This study is supported by funding from Taipei Medical University Hospital (102TMU-TMUH-19) and the Ministry of Science and Technology of Taiwan (MOST103-2320-B-038-031-MY3).

Nota: verificare che tutte le procedure che coinvolgono coltura cellulare e l&#39;infezione da virus sono condotte in cappe di biosicurezza certificate che sono appropriati per il livello di biosicurezza dei campioni essere manipolati. Per lo scopo di descrivere i protocolli, Gaussia luciferasi giornalista HCV-tag è usato come modello di virus 32. Nel contesto dei risultati rappresentativi, l&#39;acido composti chebulagic (CHLA) e punicalagin (PUG) sono utilizzati come farmaci antivirali candidati che hanno come target le interazioni glicoproteina virale con glicosaminoglicani superficie cellulare durante la ingresso del virus presto passi 31. Eparina, che è noto per interferire con l&#39;ingresso di molti virus 30,31,33,34, viene usato come trattamento controllo positivo in questo contesto. Per informazioni generali di base sulle tecniche di virologia, la propagazione di virus, determinazione del titolo del virus, e l&#39;espressione della dose infettante in unità formanti placca (PFU), si concentrano unità formanti (FFU), o molteplicità di infezione (MOI), il lettore è reFERRED di riferimento 35. Per esempi precedenti e condizioni ottimizzate utilizzati per virus mostrati nei risultati rappresentativi, si rimanda ai riferimenti 30-32,36-39 e dettagli elencati nella Tabella 1, Figura 1A, 2A e Figura. Cultura 1. Cella, Preparazione Compound, e Compound citotossicità <ol><li> Grow rispettiva linea cellulare per il sistema virus da analizzare (Tabella 1). Per HCV, far crescere le cellule Huh-7.5 in modificata mezzo di Eagle Dulbecco (DMEM) supplementato con 10% di siero fetale bovino (FBS), 200 U / ml di penicillina G, 200 mg / ml di streptomicina, e 0,5 mg / ml di amfotericina B. </li><li> Preparare i composti in esame ei controlli utilizzando rispettivi solventi: per esempio, sciogliere CHLA e PUG in dimetilsolfossido (DMSO); preparare eparina in acqua bidistillata sterile. Per tutte le diluizioni successive, utilizzare terreni di coltura. Nota: Il concent finalerazione di DMSO nei trattamenti composto in esame è inferiore all&#39;1% negli esperimenti; 1% DMSO è inclusa come trattamento controllo negativo nei saggi di confronto. </li><li> Determinare la citotossicità dei composti in esame (per esempio, CHLA e PUG) sulle cellule per l&#39;infezione virale utilizzando una vitalità cellulare determinare reagente come XTT (2,3-bis [2-metossi-4-nitro-5-solfofenil] -5-fenilammino) carbonile] idrossido -2H-tetrazolio): <ol><li> Per HCV, sementi Huh-7,5 cellule in un piatto da 96 pozzetti (1 × 10 4 cellule per pozzetto) e incubare a 37 ° C in un incubatore CO 2 5% O / N per ottenere un monostrato. </li><li> Applicare controllo DMSO (1%) e concentrazioni crescenti dei composti di prova e CHLA PUG (es. 0, 10, 50, 100, e 500 pM) ai pozzetti di coltura in triplice copia. </li><li> Incubare a 37 ° C per 72 ore, poi scarta il mezzo in piastra e lavare le cellule con 200 ml di tampone fosfato salino (PBS) due volte. </li><li> Aggiungere 100 ml di assaying soluzione dal kit in vitro tossicologia basata XTT ciascun pozzetto ed incubare le piastre a 37 ° C per altri 3 ore per permettere la produzione XTT formazan. </li><li> Determinare l&#39;assorbanza con un lettore per micropiastre alla lunghezza d&#39;onda di prova di 492 nm e una lunghezza d&#39;onda di riferimento di 690 nm. </li><li> Calcolare la percentuale di cellule sopravvissute utilizzando la seguente formula: vitalità cellulare (%) = A / As × 100%, dove &#39;A&#39; e &#39;Come&#39; consultare l&#39;assorbanza dei composti in esame e il controllo solvente (ex 1% DMSO. ) trattamenti, rispettivamente. Determinare la concentrazione di citotossicità cellulare 50% (CC 50) dei composti in esame da un software di analisi come GraphPad Prism secondo il protocollo del produttore. </li></ol></li></ol> 2. Lettura di infezione virale Nota: La lettura di infezione virale dipende dal sistema virus impiegato e può comportare metodi come saggi placca o measuring segnali giornalista da virus giornalista-tag. Il metodo per determinare l&#39;infezione giornalista-HCV in base all&#39;attività della luciferasi è descritto di seguito. <ol><li> Raccogliere i surnatanti dai pozzetti infetti e chiarire a 17.000 xg in una microcentrifuga per 5 minuti a 4 ° C. </li><li> Mescolare 20 ml di surnatante di prova a 50 ml di luciferasi substrato dal kit assay luciferasi Gaussia e misurare direttamente con un luminometro secondo le istruzioni del produttore. </li><li> Express HCV infettività come log 10 di unità di luce relativa (RLU) per determinare l&#39;inibizione virale (%) e calcolare la concentrazione del 50% efficace (CE 50) dei composti in esame contro l&#39;infezione da HCV utilizzando algoritmi da software GraphPad Prism secondo il protocollo del produttore. </li></ol> 3. disattivazione virale Assay Nota: esempi di periodo di incubazione e la dose virale per vari virus unri elencati nella Figura 1A. Concentrazioni più elevate del virus possono essere testati aumentando il MOI / PFU. <ol><li> Seme Huh-7.5 celle in una piastra a 96 pozzetti (1 × 10 4 cellule per pozzetto) e incubare a 37 ° C in un incubatore CO 2 5% O / N per ottenere un monostrato. </li><li> Incubare i composti di prova o di controllo (concentrazioni finali sono: CHLA = 50 micron; PUG = 50 micron; eparina = 1,000 mg / ml; DMSO = 1%) con le particelle di HCV a 37 ° C (Figura 1A, &#39;a lungo termine&#39; ) in un rapporto 1: 1. Ad esempio, per un inoculo del virus 100 ml contenente 1 x 10 4 FFU, aggiungere 100 ml