We thank F. Chisari for providing Huh-7.5.1 cell line. We would like to thank Justine Ho for editing the manuscript. This work was supported by Cedars-Sinai Medical Center Institutional Programmatic Research Award and National Center for Advancing Translational Sciences, Grant UL1TR000124 to V.A.

Un schéma général du protocole est illustré sur la figure 1. Une. Cells <ol><li> Préparer un milieu de croissance complet qui contient 10 à 15% de sérum bovin fœtal (FBS), 10 mM acides aminés non essentiels, Hepes 10 mM, de la pénicilline (100 unités / ml), streptomycine (100 mg / ml), et 2 mM de L-glutamine. </li><li> Maintenir les cellules Huh-7.5.1 13 dans du milieu complet de croissance contenant les suppléments ci-dessus pour l&#39;analyse in vitro de l&#39;hépatite C du cycle de réplication virale. </li><li> Culture souches virales en Huh-7.5.1 cellules avec le milieu de croissance complété spécifiée à 37 ° C avec 5% de CO 2. </li></ol> 2. Virus et constructions plasmidiques <ol><li> Générer l&#39;ADN complémentaire (ADNc) de l&#39;ARN du VHC forme et le cloner dans un vecteur plasmidique pour la facilité de la manipulation génétique. Remarque: La découverte de l&#39;hépatite fulminante japonais 1, JFH-1 (génotype 2a du VHC), isolat a été critique pour la recherche sur le VHC et estprincipalement utilisé pour étudier le cycle de 11 à 13 de la replication du VHC. Virus chimériques inter et intragénotypiques sur la base de JFH-1 du VHC sont couramment utilisés dans la recherche de 14 à 18. Construction de synthèse intragenotype 2a virus chimérique, FNX-VHC, et une souche rapporteur chimérique monocistronique a été décrit précédemment 19 et détails de construction sont au-delà du champ d&#39;application de ce protocole. </li><li> Utilisez le virus pFNX-VHC intragenotype 2a, car il contient un 5&#39;NTR, les régions et p7 structurelles, et une partie des régions non structurales NS2 (nucléotides 1 à 2878) de la souche parentale J6CF (NCBI accession no. AF177036), comme ainsi que les éléments non structuraux du JFH-1 souche virale (NCBI de no. AB047639). Remarque: FNX-HCV a été synthétisé sur la base de la séquence de HCV 2a Jc1 intragenotype chimérique signalé précédemment 15. </li><li> Générer un reporter chimérique construction virale monocistronique, la pFNX-Rluc, sur la base de la souche HCV-pFNX (ci-dessus à l&#39;étape 2.2) par insertion d&#39;un Rgène de la luciférase enilla entre l&#39;extrémité 5 &#39;NTR et le gène de la partie centrale (entre les nt 388 et 389). Ensuite, branchez le gène de la luciférase et le gène de base de cette construction en utilisant un virus de la maladie 2A de la fièvre aphteuse (F2A) de la séquence peptidique, qui fonctionnerait comme un signal de clivage. </li><li> Ingénieur de l&#39;ARN polymérase nul (Pol-) construction virale en utilisant soit le pFNX-VHC ou le pFNX-Rluc fond virale. Remplacer les polymérase NS5B résidus catalytiques, GDD (aa 2759-2761; nt 8615-8623), de l&#39;un de ces squelettes virales avec des résidus d&#39;acides aminés AAG. </li></ol> 3. L&#39;ARN du VHC in vitro de transcription <ol><li> Utiliser un virus chimérique intragenotype 2a FNX-VHC et FNX-VHC Pol null (Pol-) pour le VHC évaluation de la replication virale (Figure 2A). </li><li> Linéariser les plasmides viraux avec l&#39;enzyme de restriction XbaI, puis traiter avec la nucléase de haricot mungo à générer des extrémités franches. Purifier les plasmides digérés par chromatographie échangeuse d&#39;anions. Vérifier l&#39;intégrité of le plasmide linéarisé en soumettant l&#39;ADN à une électrophorèse sur gel d&#39;agarose (figure 2B). </li><li> Transcrire les plasmides viraux linéarisées en utilisant la polymérase ARN-T7. </li><li> Purifier l&#39;ARN traité à la DNase nouvellement synthétisé en utilisant un kit de purification d&#39;ARN. </li><li> Vérifiez la production d&#39;ARN par électrophorèse sur gel d&#39;agarose (figure 2C). </li><li> Quantifier l&#39;ARN par spectrophotométrie. </li><li> Stocker l&#39;ARN généré en -80 ° C à 10 pg aliquotes de travail. Remarque: Pour réduire au minimum la variation de la qualité de l&#39;ARN à l&#39;intérieur de chaque plan d&#39;expérience