This work has been funded by the National Medical Research Council, Singapore.

1. Alexa Fluor labeling of dengue virus
<ol>
	<li>Before the labeling reaction, purify dengue virus with sucrose cushion and prepare the necessary reagents and equipment as indicated in the protocol.</li>
	<li>Prepare fresh 0.2M sodium bicarbonate buffer, pH 8.5 (labeling buffer), and 1.5M hydroxylamine buffer, pH 8.3 (stop reagent), just before labeling and filter 
	sterilize with 0.2&mu;m syringe filters.</li>
	<li>Dilute approximately 3x108 plaque forming units (pfu) of purified dengue virus in 1ml of labeling buffer in a 2ml tube. This can be scaled up proportionally for batch labeling of virus.</li>
	<li>Reconstitute the lyophilized Alexa Fluor 594 (AF594) succinimidyl esters to 1mM in labeling buffer immediately prior to the labeling reaction. Other fluorochromes from the Alexa Fluor dye series may be used according to one&#x2019;s needs. Minimize exposure to light from this step onwards.</li>
	<li>Add 100&mu;l of the 1mM AF594 dye to the diluted virus while stirring gently with the pipette tip.</li>
	<li>Incubate the labeling reaction mix at room temperature for 1hr in the dark. Mix by gentle inversions every 15mins.</li>
	<li>Spin the tube briefly in a tabletop centrifuge and add 100&mu;l of stop reagent to the reaction mix while stirring gently with the pipette tip. </li>
	<li>Incubate at room temperature for an additional hour in the dark. Mix by gentle inversions every 15mins.</li>
</ol>
2. Purifying Alexa Fluor labeled dengue virus
<ol>
	<li>In the meantime, equilibrate the purification column with buffer of choice. In this experiment, a PD-10 column is equilibrated with 25ml of HNE buffer (5mM Hepes, 150mM NaCl, 0.1mM EDTA), pH 7.4, before use.</li>
	<li>Apply the labeled virus to the top of the column and start collecting the flow-through once the labeled virus enters the matrix. Fill the column with HNE buffer 
	once all the labeled virus has entered the matrix. Discard the first 2.5ml of flow-through and collect the next 2ml of labeled virus fraction.</li>
	<li>Aliquot and store purified AF594 labeled dengue virus in -80&deg;C, away from light source.</li>
</ol>
<ol>
	<li>Thaw one aliquot and determine the titer of the labeled virus by plaque assay before using the batch of labeled virus.</li>
	<li>Seed 5x104 per well of Vero cells, grown in M-199 growth medium, in a 4-well plate with a coverslip on the bottom of well a day before infection.</li>
	<li>Remove the culture supernatant in well and infect the cells with multiplicity of infection of 1 of the labeled virus in 100&mu;l volume (diluted in M-199 maintenance medium as required) for 10min at 37&deg;C.</li>
	<li>Remove the inoculums and wash the coverslips twice in 1xPBS.</li>
	<li>Fix the cells in 3% paraformalydehyde for 30mins.</li>
	<li>Wash the coverslips 3 times in 1xPBS.</li>
	<li>Permeabilize the cells with permeabilization solution containing 0.1% saponin and 5% BSA in 1xPBS for 30mins.</li>
	<li>Incubate the cells with undiluted centrifugation-clarified supernatant of 3H5 monoclonal antibody hybridoma culture for 1hr in a humid chamber, protected from light.</li>
	<li>Wash the coverslips 3 times in wash buffer (1xPBS containing 1mM calcium chloride, 1mM magnesium chloride and 0.1% saponin).</li>
	<li>Incubate the cells with AF488 anti-mouse IgG antibody, 1:100 diluted in permeabilization solution, for 45mins in humid chamber, protected from light.</li>
	<li>Wash the coverslips 3 times in wash buffer and rinse once in deionized water.</li>
	<li>Dab the edge of coverslip against a paper towel to drain excess water and mount on to glass slide with 8&mu;l Mowiol 4-88 containing 2.5% Dabco.</li>
	<li>Allow the mounting solution to set overnight at 4&deg;C before viewing using a Zeiss confocal microscope. Sequential acquisitions should be performed exciting one fluorophore at a time and switching between the detectors concomitantly.</li>
	<li>Images are then analyzed for co-localization of the E protein antibody staining with the labeled virus using the Zeiss LSM Zen software to estimate the degree of labeling.</li>
</ol>
3. Representative Results
An example of the yield of dengue virus labeled with AF594 dye is shown in Figure 2. Normally, less than 10-fold drop from the initial titer should be observed following successful labeling. However, it should be noted that all buffers have to be prepared fresh for the labeling to be successful and the Alexa Fluor succinimidyl esters should be used immediately upon reconstitution as they hydrolyze into nonreactive free acids in aqueous solutions8.
Next, the labeled virus has to be checked for sufficient fluorescence before use in experiments. A simple immunofluorescence assay was done on Vero cells and the degree of labeling can be estimated from the co-localization of the labeled virus with anti-E protein antibody staining. Several cells were examined and a typical confocal image is shown in Figure 3. Co-localization analysis of the images using the LSM Zen software demonstrated overlap coefficients ranging from 0.65 to 0.8, suggesting that approximately 65 to 80% of the virions were labeled with the dye.
<img src="/files/ftp_upload/3168/3168fig1.jpg" alt="Figure 1" /> 
Figure 1.Overall scheme depicting the Alexa Fluor dye labeling of dengue virus procedure. First, the relevant buffers and purified dengue virus are prepared. The Alexa Fluor dye is reconstituted and added to the dengue virus diluted in labeling buffer. The reaction is then stopped 1 hour later with the addition of stop reagent. Subsequently, the labeled virus is purified through a size exclusion column to remove free dye. Finally, the labeled virus is re-titrated by plaque assay and tested for fluorescence.
<img src="/files/ftp_upload/3168/3168fig2.jpg" alt="Figure 2" /> 
Figure 2. Mean number of viable virions (pfu/ml) as determined on a plaque assay before and after AF594 labeling. An aliquot of the AF594 labeled dengue virus is thawed and re-titrated by plaque assay and it typically shows less than 10-fold drop from the starting titer. Error bars indicate standard deviation of duplicates.
<img src="/files/ftp_upload/3168/3168fig3.jpg" alt="Figure 3" /> 
Figure 3. Co-localization of AF594 labels with dengue virus E proteins in Vero cells. cells grown on coverslips the day prior were infected with AF594 labeled dengue at MOI of 1 for 10 minutes at 37&deg;C. The cells were subsequently fixed and labeled with anti-E antibody, and examined for colocalization of E protein (green) and AF594 labeling (red). Fluorescent signals were visualized under 63X magnification using Zeiss LSM 710 confocal microscope. Scale bar is 10&mu;m. Yellow indicate areas of colocalization, as shown in the inset.

