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).

Remarque: Veiller à ce que toutes les procédures impliquant la culture cellulaire et infection par le virus sont menées dans les hottes de biosécurité certifiés qui sont appropriés pour le niveau des échantillons de biosécurité être manipulé. Aux fins de la description des protocoles, la luciférase de Gaussia VHC à marquage rapporteur est utilisé comme un modèle de virus 32. Dans le contexte des résultats représentatifs, l&#39;acide des composés de chébulagique (CHLA) et punicalagin (PUG) sont utilisés comme antiviraux candidats qui ciblent les interactions avec les glycosaminoglycanes glycoprotéine virale de surface cellulaire lors de l&#39;entrée virale premières étapes 31. L&#39;héparine, qui est connu pour interférer avec l&#39;entrée de nombreux virus 30,31,33,34, est utilisé comme un traitement de contrôle positif dans ce contexte. Pour des informations de base sur des techniques de virologie, la propagation de virus, la détermination du titre du virus, et l&#39;expression de la dose infectieuse en unités formant des plages (PFU), concentrer les unités de formage (FFU), ou multiplicité d&#39;infection (MOI), le lecteur est re35 préféré pour référencer. Pour des exemples antérieurs et des conditions optimisées utilisés pour les virus indiqués dans les résultats représentatifs, le lecteur est renvoyé aux références 30-32,36-39 ainsi que les détails figurant au tableau 1, figure 1A et la figure 2A. 1. Culture cellulaire, Préparation composé et composé de cytotoxicité <ol><li> Cultiver la lignée cellulaire respectif pour le système d&#39;infection par le virus à analyser (tableau 1). Pour le HCV, cultiver les cellules Huh-7,5 dans du milieu d&#39;Eagle modifié par Dulbecco (DMEM) supplémenté avec 10% de sérum bovin fœtal (FBS), 200 U / ml de pénicilline G, 200 ug / ml de streptomycine et 0,5 pg / ml d&#39;amphotéricine B. </li><li> Préparation des composés d&#39;essai et les témoins en utilisant leurs solvants respectifs: par exemple, dissoudre ABSC et PUG dans le diméthylsulfoxyde (DMSO); préparer l&#39;héparine dans l&#39;eau double distillée stérile. Pour tous les autres dilutions, utiliser des milieux de culture. Remarque: Le concent finaleration de DMSO dans les traitements composés de test est inférieure à 1% dans les expériences; 1% de DMSO est comprise comme traitement témoin négatif dans les tests de comparaison. </li><li> Déterminer la cytotoxicité des composés à tester (par exemple, ABSC et PUG) sur les cellules pour l&#39;infection virale en utilisant une viabilité cellulaire déterminer réactif tel que le XTT (2,3-bis [2-méthoxy-4-nitro-5-sulfophényl] -5-phénylamino) carbonyl] -2H-tétrazolium hydroxyde): <ol><li> Pour le VHC, ensemencer 7,5 Huh-cellules dans une plaque de 96 puits (1 x 10 4 cellules par puits) et incuber à 37 ° C dans un 5% de CO 2 incubateur O / N pour obtenir une monocouche. </li><li> Appliquer témoin de DMSO (1%) ou des concentrations croissantes de composés d&#39;essai ABSC et PUG (ex. 0, 10, 50, 100, et 500 pM) dans les puits de culture en triple exemplaire. </li><li> Incuber à 37 ° C pendant 72 heures, puis jeter le milieu dans la plaque et laver les cellules avec 200 pi de tampon phosphate salin (PBS) deux fois. </li><li> Ajouter 100 pi de assaying de solution du kit test in vitro en toxicologie-XTT dans chaque puits et incuber les plaques à 37 ° C pendant encore 3 heures pour permettre la production XTT formazan. </li><li> Déterminer l&#39;absorbance avec un lecteur de microplaques à une longueur d&#39;onde de test de 492 nm et une longueur d&#39;onde de 690 nm de référence. </li><li> Calculer le pourcentage de survie des cellules en utilisant la formule suivante: viabilité cellulaire (%) = A / As x 100%, où «A» et «sous» se référer à l&#39;absorbance des composés d&#39;essai et le témoin de solvant (par exemple 1% de DMSO. ) traitements, respectivement. Déterminer la concentration de 50% de cytotoxicité cellulaire (CC 50) des composés d&#39;essai à