This project was supported by grant 1R03MH08127-01 and 7R03MH08127-02 from NIH to Q. Li, and by the IMPACT funds from Department of Medicine at UAB to Q. Li. Support from the Molette Fund and Auburn University is appreciated. We also thank the technical assistances from Ms. Pulin Che and Mr. Volodymyr Musiienko during the course of the work.

1. Cells, Virus and the Antiviral Compounds
<ol>
	<li>Maintain BSR cells, a derivative of baby hamster kidney (BHK) cells10, in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) containing 5% fetal calf serum (FCS), 100U/ml penicillin and 100 &#956;g/ml streptomycin.</li>
	<li>For all three assays, plate cells in DMEM with 1% FCS, 100U/ml penicillin and 100 &#956;g/ml streptomycin, as optimized previously5. This medium is referred as assay medium for all three assays.</li>
	<li>Incubate all cells ...

<ol>
	<li>Hemati, B., 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=Bluetongue+virus+targets+conventional+dendritic+cells+in+skin+lymph.">Bluetongue virus targets conventional dendritic cells in skin lymph.</a> J. Virol. 83, 8789-8799 (2009).</li><li>Gu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Virostatic+Agents+against+Bluetongue+Virus.">Novel Virostatic Agents against Bluetongue Virus.</a> PLoS ONE. 7, e43341 (2012).</li><li>Meiswinkel, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2006+outbreak+of+bluetongue+in+northern+Europe--the+entomological+perspective.">The 2006 outbreak of bluetongue in northern Europe--the entomological perspective.</a> Prev. Vet. Med. 87, 55-63 (2008).</li><li>Szmaragd, C., 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=Mortality+and+case+fatality+during+the+recurrence+of+BTV-8+in+northern+Europe+in+2007.">Mortality and case fatality during the recurrence of BTV-8 in northern Europe in 2007.</a> Vet. Rec. 161, 571-572 (2007).</li><li>Li, Q., Maddox, C., Rasmussen, L., Hobrath, J. V., White, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+development+and+high+throughput+antiviral+drug+screening+against+Bluetongue+virus.">Assay development and high throughput antiviral drug screening against Bluetongue virus.</a> Antiviral Research. 83, 267-273 (2009).</li><li>Noah, J. W., 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=A+cell-based+luminescence+assay+is+effective+for+high-throughput+screening+of+potential+influenza+antivirals.">A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals.</a> Antiviral Res. 73, 50-59 (2007).</li><li>Che, P., Wang, L., Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development,+optimization+and+validation+of+an+assay+for+high+throughput+antiviral+drug+screening+against+Dengue+virus.">The development, optimization and validation of an assay for high throughput antiviral drug screening against Dengue virus.</a> Int. J. Clin. Exp. Med. 2, 363-373 (2009).</li><li>Li, Q., Li, H., Blitvich, B. J., 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=The+Aedes+albopictus+inhibitor+of+apoptosis+1+gene+protects+vertebrate+cells+from+bluetongue+virus-induced+apoptosis.">The Aedes albopictus inhibitor of apoptosis 1 gene protects vertebrate cells from bluetongue virus-induced apoptosis.</a> Insect Mol. Biol. 16, 93-105 (2007).</li><li>Petty, R. D., Sutherland, L. A., Hunter, E. M., Cree, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+MTT+and+ATP-based+assays+for+the+measurement+of+viable+cell+number.">Comparison of MTT and ATP-based assays for the measurement of viable cell number.</a> J. Biolumin. Chemilumin. 10, 29-34 (1995).</li><li>Buchholz, U. J., Finke, S., Conzelmann, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+bovine+respiratory+syncytial+virus+(BRSV)+from+cDNA:+BRSV+NS2+is+not+essential+for+virus+replication+in+tissue+culture,+and+the+human+RSV+leader+region+acts+as+a+functional+BRSV+genome+promoter.">Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.</a> J. Virol. 73, 251-259 (1999).