;amplification par PCR utilisant des amorces aléatoires, l&#39;incorporation de colorants fluorescents, et l&#39;hybridation biopuces, de numérisation et d&#39;analyse.

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Le Virochip est une puce pan-viral conçu pour détecter simultanément tous les virus connus ainsi que de nouveaux virus sur la base d&#39;une homologie de séquence conservée. Ici, nous montrent comment exécuter un test Virochip pour analyser des échantillons cliniques pour la présence de virus connus et inconnus.

En utilisant un dosage Microarray pan-virale (Virochip) pour cribler des échantillons cliniques pour les pathogènes viraux

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

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Research

JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

18.7K vues.  University of California, San Francisco. Le Virochip est une puce pan-viral conçu pour détecter simultanément tous les virus connus ainsi que de nouveaux virus sur la base d'une homologie de séquence conservée. Ici, nous montrent comment exécuter un test Virochip pour analyser des échantillons cliniques pour la présence de virus connus et inconnus.

Vidéo: Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

L&#39;établissement d&#39;une culture liquide recouvert de Airway humaines épithéliales polarisées cellules Calu-3 pour étudier la réponse cellulaire aux agents pathogènes respiratoires hôte<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Recherche

Immunologie et infection

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Utilisation de Click Chemistry pour mesurer l&#39;effet de l&#39;infection virale sur Host-Cell Synthèse des ARN

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Utilisation de protéines fluorescentes pour surveiller glycosome Dynamics dans le trypanosome africain

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

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

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

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

A miniaturisé Glycan Microarray Assay pour l&#39;évaluation Avidité et Spécificité du virus grippal A Hémagglutinines

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

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Mesure de la grippe neuraminidase Inhibition Titres d&#39;anticorps par-enzymatique lectine Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Utilisation de capsules pour la coloration négative des échantillons viraux dans le cadre du suivi biologique

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic meurtre dosage pour évaluer les réponses immunologiques contre les bactéries pathogènes

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Détermination de l’efficacité de l’inhibiteur chimique contre la croissance intracellulaire de Toxoplasma Gondii utilisant un essai de croissance basé sur luciferase

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Mesure des anticorps naturels acquis de phagocytose induisant des parasites de plasmodium falciparum par un essai basé sur la cytométrie de flux

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Diagnostic d’infection bactérienne respiratoire humaine par amplification isotherme

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Pleins feux sur l’auteur : Faire progresser la détection rapide des agents pathogènes respiratoires à l’aide d’une puce microfluidique 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Détection rapide d’agents pathogènes bactériens à l’origine d’infections des voies respiratoires inférieures grâce à une amplification isotherme médiée par boucle à base de puce microfluidique

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...