partir d&#39;un logiciel d&#39;analyse tel que GraphPad Prism selon le protocole du fabricant. </li></ol></li></ol> 2. Lecture de l&#39;infection virale Remarque: La lecture d&#39;une infection virale dépend du système de virus utilisé et peut impliquer des procédés tels que des dosages de plaques ou meaSuring signaux rapporteurs de virus rapporteurs marqués. Procédé pour détecter une infection par le VHC reporter sur la base de l&#39;activité rapporteur de la luciférase est décrite ci-dessous. <ol><li> Recueillir les surnageants des puits infectés et de clarifier à 17.000 xg dans une microcentrifugeuse pendant 5 min à 4 ° C. </li><li> Mélanger 20 ul de surnageant de test à 50 ul de substrat de la luciférase à partir de la trousse de dosage de la luciférase de Gaussia et de mesurer directement avec un luminomètre selon les instructions du fabricant. </li><li> Exprimer HCV infectieux tel que log 10 d&#39;unités lumineuses relatives (RLU) de détermination de l&#39;inhibition virale (%) et de calculer la concentration efficace 50% (EC50) des composés d&#39;essai contre une infection par le VHC en utilisant des algorithmes de logiciel GraphPad Prism selon le protocole du fabricant. </li></ol> 3. Inactivation virale Assay Remarque: Des exemples de période d&#39;incubation et la dose virale de différents virus aRE admises à la figure 1A. Des concentrations plus élevées de virus peuvent également être testés en augmentant le MOI / PFU. <ol><li> Graine Huh-7.5 cellules dans une plaque à 96 puits (1 x 10 4 cellules par puits) et incuber à 37 ° C dans un incubateur à CO2 à 5% O / N afin d&#39;obtenir une monocouche. </li><li> Incuber les composés ou des contrôles essai (concentrations finales sont: CHLA = 50 uM; PUG = 50 uM; héparine = 1,000 ug / ml; DMSO = 1%) avec les particules de VHC à 37 ° C (figure 1A, «à long terme» ) dans un rapport de 1: 1. Par exemple, pour un inoculum de virus de 100 pi contenant 1 x 10 4 FFU, ajouter 100 ul d&#39;une dilution de travail de 100 uM ABSC; on obtient de l&#39;ABSC traitement à une concentration finale de 50 uM. </li><li> On dilue le mélange virus-drogue de concentration (sans effet) &quot;sous-thérapeutique&quot; des composés d&#39;essai. Par exemple, la concentration inefficace de l&#39;ABSC et PUG contre le VHC est à 1 uM 31; doncceci nécessite une dilution de 50 fois du mélange virus-médicament qui peut être accompli avec 9,8 ml de milieu basal (milieu de culture cellulaire avec 2% de FBS). Remarque: La dilution à la concentration de sous-thérapeutique empêche l&#39;interaction significative entre les composés d&#39;essai et la surface de la cellule hôte et permet l&#39;examen de l&#39;effet du traitement sur les virions exempts de cellules. Notez que cette dilution est dépendante de la dose-réponse antivirale des composés d&#39;essai contre l&#39;infection virale particulière, et il est déterminé avant de réaliser cet essai particulier 31. </li><li> A titre de comparaison, le virus mélanger avec les composés d&#39;essai et dilué immédiatement (pas de période d&#39;incubation) de concentration sub-thérapeutique avant l&#39;infection (Figure 1A, «à court terme»). </li><li> Ajouter 100 pi du mélange VHC-médicament dilué sur le Huh-7,5 monocouche de cellules (la quantité de virus est maintenant à 1 x 10 2 FFU; finale MOI = 0,01) et incuber pendant 3 heures à 37 ° C pour permettre viraleadsorption. </li><li> Suite à l&#39;infection, retirer le inoculum dilué et laver délicatement les puits avec 200 pi de PBS deux fois. Remarque: Effectuez les lavages doucement pour éviter de soulever les cellules. </li><li> Appliquer 100 pi de milieu de base à chaque puits et incuber à 37 ° C pendant 72 heures. </li><li> Analyser l&#39;infection résultant en testant le surnageant pour l&#39;activité luciférase comme décrit dans &#39;2. Lecture d&#39;une infection virale. </li></ol> 4. virale Attachment Assay Remarque: Les exemples de la période d&#39;incubation et la dose virale pour divers virus sont répertoriés dans la figure 