</li><li>Phillips, T., Jenkinson, L., McCrae, C., Thong, B., Unitt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+high-throughput+human+rhinovirus+infectivity+cell-based+assay+for+identifying+antiviral+compounds.">Development of a high-throughput human rhinovirus infectivity cell-based assay for identifying antiviral compounds.</a> J. Virol. Methods. 173, 182-188 (2011).</li><li>Harvey, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracycline-inducible+packaging+cell+line+for+production+of+flavivirus+replicon+particles.">Tetracycline-inducible packaging cell line for production of flavivirus replicon particles.</a> J. Virol. 78, 531-538 (2004).</li><li>Puig-Basagoiti, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+assays+using+a+luciferase-expressing+replicon,+virus-like+particles,+and+full-length+virus+for+West+Nile+virus+drug+discovery.">High-throughput assays using a luciferase-expressing replicon, virus-like particles, and full-length virus for West Nile virus drug discovery.</a> Antimicrobial Agents and Chemotherapy. 49, 4980-4988 (2005).</li><li>Puig-Basagoiti, F., 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=Triaryl+pyrazoline+compound+inhibits+flavivirus+RNA+replication.">Triaryl pyrazoline compound inhibits flavivirus RNA replication.</a> Antimicrob. Agents Chemother. 50, 1320-1329 (2006).</li><li>Duan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+protection+against+the+highly+pathogenic+H5N1+influenza+virus+by+an+antisense+phosphorothioate+oligonucleotide.">In vitro and in vivo protection against the highly pathogenic H5N1 influenza virus by an antisense phosphorothioate oligonucleotide.</a> Antivir. Ther. 13, 109-114 (2008).</li><li>Ray, D., Shi, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+flavivirus+antiviral+drug+discovery+and+vaccine+development.">Recent advances in flavivirus antiviral drug discovery and vaccine development.</a> Recent Pat. Antiinfect. Drug Discov. 1, 45-55 (2006).</li><li>Severson, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+of+a+100,000-compound+library+for+inhibitors+of+influenza+A+virus+(H3N2).">High-throughput screening of a 100,000-compound library for inhibitors of influenza A virus (H3N2).</a> J. Biomol. Screen. 13, 879-887 (2008).</li><li>Severson, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+high-throughput+screen+for+inhibitors+of+SARS+CoV+and+its+application+in+screening+of+a+100,000-compound+library.">Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library.</a> J. Biomol. Screen. 12, 33-40 (2007).</li><li>Bolken, T. C., 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=Identification+and+characterization+of+potent+small+molecule+inhibitor+of+hemorrhagic+fever+New+World+arenaviruses.">Identification and characterization of potent small molecule inhibitor of hemorrhagic fever New World arenaviruses.</a> Antivir. Res. 69, 86-97 (2006).</li><li>Van Loock, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-throughput+cellular+screening+assay+for+the+discovery+of+HIV-1+integrase+inhibitors.">A novel high-throughput cellular screening assay for the discovery of HIV-1 integrase inhibitors.</a> J. Virol. Methods. 179, 396-401 (2012).</li><li>Kampmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+silico+screening+of+small+molecule+libraries+using+the+dengue+virus+envelope+E+protein+has+identified+compounds+with+antiviral+activity+against+multiple+flaviviruses.">In silico screening of small molecule libraries using the dengue virus envelope E protein has identified compounds with antiviral activity against multiple flaviviruses.</a> Antiviral Res. 84, 234-241 (2009).</li><li>Kirchmair, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+anti-viral+agents+using+molecular+modeling+and+virtual+screening+techniques.">Development of anti-viral agents using molecular modeling and virtual screening techniques.</a> Infect. Disord. Drug Targets. 11, 64-93 (2011).</li></ol>

