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.

Uno schema generale del protocollo è illustrato in Figura 1. 1. Cellule <ol><li> Preparare mezzi di crescita completo che contiene il 10-15% di siero bovino fetale (FBS), 10 mM aminoacidi non essenziali, Hepes 10 mM, penicillina (100 unità / ml), streptomicina (100 mg / ml), e 2 mM L-glutammina. </li><li> Mantenere Huh-7.5.1 celle 13 in mezzi di crescita completi contenenti i supplementi di cui sopra per l&#39;analisi in vitro di epatite C ciclo di replicazione virale. </li><li> Cultura ceppi virali in Huh-7.5.1 celle con i mezzi di crescita integrata specificato a 37 ° C con 5% di CO 2. </li></ol> 2. Virus e plasmidi costrutti <ol><li> Generare la forma complementare DNA (cDNA) di HCV RNA e clonare in un vettore plasmidico per facilità di manipolazione genetica. Nota: La scoperta del giapponese epatite fulminante 1, JFH-1 (genotipo HCV 2 bis), isolare è stato fondamentale per la ricerca HCV ed èutilizzato principalmente per studiare il ciclo 11-13 replicazione di HCV. Virus chimerici-Inter e intragenotypic basato su JFH-1 HCV sono comunemente utilizzati nella ricerca 14-18. Costruzione di intragenotype sintetico 2a virus chimerico, FNX-HCV, e un monocistronic ceppo giornalista chimerico è stato descritto in precedenza i 19 ei dettagli costruttivi sono oltre la portata di questo protocollo. </li><li> Utilizzare il pFNX-HCV virus intragenotype 2a in quanto contiene un 5&#39;NTR, regioni strutturali e p7, e parte delle regioni non strutturali NS2 (nucleotidi 1-2878) del ceppo parentale J6CF (NCBI adesione n. AF177036), come nonché i componenti non strutturali del JFH-1 ceppo virale (NCBI adesione n. AB047639). Nota: FNX-HCV è stato sintetizzato basandosi sulla sequenza di Jc1 intragenotype 2a HCV chimerico riferito in precedenza 15. </li><li> Generare un costrutto chimerico monocistronic giornalista virale, la pFNX-Rluc, basato sul ceppo pFNX-HCV (sopra al punto 2.2) inserendo un Renilla gene luciferasi tra il 5 &#39;NTR e gene nucleo (nt tra 388 e 389). Successivamente, collegare il gene della luciferasi e nucleo gene di questo costrutto utilizzando un 2A virus dell&#39;afta epizootica (F2A) sequenza peptidica, che funzioni come un segnale di scissione. </li><li> Ingegnere il costrutto virale RNA polimerasi-null (Pol-) utilizzando il pFNX-HCV o lo sfondo virale pFNX-Rluc. Sostituire i residui catalitici NS5B polimerasi, GDD (aa 2759-2761; nt 8615-8623), di una di queste dorsali virali con residui di acido AAG amino. </li></ol> 3. In Vitro HCV RNA Trascrizione <ol><li> Utilizzare un intragenotype 2a virus chimerico FNX-HCV e FNX-HCV Pol null (Pol-) HCV per la valutazione della replicazione virale (Figura 2A). </li><li> Linearizzare i plasmidi virali con XbaI enzima di restrizione e poi trattare con mung bean nucleasi per generare estremità smussata. Purificare i plasmidi digeriti mediante cromatografia a scambio anionico. Verificare l&#39;integrità of plasmide linearizzato sottoponendo DNA per elettroforesi su gel di agarosio (Figura 2B). </li><li> Trascrivere i plasmidi virali linearizzate utilizzando la polimerasi T7-RNA. </li><li> Purificare il RNA DNasi trattati nuova sintesi utilizzando un kit di purificazione dell&#39;RNA. </li><li> Verificare la produzione di RNA mediante elettroforesi su gel di agarosio (Figura 2C). </li><li> Quantificare l&#39;RNA per spettrofotometria. </li><li> Conservare il RNA generati in -80 ° C in aliquote di 10 mcg lavorativi. Nota: Per ridurre al minimo la variazione di qualità RNA in ciascun disegno sperimentale trascrivendo tutto RNA per ogni campione e controllo virale allo stesso tempo. </li></ol> 4. HCV RNA Transfezione e raccolta dei campioni <ol><li> Raccogliere le cellule Huh-7.5.1 utilizzando tripsina. </li><li> Per lavare