2A, «attachement». Des concentrations plus élevées de virus peuvent également être testés en augmentant le MOI / PFU. <ol><li> Graine Huh-7.5 cellules dans une plaque à 96 puits (1 x 10 4 cellules par puits) et incuber à 37 ° C dans un incubateur à CO2 à 5% O / N afin d&#39;obtenir une monocouche. </li><li> Pré-refroidir les monocouches de cellules dans des plaques à 4 ° C for 1 h. </li><li> Co-traiter les cellules infectées par le VHC inoculum (MOI = 0,01) et les composés d&#39;essai ou de contrôle (concentrations finales sont: CHLA = 50 uM; PUG = 50 uM; héparine = 1,000 ug / ml; DMSO = 1%) à 4 ° C pour 3 h. Par exemple, pour un inoculum de virus de 90 ul contenant 1 x 10 2 FFU, ajouter 10 ul d&#39;une dilution de travail de 500 uM ABSC; on obtient de l&#39;ABSC traitement à une concentration finale de 50 uM et une infection par le VHC à une MOI = 0,01 sur la monocouche cellulaire. Remarque: Il est important de réaliser l&#39;expérience à 4 ° C, car elle permet la liaison du virus mais empêche l&#39;entrée qui se produit le plus efficacement à 37 ° C. Effectuer l&#39;addition de composés de virus et de test sur de la glace et l&#39;incubation qui a suivi dans un réfrigérateur à 4 C ° pour garantir que la température est maintenue à 4 ° C. </li><li> Retirer le surnageant et laver délicatement la monocouche cellulaire avec 200 pi de PBS glacé deux fois. Remarque: Effectuez les lavages doucement pour éviter de soulever les cellules &lt;./ li&gt; <li> Appliquer 100 pi de milieu de base à chaque puits et incuber à 37 ° C pendant 72 heures. </li><li> Analyser l&#39;infection résultant en testant le surnageant pour l&#39;activité luciférase comme décrit dans &#39;2. Lecture d&#39;une infection virale. </li></ol> 5. virale Entrée / Fusion Assay Remarque: Exemple de périodes d&#39;incubation et dose virale pour divers virus sont répertoriés dans &#39;Entrée / Fusion&#39; Figure 2A. Des concentrations plus élevées de virus peuvent également être testés en augmentant le MOI / PFU. <ol><li> Graine Huh-7.5 cellules dans une plaque à 96 puits (1 x 10 4 cellules par puits) et incuber à 37 ° C dans un incubateur à CO2 à 5% O / N afin d&#39;obtenir une monocouche. </li><li> Pré-refroidir les monocouches de cellules dans des plaques à 4 ° C pendant 1 heure. </li><li> Infecter les cellules infectées par le VHC (MOI = 0,01) à 4 ° C pendant 3 heures. Par exemple, utiliser un inoculum viral 100 ul contenant 1 x 10 2 FFU. Remarque: Effectuez l&#39;Addition de l&#39;inoculum viral sur de la glace et l&#39;incubation qui a suivi dans un réfrigérateur à 4 C ° pour maintenir la température à 4 ° C, ce qui permet l&#39;entrée virale, mais non obligatoire. </li><li> Retirer le surnageant et laver délicatement les monocouches de cellules avec 200 pi de PBS glacé deux fois. Remarque: Effectuez les lavages doucement pour éviter de soulever les cellules. </li><li> Traiter les puits avec les composés d&#39;essai ou de contrôle (concentrations finales sont les suivantes: 50 uM = ABSC; PUG = 50 uM; héparine = 1,000 ug / ml; DMSO = 1%) et incuber à 37 ° C pendant 3 heures. Par exemple, ajouter 10 ul d&#39;une dilution de travail 500 um CHLA à 90 pi de milieu, mélanger, et de traiter les puits; on obtient de l&#39;ABSC traitement à une concentration finale de 50 uM. Remarque: Le passage de 4 ° C à 37 ° C facilite désormais l&#39;événement d&#39;entrée / fusion virale et permet donc l&#39;évaluation de l&#39;effet de composés de test sur cette étape particulière. </li><li> Aspirer le surnageant contenant le médicament et enlever non intérioriséles virus extracellulaires, soit par lavage avec 200 ul de tampon citrate (citrate de sodium 50 mM, chlorure de potassium 4, pH 3,0) ou du PBS. Appliquer 100 ul de milieu de base avant l&#39;incubation à 37 ° C pendant 72 h. </li><li> Analyser l&#39;infection résultant en testant le surnageant pour l&#39;activité luciférase comme décrit dans &#39;2. Lecture d&#39;une infection virale. </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.