di una diluizione di lavoro 100 mM CHLA; questo produce trattamento CHLA ad una concentrazione finale di 50 mM. </li><li> Diluire la miscela virus-farmaco (inefficace) concentrazione &quot;sub-terapeutica&quot; dei composti in esame. Ad esempio, la concentrazione di CHLA inefficace e PUG contro l&#39;HCV è a 1 pM 31; dunquequesto richiede una diluizione di 50 volte della miscela virus farmaco che può essere realizzato con 9,8 ml di mezzo basale (medium di coltura delle cellule con 2% FBS). Nota: La diluizione a concentrazione sub-terapeutica impedisce significativa interazione tra i composti di prova e la superficie della cellula ospite e consente l&#39;esame degli effetti del trattamento sui virioni cell-free. Si noti che questa diluizione è dipendente dalla dose-risposta antivirale dei composti in esame contro la particolare infezione virale, ed è determinata prima di eseguire questo particolare test 31. </li><li> Per confronto, mescolare il virus con i composti di test e immediatamente diluito (senza periodo di incubazione) alla concentrazione sub-terapeutica prima di infezione (Figura 1A, &#39;a breve termine&#39;). </li><li> Aggiungere 100 pl di miscela HCV-farmaco diluito sulla Huh-7.5 monostrato di cellule (la quantità di virus è ora a 1 x 10 2 FFU; finale MOI = 0.01) e incubare per 3 ore a 37 ° C per consentire viraleadsorbimento. </li><li> Seguendo l&#39;infezione, rimuovere il inoculi diluito e lavare delicatamente i pozzetti con 200 ml di PBS due volte. Nota: Eseguire le lava delicatamente per evitare di sollevare le cellule. </li><li> Applicare 100 ml di terreno di base a ciascun pozzetto e incubare a 37 ° C per 72 ore. </li><li> Analizzare l&#39;infezione risultante analizzando il surnatante per l&#39;attività luciferasi come descritto in &#39;2. Lettura di infezione virale &#39;. </li></ol> 4. Viral Allegato Assay Nota: esempi di periodo di incubazione e la dose virale per vari virus sono elencati nella Figura 2A, &#39;allegato&#39;. Concentrazioni più elevate del virus possono essere testati aumentando il MOI / PFU. <ol><li> Seme Huh-7.5 celle in una piastra a 96 pozzetti (1 × 10 4 cellule per pozzetto) e incubare a 37 ° C in un incubatore CO 2 5% O / N per ottenere un monostrato. </li><li> Pre-raffreddare i monostrati cellulari in piastre a 4 ° C for 1 ora. </li><li> Co-trattare le cellule con HCV inoculo (MOI = 0.01) e composti di prova o controlli (concentrazioni finali sono: CHLA = 50 mM; PUG = 50 mM; eparina = 1,000 g / ml; DMSO = 1%) a 4 ° C per 3 ore. Ad esempio, per un inoculo del virus 90 ml contenente 1 x 10 2 FFU, aggiungere 10 ml di una diluizione di lavoro 500 mM CHLA; questo produce trattamento CHLA ad una concentrazione finale di 50 mM e un&#39;infezione da HCV a MOI = 0.01 sul monostrato cellulare. Nota: È importante effettuare l&#39;esperimento a 4 ° C in quanto consente per il legame del virus, ma preclude ingresso che si verifica più efficiente a 37 ° C. Effettuare l&#39;aggiunta di composti di virus e di test su ghiaccio e la conseguente incubazione in un 4 ° C frigorifero per garantire che la temperatura è mantenuta a 4 ° C. </li><li> Rimuovere il surnatante e lavare delicatamente il monostrato cellulare con 200 ml di ghiacciata PBS due volte. Nota: Eseguire le lava delicatamente per evitare di sollevare le cellule &lt;./ li&gt; <li> Applicare 100 ml di terreno di base a ciascun pozzetto e incubare a 37 ° C per 72 ore. </li><li> Analizzare l&#39;infezione risultante analizzando il surnatante per l&#39;attività luciferasi come descritto in &#39;2. Lettura di infezione virale &#39;. </li></ol> 5. Viral Entrata / Fusion Assay Nota: Esempio di periodi di incubazione e la dose virale per vari virus sono elencati nella Figura 2A &#39;Entry / fusione&#39;. Concentrazioni più elevate del virus possono essere testati aumentando il MOI / PFU. <ol><li> Seme Huh-7.5 celle in una piastra a 96 pozzetti (1 × 10 4 cellule per pozzetto) e incubare a 37 ° C in un incubatore CO 2 5% O / N per ottenere un monostrato. </li><li> Pre-raffreddare i monostrati cellulari in piastre a 4 ° C per 1 ora. </li><li> Infettare le cellule con HCV (MOI = 0,01) a 4 ° C per 3 ore. Ad esempio, utilizzare un inoculo del virus 100 ml contenente 1 x 10 2 FFU. Nota: Eseguire la addizione dell&#39;inoculo virale su ghiaccio e la conseguente incubazione in un 4 ° C frigorifero per mantenere la temperatura a 4 ° C, che permette virale entrata ma non vincolante. </li><li> Rimuovere il surnatante e lavare delicatamente i monostrati cellulari con 200 ml di ghiacciata PBS due volte. Nota: Eseguire le lava delicatamente per evitare di sollevare le cellule. </li><li> Trattare i pozzetti con i composti in esame o controlli (concentrazioni finali sono: CHLA = 50 mM; PUG = 50 mM; eparina = 1,000 g / ml; DMSO = 1%) e incubare a 37 ° C per 3 ore. Ad esempio, aggiungere 10 ml di una diluizione di lavoro di 500 micron CHLA a 90 ml di media, mescolare, e trattare i pozzetti; questo produce trattamento CHLA ad una concentrazione finale di 50 mM. Nota: Il passaggio da 4 ° C a 37 ° C facilita ora l&#39;evento di ingresso / fusione virale e quindi permette la valutazione dell&#39;effetto composti di prova &#39;su questo particolare punto. </li><li> Aspirare il surnatante contenente droga e rimuovere non interiorizzatovirus extracellulari né dal lavaggio con 200 ml di tampone citrato (citrato di sodio 50 mM, cloruro di potassio 4 mM, pH 3,0) o PBS. Applicare 100 ml di terreno di base prima di incubazione a 37 ° C per 72 ore. </li><li> Analizzare l&#39;infezione risultante analizzando il surnatante per l&#39;attività luciferasi come descritto in &#39;2. Lettura di infezione virale &#39;. </li></ol>

