This work was supported in part by the Research Program on Hepatitis from Japan Agency for Medical Research and Development (AMED) and by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

1. Produzione di ricombinante HBV codifica per la proteina Reporter <ol><li> Preparazione delle cellule HepG2 <ol><li> Preparare terreno di coltura cellulare (mezzo di Dulbecco modificato Eagle (DMEM) supplementato con 10% siero bovino fetale (FBS), 100 U / ml di penicillina, 100 ug / ml di streptomicina e 100 ml aminoacidi U / non essenziali). </li><li> Piastra 4 x 10 6 cellule HepG2 in 10 cm Piatto collagene rivestite in 10 ml di mezzo di coltura il giorno prima della transfezione. Incubare le cellule HepG2 a 37 ° C in un umidificata al 5% CO 2 incubatore. NOTA: Quando circa 4 x 10 6 cellule HepG2 vengono piastrate in 10 cm piatto collagene rivestite in 10 ml di mezzo di coltura, saranno 70-90% confluenti il giorno successivo. </li></ol></li><li> trasfezione <ol><li> Cellule trasfezione HepG2 con 5 mg di pUC1.2HBV Delta Epsilon 15 e 5 mg di pUC1.2HBV / NL 15 utilizzando una trasfezioneReattivo secondo le istruzioni del produttore. </li><li> Il giorno successivo, rimuovere il terreno di coltura e aggiungere 10 ml di terreno di coltura fresco. </li><li> Una settimana dopo la trasfezione, trasferire il terreno di coltura contenente l&#39;HBV ricombinante ad un ml tubo 50 e procedere al punto 1.3.1. Aggiungere 10 ml di terreno di coltura fresco alla piastra contenente le cellule trasfettate. NOTA: Produzione del HBV ricombinante viene mantenuta per 4 settimane. Il mezzo di coltura contenente HBV ricombinante può essere conservato a 4 ° C per 1 mese. </li></ol></li><li> Purificazione di HBV ricombinante <ol><li> Rimuovere detriti cellulari dal mezzo di coltura contenente HBV ricombinante mediante centrifugazione (2300 xg per 5 min). </li><li> Passare il surnatante attraverso un filtro da 0,45 micron membrana. </li><li> Aggiungere un volume uguale del 26% PEG / 1,5 M NaCl (polietilenglicole 6000: 130 g, NaCl, 49 g, 1 ml di 0,5 M EDTA pH 8,0 e 5 ml di 1 M HEPES pH 7.6 a 500 ml) al mezzo di coltura contenente ricombinanteHBV e mescolare delicatamente. Incubare per una notte a 4 ° C. </li><li> Centrifugare a 2300 xg per 20 min a 4 ° C. </li><li> Eliminare il supernatante e sciogliere il precipitato in 0,5 ml di TNE (10 mm Tris, 50 mM NaCl, 1 mM EDTA). </li><li> Rimuovere i detriti per centrifugazione a 2.300 xg per 5 min. </li><li> Carico 0.5 ml di TNE contenente HBV ricombinante su 0,8 ml di 20% di saccarosio in TNE. </li><li> Centrifugare a 100.000 xg per 3 ore a 15 ° C. </li><li> Scartare tanto del surnatante possibile, e salvare il pellet. Risospendere in 1 ml di siero DMEM senza per 40 ml di mezzo di coltura di partenza. </li><li> Incubare per una notte a 4 ° C. </li><li> Filtrare attraverso un filtro da 0,45 micron. Preparare 0,5 ml aliquote e conservare a -80 ° C. NOTA: Se si osserva la contaminazione proteina reporter del campione virus originale, di purificare il virus dal gradiente di densità ultra-centrifugazione del 5-30% di saccarosio in TNE a 100.000 xg per 2 ore, o CsCl densità equilibrata centrifugazione from 1.1-1.6 g / ml a 150.000 xg per 50 ore. </li></ol></li></ol> 2. L&#39;infezione di HBV ricombinante <ol><li> Cellule Culture HepG2 stabilmente esprimenti NTCP (HepG2 / NTCP) 15 che sono suscettibili di infezione da HBV in DMEM supplementato con 10% FBS, 100 U / ml penicillina, 100 ug / ml di streptomicina, 250 ug / ml di G-418 e 100 U / ml aminoacidi non essenziali a 37 ° C in un umidificata al 5% CO 2 incubatore. </li><li> Un giorno prima dell&#39;infezione, piatto di circa 5 x 10 4 HepG2 / cellule NTCP 14 in una piastra ricoperta da 96 pozzetti di collagene in 0,1 ml di terreno di coltura. </li><li> Scongelare HBV ricombinante in un bagnomaria C 37 ° fino a quando c&#39;è un piccolo pezzo di ghiaccio che resta nel flaconcino. </li><li> Preparare il mezzo di infezione combinando i seguenti: 10 ml di 40% PEG8000 in 1x tampone fosfato salino (PBS), 2 ml di dimetilsolfossido (DMSO), 10 ml di HBV ricombinante e 78 ml di terreno di coltura frescoper pozzetto di una piastra a 96 pozzetti. </li><li> Aggiungere 100 pl di soluzione di HBV ricombinante ad un pozzetto della piastra a 96 pozzetti. </li><li> Un giorno dopo l&#39;infezione, lavare le cellule infettate 3 volte con 300 ml di PBS per pozzetto per rimuovere la proteina contaminante giornalista dalla frazione virus. </li><li> Incubare le cellule infette in 200 ml di terreno di coltura contenente 2% DMSO per una settimana o meno. </li></ol> 3. Analisi <ol><li> Una settimana dopo l&#39;infezione, lavare le cellule infettate 3 volte con PBS. </li><li> Aggiungere 50 ml di tampone di lisi delle cellule infette. </li><li> Oscillare la piastra di coltura per 5 minuti, quindi si centrifuga a 2000 xg per 5 min. </li><li> Aggiungere 50 ml di substrato giornalista nella piastra luminometro. </li><li> Aggiungere 20 ml di lisato cellulare ad una piastra luminometro contenente il substrato giornalista. Mescolare vortexando brevemente. </li><li> Porre la piastra in luminometro e avviare la lettura 15. </li></ol>