en transcrivant tout l&#39;ARN de chaque échantillon et témoin viral dans le même temps. </li></ol> 4. L&#39;ARN du VHC transfection et prélèvement d&#39;échantillons <ol><li> Récolter les cellules Huh-7.5.1 en utilisant la trypsine. </li><li> Pour rincer les cellules, centrifugeuse et remettre la suspension à deux reprises avec les médias froid sérique bas. Remettre en suspension les cellules dans un milieu de sérum faible à 1 x 10 7 cellules par ml. </li><li> Mélanger un total de 10 &amp; #956; g d&#39;ARN viral transcrit avec 400 ul de cellules remises en suspension (4 x 10 6 cellules) dans une cuvette d&#39;électroporation de 0,4 cm de large. Transfecter les cellules par électroporation à 270 V, 100 Ω, et 950 pF. </li><li> Remettre en suspension les cellules par électroporation dans 10 ml de milieux de culture avec 15% de FBS. Remarque: Augmentation de la capacité de survie des cellules par électroporation est observée lorsque les cellules Huh-7.5.1 ont été cultivées dans 15% de sérum bovin fœtal. </li><li> Plaquer les cellules dans les deux flacons T-25 (1,2 x 10 ~ 6 cellules par flacon) et des plaques de 48 puits (1 x 10 4 cellules par puits) pour chacun des points de temps suivants: 4, 48 et 96 h. </li><li> Remplacez le support à 4-8 heures après la transfection avec les médias frais de croissance supplémenté avec 10% de FBS pour enlever les débris de cellules mortes des flacons et des plaques de culture. </li><li> Les surnageants de culture de cellules de récolte à la 48, 96 points de temps d&#39;heure et éliminer les débris cellulaires à partir d&#39;échantillons recueillis par centrifugation des cellules à 1500 rpm pendant 10 min à 4 º C. </li><li> Conserver les surnageants acellulaires à -80 º C. Lyser les cellules pour la protéine et l&#39;analyse d&#39;ARN par transfert de Western et inverse-PCR quantitative transcription au niveau des points de temps indiqués. </li></ol> 5. Transcription inverse-PCR quantitative (RT-qPCR) pour l&#39;évaluation des copies VHC génomique <ol><li> Effectuer un deux-étape de RT-qPCR pour déterminer la génomique nombre de copies d&#39;ARN du VHC. </li><li> Transcrire inverse 1 pg d&#39;ARN cellulaire total en utilisant la transcriptase inverse et une amorce spécifique du VHC brin sens qui se lie à 5&#39;NTR (JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3 &#39;) ou une amorce spécifique de gène de ménage peptidylprolyl isomérase G (R ppig : 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3 &#39;). Inverser aussi transcrire FNX-ARN du VHC (généré à l&#39;étape 3) de copies du génome connus (10 janvier-10 septembre étalon) de VHC brin sens primaire. </li><li> Effectuer qPCR en utilisant 50 ng de l&#39;ADNc transcrit résultant en utilisant des amorces spécifiques du VHC (JFH RTQF: 5&#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3 &#39;) et liaison à l&#39;ADN colorant vert contenant qPCR superbe mélange. Effectuer qPCR pour le ménage gène ppig ainsi (amorces ppig F: 5&#39;-GAAGAGTGCGATCAAGAACCCATGAC-3 &#39;; ppig R: 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#39;) Remarque: Pour le calcul du niveau de l&#39;ARN du VHC intracellulaire précise à travers des échantillons, utiliser gène de ménage cellulaire, ppig , le niveau d&#39;expression (le cycle Ct) pour la normalisation. Utilisez le nombre de copies du génome FNX-VHC comme la norme pour la détermination du nombre de copies. </li><li> Utiliser les conditions suivantes lors de l&#39;exécution qPCR pour déterminer le nombre de copies d&#39;ARN du VHC: 95 º C pendant 15 secondes et 60 &#160; º C pendant 30 secondes (40 cycles) en utilisant le système de PCR en temps réel. </li><li> Voir les figures 3A et 3B pour des résultats de réplication du génome. </li></ol> 6. Western Blot L&#39;analyse pour la détection du VHC Protein Expression (figure 3C) <ol><li> Utilisez lysats cellulaires frARN viral om transfectées à 96 heures après transfection de protéines analyse western blot. </li><li> Résoudre le lysat cellulaire en utilisant une SDS-PAGE et transfert de polyfluorure de vinylidène (PVDF). </li><li> Bloquer la membrane en utilisant une solution de blocage contenant 5% de lait