le celle, centrifugare e risospendere la sospensione per due volte con il freddo i media siero basso. Risospendere le cellule in mezzi siero partire a 1 x 10 7 cellule per ml. </li><li> Mescolare un totale di 10 &amp; #956; g di RNA virale trascritto con 400 microlitri di cellule risospese (4 x 10 6 cellule) in 0,4-cm elettroporazione cuvetta. Transfettare le cellule tramite elettroporazione a 270 V, 100 Ω e 950 uF. </li><li> Risospendere le cellule elettroporate in 10 ml di media crescita completa con il 15% FBS. Nota: Aumento di sopravvivenza delle cellule elettroporate si vede quando le cellule Huh-7.5.1 sono state coltivate in 15% di siero fetale bovino. </li><li> Piastra le cellule in entrambe T-25 palloni (~ 1,2 x 10 6 cellule per pallone) e piastre a 48 pozzetti (1 x 10 4 cellule per pozzetto) per ciascuno dei seguenti punti temporali: 4, 48 e 96 ore. </li><li> Sostituire i media a 4-8 ore post-trasfezione con fresca mezzi di crescita supplementato con 10% FBS per rimuovere i detriti cellulari morti palloni in coltura e piatti. </li><li> Surnatanti di coltura cellulare Harvest ai 48, 96 punti di tempo hr e rimuovere detriti cellulari da campioni raccolti mediante centrifugazione di cellule a 1.500 rpm per 10 minuti a 4 º C. </li><li> Conservare il surnatante cell-free a -80 ° C. Lisare le cellule per l&#39;analisi delle proteine ​​e RNA da Western blot e trascrizione inversa-PCR quantitativa nei punti all&#39;ora indicata. </li></ol> 5. Trascrizione inversa-PCR quantitativa (RT-qPCR) per la valutazione delle copie HCV Genome <ol><li> Eseguire un due fasi RT-qPCR per determinare il numero di copie di RNA genomico di HCV. </li><li> Trascrivere Reverse 1 mg di RNA cellulare totale utilizzando la trascrittasi inversa e un primer specifico per HCV senso filo che lega 5&#39;NTR (JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3 &#39;) o un primer specifico per il gene housekeeping peptidylprolyl isomerasi G (ppig R : 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3 &#39;). Anche invertire trascrivere FNX-HCV RNA (generato nel passaggio 3) di copie del genoma noti (10 gennaio - 10 settembre di serie) utilizzando senso HCV filamento primer. </li><li> Effettuare qPCR utilizzando 50 ng del risultante cDNA trascritto utilizzando specifici primer HCV (JFH RTQF: 5&#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3 &#39;) e il legame al DNA colorante verde contenente qPCR eccellente mix. Eseguire qPCR per le pulizie gene ppig così (primer ppig F: 5&#39;-GAAGAGTGCGATCAAGAACCCATGAC-3 &#39;; ppig R: 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#39;) Nota: Per il calcolo esatto livello intracellulare di RNA di HCV di tutti i campioni, usare cellulari gene housekeeping, ppig , livello di espressione (ciclo Ct) per la normalizzazione. Utilizzare il numero di copie del genoma FNX-HCV come standard per la determinazione del numero di copie. </li><li> Utilizzare le seguenti condizioni durante l&#39;esecuzione qPCR per la determinazione del numero di copie di HCV RNA: 95 º C per 15 sec e 60 &#160; ° C per 30 sec (40 cicli) utilizzando il sistema di PCR in tempo reale. </li><li> Vedere le Figure 3A e 3B per i risultati replicazione del genoma. </li></ol> 6. Western Blotting analisi per il rilevamento di HCV Protein Expression (Figura 3C) <ol><li> Utilizzare lisati cellulari frRNA virale om transfettata a 96 ore dopo la trasfezione di proteine ​​western blotting. </li><li> Risolvere il lisato cellulare mediante SDS-PAGE e trasferimento a polivinilidene (PVDF) membrana. </li><li> Bloccare la membrana con una soluzione bloccante contenente latte scremato 5%, 0,2% Tween 20 in tampone fosfato salino (PBS). </li><li> Incubare la membrana con l&#39;anticorpo monoclonale di topo primario NS3 (1 in 1.000 diluizione) e