Dans la figure 1, le «test d&#39;inactivation virale» a été réalisée pour examiner si deux composés naturels CHLA et PUG spécifiques pourraient inactiver les virus enveloppés dans différents état ​​libre-cellulaire et prévenir l&#39;infection ultérieure. La réponse à la dose antivirale et la cytotoxicité de ces composés ont été déterminées avant l&#39;exécution de l&#39;étude mécaniste 31. Les virus ont été pré-traitées avec les composés d&#39;essai et ensuite les mélanges virus-médicament ont été dilués à des concentrations sous-thérapeutiques avant l&#39;inoculation sur la monocouche de cellule respectif pour chaque système de virus. Comme le montre la figure 1, les deux ABSC et PUG sont apparus pour interagir avec les virions exempts de cellules, ce qui entraîne des effets irréversibles qui protégeaient la monocouche de cellules à partir de l&#39;infection ultérieure. Les deux composés d&#39;essai réalisé un près de 100% d&#39;inhibition contre le HCMV, le VHC et DENV-2, alors qu&#39;un 60 - bloc 80% a été observée contre MV et RSV. Ces résultats SuggEst ce que l&#39;ABSC et PUG ont un impact direct sur ces particules virales libres en les neutralisant l&#39;inactivation et leur infectivité. Dans la figure 2, l&#39;attachement et d&#39;entrée / essais de fusion ont été réalisées pour étudier l&#39;effet de l&#39;ABSC et PUG contre ces événements virales précoces liées entrée-de HCMV, VHC, DENV-2, MV, et RSV. Les deux CHLA et PUG efficacement empêché liaison des virus étudiés sur la cellule hôte respective comme indiqué par l&#39;inhibition de l&#39;infection virale résultante (Figure 2, «attachement»: barres gris clair). L&#39;effet inhibiteur sur l&#39;attachement du virus par les deux composés était semblable contre HCMV (figure 2B), le VHC (figure 2C), DENV-2 (figure 2D), et RSV (Figure 2F), allant de 90 - 100%. D&#39;autre part, PUG semblait être plus efficace que l&#39;ABSC contre MV liaison (Figure 2E), le taux de la tw d&#39;inhibitioncomposés o variant entre 50 - 80%. L&#39;héparine de traitement de commande, qui est connu pour bloquer l&#39;entrée de nombreux virus, aussi inhibé la fixation de HCMV, DENV-2, RSV, annonce MV, mais est moins efficace contre le VHC. Le suivi &#39;essai entrée / fusion virale&#39; examiner si CHLA et PUG ont conservé leur activité au cours de la phase entrée du virus / fusion (Figure 2, &#39;Entrée / Fusion &quot;: barres gris foncé). Encore une fois, les deux ABSC et PUG ont été observés à altérer efficacement l&#39;étape d&#39;entrée / de fusion virale des virus examinés (Figure 2B - F), ce qui donne un 50 - effet protecteur de 90% sur la monocouche de cellule respectif. Héparine aussi puissamment inhibé entrée / fusion dans DENV-2 et les infections à VRS, mais était moins efficace contre le HCMV, le VHC et MV (&lt;40% d&#39;inhibition en moyenne). <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Virus </td><td> Type de cellule </td></tr><tr><td> HCMV </td> &lt;td&gt; HEL </td></tr><tr><td> VHC </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> Tableau 1:. Hôte type de cellule à une infection virale du type de cellule utilisé pour chaque système d&#39;infection virale décrit dans les résultats représentatifs est indiquée. Des détails supplémentaires concernant les cellules peuvent être trouvés dans la référence 31. <img alt="Figure 1" src="/files/ftp_upload/53124/53124fig1.jpg" /> Figure 1. L&#39;inactivation des infections virales par les composés de test CHLA et PUG Différents virus ont été traités avec les composés d&#39;essai pendant une longue période. (Incubé pendant 1,5 à 3 h avant le titrage; barres gris clair) ou une période courte (dilué immédiatement; gris foncé bars) à 37 ° C avant une dilution de concentration sub-thérapeutiquetion et l&#39;analyse ultérieure de l&#39;infection sur les cellules hôtes respectives. (A) Schéma de l&#39;expérience (montré sur la gauche) avec la concentration de virus final (PFU / puits ou MOI), à long terme période virus médicament incubation (i), et le temps d&#39;incubation subséquente (ii) indiqué pour chaque virus dans le tableau sur la droite. Les analyses de (B) de HCMV, (C) le VHC, (D) DENV-2, (E) MV, et (F) de RSV sont indiqués dans chaque panneau supplémentaire. Les résultats sont tracées en fonction du traitement de contrôle négatif DMSO pour infection par le virus ainsi que les données représentées sont les moyennes ± l&#39;erreur-type de la moyenne (SEM) à partir de trois expériences indépendantes. Ce chiffre a été modifié de la référence 31. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig1highres.jpg" target="_blank">S&#39;il vous plaît cliquez ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 2" src="/files/ftp_upload/53124/53124fig2.jpg" /> Figure 2. L&#39;évaluation des activités antivirales de composés d&#39;essai ABSC et PUG contre l&#39;attachement du virus et l&#39;entrée / fusion. (A) Le mode opératoire expérimental, la concentration en virus (PFU / puits ou MOI), et le moment de l&#39;addition et le traitement avec les composés d&#39;essai (i, ii, iii) sont présentés pour chaque virus dans les schémas et les tableaux associés. Dans l&#39;analyse de fixation de virus (barres gris clair), des monocouches de différents types de cellules ont été pré-refroidi à 4 ° C pendant 1 heure, puis co-traités avec les virus respectifs et les composés d&#39;essai à 4 ° C (1,5 - 3 h, i) avant de rincer les inoculats et les composés d&#39;essai à une incubation ultérieure (37 ° C, ii) et de l&#39;examen de l&#39;infection par le virus. Dans l&#39;entrée du virus / analyse de fusion (barres gris foncé), des monocouches de cellules ensemencées ont été pré-refroidi à 4 ° C pendant 1 h et ensuite provoqués avec chacun des virus à 4 ° C pendant 1,5 - 3 h (i). Les cellules ont ensuitelavé et traité avec des composés d&#39;essai pendant une période d&#39;incubation supplémentaire (ii) au cours de laquelle la température a été décalé à 37 ° C pour faciliter l&#39;événement d&#39;entrée / fusion virale. A la fin de l&#39;incubation, les virus extracellulaires ont été retirés soit par un tampon citrate (pH 3,0) ou lavages au PBS et les cellules ont été encore incubées (iii) pour l&#39;analyse d&#39;une infection par virus. Résultats pour (B) HCMV, (C) le VHC, (D) DENV-2, (E) MV, et (F) RSV sont indiqués dans chaque panneau supplémentaire. Les données sont tracées en fonction du traitement de contrôle négatif de DMSO infection par le virus et sont présentées comme moyenne ± SEM à partir de trois expériences indépendantes. Ce chiffre a été modifié de la référence 31. <a href="https://www.jove.com/files/ftp_upload/53124/53124fig2highres.jpg" target="_blank">S&#39;il vous plaît cliquez ici pour voir une version plus grande de cette figure.</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.