<ol>
	<li>Olenych, S. G., Claxton, N. S., Ottenberg, G. K. &. a. m. p. ;. a. m. p., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fluorescent+Protein+Color+Palette.">The Fluorescent Protein Color Palette.</a> Current Protocols in Cell Biology. 21.5, 1-34 (2007).</li><li>Panchuk-Voloshina, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alexa+dyes,+a+series+of+new+fluorescent+dyes+that+yield+exceptionally+bright,+photostable+conjugates.">Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.</a> J Histochem Cytochem. 47, 1179-1188 (1999).</li><li>Shaner, N. C., Patterson, G. H., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+fluorescent+protein+technology.">Advances in fluorescent protein technology.</a> J Cell Sci. 120, 4247-4260 (2007).</li><li>Lippincott-Schwartz, J., Patterson, G. H. <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+use+of+fluorescent+protein+markers+in+living+cells.">Development and use of fluorescent protein markers in living cells.</a> Science. 300, 87-91 (2003).</li><li>Kuhn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+dengue+virus:+implications+for+flavivirus+organization,+maturation,+and+fusion.">Structure of dengue virus: implications for flavivirus organization, maturation, and fusion.</a> Cell. 108, 717-725 (2002).</li><li>Aguilar, J. S., Roy, D., Ghazal, P., Wagner, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethyl+sulfoxide+blocks+herpes+simplex+virus-1+productive+infection+in+vitro+acting+at+different+stages+with+positive+cooperativity.+Application+of+micro-array+analysis.">Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis.</a> BMC Infect Dis. 2, 9-9 (2002).</li><li>Zhang, S. L., Tan, H. C., Hanson, B. J., Ooi, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+Alexa+Fluor+dye+labelling+of+dengue+virus.">A simple method for Alexa Fluor dye labelling of dengue virus.</a> J Virol Methods. 167, 172-177 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Alexa Fluor Succinimidyl Esters (Molecular Probes. , (2009).</li><li>Huang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+proteins+stained+by+Alexa+dyes.">Analysis of proteins stained by Alexa dyes.</a> Electrophoresis. 25, 779-784 (2004).</li><li>Freistadt, M. S., Eberle, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+poliovirus+for+flow+cytometric+cell+surface+binding+studies.">Fluorescent poliovirus for flow cytometric cell surface binding studies.</a> J Virol Methods. 134, 1-7 (2006).</li></ol>