beta-actina (1 in 5.000 diluizione). </li><li> Aggiungi IgG di capra anti-topo coniugata con perossidasi di rafano (1 in 5.000 diluizione) e rilevare da chemiluminescenza. </li></ol> 7. Immunofluorescenza Assay (IFA) <ol><li> Fissare le cellule con infezione da HCV e trasfettate con metanolo per 30 minuti a -20 ° C per l&#39;analisi di immunofluorescenza. </li><li> Lavare le cellule con PBS per tre volte e bloccare con IFA tampone di bloccaggio. </li><li> Utilizzare policlonale di coniglio anti-NS5A principalmente anticorpo (gentilmente fornito dal Dr. Dasgupta, UCLA) o il mouse monoclonale anti-DSRNA anticorpo J2 alla diluizione 1:200 (1 mg / ml) e incubare per 5 ore a tutta la notte a 4 º C. </li><li> Lavare le cellule con PBS per tre volte dopo l&#39;anticorpo primario. </li><li> Aggiungere goat anti-rabbit IgG-488 policlonale anticorpo secondario o di capra anti-topo IgG-594 anticorpo secondario policlonale ad una diluizione 1:1000 (1 mg / ml) e incubare per 1 ora a temperatura ambiente. </li><li> Lavare le cellule con PBS per tre volte e nuclei macchia con Hoechst colorante e vista utilizzando microscopio a fluorescenza (Figura 3D). </li></ol> 8. Virus misura Titer <ol><li> Piatto ingenui Huh-7.5.1 cellule a circa 3 x 10 3 cellule / pozzetto utilizzando una piastra a 96 pozzetti. Il giorno successivo, eseguire diluizioni seriali di 10 volte del cell-free cultura surnatante raccolto da RNA transfettate cellule HCV utilizzo dei supporti di crescita e di inoculare in triplice copia su Huh-7.5.1 cellule. </li><li> Fissare le cellule a 72 ore dopo l&#39;infezione con metanolo (per 30 minuti a -20 </sup&gt; º C) e immunostain per HCV proteine ​​NS5A come indicato nella sezione 7. </li><li> Usare la massima diluizione di contare per i fuochi positivo NS5A e calcolare il numero medio di messa a fuoco unità di formazione (FFU) per millilitro. Vedere Figura 4 per HCV titolo. </li></ol> 9. Renilla Luciferase Reporter Assay per Viral Genome Replica e Infettività <ol><li> Per valutare la replicazione del genoma virale, piastra HCV RNA-elettroporato Huh-7.5.1 cellule in triplice copia in piastre 48 pozzetti (1 x 10 4 cellule / pozzetto). </li><li> Lisare le cellule con 75 ml di tampone di lisi passiva al 6 ore, 48 ore e 96 ore dopo la trasfezione. </li><li> Oscillare delicatamente le piastre per 15 minuti a temperatura ambiente e conservare a -80 ° C. </li><li> Per misurare l&#39;attività enzimatica Renilla luciferasi, scongelare il lisato proteico e Renilla luciferasi i reagenti a temperatura ambiente. </li><li> Aggiungere 20 ml di lisato proteico per pozzetto di una 96-wel<html> l piatto luminescenza bianca e mettere il piatto in un luminometro. </li><li> Dispensare 100 ml di Renilla luciferasi di reagenti del dosaggio (coelenterazina substrato + tampone) per bene e dopo un ritardo di 2 secondi di pre-lettura; integrare la luce emessa per 10 sec. </li><li> Calcolare la media e la deviazione standard per ciascun campione dai valori di luciferasi triplicato biologici e sottoporre i dati per l&#39;analisi statistica (Figura 5). </li><li> Per valutare l&#39;infettività, inoculare 500 ml di surnatante cell-free ottenuti da cellule RNA-HCV transfettate per i punti temporali indicati (48 ore e 96 ore) in triplice copia su naïve Huh-7.5.1 cellule in piastre 48 pozzetti. </li><li> Sostituire l&#39;inoculo virale con 500 ml di mezzo fresco per bene dopo 6 ore dopo l&#39;infezione. </li><li> Lisare le cellule a 48 ore dopo l&#39;infezione e sono soggetti a Renilla saggio luciferasi come descritto nei passaggi 8,2-8,7 (Figura 5). </li></ol>DENOMINAZIONE &quot;&gt; 10. Analisi statistica <ol><li> Le barre di errore nei grafici indicano deviazioni standard (SD). I valori di P sono stati calcolati con il test t spaiato. </li></ol>