écrémé, 0,2% de Tween 20 dans du tampon phosphate salin (PBS). </li><li> Incuber la membrane avec l&#39;anticorps monoclonal de souris primaire NS3 (une dilution à 1 000) et de la bêta-actine (une dilution à 5000). </li><li> Ajouter IgG de chèvre anti-souris conjugué à la peroxydase de raifort (1 en 5000 dilution) et de détecter par chimiluminescence. </li></ol> 7. Immunofluorescence (IFA) <ol><li> Fixer les cellules infectées par le VHC et transfectées avec du méthanol pendant 30 min à -20 ° C pour immunofluorescence. </li><li> Laver la cellule avec du PBS à trois reprises et bloquer avec IFA tampon de blocage. </li><li> Utilisez polyclonaux de lapin anti-NS5A principalement anticorps (aimablement fourni par le Dr Dasgupta, UCLA) ou monoclonal de souris anti-DSRNA J2 anticorps à une dilution de 1:200 (1 pg / ml) et incuber pendant 5 heures à une nuit à 4 º C. </li><li> Laver les cellules avec du PBS à trois reprises après l&#39;anticorps primaire. </li><li> Ajouter de chèvre anti-IgG de lapin-488 ou l&#39;anticorps de chèvre anti-IgG de souris-594 anticorps polyclonal secondaire à une dilution de 1:1000 (1 pg / ml) polyclonal secondaire et incuber pendant 1 heure à température ambiante. </li><li> Laver les cellules avec du PBS trois fois et les noyaux des taches à l&#39;aide de colorant Hoechst et vue en utilisant un microscope fluorescent (figure 3D). </li></ol> 8. Mesure Virus Titer <ol><li> Plate naïfs cellules Huh-7.5.1 à environ 3 x 10 3 cellules / puits en utilisant une plaque de 96 puits. Le lendemain, effectuer des dilutions en série de 10 fois de surnageant de culture sans cellules récoltées à partir de cellules transfectées avec de l&#39;ARN du VHC en utilisant des milieux de croissance et inoculer en triple sur les cellules Huh-7.5.1. </li><li> Fixer les cellules à 72 heures après l&#39;infection en utilisant du méthanol (pendant 30 min à -20 </sup&gt; º C) et immunomarquage pour le VHC protéine NS5A comme indiqué dans la section 7. </li><li> Utilisez la plus forte dilution de compter pour les foyers positif NS5A et calculer le nombre moyen de se concentrer unité de formation (FFU) par millilitre. Voir Figure 4 pour le VHC titre. </li></ol> 9. Renilla Reporter Essai de génome viral de réplication et de l&#39;infectiosité <ol><li> Pour l&#39;évaluation de la réplication du génome viral, l&#39;ARN du VHC-plaque électroporation des cellules Huh-7.5.1, en ​​triple exemplaire dans des plaques de 48 puits (1 x 10 4 cellules / puits). </li><li> Lyse des cellules avec 75 ul de tampon de lyse passive au 6 h, 48 h et 96 h post-transfection. </li><li> Basculez les plaques doucement pendant 15 min à température ambiante et conserver à -80 ° C. </li><li> Pour mesurer l&#39;activité enzymatique de la luciférase de Renilla, décongeler le lysat de protéine luciférase de Renilla et les réactifs de dosage à la température ambiante. </li><li> Ajouter 20 ul de lysat de protéine par puits d&#39;une 96-wel<html> l plaque de luminescence blanche et placer la plaque dans un luminomètre. </li><li> Distribuer 100 ul de Renilla réactif de dosage (coelentérazine substrat + tampon) par puits et après un délai pré-lire 2 sec; intégrer la lumière émise pendant 10 secondes. </li><li> Calculer la moyenne et l&#39;écart type pour chaque échantillon à partir des valeurs de luciférase de triplicats biologiques et soumettre les données à l&#39;analyse statistique (figure 5). </li><li> Pour évaluer l&#39;infectiosité, inoculer 500 pi de surnageant acellulaire obtenus à partir de cellules transfectées ARN VHC pour les points de temps indiqués (48 h et 96 h), en trois exemplaires sur naïfs cellules Huh-7.5.1 dans des plaques à 48 puits. </li><li> Remplacer l&#39;inoculum viral avec 500 pi de milieu frais par puits après 6 heures après l&#39;infection. </li><li> Lyse des cellules à 48 heures après l&#39;infection et sous réserve de Renilla dosage de la luciférase comme décrit dans les étapes 08.02 à 08.07 (figure 5). </li></ol>itre &quot;. Analyse statistique&gt; 10 <ol><li> Les barres d&#39;erreur dans les graphiques indiquent les écarts-types (SD). Les valeurs de p ont été calculées par le test t non apparié. </li></ol>

