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).

Hinweis: Stellen Sie sicher, dass alle Verfahren, die Zellkultur und Virusinfektion sind in zertifizierten biologischen Sicherheit Hauben, die für die Sicherheitsstufe der Proben entsprechende gehandhabt werden durchgeführt. Für den Zweck der Beschreibung der Protokolle wird Gaussia-Luciferase-Reporter-markierten HCV als Modellvirus 32 benutzt. Im Zusammenhang mit den repräsentativen Ergebnissen, sind die Verbindungen chebulagic Säure (CHLA) und Punicalagin (PUG) als Kandidaten Virostatika, die virale Glycoprotein Wechselwirkungen mit Zelloberflächen Glycosaminoglycane Target während der frühen viralen Eintritt Stufen 31 verwendet. Heparin, das dafür bekannt ist, mit der Eingabe von vielen Viren 30,31,33,34 eingreifen, wird als positive Kontrolle der Behandlung in diesem Kontext verwendet wird. Für grundlegende Hintergrund Virologie Techniken Vermehrung von Viren, die Bestimmung der Virustiter und die Expression von infektiösen Dosis in plaquebildenden Einheiten (PFU), Fokus-bildenden Einheiten (FFU) oder Multiplizität der Infektion (MOI) ist, worin Re der Leserübertragen auf Bezugs 35. Der vorherigen Beispiele und Viren in den repräsentativen gezeigten Ergebnisse verwendet optimierten Bedingungen wird der Leser auf die in Tabelle 1, 1A und 2A aufgeführten Referenzen 30-32,36-39 sowie Einzelheiten bezeichnet. 1. Zellkultur, Compound Vorbereitung, und die Verbindung Zytotoxizität <ol><li> Wachsen die jeweilige Zelllinie für die Virusinfektion zu analysierende System (Tabelle 1). Für HCV, wachsen die Huh-7,5-Zellen in Dulbeccos modifiziertem Eagle-Medium (DMEM) mit 10% fötalem Rinderserum (FBS), 200 U / ml Penicillin G, 200 ug / ml Streptomycin und 0,5 ug / ml Amphotericin B. </li><li> Bereiten Sie die Testverbindungen und Kontrollen unter Verwendung ihres jeweiligen Lösemittel: beispielsweise aufzulösen CHLA und PUG in Dimethylsulfoxid (DMSO); vorzubereiten Heparin in sterilem doppelt destilliertem Wasser. Für alle nachfolgenden Verdünnungen verwenden Kulturmedien. Hinweis: Die endgültige concentRation von DMSO in der Testverbindung Behandlungen ist weniger als 1% in den Experimenten; 1% DMSO als ein negativer Kontrollbehandlung in den Tests zum Vergleich eingeschlossen. </li><li> Bestimmung der Zytotoxizität der Testverbindungen (zB CHLA und PUG) auf die Zellen für die Virusinfektion durch die Verwendung eines Zell-Lebensfähigkeit Bestimmen Reagens wie XTT (2,3-Bis [2-methoxy-4-nitro-5-sulfophenyl] -5-phenylamino) carbonyl] -2H-tetrazolium Hydroxid): <ol><li> Für HCV, Samen Huh-7,5-Zellen in einer Platte mit 96 Vertiefungen (1 × 10 4 Zellen pro Vertiefung) und bei 37 ° C in einem 5% CO 2 -Inkubator O / N um eine Monoschicht zu erhalten. </li><li> Gelten DMSO-Kontrolle (1%) oder steigenden Konzentrationen der Testverbindungen CHLA und PUG (ex. 0, 10, 50, 100, und 500 &amp; mgr; M) zu den Kulturvertiefungen in dreifacher Ausführung. </li><li> Bei 37 ° C für 72 Stunden und wirf dann das Medium in der Platte und waschen Sie die Zellen mit 200 ul phosphatgepufferter Kochsalzlösung (PBS) zweimal. </li><li> 100 l assaying Lösung aus dem XTT-basierte in vitro-Toxikologie-Assay-Kit, um jede Vertiefung und inkubieren Sie die Platten bei 37 ° C für weitere 3 h auf XTT ermöglichen Formazan-Produktion. </li><li> Die Optische mit einem Mikroplattenleser bei einer Testwellenlänge von 492 nm und einer Referenzwellenlänge von 690 nm. </li><li> Der prozentuale Anteil der überlebenden Zellen nach der folgenden Formel: Zelllebensfähigkeit (%) = An / Wie × 100%, wobei &#39;An&#39; und &#39;Wie&#39; beziehen sich auf die Absorption der Testverbindungen und die Lösungsmittelkontrolle (ex 1% DMSO. ) Behandlungen sind. Bestimmung der Konzentration von 50% zelluläre Zytotoxizität (CC 50) der Testverbindungen aus