<ol>
	<li>Alter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.+Epidemiology+of+hepatitis+C.">J. Epidemiology of hepatitis C.</a> Hepatology.. 26(3 Suppl 1), (1997).</li><li>Alter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.+Epidemiology+of+hepatitis+C+virus+infection.">J. Epidemiology of hepatitis C virus infection.</a> World J Gastroenterol. 13 (17), 2436-2441 (2007).</li><li>Lavanchy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+of+hepatitis+C.">The global burden of hepatitis C.</a> Liver Int. 29 (Suppl 1), 74-81 (2009).</li><li>Lindenbach, B. D., Thiel, H. J., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Flaviviridae: viruses and their replication in Fields Virology. , 1101-1152 (2007).</li><li>Ciesek, S., Manns, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+in+2010:+the+dawn+of+a+new+era+in+HCV+therapy.">Hepatitis in 2010: the dawn of a new era in HCV therapy.</a> Nat Rev Gastroenterol Hepatol. 8 (2), 69-71 (2011).</li><li>Ghany, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+treatment+of+genotype+1+chronic+hepatitis+C+virus+infection:+2011+practice+guideline+by+the+American+Association+for+the+Study+of+Liver+Diseases.">An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 practice guideline by the American Association for the Study of Liver Diseases.</a> Hepatology. 54 (4), 1433-1444 (2011).</li><li>Choo, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+cDNA+clone+derived+from+a+blood-borne+non-A,+non-B+viral+hepatitis+genome.">Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.</a> Science. 244 (4902), 359-362 (1989).</li><li>Kolykhalov, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+hepatitis+C+by+intrahepatic+inoculation+with+transcribed+RNA.">Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.</a> Science. 277 (5325), 570-574 (1997).</li><li>Lohmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+subgenomic+hepatitis+C+virus+RNAs+in+a+hepatoma+cell+line.">Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.</a> Science. 285 (5424), 110-113 (1999).</li><li>Blight, K. J., Kolykhalov, A. A., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+initiation+of+HCV+RNA+replication+in+cell+culture.">Efficient initiation of HCV RNA replication in cell culture.</a> Science. 290 (5498), 1972-1974 (2000).</li><li>Lindenbach, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+replication+of+hepatitis+C+virus+in+cell+culture.">Complete replication of hepatitis C virus in cell culture.</a> Science. 309 (5734), 623-626 (2005).</li><li>Wakita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+infectious+hepatitis+C+virus+in+tissue+culture+from+a+cloned+viral+genome.">Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.</a> Nat Med. 11 (7), 791-796 (2005).</li><li>Zhong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+hepatitis+C+virus+infection+in+vitro.">Robust hepatitis C virus infection in vitro.</a> Proc Natl Acad Sci U S A. 102 (26), 9294-9299 (2005).</li><li>Yi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensatory+mutations+in+E1,+p7,+NS2,+and+NS3+enhance+yields+of+cell+culture-infectious+intergenotypic+chimeric+hepatitis+C+virus.">Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus.</a> J Virol. 81 (2), 629-638 (2007).</li><li>Pietschmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+infectious+intragenotypic+and+intergenotypic+hepatitis+C+virus+chimeras.">Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras.</a> Proc Natl Acad Sci U S A. 103 (19), 7408-7413 (2006).