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The authors have nothing to disclose.

Cette illustration décrit un procédé d&#39;analyse du cycle de réplication du virus de l&#39;hépatite C. VHC est un agent pathogène humain et le protocole sur la biosécurité prescrit devra être strictement suivies. Les systèmes de culture de cellules de VHC infectieuses ont été décrites précédemment 11-13,16,17. Il ya quelques points essentiels nous mettons en œuvre en suivant le protocole illustré. Premièrement, il est d&#39;une grande importance d&#39;avoir une bonne qualité de l&#39;ARN ...

L&#39;hépatite C (VHC) provoque une cirrhose et un cancer du foie. Elle touche 170 millions de personnes dans le monde avec 350 000 personnes meurent chaque année 1-3. Le VHC est un virus à ARN à brin positif ayant une taille de génome de 9,6 kb. Le génome du VHC est traduit en une seule polyprotéine de ~ 3000 résidus d&#39;acides aminés qui est clivée de manière protéolytique par diverses proteases cellulaires et virales en 10 polypeptides. Le VHC est le virus prototype du genre Hepacivirus et appartient à la famille des Flaviviridae 4. Lors de l&#39;exposition, le VHC établit une infection chronique chez 80% des individus. L&#39;infection est souvent asymptomatique et en temps opportun diagnostic peut permettre une intervention thérapeutique pour prévenir la détérioration du foie. Le traitement actuel est sous-optimale et aucun vaccin n&#39;est disponible 5,6. L&#39;étiologie de l&#39;hépatite C a été décrite pour la première en 1989 7. Etudier la réplication du VHC est important pour l&#39;hépatite C vaccin et la recherche de traitement, mais il avait étélongue entravée par l&#39;absence d&#39;un système de culture virale efficace. Un clone moléculaire du VHC a été montré pour être infectieux chez les chimpanzés sur inoculation intrahépatique 8. Par la suite, des réplicons du VHC sub-génomiques ont été décrites, qui ont permis de disséquer l&#39;étape de réplication du génome viral dans un système de 9,10 de la culture cellulaire. Découverte d&#39;un VHC de génotype 2a isoler JFH-1 (hépatite fulminante-1 japonais), capable d&#39;infecter la culture cellulaire a ouvert de nouvelles voies pour la recherche de la réplication du VHC 11-13. Souche de génotype 2a JFH-1 des systèmes de culture à base infectieuses basé virus chimériques inter-et intra-génotypiques et VHC de génotype 1 sont disponibles ainsi 14-18. Nous avons utilisé avec succès JFH-1 et le VHC souche intragenotype 2a virus chimère pour obtenir la haute résolution profilage fonctionnelle carte de domaines protéiques et éléments agissant en cis d&#39;ARN 19,20. Selon cette étude, nous décrivons ici un système de culture efficace couramment utilisé qui permetl&#39;étude de différentes étapes du cycle de replication du VHC et de l&#39;interaction hôte-pathogène. Nous présentons des tests virologiques pour évaluer la réplication du génome viral et de l&#39;infectiosité de novo intragenotype 2a du VHC et un VHC de journaliste en Renilla.