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	<li>Munier, C. M., Andersen, C. R., Kelleher, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+vaccines:+progress+to+date.">HIV vaccines: progress to date.</a> Drugs. 71, 387-414 (2011).</li><li>Rothman, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+to+dengue+virus:+a+tale+of+original+antigenic+sin+and+tropical+cytokine+storms.">Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms.</a> Nat Rev Immunol. 11, 532-543 (2011).</li><li>Sung, H., Schleiss, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+the+current+status+of+cytomegalovirus+vaccines.">Update on the current status of cytomegalovirus vaccines.</a> Expert Rev Vaccines. 9, 1303-1314 (2010).</li><li>Torresi, J., Johnson, D., Wedemeyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+development+of+preventive+and+therapeutic+vaccines+for+hepatitis+C+virus.">Progress in the development of preventive and therapeutic vaccines for hepatitis C virus.</a> J Hepatol. 54, 1273-1285 (2011).</li><li>Wright, M., Piedimonte, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+syncytial+virus+prevention+and+therapy:+past,+present,+and+future.">Respiratory syncytial virus prevention and therapy: past, present, and future.</a> Pediatr Pulmonol. 46, 324-347 (2011).</li><li>Christou, L. <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+bacterial+and+viral+zoonotic+infections.">The global burden of bacterial and viral zoonotic infections.</a> Clin Microbiol Infect. 17, 326-330 (2011).</li><li>Cascio, A., Bosilkovski, M., Rodriguez-Morales, A. J., Pappas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+socio-ecology+of+zoonotic+infections.">The socio-ecology of zoonotic infections.</a> Clin Microbiol Infect. 17, 336-342 (2011).</li><li>Grais, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measles+vaccination+in+humanitarian+emergencies:+a+review+of+recent+practice.">Measles vaccination in humanitarian emergencies: a review of recent practice.</a> Confl Health. 5, 21 (2011).</li><li>Gautret, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+viral+respiratory+tract+infections-environmental+risk+factors+and+transmission.">Emerging viral respiratory tract infections-environmental risk factors and transmission.</a> Lancet Infect Dis. 14, 1113-1122 (2014).</li><li>Sampathkumar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+East+respiratory+syndrome:+what+clinicians+need+to+know.">Middle East respiratory syndrome: what clinicians need to know.</a> Mayo Clin Proc. 89, 1153-1158 (2014).</li><li>Burd, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ebola+Virus:+a+Clear+and+Present+Danger.">Ebola Virus: a Clear and Present Danger.</a> J Clin Microbiol. 53, 4-8 (2015).</li><li>Bishop, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+and+Emerging+Treatment+Options+for+Ebola+Virus+Disease.">Potential and Emerging Treatment Options for Ebola Virus Disease.</a> Ann Pharmacother. , (2014).</li><li>Arduino, P. G., Porter, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+and+perioral+herpes+simplex+virus+type+1+(HSV-1)+infection:+review+of+its+management.">Oral and perioral herpes simplex virus type 1 (HSV-1) infection: review of its management.</a> Oral Dis. 12, 254-270 (2006).</li><li>Mitrasinovic, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+structure-based+design+of+the+influenza+A+neuraminidase+inhibitors.">Advances in the structure-based design of the influenza A neuraminidase inhibitors.</a> Curr Drug Targets. 11, 315-326 (2010).</li><li>Soriano, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directly+acting+antivirals+against+hepatitis+C+virus.">Directly acting antivirals against hepatitis C virus.</a> J Antimicrob Chemother. 66, 1673-1686 (2011).</li><li>Haqqani, A. A., Tilton, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+inhibitors+and+their+use+in+the+treatment+of+HIV-1+infection.">Entry inhibitors and their use in the treatment of HIV-1 infection.</a> Antiviral Res. 98, 158-170 (2013).</li><li>Melby, T., Westby, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+viral+entry.">Inhibitors of viral entry.</a> Handb Exp Pharmacol. , 177-202 (2009).</li><li>Vanderlinden, E., Naesens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+antiviral+strategies+to+interfere+with+influenza+virus+entry.">Emerging antiviral strategies to interfere with influenza virus entry.</a> Med Res Rev. 34, 301-339 (2014).</li><li>Antoine, T. E., Park, P. J., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+targeted+therapeutics:+a+new+era+of+anti-herpes+simplex+virus-1+therapeutics.">Glycoprotein targeted therapeutics: a new era of anti-herpes simplex virus-1 therapeutics.</a> Rev Med Virol. 23, 194-208 (2013).</li><li>Pawlotsky, J. M., Chevaliez, S., McHutchison, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hepatitis+C+virus+life+cycle+as+a+target+for+new+antiviral+therapies.">The hepatitis C virus life cycle as a target for new antiviral therapies.</a> Gastroenterology. 132, 1979-1998 (2007).</li><li>Beyleveld, G., White, K. M., Ayllon, J., Shaw, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New-generation+screening+assays+for+the+detection+of+anti-influenza+compounds+targeting+viral+and+host+functions.">New-generation screening assays for the detection of anti-influenza compounds targeting viral and host functions.</a> Antiviral Res. 100, 120-132 (2013).</li><li>Kilianski, A., Baker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+antiviral+screening+against+coronaviruses:+developing+virus-specific+and+broad-spectrum+inhibitors.">Cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors.</a> Antiviral Res. 101, 105-112 (2014).</li><li>Caillet-Saguy, C., Lim, S. P., Shi, P. Y., Lescar, J., Bressanelli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerases+of+hepatitis+C+viruses+and+flaviviruses:+structural+and+mechanistic+insights+and+drug+development.">Polymerases of hepatitis C viruses and flaviviruses: structural and mechanistic insights and drug development.</a> Antiviral Res. 105, 8-16 (2014).