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+full-length+hepatitis+C+virus+genotype+1+(strain+TN)+infectious+culture+system.">Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system.</a> Proc Natl Acad Sci U S A. 109 (48), 19757-19762 (2012).</li><li>Yi, M., Lemon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+1a+HCV+(H77S)+infection+system.">Genotype 1a HCV (H77S) infection system.</a> Methods Mol Biol. 510, 337-346 (2009).</li><li>Gottwein, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+hepatitis+C+reporter+viruses+with+genotype+1+to+7+core-nonstructural+protein+2+(NS2)+expressing+fluorescent+proteins+or+luciferase+in+modified+JFH1+NS5A.">Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A.</a> J Virol. 85 (17), 8913-8928 (2011).</li><li>Arumugaswami, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+functional+profiling+of+hepatitis+C+virus+genome.">High-resolution functional profiling of hepatitis C virus genome.</a> PLoS Pathog. 4 (10), (2008).</li><li>Chu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+enhancer+and+critical+cis-acting+RNA+elements+in+the+protein-encoding+region+of+the+hepatitis+C+virus+genome.">Systematic analysis of enhancer and critical cis-acting RNA elements in the protein-encoding region of the hepatitis C virus genome.</a> J Virol. 87 (10), 5678-5696 (2013).</li><li>Salloum, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab18+binds+to+hepatitis+C+virus+NS5A+and+promotes+interaction+between+sites+of+viral+replication+and+lipid+droplets.">Rab18 binds to hepatitis C virus NS5A and promotes interaction between sites of viral replication and lipid droplets.</a> PLoS Pathog. 9 (8), (2013).</li><li>Gonzalez, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+an+optimal+RNA+transfection+reagent+and+comparison+to+electroporation+for+the+delivery+of+viral+RNA.">Selection of an optimal RNA transfection reagent and comparison to electroporation for the delivery of viral RNA.</a> J Virol Methods. 145 (1), 14-21 (2007).</li><li>Phan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+NS2+protein+contributes+to+virus+particle+assembly+via+opposing+epistatic+interactions+with+the+E1-E2+glycoprotein+and+NS3-NS4A+enzyme+complexes.">Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes.</a> J Virol. 83 (17), 8379-8395 (2009).</li><li>Schaller, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+hepatitis+C+virus+superinfection+exclusion+by+using+novel+fluorochrome+gene-tagged+viral+genomes.">Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes.</a> J Virol. 81 (9), 4591-4603 (2007).</li><li>Li, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+hepatitis+C+virus+lacking+the+envelope-encoding+genes+for+single-cycle+infection+by+providing+homologous+envelope+proteins+or+vesicular+stomatitis+virus+glycoproteins+in+trans.">Production of hepatitis C virus lacking the envelope-encoding genes for single-cycle infection by providing homologous envelope proteins or vesicular stomatitis virus glycoproteins in trans.</a> J Virol. 85 (5), 2138-2147 (2011).</li><li>Jones, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+p7+and+NS2+proteins+are+essential+for+production+of+infectious+virus.">Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus.</a> J Virol. 81 (16), 8374-8383 (2007).</li><li>Steinmann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+p7+protein+is+crucial+for+assembly+and+release+of+infectious+virions.">Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions.</a> PLoS Pathog. (7), (2007).</li></ol>