For the initial identification of antiviral hits, one of the key steps for antiviral drug discovery and development is to develop robust assays, which includes selecting a quantifiable marker, developing a simple protocol, obtaining sufficient signals and less than 10% CV. Most biochemical or cell-based screens are designed to provide a chemical starting point based upon the most robust, simple and inexpensive assay, due to the required reproducibility in the screening process and the potentially large number of molecule...

BTV is a prototype double-stranded RNA virus in the genus Orbivirus, family Reoviridae. BTV is one of the most important diseases of domestic livestock, including sheep, goat, cattle and other domestic animals, with $3 billion/year loss worldwide1,2. The exotic BTV serotype is an important animal pathogen listed in the &#34;USDA High Consequence Livestock Pathogens.&#34; Recently, the re-emerging of BTV has caused a major outbreak of disease in cattle and sheep in several countries across Northern Europe3,4. As a result of its economic significance and as a model system, BTV has been the subject of extensive molecular, genetic and structural studies, and several vaccines have been developed. However, due to the lack of proper assays for antiviral drug discovery, there are no antivirals available against BTV.
In a recent high throughput screening (HTS) campaign using BTV as the model system, we developed, optimized and validated a CPE-based assay to identify potential broad-spectrum antivirals against arboviruses5. CPE-based assay is a well-recognized assay that has been used in antiviral drug discovery against a number of viruses that induced rapid and observable CPE/apoptosis5-7. In our system, post BTV infection, CPE is evident in vertebrate cells, including HeLa, BSR, and HEK 293T8. BTV-induced CPE could be monitored and quantified using various cell viability detection methods, including the CellTiter Glo cell viability reagent kit (CTG kit)9. This kit determines the number of viable cells in culture based on quantitation of cellular ATP presented, which signals the presence of metabolically active living cells. Under optimized conditions, the CPE-based assay presented here showed its feasibility with the &#34;mix and measure&#34; one step protocol, and flexibility with stable luminescent signals. Meanwhile, toxic compounds reducing cell viability will be excluded in this CPE-based assay. The CPE-based assay showed its robustness and reliability for antiviral drug discovery against BTV, and has been used to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR), which leads to the identification of six novel cluster of potential antiviral lead compound(s)5.
When a potential antiviral compound has been identified using the CPE-based assay, it will need to be subjected to the ten-concentration dose-response assay to determine the range of antiviral efficacy and cytotoxicity2. The antiviral efficacy, represented as the 50% inhibitory concentration (IC50) or the 50% effective concentration (EC50), is the concentration of a drug which inhibits virus-induced CPE halfway between the baseline and maximum. The cytotoxicity of the antivirals, i.e.&#160;the 50% cytotoxicity concentration (CC50), is the concentration of a drug inducing 50% of cytotoxicity between the baseline and maximum. The selective index (SI), denoted as 50% SI (SI50) is calculated from CC50/IC50 which determines the specificity of the antiviral against virus-induced CPE. The IC50 (or EC50), CC50 and SI50 values are critical measures to determine whether an antiviral compound is potent and selective for further drug development.
When an antiviral showed no overt toxicity in vitro, yet prevented virus induced CPE and the productive viral life-cycle, it is important to characterize its MoA2. We initiated such characterization by carrying out ToA assay to determine the possible step(s) of viral life-cycle that is affected by the antiviral. Generally, antiviral compound were added to cells at different times pre- or post-virus infection. If the antivirals were added to the infected cells post to its target step during the course of infection, it would result in lower activity when compared to the one which was added prior to the step. Thus, ToA study is critical for determining the antiviral efficacy of a compound, and its potential target, either on the viral life-cycle or the host machinery involved in the viral life-cycle.
For all three assays, cell viability was determined using the CTG kit following manufacturer&#39;s instruction5. This detection system outputs adequate luminescence signals that could be analyzed using various in-house software. Each assay was validated and performed at least in triplicate with eight replicas. For all the obtained data, three parameters, including mean value (AVE), standard deviation (STDEV), and co-efficient variation (CV) were analyzed to determine the robustness of the assay. Once the robustness of the assay has been determined, the data will be further analyzed and plotted using various biostatics and graphic tools2.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM medium</td>
			<td>Gibco</td>
			<td>1134218</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>1000185</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gbico</td>
			<td>1049769</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>CellTiter-Glo (CTG) kit</td>
			<td>Promega</td>
			<td>TB288</td>
			<td>For cell viability measurement</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Fisher</td>
			<td>S25309B</td>
			<td>Diluted from 95%</td>
		</tr>
		<tr>
			<td>Antiviral huashilcompounds</td>
			<td>NIH MLSMR and de novo synthesis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BTV-10</td>
			<td>ATCC</td>
			<td>VR-187</td>
			<td></td>
		</tr>
		<tr>
			<td>BSR cell</td>
			<td>Developed in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy-II multi-mode microplate reader</td>
			<td>BioTek</td>
			<td></td>
			<td>For luminescent signal reading</td>
		</tr>
		<tr>
			<td>MicroFlo select dispenser</td>
			<td>BioTek</td>
			<td></td>
			<td>Adding cells, virus, and reagents</td>
		</tr>
		<tr>
			<td>384-well flat-bottom microplate</td>
			<td>CORNING</td>
			<td>28908031</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Gen. 5 software</td>
			<td>BioTek</td>
			<td></td>
			<td>For analysis of reading outputs from Synergy-II multi-mode microplate reader</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad</td>
			<td>Version 5</td>
			<td>For biostatic analysis and plot</td>
		</tr>
	</tbody>
</table>

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.

1. Antiviral efficacy of compound
The cell-based CPE assay was developed, optimized and validated in vitro using the luminescent-based CTG kit to identify novel antivirals against BTV as described previously2,5. The ten-dose response assay was employed to reflect the antiviral efficacy and cytotoxicity of an identified lead compound by measuring the number of metabolically viable cells in culture based on quantitation of cellular ATP presented in the living cells5,11</sup...

Watch this Scientific Journal Video about Assays for the Identification of Novel Antivirals against Bluetongue Virus at JoVE.com

Assays for the Identification of Novel Antivirals against Bluetongue Virus

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.

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

assays-for-identification-novel-antivirals-against-bluetongue

Research

JoVE Journal

Immunology and Infection

13.9K vues.  University of Alabama at Birmingham.  Trois essais, y compris l'effet cytopathique (CPE) à base de test, essai dose-réponse et (TOA) test à temps de Addition ont été développés, optimisés, validés et utilisés pour identifier de nouveaux antiviraux contre le virus de la fièvre catarrhale (BTV), ainsi à déterminer le Mécanisme d'action-de-possible (MOA) pour antiviraux nouvellement identifiés. 

Vidéo: Assays for the Identification of Novel Antivirals against Bluetongue Virus

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.

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

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

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.

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

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

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.

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

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

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

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

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

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

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

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