</li><li>Frey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+cell-cell+fusion+induced+by+the+envelope+glycoprotein+of+human+immunodeficiency+virus+type+1.">Temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1.</a> J Virol. 69, 1462-1472 (1995).</li><li>Tscherne, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-+and+temperature-dependent+activation+of+hepatitis+C+virus+for+low-pH-triggered+entry.">Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry.</a> J Virol. 80, 1734-1741 (2006).</li><li>Madan, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+umbrellas:+a+novel+class+of+candidate+topical+microbicides+to+prevent+human+immunodeficiency+virus+and+herpes+simplex+virus+infections.">Molecular umbrellas: a novel class of candidate topical microbicides to prevent human immunodeficiency virus and herpes simplex virus infections.</a> J Virol. 81, 7636-7646 (2007).</li><li>Haywood, A. M., Boyer, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+and+temperature+dependence+of+influenza+virus+membrane+fusion+at+neutral+pH.">Time and temperature dependence of influenza virus membrane fusion at neutral pH.</a> J Gen Virol. 67 (Pt 12), 2813-2817 (1986).</li><li>Haywood, A. M., Boyer, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sendai+virus+membrane+fusion:+time+course+and+effect+of+temperature,+pH,+calcium,+and+receptor+concentration).">Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration).</a> Biochemistry. 21, 6041-6046 (1982).</li><li>Wang, G., Hernandez, R., Weninger, K., Brown, D. T. <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+cells+by+Sindbis+virus+at+low+temperature.">Infection of cells by Sindbis virus at low temperature.</a> Virology. 362, 461-467 (2007).</li><li>Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrolyzable+tannins+(chebulagic+acid+and+punicalagin)+target+viral+glycoprotein-glycosaminoglycan+interactions+to+inhibit+herpes+simplex+virus+1+entry+and+cell-to-cell+spread.">Hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread.</a> J Virol. 85, 4386-4398 (2011).</li><li>Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad-spectrum+antiviral+activity+of+chebulagic+acid+and+punicalagin+against+viruses+that+use+glycosaminoglycans+for+entry.">Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry.</a> BMC Microbiol. 13, 187 (2013).</li><li>Marukian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture-produced+hepatitis+C+virus+does+not+infect+peripheral+blood+mononuclear+cells.">Cell culture-produced hepatitis C virus does not infect peripheral blood mononuclear cells.</a> Hepatology. 48, 1843-1850 (2008).</li><li>Baba, M., Snoeck, R., Pauwels, R., de Clercq, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfated+polysaccharides+are+potent+and+selective+inhibitors+of+various+enveloped+viruses,+including+herpes+simplex+virus,+cytomegalovirus,+vesicular+stomatitis+virus,+and+human+immunodeficiency+virus.">Sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus.</a> Antimicrob Agents ChemotheR. 32, 1742-1745 (1988).</li><li>Barth, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+binding+of+hepatitis+C+virus+envelope+glycoprotein+E2+requires+cell+surface+heparan+sulfate.">Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.</a> J Biol CheM. 278, 41003-41012 (2003).</li><li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. 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> Principles of Virology. , (2008).</li><li>Brown, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+caspase-dependent+apoptosis+in+antibody-enhanced+dengue+virus+infection+of+human+mast+cells.">Dramatic caspase-dependent apoptosis in antibody-enhanced dengue virus infection of human mast cells.</a> J Leukoc Biol. 85, 71-80 (2009).</li><li>Huang, Y., Cyr, S. L., Burt, D. S., Anderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+host+responses+to+respiratory+syncytial+virus+(RSV)+following+intranasal+administration+of+a+Protollin-adjuvanted,+epitope-enhanced+recombinant+G+protein+vaccine.">Murine host responses to respiratory syncytial virus (RSV) following intranasal administration of a Protollin-adjuvanted, epitope-enhanced recombinant G protein vaccine.</a> J Clin Virol. 44, 287-291 (2009).</li><li>Isaacson, M. K., Compton, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cytomegalovirus+glycoprotein+B+is+required+for+virus+entry+and+cell-to-cell+spread+but+not+for+virion+attachment,+assembly,+or+egress.">Human cytomegalovirus glycoprotein B is required for virus entry and cell-to-cell spread but not for virion attachment, assembly, or egress.</a> J Virol. 83, 3891-3903 (2009).</li><li>Leonard, V. 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=Measles+virus+blind+to+its+epithelial+cell+receptor+remains+virulent+in+rhesus+monkeys+but+cannot+cross+the+airway+epithelium+and+is+not+shed.">Measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed.</a> J Clin Invest. 118, 2448-2458 (2009).</li><li>Ciesek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+tea+polyphenol,+epigallocatechin-3-gallate,+inhibits+hepatitis+C+virus+entry.">The green tea polyphenol, epigallocatechin-3-gallate, inhibits hepatitis C virus entry.</a> Hepatology. 54, 1947-1955 (2011).</li><li>Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saikosaponin+b2+is+a+naturally+occurring+terpenoid+that+efficiently+inhibits+hepatitis+C+virus+entry.">Saikosaponin b2 is a naturally occurring terpenoid that efficiently inhibits hepatitis C virus entry.</a> J Hepatol. 62, 541-548 (2015).</li><li>Atkins, C., Evans, C. W., White, E. L., Noah, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+methods+for+influenza+antiviral+drug+discovery.">Screening methods for influenza antiviral drug discovery.</a> Expert Opin Drug Discov. 7, 429-438 (2012).</li><li>Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+virus+inhibitors+by+influenza+A+virus+specific+reporter+cell+based+screening.">Identification of novel virus inhibitors by influenza A virus specific reporter cell based screening.</a> Antiviral Res. 93, 48-54 (2012).</li></ol>