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			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">10-017-CV</td>
			<td></td>
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			<td height="21" style="height:21px;width:569px;">Non essential amino acid</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">MT25025CI</td>
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			<td height="21" style="height:21px;width:569px;">HEPES</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">15630080</td>
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			<td height="21" style="height:21px;width:569px;">Glutamax</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
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			<td height="21" style="height:21px;width:569px;">Opti-MEM Reduced Serum Medium,no Phenol Red</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11058-021</td>
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			<td height="21" style="height:21px;width:569px;">Huh-7.5.1</td>
			<td style="width:267px;">The Scripps Research Institute</td>
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			<td>The cell line was kindly provided by Dr. Francis Chisari to Dr. Arumugaswami under executed MTA between The Scripps Research Institute and Cedars-Sinai Medical Center</td>
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			<td height="23" style="height:23px;width:569px;">Plasmids (pFNX-HCV, pFNX-HCV Pol null, pFNX-Rluc, and pFNX-Rluc Pol null)&#160;</td>
			<td style="width:267px;">Cedars-Sinai Medical Center</td>
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			<td>The HCV plasmids were synthesized by Dr. Arumugaswami using overlapping oligo-nucleotides.&#160;</td>
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			<td height="21" style="height:21px;width:569px;">XbaI</td>
			<td>New England Biolabs Inc.</td>
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			<td height="21" style="height:21px;width:569px;">Mung Bean Nuclease</td>
			<td>New England Biolabs Inc.</td>
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			<td height="21" style="height:21px;width:569px;">T7 RiboMAX Express Large Scale RNA Production System</td>
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			<td height="21" style="height:21px;width:569px;">Rneasy Mini Kit</td>
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			<td height="21" style="height:21px;width:569px;">Nanodrop 2000</td>
			<td style="width:267px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
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			<td height="21" style="height:21px;width:569px;">Electroporation Cuvette (4 mm)</td>
			<td style="width:267px;">Bioexpress</td>
			<td>E-5010-4</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Gene Pulser Xcell Total System</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">165-2660</td>
			<td></td>
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			<td height="21" style="height:21px;">mouse monoclonal anti-dsRNA antibody J2&#160;</td>
			<td>English &#38; Scientific Consulting Kft.</td>
			<td style="width:119px;">10010200</td>
			<td></td>
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			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 488</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11008</td>
			<td></td>
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			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 594</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11020</td>
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			<td height="21" style="height:21px;">PVDF membrane package</td>
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			<td height="21" style="height:21px;width:569px;">Blotting Grade Blocker Non Fat Dry Milk</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6404XTU</td>
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			<td height="21" style="height:21px;width:569px;">Tween-20</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6531XTU</td>
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			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [8 G-2]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab65407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [H23]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab13830</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td style="width:119px;">115-035-003</td>
			<td></td>
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			<td height="21" style="height:21px;">Amersham ECL Prime Western Blotting Detection Reagents&#160;</td>
			<td style="width:267px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2236</td>
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			<td height="21" style="height:21px;width:569px;">SUPERSCRIPT III RT&#160;</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">18080085</td>
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			<td height="21" style="height:21px;width:569px;">SYBR QPCR SUPERMIX W/ROX</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11744500</td>
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			<td height="21" style="height:21px;">ViiA 7 real-time PCR system</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">NA</td>
			<td></td>
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			<td height="21" style="height:21px;">Renilla Luciferase Assay System kit</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">E2810</td>
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			<td height="21" style="height:21px;width:569px;">RNase-Free DNase</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">M6101</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:569px;">GloMax-Multi Detection System (Luminometer)</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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a protocol for analyzing hepatitis c virus replication