einer analytischen Software wie GraphPad Prism nach dem Protokoll des Herstellers. </li></ol></li></ol> 2. Auslesen der Virusinfektion Hinweis: Das Auslesen der Virusinfektion hängt von der Virus-System verwendet und können Methoden wie Plaque-Assays oder mea beinhaltensuring Reporter Signale von Reporter-markierten Viren. Methode für das Erfassen Reporter-HCV-Infektion auf der Basis der Luciferase-Reporteraktivität wird nachstehend beschrieben. <ol><li> Sammeln der Überstände aus den infizierten Vertiefungen und Klärung bei 17.000 xg in einer Mikrozentrifuge für 5 min bei 4 ° C. </li><li> Mix 20 ul Testüberstand wurde in 50 ul Luciferase-Substrat aus der Gaussia-Luciferase-Assay-Kits und direkt mit einem Luminometer gemessen nach den Angaben des Herstellers. </li><li> Auszudrücken HCV Infektiosität als log 10 der relativen Lichteinheiten (RLU) zum Bestimmen virale Inhibition (%) und die Berechnung der 50% effektive Konzentration (EC 50) der Testverbindungen gegen die HCV-Infektion unter Verwendung von Algorithmen von GraphPad Prism Software gemäß dem Protokoll des Herstellers. </li></ol> 3. Vireninaktivierung Assay Hinweis: Beispiele für Inkubationszeit und Virusdosis für verschiedene Viren einwieder in 1A aufgeführt. Höhere Konzentrationen des Virus kann auch durch Erhöhung der MOI / PFU getestet werden. <ol><li> Samen Huh-7,5-Zellen in einer Platte mit 96 Vertiefungen (1 × 10 4 Zellen pro Vertiefung) und bei 37 ° C in einem 5% CO 2 -Inkubator O / N um eine Monoschicht zu erhalten. </li><li> Inkubieren Sie die Testverbindungen oder Kontrollen (Endkonzentrationen: CHLA = 50 &amp; mgr; M; PUG = 50 &amp; mgr; M; Heparin = 1.000 ug / ml; DMSO = 1%) mit den HCV-Partikel bei 37 ° C (1A, &quot;Long-Term &#39; ) in einem Verhältnis 1: 1. Beispielsweise auf einen 100 &amp; mgr; Virusinokulum enthaltend 1 x 10 4 FFU wird mit 100 ul einer 100 uM CHLA Arbeitsverdünnung; Dies ergibt CHLA Behandlung bei einer Endkonzentration von 50 uM. </li><li> Verdünnt das virus-Wirkstoff-Mischung &quot;sub-therapeutische&quot; (unwirksam) Konzentration der Testverbindungen. Zum Beispiel ist die unwirksame Konzentration CHLA und PUG gegen HCV bei 1 &amp; mgr; M 31; deshalbdies erfordert eine 50-fache Verdünnung des Virus-Wirkstoff-Mischung, die mit 9,8 ml Basalmedium (Zellkulturmedium mit 2% FBS) erreicht werden kann. Anmerkung: Die Verdünnung auf subtherapeutische Konzentration verhindert erhebliche Wechselwirkung zwischen der Testverbindungen und der Oberfläche der Wirtszelle und ermöglicht die Untersuchung der Wirkung der Behandlung auf den zellfreien Virionen. Beachten Sie, dass diese Verdünnung hängt von der antivirale Dosis-Antwort der Testverbindungen gegen die insbesondere virale Infektion und vor der Durchführung dieses bestimmten Test 31 ermittelt. </li><li> Zum Vergleich, mischen das Virus mit den Testverbindungen und sofort zu verdünnen (kein Inkubationszeit), um Unter therapeutische Konzentration vor der Infektion (1A, &quot;Short-Term). </li><li> Zugabe von 100 ul der verdünnten HCV-Wirkstoff-Mischung auf die Huh-7,5 Zellmonolayer (die Virusmenge ist nun auf 1 x 10 2 FFU; final MOI = 0,01) und Inkubation für 3 Stunden bei 37 ° C viral, damitAdsorption. </li><li> Nach der Infektion, entfernen Sie die Inokula verdünnt und vorsichtig waschen die Vertiefungen zweimal mit 200 ul PBS. Hinweis: Führen Sie die wäscht vorsichtig, um zu vermeiden, heben Sie die Zellen. </li><li> Gelten 100 ul Basalmedium in jede Vertiefung und Inkubieren bei 37 ° C für 72 Std. </li><li> Analyse des resultierenden Infektion durch Untersuchung des Überstandes auf Luciferase-Aktivität wie in &#39;2 beschrieben. Auslesen der Virusinfektion. </li></ol> 4. Viral Befestigung Assay Hinweis: Beispiele für Inkubationszeit und Virusdosis für verschiedene Viren sind in 2A aufgeführt, &quot;Anhang&quot;. Höhere