The authors declare that no competing interests exist.

In this report the methods to identify and evaluate antiviral compounds based on a mechanistic approach of dissecting the early viral entry events were described. Specifically, the assays allowed us to examine the effect of test compounds on free virus particles, viral attachment, and viral entry/fusion. Critical steps were implemented to distinctly evaluate the drug effect on the specific stage of early viral entry. For instance, in the &#8216;viral inactivation assay&#8217;, the dilution of the virus-drug mixture to sub-therapeutic concentration prevents significant interaction between the test compound and the host cell surface by &#8216;titrating out&#8217; the drug. This ensures that the inhibitory effect observed on the subsequent infection of the host cell is due to a direct interaction between the test compound and the cell-free virions, rather than an effect from the test compound on host cell membrane or membrane-associated molecules, including viral receptors30. Similarly, the shift in temperature between 4 &#176;C (which allows for virus binding but not entry) and 37 &#176;C (which facilitates virus entry/fusion) in the &#8216;viral attachment assay&#8217; and &#8216;viral entry/fusion assay&#8217; are crucial to determine the test compound&#8217;s effect on each of these specific events. This is feasible due to the temperature sensitivity of enveloped viruses during these steps in the infection24-29. It is therefore important that the assays are performed at the indicated temperature to ensure the accuracy of the results; for example, by carrying out the experiment on ice to maintain at 4 &#176;C and by placing the sample directly in a 37 &#176;C incubator for the temperature shift. In addition, the use of negative (ex. DMSO solvent for drug preparation) and positive (ex. heparin treatment) controls also help further establish the assays&#8217; accuracy. The utility and applicability of such methods have been demonstrated in many antiviral studies26,30,31,40,41. Note that while heparin is included as a control for all three assays in the context of the representative results, it typically blocks the initial virus binding rather than the ensuing fusion/entry step (as reflected by the data in Figure 2). Additional controls could also be used, such as neutralizing antibodies directed against the virus (for viral inactivation assay), antibodies that mask the cell surface receptors for the virus (for viral attachment assay), and membrane fusion inhibitors (for viral fusion/entry assay). 
The assays described in this report, which are specific to the early stages of the viral infection, are useful in terms of application as secondary tests to characterize the mechanism of action of candidate drugs from primary screens which typically target the viral infection more broadly. Alternatively, they could also be incorporated in primary screens if one is specifically looking for inhibitors of early viral entry, including virus inactivating agents, viral attachment antagonists, and inhibitors to viral entry/fusion. In this case, their use allows a more focused and precise screen analysis for the identification of mechanism-specific antiviral candidates, which, in turn, would expedite downstream drug development. 
The use of cell-based assays in identifying antiviral agents provides several important advantages compared to biochemical assays, including revealing potential off-target effects (such as cytotoxicity) and adding physiological relevance to the bioactivity of the test agents42. These issues are important considerations for deciding whether a candidate agent is of value for continuation in subsequent phases of drug development. Similarly, the early viral entry-specific assays described in this report allow examination of the drug effect on the distinct viral entry stage at the cellular level, and more specifically in the context of an authentic viral infection in vitro. The results obtained from such assays would therefore help better predict the antiviral efficacy of the test compounds and also identify potentially unwanted off-target effects against the host cell. One potential limitation though, is that an in vitro cell-based assay may not completely reflect the actual in vivo entry step in the context of a natural viral infection. Nonetheless, the assays presented in this protocol do serve as an analytical platform for mechanism-based identification and evaluation of novel antiviral agents.
The development of reporter viruses or reporter cell systems to quantitate the amount of viral infection has greatly facilitated cell-based screening and evaluation of antiviral compounds. Examples include the use of recombinant viruses carrying a reporter gene or by means of recombinant human cell lines containing a reporter gene driven by the specific virus promoter31,43. In this report, the infection from luciferase-tagged HCV can be easily monitored by quantitating the reporter signal, thus facilitating data analysis. By incorporating these useful reporter-based tools, the early viral entry assays described here can essentially be adapted into high-throughput format for mechanism-based screening of small molecule libraries.
In conclusion, a protocol was described for assays dissecting the early viral entry as a means of identifying and evaluating mechanism-specific antiviral compounds. Such assays would be useful for discovering novel antagonists/inhibitors to viral entry and help expand the scope of antiviral agents for development as prophylactic and/or therapeutic treatments.