We have nothing to disclose.

Although AF594 dye was used in this report, a wide range of fluorophores in the Alexa Fluor succinimidyl esters series is available with similar labeling chemistry. This could extend the labeling application beyond imaging. Flow cytometry can be used as an alternative to confocal microscopy for estimating the degree of labeling for fluorophores that can be excited and detected by the FACS machine.
Alexa Fluor dyes are small molecules that react with free amino groups, primarily arginine and ly...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hydroxylamine</td>
			<td>Sigma-Aldrich</td>
			<td>159417</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Merck</td>
			<td>106498</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AF594 succinimidyl esters</td>
			<td>Molecular Probes, Invitrogen</td>
			<td>A20004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD-10 column</td>
			<td>GE Healthcare</td>
			<td>17-0851-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H6147</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>M-199</td>
			<td>Invitrogen</td>
			<td>11150</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-well plate</td>
			<td>Nunc</td>
			<td>176740</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Einst</td>
			<td>0111520</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Sail Brand</td>
			<td>7105</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3H5 hybridoma</td>
			<td>ATCC</td>
			<td>HB46</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>1st Base</td>
			<td>BUF-2040-10X1L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>S4521</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehye</td>
			<td>Sigma-Aldrich</td>
			<td>15,812-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dabco</td>
			<td>Sigma-Aldrich</td>
			<td>D27802</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tabletop centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 710</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

To prepare M-199 growth medium, add 50ml of FBS, 5ml of sodium pyruvate and 5ml of non-essential amino acids to 500ml of M-199, sterile filter. 
To prepare M-199 maintenance medium, add 15ml of FBS, 5ml of sodium pyruvate and 5ml of non-essential amino acids to 500ml of M-199, sterile filter.

visualizing dengue virus through alexa fluor labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Watch this Scientific Journal Video about Visualizing Dengue Virus through Alexa Fluor Labeling at JoVE.com

Visualizing Dengue Virus through Alexa Fluor Labeling

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

visualizing-dengue-virus-through-alexa-fluor-labeling

Research

JoVE Journal

Immunology and Infection

13.7K vistas.  DSO National Laboratories. Aprovechando los avances en el desarrollo de fluoróforo y la tecnología de imágenes, un método sencillo de Alexa Fluor etiquetado de los virus del dengue se ideó para visualizar las primeras interacciones entre el virus y la célula.

Video: Visualizing Dengue Virus through Alexa Fluor Labeling

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

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 ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

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 ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...