Konzentrationen des Virus kann auch durch Erhöhung der MOI / PFU getestet werden. <ol><li> Samen Huh-7,5-Zellen in einer Platte mit 96 Vertiefungen (1 × 10 4 Zellen pro Vertiefung) und bei 37 ° C in einem 5% CO 2 -Inkubator O / N um eine Monoschicht zu erhalten. </li><li> Pre-Chill die Zellmonoschichten in Platten bei 4 ° C for 1 Std. </li><li> Co-Behandlung der Zellen mit HCV Inokulum (MOI = 0,01) und die Testverbindungen oder Kontrollen (Endkonzentrationen: CHLA = 50 uM; PUG = 50 uM; Heparin = 1,000 &amp; mgr; g / ml; DMSO = 1%) bei 4 ° C für 3 Std. Zum Beispiel, um eine 90 &amp; mgr; l Virusinokulum mit 1 x 10 2 FFU, fügen Sie 10 ul einer 500 uM CHLA Arbeitsverdünnung; Dies ergibt CHLA Behandlung bei einer Endkonzentration von 50 uM und eine Infektion durch HCV bei MOI = 0,01 auf der Zell-Monoschicht. Hinweis: Es ist wichtig, für die Durchführung des Experiments bei 4 ° C, da es eine Virusbindung, sondern schließt Eintrag, der am effizientesten bei 37 ° C auftritt. Führen die Zugabe des Virus und die Testverbindungen auf Eis und der anschließenden Inkubation in einem 4ºC Kühlraum, um sicherzustellen, dass die Temperatur auf 4 ° C gehalten. </li><li> Entfernen Sie den Überstand und waschen Sie sich leicht die Zellmonoschicht mit 200 ul eiskaltem PBS zweimal. Hinweis: Führen Sie die wäscht vorsichtig, um zu vermeiden, heben Sie die Zellen &lt;./ li&gt; <li> Gelten 100 ul Basalmedium in jede Vertiefung und Inkubieren bei 37 ° C für 72 Std. </li><li> Analyse des resultierenden Infektion durch Untersuchung des Überstandes auf Luciferase-Aktivität wie in &#39;2 beschrieben. Auslesen der Virusinfektion. </li></ol> 5. Viral Entry / Fusion Assay Hinweis: Beispiel für Inkubationszeiten und Virusdosis für verschiedene Viren sind in 2A &#39;Entry / Fusion &quot;aufgeführt. Höhere Konzentrationen des Virus kann auch durch Erhöhung der MOI / PFU getestet werden. <ol><li> Samen Huh-7,5-Zellen in einer Platte mit 96 Vertiefungen (1 × 10 4 Zellen pro Vertiefung) und bei 37 ° C in einem 5% CO 2 -Inkubator O / N um eine Monoschicht zu erhalten. </li><li> Pre-Chill die Zellmonoschichten in Platten bei 4 ° C für 1 Stunde. </li><li> Infizieren die Zellen mit HCV (MOI = 0,01) bei 4 ° C für 3 Stunden. Verwenden Sie beispielsweise eine 100 ul Virusinokulum mit 1 x 10 2 FFU. Hinweis: Führen Sie die addition des viralen Inokulum auf Eis und der anschließenden Inkubation in einem 4ºC Kühlraum, die Temperatur auf 4 ° C, die die virale Bindung jedoch keine Ausgabe ermöglicht aufrechtzuerhalten. </li><li> Entfernen Sie den Überstand und waschen Sie sich leicht die Zellmonoschichten zweimal mit 200 ul eiskaltem PBS. Hinweis: Führen Sie die wäscht vorsichtig, um zu vermeiden, heben Sie die Zellen. </li><li> Behandeln Sie die Vertiefungen mit den Testverbindungen oder Kontrollen (Endkonzentrationen: CHLA = 50 &amp; mgr; M; PUG = 50 &amp; mgr; M; Heparin = 1.000 ug / ml; DMSO = 1%) und bei 37 ° C für 3 Stunden. Fügen Sie beispielsweise 10 &amp; mgr; l einer 500 &amp; mgr; M CHLA Arbeitsverdünnung auf 90 ul Medium, zu mischen und zu behandeln, die Brunnen; Dies ergibt CHLA Behandlung bei einer Endkonzentration von 50 uM. Hinweis: Der Wechsel von 4 ° C bis 37 ° C ermöglicht nun den Viruseintritt / Fusion Veranstaltung und ermöglicht somit Beurteilung der Testverbindungen &quot;-Effekt an diesem Schritt. </li><li> Saugen Sie das Arzneimittel enthaltende Überstand und entfernen Sie nicht internalisiertextrazellulären Viren durch Waschen mit 200 ul Citratpuffer (50 mM Natriumcitrat, 4 mM Kaliumchlorid, pH 3.0) oder PBS. Gelten 100 ul des Grundmediums vor der Inkubation bei 37 ° C für 72 Std. </li><li> Analyse des resultierenden Infektion durch Untersuchung des Überstandes auf Luciferase-Aktivität wie in &#39;2 beschrieben. Auslesen der Virusinfektion. </li></ol>