Viral infections are a constant threat to the public health and a significant cause of epidemic diseases, morbidity, and deaths worldwide. Specific modes of control against viral infections include vaccine development and antiviral therapies. While vaccine efforts have proven successful in immunizing against several viruses, many viral pathogens remain without a protective vaccine including dengue virus (DENV), human cytomegalovirus (HCMV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV)1-5. Antivirals, on the other hand, play an important role for the management of these viral infections when prophylactic vaccines are unavailable. However, to date, only few licensed and cost-effective antiviral drugs are available compared to the number of viral pathogens that threatens the public health. More importantly, due to an increase in global travel and rapid urbanization, the situation is aggravated by risks of epidemic outbreaks from emerging/re-emerging viral infections that are being introduced into non-indigenous areas6. Recent outbreaks caused by severe acute respiratory syndrome (SARS) virus, influenza viruses (H1N1, H5N1, H3N2, and H7N9), DENV, West Nile virus (WNV), measles virus (MV), Middle East Respiratory Syndrome (MERS) virus, and Ebola virus6-12 are among the examples reflecting the need for antivirals development when immunization and/or therapeutics are unavailable. In addition, there is always a potential risk of generating drug-resistant infections with currently used antivirals. Thus, the continuous development and expansion of the scope of antivirals to these emerging/re-emerging viral infections are necessary to provide better management strategies and safeguard the public health.
Most antiviral therapies consist of direct acting antivirals (DAAs) which target a specific viral protein or cofactor that mediates important steps in the viral life cycle. For example, the nucleoside analogue Acyclovir inhibits herpesvirus DNA polymerase, protease inhibitors Boceprevir and Telaprevir antagonize the HCV NS3, and Oseltamivir and Zanamivir are neuraminidase inhibitors that block the release of influenza virus particles from infected cells13-15. There are however very few licensed viral entry inhibitors including Enfuvirtide, which targets HIV gp41 to prevent fusion, and Maraviroc, which blocks the HIV co-receptor CCR5, thereby preventing viral entry16. Exploring novel antagonists/inhibitors to viral entry could help provide additional therapeutics for prophylactic or therapeutic use, such as in combination with other antivirals with a different mechanism of action to better manage viral infections17-19. 
Identification of antivirals can involve structure-guided drug design and candidate drug screening-based strategy. Methods for assessing antiviral activity of test agents include biochemical assays of enzymatic activity and evaluation by cell-based systems20-23. In cell-based systems, the viral life cycle can be dissected into distinct stages of infection, such as entry events (attachment, fusion, uncoating), the replication phase (viral genome replication and protein translation), and virion egress (assembly, maturation, and release). Since the assays can be adapted to investigate each specific stage using various tools and methods, this approach allows identification/examination of potential candidate antivirals with a specific mechanism of action targeting the distinct stage analyzed. For instance, to analyze drug effect specifically on the free virus particles prior to binding to the host cell, a &#8216;viral inactivation assay&#8217; can be performed. In this assay, the virus is allowed to incubate with the test drug and then diluted to titrate out the drug before infecting a cell monolayer. Additional steps such as viral attachment and entry/fusion stages can also be analyzed individually, by shifting the temperature during the infection. For many enveloped viruses, viral entry/fusion at the host cell membrane is greatly facilitated at 37 &#176;C, but is typically precluded at 4 &#176;C which does not affect virus binding24-29. Finally, the use of reporter viruses or cell systems could facilitate these studies and permit a high-throughput analysis.
We previously employed the cell-based approach and dissected the early entry of various enveloped viruses for the examination of antiviral compounds that potentially antagonize viral entry30,31. Herein, the various methods used, including viral inactivation, viral attachment, and viral entry/fusion assays, are described.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>GIBCO</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>In vitro toxicology assay kit, XTT-based</td>
			<td>Sigma</td>
			<td>TOX2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4&#160;</td>
			<td>GIBCO</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Bio-Tek Instrument, Inc.</td>
			<td>ELx800&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002420</td>
			<td></td>
		</tr>
		<tr>
			<td>BioLux Gaussia luciferase assay kit</td>
			<td>New England Biolabs</td>
			<td>E3300L&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax-20/20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate, dihydrate</td>
			<td>Sigma</td>
			<td>71402</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
	</tbody>
</table>