<ol>
	<li>Fattovich, G., Stroffolini, T., Zagni, I., Donato, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma+in+cirrhosis:+incidence+and+risk+factors.">Hepatocellular carcinoma in cirrhosis: incidence and risk factors.</a> Gastroenterology. 127 (5), 35-50 (2004).</li><li>Revill, P., Testoni, B., Locarnini, S., Zoulim, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+strategies+are+required+to+cure+and+eliminate+HBV+infection.">Global strategies are required to cure and eliminate HBV infection.</a> Nat. Rev. Gastroenterol. Hepatol. 13 (4), 239-248 (2016).</li><li>Wursthorn, K., 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=Peginterferon+alpha-2b+plus+adefovir+induce+strong+cccDNA+decline+and+HBsAg+reduction+in+patients+with+chronic+hepatitis+B.">Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.</a> Hepatology. 44 (3), 675-684 (2006).</li><li>Stuyver, L. 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=Nomenclature+for+antiviral-resistant+human+hepatitis+B+virus+mutations+in+the+polymerase+region.">Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region.</a> Hepatology. 33 (3), 751-757 (2001).</li><li>Ochiya, T., Tsurimoto, T., Ueda, K., Okubo, K., Shiozawa, M., Matsubara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+system+for+infection+with+hepatitis+B+virus+that+uses+primary+human+fetal+hepatocytes.">An in vitro system for infection with hepatitis B virus that uses primary human fetal hepatocytes.</a> Proc. Natl. Acad. Sci. USA. 86 (6), 1875-1879 (1989).</li><li>Galle, P. R., Hagelstein, J., Kommerell, B., Volkmann, M., Schranz, P., Zentgraf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+experimental+infection+of+primary+human+hepatocytes+with+hepatitis+B+virus.">In vitro experimental infection of primary human hepatocytes with hepatitis B virus.</a> Gastroenterology. 106 (3), 664-673 (1994).</li><li>Gripon, P., 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=Infection+of+a+human+hepatoma+cell+line+by+hepatitis+B+virus.">Infection of a human hepatoma cell line by hepatitis B virus.</a> Proc. Natl. Acad. Sci. USA. 99 (24), 15655-15660 (2002).</li><li>Murakami, 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=Discovering+novel+direct+acting+antiviral+agents+for+HBV+using+in+silico+screening.">Discovering novel direct acting antiviral agents for HBV using in silico screening.</a> Biochem. Biophys. Res. Commun. 456 (1), 20-28 (2015).</li><li>Ladner, S. K., 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=Inducible+expression+of+human+hepatitis+B+virus+(HBV)+in+stably+transfected+hepatoblastoma+cells:+a+novel+system+for+screening+potential+inhibitors+of+HBV+replication.">Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.</a> Antimicrob. Agents. Chemother. 41 (8), 1715-1720 (1997).</li><li>Yan, H., 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=Sodium+taurocholate+cotransporting+polypeptide+is+a+functional+receptor+for+human+hepatitis+B+and+D+virus.">Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.</a> Elife. 3, (2012).</li><li>Wang, Z., 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-competent+infectious+hepatitis+B+virus+vectors+carrying+substantially+sized+transgenes+by+redesigned+viral+polymerase+translation.">Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.