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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.

In Abbildung 1 wurde die &quot;Virus-Inaktivierung Test&quot; durchgeführt, um zu prüfen, ob zwei bestimmte natürliche Verbindungen CHLA und PUG könnten die verschiedenen umhüllten Viren in zellfreien Zustand zu inaktivieren und zu verhindern nachfolgende Infektion. Die Zytotoxizität und antivirale Dosis-Antwort dieser Verbindungen vor der Durchführung der mechanistische Studie 31 bestimmt. Die Viren wurden mit den Testverbindungen vorbehandelt und anschließend die Virus-Wirkstoff-Mischungen wurden zu subtherapeutischen Konzentrationen vor der Impfung auf die jeweilige Zellmonoschicht für jede Virus-System verdünnt. Wie in 1 gezeigt, sowohl CHLA und PUG schienen mit den zellfreien Virionen interagieren, was irreversible Effekte, die die Zell-Monoschicht von der nachfolgenden Infektion geschützt. Die beiden Testverbindungen erreicht eine nahezu 100% ige Hemmung gegen HCMV, HCV und DENV-2, während eine 60 bis 80% Block wurde gegen MV und RSV beobachtet. Diese Ergebnisse suggest, dass CHLA und PUG haben direkten Einfluss auf diese freie Viruspartikel durch Inaktivierung von ihnen und zur Neutralisierung ihrer Infektiosität. In 2 wurden die Befestigung und Ein- / Fusion-Assays durchgeführt, um die Wirkung von CHLA und PUG gegen diesen frühen Viruseintritt bezogene Ereignisse von HCMV, HCV, DENV-2, MV und RSV erkunden. Sowohl CHLA und PUG wirksam verhindert die Bindung der untersuchten Viren auf die jeweilige Wirtszelle, wie durch Hemmung auf die resultierende Virusinfektion (Abbildung 2, &quot;Anhänge&quot;: hellgraue Balken) gezeigt. Die inhibitorische Wirkung auf die Virus Befestigung von beiden Verbindungen war ähnlich gegen HCMV (2B), HCV (2C), DENV-2 (2D) und RSV (2F), im Bereich von 90 - 100%. Andererseits erschien PUG wirksamer als CHLA gegen MV Bindung (2E) zu sein, wobei der Hemmungsrate von twO-Verbindungen zwischen 50 - 80%. Die Kontrollbehandlung Heparin, von dem bekannt ist, um den Eintritt von vielen Viren, inhibierte auch die Anbringung von HCMV, DENV-2, RSV, ad MV blockieren, war aber weniger wirksam gegen HCV. Die darauf folgende &#39;Viruseintritt / Fusionstest &quot;untersucht, ob CHLA und PUG während der Viruseintritt / Schmelzphase behielten ihre Aktivität (Abbildung 2,&quot; Entry / Fusion &quot;: dunkelgraue Balken). Auch hier sowohl CHLA und PUG wurden beobachtet, um das Eindringen des Virus / Fusionsschritt der untersuchten Viren (2B - F) wirksam zu beeinträchtigen, was zu einer 50 bis 90% schützende Wirkung auf die jeweilige Zellmonolayer. Heparin auch potent gehemmt Eintrag / Fusion in DENV-2 und RSV-Infektionen, war aber gegen HCMV, HCV, und MV (&lt;40% ige Hemmung im Durchschnitt) weniger wirksam. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Virus </td><td> Zellen-Art </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> Tabelle 1:. Wirtszelltyp für die virale Infektion der Zelltyp für jeden der repräsentativen Ergebnisse beschriebenen viralen Infektionssystem verwendet wird, angegeben. Weitere Einzelheiten zu den Zellen in Referenz 31 gefunden werden. <img alt="Abbildung 1" src="/files/ftp_upload/53124/53124fig1.jpg" /> Abbildung 1. Inaktivierung von Virusinfektionen durch die Testverbindungen CHLA und PUG Verschiedene Viren wurden mit den Testverbindungen für einen langen Zeitraum behandelt werden. (1,5 inkubiert - 3 Stunden vor der Titration, hellgraue Balken) oder kurzen Zeitraum (sofort verdünnt; dunkelgrau bar) bei 37 ° C, bevor eine Verdünnung auf subtherapeutischen Konzentrationention und anschließender Analyse der Infektion auf den jeweiligen Wirtszellen. (A) Schematische Darstellung des Experiments (links dargestellt) mit der endgültigen Viruskonzentration (PFU / Vertiefung oder MOI), Langzeit-Virus-Medikament Inkubationszeit (i), und anschließender Inkubation (ii) angegeben für jedes Virus in der Tabelle auf der rechten Seite. Die Analysen für (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, und (F) RSV in jedem zusätzlichen Platte angezeigt. Ergebnisse werden mit dem DMSO negative Kontrolle Behandlung für Virusinfektion und die gezeigten Daten wurden die Mittelwerte ± Standardfehler des Mittelwerts (SEM) aus drei unabhängigen Experimenten aufgetragen sind. Diese Zahl hat sich von Referenz 31 modifiziert worden ist. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig1highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/53124/53124fig2.jpg" /> Abbildung 2. Evaluierung von antiviralen Aktivitäten der Testverbindungen CHLA und PUG Viren Befestigung und Ein- / Fusion. (A) Die experimentelle Vorgehensweise, Viruskonzentration (PFU / Vertiefung oder MOI), und der Zeitpunkt der Zugabe und die Behandlung mit den Testverbindungen (i, ii, iii) für jedes Virus in den Schemata und den zugehörigen Tabellen dargestellt. In Virusanheftung Analyse (hellgraue Balken) Monoschichten aus verschiedenen Zelltypen wurden bei 4 ° C für 1 h vorgekühlt, wobei die jeweiligen Viren und Testverbindungen dann mitbehandelt bei 4 ° C (1,5 - 3 h, i) vor dem Waschen die Inokulate und die Testverbindungen für die anschließende Inkubation (37 ° C, ii) und Prüfung der Virusinfektion. In virus entry / Schmelzanalyse (dunkelgraue Balken), wurden ausgesät Zellmonoschichten vorgekühlte bei 4 ° C für 1 Stunde und dann mit den entsprechenden Viren bei 4 ° C für 1,5 in Frage gestellt - 3 h (i). Zellen wurden danngewaschen und mit den Testverbindungen für eine weitere Inkubationsperiode (ii), während der die Temperatur auf 37 ° C verschoben, um das Eindringen des Virus / Fusionsereignis erleichtern behandelt. Am Ende der Inkubation wurden die extrazellulären Viren entweder durch Citratpuffer (pH 3.0) oder PBS Waschschritten entfernt und die Zellen wurden für weitere Analyse der Virus-Infektion inkubiert (iii). Ergebnisse für (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, und (F) RSV in jedem zusätzlichen Platte angezeigt. Daten werden gegen die DMSO negative Kontrolle Behandlung einer Virusinfektion, aufgetragen und werden als Mittel ± SEM von drei unabhängigen Experimenten. Diese Zahl hat sich von Referenz 31 modifiziert worden ist. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig2highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