early viral entry assays for the identification and evaluation of antiviral compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Nella figura 1, il &#39;inattivazione virale test&#39; stata effettuata per verificare se due specifici composti CHLA naturale e PUG potrebbero inattivare i diversi virus con involucro in stato senza cellulare e prevenire l&#39;infezione successiva. La citotossicità e antivirale dose-risposta di questi composti sono stati determinati prima di eseguire lo studio meccanicistica 31. I virus sono stati pretrattati con composti in esame e poi le miscele virus-farmaco sono stati diluiti a concentrazioni sub-terapeutiche prima dell&#39;inoculazione sul rispettivo monostrato cellulare per ogni sistema virus. Come mostrato in Figura 1, sia CHLA e PUG sembravano interagire con i virioni cellule libere, con gravi effetti irreversibili che proteggeva il monostrato cellulare dalla successiva infezione. I due composti in esame ha realizzato un vicino di inibizione 100% contro HCMV, HCV e DENV-2, mentre un 60 - blocco 80% è stata osservata contro MV e RSV. Questi risultati Suggest che CHLA e PUG hanno un impatto diretto su queste particelle virali libere inattivando loro e neutralizzando la loro infettività. Nella figura 2, l&#39;allegato e di ingresso / test di fusione sono stati eseguiti per esplorare l&#39;effetto di CHLA e PUG contro questi eventi rapida entrata correlati virali da HCMV, HCV, DENV-2, MV, e RSV. Sia CHLA e PUG efficacemente impedito vincolante del virus esaminati sui rispettivi cellula ospite come mostrato dalla inibizione virale risultante (Figura 2, &#39;Allegato&#39;: luce barre grigie). L&#39;effetto inibitorio sulla attaccamento virus da entrambi i composti era simile contro HCMV (Figura 2B), HCV (Figura 2C), DENV-2 (Figura 2D), e RSV (Figura 2F), che vanno da 90 - 100%. D&#39;altra parte, PUG sembrava essere più efficace contro CHLA MV assorbente (figura 2E), con il tasso di inibizione della two composti variabili tra 50 - 80%. L&#39;eparina trattamento di controllo, che è noto per impedire l&#39;ingresso di molti virus, anche inibito attaccamento di HCMV, DENV-2, RSV, annuncio MV, ma era meno efficace contro HCV. La conseguente &#39;test di ingresso / fusione virale&#39; esaminato se CHLA e PUG conservato la loro attività durante la fase di entrata del virus / fusione (Figura 2, &#39;Entry / Fusion&#39;: barre grigio scuro). Ancora una volta, sia CHLA e PUG stati osservati compromettere efficacemente l&#39;ingresso virale passo / fusione dei virus esaminati (Figura 2B - F), ottenendo un 50 - effetto protettivo 90% corrisponde monostrato cellulare. Eparina anche potentemente inibito l&#39;ingresso / fusione DENV-2 e infezioni da RSV, ma era meno efficace contro HCMV, HCV, e MV (&lt;40% di inibizione in media). <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Virus </td><td> Tipo di cella </td></tr><tr><td> HCMV </td> &lt;td&gt; HEL </td></tr><tr><td> HCV </td><td> Huh-7.5 </td></tr><tr><td> DENV-2 </td><td> Vero </td></tr><tr><td> MV </td><td> CHO-SLAM </td></tr><tr><td> RSV </td><td> HEp-2 </td></tr></tbody></table> Tabella 1:. Tipo cellula ospite per infezione virale è indicato il tipo cellulare utilizzato per ogni sistema infezione virale descritto nei risultati rappresentativi. Ulteriori dettagli riguardanti le cellule possono essere trovati in riferimento 31. <img alt="Figura 1" src="/files/ftp_upload/53124/53124fig1.jpg" /> Figura 1. L&#39;inattivazione di infezioni virali da parte CHLA composti di prova e PUG diversi virus sono stati trattati con i composti di prova per un lungo periodo. (Incubate per 1,5-3 ora prima titolazione; luce barre grigie) o breve periodo (immediatamente diluita; grigio scuro bar) a 37 ° C prima di una diluizione di concentrazione sub-terapeuticizione e successiva analisi di infezione delle cellule rispettivo ospitanti. (A) Schema dell&#39;esperimento (mostrato a sinistra) con la concentrazione di virus finale (PFU / pozzetto o MOI), il periodo di incubazione del virus farmaco-lungo termine (i), e tempo di incubazione successivo (ii) indicato per ciascun virus nella tabella a destra. Analisi per (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, e (F) RSV sono indicati in ogni pannello supplementare. I risultati sono riportati contro il trattamento di controllo negativo DMSO per infezione da virus ed i dati indicati sono il mezzo ± errori standard della media (SEM) da tre esperimenti indipendenti. Questa cifra è stata modificata da riferimento 31. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig1highres.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53124/53124fig2.jpg" /> Figura 2. Valutazione delle attività antivirale del CHLA composti di prova e PUG contro virus e attaccamento entrata / fusion. (A) La procedura sperimentale, concentrazione virale (PFU / pozzetto o MOI), e il tempo di aggiunta e trattamento con i composti in esame (i, ii, iii) sono presentati per ogni virus negli schemi e le tabelle associate. Nell&#39;analisi allegato virus (luce barre grigie), monostrati di diversi tipi cellulari sono stati pre-raffreddato a 4 ° C per 1 ora, poi co-trattati con rispettivi virus e composti di prova a 4 ° C (1.5 - 3 ore; i) prima di lavare via i monostrati e composti di prova per la successiva incubazione (37 ° C; ii) e l&#39;esame di infezione da virus. In entrata virus / analisi fusione (barre grigio scuro), monostrati di cellule sono stati seminati pre-raffreddata a 4 ° C per 1 ora e poi sfidato con rispettivi virus a 4 ° C per 1,5 - 3 ore (i). Le cellule sono state poilavata e trattata con i composti di prova per un periodo di incubazione addizionale (ii) durante il quale la temperatura è stata spostata a 37 ° C per facilitare l&#39;evento di ingresso / fusione virale. Al termine dell&#39;incubazione, virus extracellulari sono stati rimossi da uno tampone citrato (pH 3,0) o lavaggi PBS e le cellule sono state ulteriormente incubate (iii) per l&#39;analisi di virus. Risultati per (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, e (F) RSV sono indicati in ogni pannello supplementare. I dati sono riportati contro il trattamento di controllo negativo DMSO di infezione da virus e sono presentati come media ± SEM di tre esperimenti indipendenti. Questa cifra è stata modificata da riferimento 31. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig2highres.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