The authors have nothing to disclose.

Questa illustrazione descrive un metodo per analizzare il ciclo di replicazione del virus dell&#39;epatite C. HCV è un agente patogeno umano e il protocollo sulla biosicurezza prescritta dovrà essere seguite scrupolosamente. Sistemi di coltura di cellule HCV infettive sono state descritte in precedenza 11-13,16,17. Ci sono alcuni punti cruciali attuiamo quando seguendo il protocollo illustrato. In primo luogo, è di grande importanza per avere una buona qualità di intatta tutta la lunghezza RNA genomico vi...

Virus dell&#39;epatite C (HCV) causa cirrosi e cancro del fegato. Essa colpisce 170 milioni di persone in tutto il mondo con 350.000 persone muoiono ogni anno 1-3. HCV è un virus a RNA filamento positivo con un genoma di 9,6 kb. Il genoma di HCV è tradotto come singola poliproteina di ~ 3000 residui amminoacidici che viene scissione proteolitica da varie proteasi cellulari e virali in 10 polipeptidi. HCV è il virus prototipo in genere Hepacivirus e appartiene alla famiglia Flaviviridae 4. Dopo l&#39;esposizione, HCV stabilisce infezione cronica nel 80% degli individui. L&#39;infezione è prevalentemente asintomatica e tempestiva diagnosi può consentire l&#39;intervento terapeutico per prevenire il deterioramento del fegato. Il trattamento attuale è ottimale e nessun vaccino è disponibile 5,6. L&#39;eziologia di epatite C è stata descritta per la prima nel 1989 7. Studiare la replica di HCV è importante per il vaccino contro l&#39;epatite C e la ricerca del trattamento, ma era statolungo ostacolata dalla mancanza di un efficiente sistema di coltura virale. Un clone molecolare di HCV ha dimostrato di essere contagiosa negli scimpanzé su inoculazione intraepatica 8. Successivamente, sono stati descritti repliconi HCV sub-genomici, che ha permesso di sezionare la fase di replicazione del genoma virale in un sistema di coltura cellulare 9,10. Scoperta di un HCV genotipo 2a isolare JFH-1 (Giapponese epatite fulminante-1), in grado di infettare colture cellulari ha aperto nuove strade per la ricerca replicazione di HCV 11-13. Ceppo genotipo 2a JFH-1 sistemi di coltura infettive basati basati virus chimerici inter-e intra-genotipiche e genotipo 1 HCV sono disponibili pure 14-18. Abbiamo utilizzato con successo JFH-1 ceppo e HCV intragenotype 2a virus chimerico avere la ad alta risoluzione profili funzionali mappa di domini di proteine ​​e cis-acting elementi RNA 19,20. In base a questo, qui si descrive un sistema efficace cultura utilizzato di routine che permettestudiando varie fasi del ciclo di replicazione di HCV e l&#39;interazione ospite-patogeno. Vi presentiamo saggi virologici per valutare la replicazione del genoma virale e infettività de novo di intragenotype 2a HCV e un giornalista HCV basata Renilla luciferasi.

<table border="0" cellpadding="0" cellspacing="0" width="1121">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Name of Material/ Equipment</td>
			<td style="width:267px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">HEPES</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">15630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Glutamax</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Huh-7.5.1</td>
			<td style="width:267px;">The Scripps Research Institute</td>
			<td style="width:119px;"></td>
			<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>
		</tr>
		<tr height="23">
			<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>
			<td style="width:119px;"></td>
			<td>The HCV plasmids were synthesized by Dr. Arumugaswami using overlapping oligo-nucleotides.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">XbaI</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">R0145S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Mung Bean Nuclease</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">M0250S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">T7 RiboMAX Express Large Scale RNA Production System</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">P1320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Rneasy Mini Kit</td>
			<td style="width:267px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVDF membrane package</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">162-0263</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Renilla Luciferase Assay System kit</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">E2810</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
	</tbody>
</table>