</a> PLoS. One. 8, 60306 (2013).</li><li>Chaisomchit, S., Tyrrell, D. L., Chang, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+replicative+and+nonreplicative+hepatitis+B+virus+vectors.">Development of replicative and nonreplicative hepatitis B virus vectors.</a> Gene. Ther. 4 (12), 1330-1340 (1997).</li><li>Hong, 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=Novel+recombinant+hepatitis+B+virus+vectors+efficiently+deliver+protein+and+RNA+encoding+genes+into+primary+hepatocytes.">Novel recombinant hepatitis B virus vectors efficiently deliver protein and RNA encoding genes into primary hepatocytes.</a> J. Virol. 87 (12), 6615-6624 (2013).</li><li>Hall, M. P., 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=Engineered+luciferase+reporter+from+a+deep+sea+shrimp+utilizing+a+novel+imidazopyrazinone+substrate.">Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.</a> ACS. Chem. Biol. 7 (11), 1848-1857 (2012).</li><li>Nishitsuji, H., 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=Novel+reporter+system+to+monitor+early+stages+of+the+hepatitis+B+virus+life+cycle.">Novel reporter system to monitor early stages of the hepatitis B virus life cycle.</a> Cancer. Sci. 106 (11), 1616-1624 (2015).</li><li>Sugiyama, 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=Influence+of+hepatitis+B+virus+genotypes+on+the+intra-+and+extracellular+expression+of+viral+DNA+and+antigens.">Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens.</a> Hepatology. 44 (4), 915-924 (2006).</li><li>Seeger, C., Zoulim, F., Mason, W., Knipe, D., Howley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepadnaviruses.">Hepadnaviruses.</a> Field Virology. Volume 1. , 2185-2221 (2013).</li><li>Moolla, N., Kew, M., Arbuthnot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+elements+of+hepatitis+B+virus+transcription.">Regulatory elements of hepatitis B virus transcription.</a> J. Viral. Hepat. 9 (5), 323-331 (2002).</li><li>Cattaneo, R., Will, H., Schaller, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus+transcription+in+the+infected+liver.">Hepatitis B virus transcription in the infected liver.</a> EMBO. J. 3 (9), 2191-2196 (1984).</li><li>Enders, G. H., Ganem, D., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+major+transcripts+of+ground+squirrel+hepatitis+virus:+the+presumptive+template+for+reverse+transcriptase+is+terminally+redundant.">Mapping the major transcripts of ground squirrel hepatitis virus: the presumptive template for reverse transcriptase is terminally redundant.</a> Cell. 42 (1), 297-308 (1985).</li><li>Haines, K. M., Loeb, D. 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+sequence+of+the+RNA+primer+and+the+DNA+template+influence+the+initiation+of+plus-strand+DNA+synthesis+in+hepatitis+B+virus.">The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus.</a> J. Mol. Biol. 370 (3), 471-480 (2007).</li><li>Hirsch, R. C., Lavine, J. E., Chang, L. J., Varmus, H. E., Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+gene+products+of+hepatitis+B+viruses+are+required+for+genomic+RNA+packaging+as+well+as+for+reverse+transcription.">Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as well as for reverse transcription.</a> Nature. 344 (6266), 552-555 (1990).</li></ol>