L&#39;hépatite C (VHC) affecte 3% de la population du monde et provoque des maladies graves du foie comme l&#39;hépatite chronique, la cirrhose et le carcinome hépatocellulaire. Le VHC est un virus à ARN enveloppé de la famille des Flaviviridae. Le traitement actuel n&#39;est pas pleinement efficace et provoque des effets secondaires indésirables. Il n&#39;ya pas de vaccin disponible VHC. Ainsi, la poursuite des efforts est nécessaire pour développer un vaccin et une meilleure thérapie. Un système de culture cellulaire du VHC est essentielle pour l&#39;étude des diverses étapes de la croissance de VHC, y compris l&#39;entrée virale, la réplication du génome, à l&#39;emballage, et la sortie. Dans la procédure actuelle présentée, nous avons utilisé un virus de type sauvage intragenotype 2a chimérique, FNX-VHC, et un virus recombinant FNX-Rluc portant un gène rapporteur de la luciférase Renilla pour étudier la réplication du virus. Une lignée cellulaire d&#39;hépatome humain (Huh-7 repose) a été utilisé pour la transfection in vitro de transcrits ARN génomiques du VHC. Surnageants de culture sans cellules, des lysats de protéines et total ARN ont été récoltés à divers moments post-transfection pour évaluer la croissance du VHC. Génome HCV état de réplication a été évaluée par RT-PCR quantitative et la visualisation de la présence de HCV ARN double brin. L&#39;expression de la protéine du VHC a été vérifiée par blot et immunofluorescence dosages Western en utilisant des anticorps spécifiques des protéines du VHC NS3 et NS5A. les cellules transfectées de l&#39;ARN du VHC libérés des particules infectieuses dans le surnageant de culture et le titre viral a été mesuré. dosages de la luciférase ont été utilisées pour évaluer le niveau de réplication et l&#39;infectiosité de reporter le VHC. En conclusion, nous présentons diverses analyses virologiques pour caractériser les différentes étapes du cycle de réplication du VHC.

Virus de l&#39;hépatite C est un virus à ARN. Ainsi, à des fins de manipulation génétique, l&#39;ADNc génomique du VHC a été cloné dans un vecteur plasmidique bactérien. Une séquence de promoteur de l&#39;ARN polymerase T7 a été introduit juste avant l&#39;extrémité 5 &#39;du génome de HCV. Un schéma général de l&#39;analyse du HCV flux de travail est présentée en figure 1. Pour générer l&#39;ARN génomique du VHC avec précision l&#39;extrémité 3 &#39;, le génome du VHC conten...

Watch this Scientific Journal Video about A Protocol for Analyzing Hepatitis C Virus Replication at JoVE.com

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Un protocole pour l&#39;analyse de l&#39;hépatite C de réplication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

a-protocol-for-analyzing-hepatitis-c-virus-replication

Research

JoVE Journal

Immunology and Infection

23.9K vues.  Cedars-Sinai Medical Center. Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle. 

Vidéo: A Protocol for Analyzing Hepatitis C Virus Replication

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Utiliser l&#39;imagerie Bioluminescent chargé d&#39;enquêter sur une synergie entre Streptococcus pneumoniae Et virus Influenza A de souriceaux

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Recherche

Immunologie et infection

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Dissection hôte-virus interaction dans la réplication lytique d&#39;un virus de l&#39;herpès modèle

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Un système de neurones primaires de la culture pour l&#39;étude de l&#39;herpès simplex virus de latence et de réactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

À haut débit de dosage de phénotype<em&gt; Salmonella enterica</em&gt; Association Typhimurium, Invasion, et la réplication dans les macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Immunofluorescence de suivi de l&#39;absorption cellulaire de la lactoferrine humaine et de son activité associée antivirale contre le virus de l&#39;hépatite C

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

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 ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Développement d&#39;un système de virus de l&#39;hépatite B Reporter pour surveiller les premiers stades du cycle de réplication

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Le sauvetage et la caractérisation du virus recombinant à partir d&#39;un nouveau clone infectieux du virus Zika du monde

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

« Foie-on-a-Chip » Cultures d’hépatocytes primaires et des cellules de Kupffer pour Infection de Virus de l’hépatite B

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identification de codage et de Classes d’ARN Non codants exprimée chez le porc sang total

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...