Early Viral entrée dosages pour l&#39;identification et l&#39;évaluation des composés antiviraux

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

Vidéo: 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.

Analyses quantitatives de tous influenza de type A hémagglutinines virales et les neuraminidases utilisant des anticorps Universal en simples essais 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 ...

Recherche

Immunologie et infection

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.

Amplifier et quantifier l&#39;ARN VIH-1 chez les individus infectés par le VIH ayant une charge virale sous le seuil de détection par Standard tests cliniques

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.

Les tests pour l&#39;identification de nouveaux antiviraux contre la fièvre catarrhale Virus

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.

Détermination de la concentration virale par Plaque Essais: Systèmes calque à l&#39;aide traditionnels et nouveaux

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.

Haut-débit quantitative en temps réel RT-PCR analyse pour déterminer les profils d&#39;expression de types I et III sous-types d&#39;interféron

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.

Les analyses qualitatives et quantitatives pour la détection et la caractérisation des antimicrobiens en protéines

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.

Le développement de lyophilisé boucle médiées isothermes Amplification Réactifs pour la détection des<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 Méthode d&#39;échantillonnage pour la détection des norovirus humains sur les surfaces

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.

Purification de l’ADN Viral pour l’Identification des protéines virus et cellulaires associées

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.

Évaluation des réponses de cellules T spécifiques du Virus Zika dans les organes Immunoprivileged des Ifnar1 infecté les souris- / -

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