The authors declare that they have no competing financial interests.

Il genoma HBV ha quattro principali fasi di lettura aperti (ORF), tra cui il nucleo, polimerasi, di superficie e X ORF (Figura 1). Trascrizione di questi quattro HBV ORF è strettamente regolata da quattro promotori: il promotore precore / core contenenti enhancer II, S1 promotore, promotrice S2 e X promotore contenente enhancer I 18. I 3,5 kb e 3,4 kb mRNA sono tradotti in precore e Core proteine, rispettivamente. La grande proteina busta (L) è prodotto dal più grande mRNA sub...

L&#39;infezione cronica da virus dell&#39;epatite B (HBV) è un importante fattore di rischio per le malattie croniche del fegato 1. Sebbene strategie terapeutiche attuali si basano su analoghi nucleotidici che inibiscono la funzione HBV pol e / o la somministrazione di tipo I interferon che attiva risposte immunitarie negli individui infetti nonché indirettamente sopprimere la proliferazione HBV attraverso funzioni genici stimolati 2, questi trattamenti non possono eliminare HBV DNA completamente 3. Inoltre, l&#39;emergere di HBVs che sono resistenti agli agenti anti-Pol è di preoccupazione 4. La somministrazione di agenti anti-virali combinati rivolti direttamente le varie fasi del virus dell&#39;immunodeficienza umana (HIV) o il ciclo di vita del virus dell&#39;epatite C (HCV) è stato dimostrato che sopprimere o eliminare il virus (es) con successo. Simile a questa idea, lo sviluppo di anti-HBV agent (s) che agiscono direttamente sulle diverse fasi di HBV ciclo di vita è importante per stabilire le future terapie HBV. In generale, la creazione di un semplice sistema di coltura in vitro del virus destinazione facilita lo sviluppo di agenti anti-virus. Tuttavia, ci sono almeno due ostacoli allo sviluppo di sistemi di coltura in vitro per lo screening agenti anti-HBV. La prima è la mancanza di una comoda vitro sistema di coltura cellulare a HBV / proliferazione. A differenza di altri virus, come l&#39;HIV e HCV, che si propagano in linee cellulari stabilizzate, è difficile da coltivare HBV in vitro a causa delle limitazioni sperimentali compresa una gamma ristretta ospitante. L&#39;uso di sistemi di coltura cellulare specifici quali la linea di cellule di epatoma umano HepaRG, che è suscettibile di infezione da HBV, 5, 6, 7 sono state sviluppate per superare questi problemi. Inoltre, le cellule PXB, isolate da urochinasi-tyPE attivatore del plasminogeno transgenici / topi SCID inoculati con primari epatociti umani (PHH), hanno dimostrato di essere suscettibili di infezione da HBV e la replicazione 8. Tuttavia, i livelli di replicazione dell&#39;HBV in HepaRG dipendono dallo stato differenziazione cellulare dopo la cultura, che può causare risultati incoerenti e non riproducibili su livelli di infezione / replicazione dell&#39;HBV. PXB è comunemente utilizzato per esperimenti di infezione da HBV, ma è limitata dalla sua disponibilità. Una linea di cellule espressione inducibile HBV tetraciclina, HepAD38, è stato ampiamente utilizzato per studiare la replicazione dell&#39;HBV, ma questo sistema consente solo di valutazione dopo la trascrizione e non al passo ingresso di infezione da HBV 9. Recentemente, l&#39;identificazione di sodio taurocolato cotransporting polipeptide (NTCP) come un recettore funzionale per HBV ha consentito lo sviluppo di un sistema di coltura variabile di HBV 10. In effetti, l&#39;espressione NTCP in cellule di epatocarcinoma non sensibili, come Huh7 eHepG2 consente HBV 10 e, quindi, la scelta delle linee cellulari sensibili HBV è stata ampliata, risolvere molti dei limiti sperimentali. Il secondo problema è la mancanza di un semplice sistema di test per valutare HBV e la replicazione. Valutazione della HBV è solitamente condotta analizzando HBV DNA, RNA e proteine. Tuttavia, la quantificazione di questi marcatori virali è che richiede tempo, spesso costosi e non sempre semplice. Pertanto, lo sviluppo di un semplice sistema di analisi, ad esempio utilizzando un gene reporter, potrebbe superare i problemi associati ai sistemi di dosaggio HBV. Tuttavia, poiché la dimensione del genoma che può essere confezionato in un capside HBV è limitata - meno di 3,7 kb 11 - la dimensione di un gene reporter deve essere il più breve possibile. Inoltre, la presenza di più elementi cis sparsi in tutto il genoma, che sono essenziali per la replicazione virale, limita le posizioni disponibili dal<html> serimento del gene reporter nel genoma. Diversi studi hanno tentato