a protocol for analyzing hepatitis c virus replication

Virus dell&#39;epatite C (HCV) colpisce il 3% della popolazione mondiale e provoca gravi disturbi epatici, tra cui epatite cronica, cirrosi e carcinoma epatocellulare. HCV è un virus a RNA enveloped appartenente alla famiglia Flaviviridae. Terapia attuale non è pienamente efficace e provoca effetti collaterali negativi. Non esiste un vaccino HCV disponibili. Pertanto, è necessaria continuato sforzo per sviluppare un vaccino e migliore terapia. Un sistema di coltura cellulare HCV è fondamentale per studiare varie fasi di crescita HCV tra cui l&#39;ingresso del virus, la replicazione del genoma, imballaggio e uscita. Nella procedura attuale presentata, abbiamo utilizzato un virus wild-type intragenotype 2a chimerico, FNX-HCV, e un virus ricombinante FNX-Rluc portando un Renilla luciferasi gene reporter per studiare la replicazione del virus. Una linea di cellule di epatoma umano (Huh-7 riferiscono) è stato utilizzato per la trasfezione in vitro di trascritti HCV RNA genomici. Surnatanti di coltura privi di cellule, lisati proteici e tRNA otale sono state raccolte in vari momenti punti post-trasfezione per valutare la crescita HCV. HCV stato replicazione del genoma è stata valutata mediante RT-PCR e visualizzando la presenza di HCV RNA a doppio filamento. L&#39;espressione della proteina di HCV è stata verificata da blot e immunofluorescenza saggi occidentali utilizzando anticorpi specifici per le proteine ​​HCV NS3 e NS5A. Cellule trasfettate HCV RNA rilasciate particelle infettive nella cultura surnatante e il titolo virale è stato misurato. Saggi di luciferasi sono stati utilizzati per valutare il livello di replicazione e infettività di HCV giornalista. In conclusione, vi presentiamo vari saggi virologici per la caratterizzazione delle diverse fasi del ciclo di replicazione di HCV.

Il virus dell&#39;epatite C è un virus a RNA. Così per scopo di manipolazione genetica, l&#39;HCV cDNA genomico è stato clonato in un plasmide vettore batterico. Una sequenza del promotore T7 RNA polimerasi è stata introdotta immediatamente prima estremità 5 &#39;del genoma di HCV. Uno schema generale di workflow analisi HCV è presentato nella figura 1. Per generare HCV RNA genomico con precisione 3 &#39;, il genoma di HCV contenente plasmide è tagliato con l&#39;enzima di restrizione XbaI</e...

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 protocollo per l&#39;analisi dell&#39;epatite C replicazione del virus

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 Views.  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. 

Video: 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.

Utilizzando Imaging Bioluminescent per Indagare il sinergismo tra Streptococcus pneumoniae E Influenza A virus in topi neonati

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

Ricerca

Immunologia e infezioni

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.

Dissezione Host-virus Interazione in replica litico di un Herpesvirus modello

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.

A primaria del sistema cultura Neuron per lo Studio della latenza del virus Herpes Simplex e la riattivazione

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.

High-throughput Assay per Fenotipo<em&gt; Salmonella enterica</em&gt; Typhimurium Associazione, Invasion, e replica nei macrofagi

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.

Immunofluorescenza per Monitorare il Cellular assorbimento di Lattoferrina umana e la sua associata attività antivirale contro il virus dell&#39;epatite 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.

Un miniaturizzato Glycan microarray test per la valutazione avidità e Specificità del virus influenzale A emoagglutinine

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.

Sviluppo di un virus System Reporter dell&#39;epatite B per monitorare la fasi iniziali del ciclo di replicazione

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.

Salvataggio e caratterizzazione del virus ricombinante da un nuovo mondo Clone infettivo del virus Zika

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.

"Fegato-on-a-Chip" colture di epatociti primari e cellule di Kupffer per infezione da Virus dell'epatite 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.

Identificazione di Coding e classi di RNA Non codificanti espressa in suina sangue intero

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