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Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

All&#39;inizio virali ingresso saggi per l&#39;identificazione e la valutazione di composti antivirali

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

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Immunology and Infection

30.0K Views.  Taipei Medical University Hospital. Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Video: Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Analisi quantitativa di tutti influenza di tipo A Hemagglutinins e neuraminidasi virale utilizzando anticorpi Universale di saggi semplice Slot Blot

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

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Immunologia e infezioni

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplificazione e quantificazione di HIV-1 RNA in soggetti affetti da HIV con una carica virale inferiore il limite di rilevamento da parte di Standard saggi clinici

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

Saggi per l&#39;individuazione di nuovi antivirali contro virus della Bluetongue

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Viral Determinazione Concentrazione Attraverso placca saggi: Sistemi overlay per mezzo di tradizionali e nuovi

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

High-throughput Quantitative Real-time RT-PCR test per la determinazione di espressione Profili di tipo I e III interferone sottotipi

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Saggi qualitativi e quantitativi per il rilevamento e la caratterizzazione di proteine ​​antimicrobici

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Lo sviluppo del liofilizzato Loop-mediate isotermici amplificazione reagenti per la rilevazione di<em&gt; Burnetii Coxiella</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Tampone Metodo di campionamento per la rilevazione di Norovirus umana sulle superfici

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Purificazione di DNA virale per l'identificazione delle proteine virali e cellulari associate

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Valutazione delle risposte a cellula T Zika Virus-specifici in organi di nuova di Ifnar1 infetti- / - Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...