di inserire geni estranei, compreso l&#39;HIV-1 Tat, proteina fluorescente verde e DsRed, in HBV genoma 11, 12, 13. Tuttavia, questi HBVs ricombinanti non sono utili per lo screening HBV / replica, o per l&#39;high-throughput screening dei fattori che influenzano l&#39;infezione da HBV / replica. Questo è principalmente a causa della bassa produttività dei virus ricombinanti e la ridotta intensità di espressione del gene reporter causato dalla produzione di virus inefficiente. Per superare questi problemi, abbiamo costruito un HBV giornalista con un alto rendimento di produzione di virus. Questo virus è altamente sensibile per monitorare le prime fasi del ciclo di replicazione dell&#39;HBV, dall&#39;entrata alla trascrizione. Per raggiungere questo obiettivo, NanoLuc (NL) è stato scelto come gene marcatore perché è un piccolo (171 amminoacidi) progettati giornalista luminescenticlass = &quot;xref&quot;&gt; 14. Inoltre, NL è di circa 150 volte più luminoso di lucciola o Renilla luciferasi, e la reazione luminescenti è ATP-indipendenti, il che suggerisce che il tasso di successo falso sarà basso per lo screening ad alto rendimento. L&#39;efficienza produttiva del HBV ricombinante è pari a circa 1/5 del genitore HBV, e simile a livelli riportati per precedenti virus ricombinanti HBV; tuttavia, la luminosità NL supera problemi di produttività virus in modo che possa essere utilizzato per lo screening di massa di agenti anti-HBV. Proiezione di agenti anti-HBV utilizzando epatociti primari, HepaRG, HepAD38 e epatociti NTCP-trasdotte potrebbe essere utile per lo screening di agenti anti-HBV con il metodo convenzionale (s). Tuttavia, il sistema qui descritto presenta diversi vantaggi, quali una semplice manipolazione, ad alta sensibilità e basso costo per lo screening. Questi vantaggi sono adatti per dosaggi elevati di throughput per lo sviluppo e identificare nuovi agenti HBV per scopi terapeutici.

<table border="0" cellpadding="0" cellspacing="0" width="781">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Nano-Glo Luciferase Assay Regent</td>
			<td style="width:201px;">Promega</td>
			<td style="width:160px;">N1110</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin Mixed Solution</td>
			<td>Nacalai tesque</td>
			<td>09367-34&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:332px;">100 mm/collagen-coated dish</td>
			<td>Iwaki</td>
			<td>4020-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 3000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000001&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol (PEG) 6000&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>81255</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol (PEG) 8000&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Nacalai tesque</td>
			<td>31319-45&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 mol/l-EDTA Solution</td>
			<td>Nacalai tesque</td>
			<td>06894-14&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>Nacalai tesque</td>
			<td>35434-21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millex-HP, 0.45 &#956;m, polyethethersulfone, filter</td>
			<td>Merck Millipore</td>
			<td>SLHP033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagen coated 96-well plate</td>
			<td>Corning</td>
			<td>NO3585</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Passive Lysis 5xBuffer</td>
			<td>Promega</td>
			<td>E1941</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GloMax 96 Microplate Luminometer</td>
			<td>Promega</td>
			<td>E6501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Nacalai tesque</td>
			<td>30403-55</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luminometer plate</td>
			<td>Greiner bio-one</td>
			<td>655075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HepG2-NTCP1-myc-clone22</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC1.2HBV delta epsilon</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC1.2HBV/NL</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tube</td>
			<td>Violamo</td>
			<td>1-3500-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Myc antibody</td>
			<td>Sigma-Aldrich</td>
			<td>C3956</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBIG</td>
			<td>Japan Blood Products Organization</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IFN-&#946;</td>
			<td>Mochida Pharmaceutical</td>
			<td>14987224005413</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3393</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