Watch this Scientific Journal Video about Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds at JoVE.com

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.

Frühe das Eindringen des Virus-Assays für die Ermittlung und Bewertung von antiviralen Verbindungen

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

Serie: 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.

Quantitative Analysen aller Influenza Typ A Viral Hämagglutininen und Neuraminidasen mit Universal Antikörper in Einfache 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 ...

Forschung

Immunologie und Infektion

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.

Verstärkung und Quantifizierung von HIV-1 RNA in HIV-infizierten Personen mit Viruslast unter der Nachweisgrenze von 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 ...

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.

Tests für die Identifizierung neuer Antivirale gegen Blauzungenvirus

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 Entschlossenheit Konzentration Durch Plaque-Assays: Mit Tradition und neuen Überzugssysteme

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.

Hochdurchsatz-quantitative Echtzeit-RT-PCR-Assay zur Bestimmung Expressionsprofile der Typen I und III Interferon-Formationsglieder

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.

Qualitative und quantitative Assays zur Detektion und Charakterisierung von Protein Antimikrobielle

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.

Die Entwicklung der Lyophilisierte Loop-mediated Isothermal Amplification Reagenzien zum Nachweis von<em&gt; Coxiella burnetii</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.

Swab Sampling Verfahren zum Nachweis von humanem Norovirus auf Oberflächen

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.

Reinigung der viralen DNA für die Identifizierung von assoziierten viralen und zellulären Proteinen

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.

Bewertung der Zika-Virus-spezifischen T-Zell-Reaktionen in Organe der Immunabwehr von infizierten Ifnar1- / - Mäusen

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