La figura 1 mostra uno schema del genoma HBV, RNA trascritti, proteine del virus e la cui regione codificante sul genoma. Il gene reporter e la sua posizione nel genoma sono anche indicati. La Figura 2 mostra l&#39;HBV plasmide reporter e helper plasmide contenente il gene reporter. Il reporter pUC1.2HBV / NL è stato costruito eliminando posizioni nucleotidiche 223-811 dal sito inizio della trascrizione del mRNA precore di pUC1.2HBV 16...

Watch this Scientific Journal Video about Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle at JoVE.com

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

Qui si descrive un virus dell&#39;epatite B recentemente sviluppato (HBV) sistema reporter per monitorare le prime fasi del ciclo di vita HBV. Questo semplificata nel sistema vitro sarà di aiuto nella proiezione di agenti anti-HBV utilizzando una strategia ad alto rendimento.

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), ...

development-hepatitis-b-virus-reporter-system-to-monitor-early-stages

Research

JoVE Journal

Immunology and Infection

13.1K Views.  National Center for Global Health and Medicine.  Qui si descrive un virus dell'epatite B recentemente sviluppato (HBV) sistema reporter per monitorare le prime fasi del ciclo di vita HBV. Questo semplificata nel sistema vitro sarà di aiuto nella proiezione di agenti anti-HBV utilizzando una strategia ad alto rendimento. 

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

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Sviluppo di un marcatore negativo selezionabile per Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

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 Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

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 ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Mouse fetale Fegato Cultura di sistema per sezionare Funzioni gene bersaglio nelle fasi precoci e tardivi del Terminal eritropoiesi

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Analisi Vitro di Myd88 mediata Cellular Immune Response to West Nile virus mutante Strain Infezione

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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 ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

impennata<em&gt; Ixodes scapularis,</em&gt; Il Tick Black-legged: Alimentazione stadi immaturi sui topi

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nuovo sistema privo di alimentatore per la produzione di massa di cellule di assassino naturali murini In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

Assemblaggio e purificazione del prototipo Virus schiumoso Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Rapido, sicuro e semplice manuale sul comodino estrazione dell'acido nucleico per la rilevazione del Virus in campioni di sangue intero

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Sviluppo di un virus System Reporter dell'epatite B per monitorare la fasi iniziali del ciclo di replicazione

Riepilogo

Esplora Altri Video

Capitoli in questo video

Sviluppo di un marcatore negativo selezionabile per Entamoeba histolytica

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

Un protocollo per l'analisi dell'epatite C replicazione del virus

Mouse fetale Fegato Cultura di sistema per sezionare Funzioni gene bersaglio nelle fasi precoci e tardivi del Terminal eritropoiesi

In Analisi Vitro di Myd88 mediata Cellular Immune Response to West Nile virus mutante Strain Infezione

Immunofluorescenza per Monitorare il Cellular assorbimento di Lattoferrina umana e la sua associata attività antivirale contro il virus dell'epatite C

impennata

Un nuovo sistema privo di alimentatore per la produzione di massa di cellule di assassino naturali murini In Vitro

Assemblaggio e purificazione del prototipo Virus schiumoso Intasomes

Rapido, sicuro e semplice manuale sul comodino estrazione dell'acido nucleico per la rilevazione del Virus in campioni di sangue intero