We thank David Wang, Anatoly Urisman, Amy Kistler, Kael Fischer, Patrick Tang, and Alexander Greninger for the initial development and optimization of the Virochip protocol. This work is supported by an NIH K08 grant and Abbott Discovery Award (to CC) and the Howard Hughes Medical Institute (to JLD).

The steps of the Virochip assay include (1) nucleic acid extraction from clinical samples, (2) reverse transcription of extracted RNA and 2nd-strand cDNA synthesis, (3) PCR amplification of randomly primed cDNA, (3) Cy3 fluorescent dye incorporation, (4) hybridization of labeled material to the Virochip microarray, and (5) scanning and analysis (Figure 1). The protocol shown below will demonstrate the use of the Virochip assay on a nasal swab sample from a child with an influenza-like illness.
Primer Sequences
Primer A 5'-GTTTCCCAGTCACGATA-(N9)-3'
Primer B 5'-GTTTCCCAGTCACGATA-3'
1. Nucleic Acid Extraction
<ol>
	<li>Nucleic acid extraction from clinical samples should be performed under Biosafety Level 2 (BSL-2) or higher conditions, as these samples are potentially infectious.</li>
	<li>The nasal swab sample is transported in viral holding medium. Aliquot 200 &mu;L of the sample into a 1.5 mL Eppendorf microcentrifuge tube.</li>
	<li>Optional: pass sample through a 0.22 &mu;m filter (to purify for viral particles)</li>
	<li>Optional: pretreat sample with DNAse (to degrade host genomic DNA and purify for protected, encapsidated viral nucleic acid) using the Turbo DNAse kit (Ambion) per the manufacturer's instructions</li>
	<li>Extract the sample using the Zymo ZR Viral RNA Extraction Kit or Qiagen Ultrasens Virus Kit per the manufacturer's instructions.</li>
</ol>
2. Reverse Transcription and 2nd-strand CDNA Synthesis ("Round A")
<ol>
	<li>Add 1 uL 40 pmol / &mu;L Primer A to 4 uL extracted RNA. Heat to 65&deg;C for 5 min in a thermocycler (e.g. GeneAmp PCR System 9700) and then let cool at room temp x 5 min.</li>
	<li>Add 5 &mu;L of a master mix consisting of 2 &mu;L 5X RT buffer, 1 &mu;L 12.5 mM dNTP, 1 &mu;L water, 0.5 &mu;L 0.1M DTT, and 0.5 &mu;L SSIII RT (Invitrogen). Incubate at 42&deg;C x 60 minutes.</li>
	<li>In a programmable thermocycler, heat to 94&deg;C x 2 min (to abort the reverse transcriptase reaction), cool to 10&deg;C, pulse centrifuge, and then add 5 &mu;L Sequenase mix consisting of 1 &mu;L 5X Sequenase buffer, 3.8 &mu;L water, and 0.15 &mu;L Sequenase (USB Corporation). For 2nd strand synthesis, ramp up from 10&deg;C to 37&deg;C over 8 min, hold 8 min, then heat to 94&deg;C x 2 min (to inactivate Sequenase) and cool to 10&deg;C (hold).</li>
	<li>Optional : Rd A sample cDNA can be stored at this point at -20&deg;C.</li>
</ol>
3. PCR Amplification of Randomly Primed CDNA ("Round B")
<ol>
	<li>Add 5 uL of Rd A sample to 45 &mu;L of a master mix consisting of 5 &mu;L 10X PCR buffer, 1 &mu;L 12.5 mM dNTP, 1 &mu;L 100 pmol / &mu;L primer B, 1 &mu;L KlenTaq LA (Sigma), and 37 &mu;L water.</li>
	<li>Run PCR protocol as follows: 94&deg;C, 2 min &rarr;(25 cycles of 94&deg;C, 30 s / 50&deg;C, 45 s / 72&deg;C, 1 min) &rarr;72&deg;C, 5 min &rarr;10&deg;C (hold)</li>
	<li>Check the Rd B sample on a 1.5% agarose electrophoresis gel. A smear from approximately 200 - 1000 bp should be visualized (Figure 2A)</li>
</ol>
4. Cy3 Fluorescent Dye Incorporation
<ol>
	<li>Optional : another sample can be labeled with the Cy5 fluorescent dye and hybridized to the same microarray.</li>
	<li>To incorporate aa-dNTP, add 5 uL of Rd B sample to 45 uL of a master mix consisting of 5 uL 10X PCR buffer, 1 uL 12.5 mM aa-dNTP(Invitrogen), 1 uL 100 pmol / uL primer B, 1, 1 uL KlenTaq LA (Sigma), and 37 uL water. Run PCR protocol as follows: 94&deg;C, 2 min &rarr;(15cycles of 94&deg;C, 30 s / 50&deg;C, 45 s / 72&deg;C, 1 min) &rarr;72&deg;C, 5 min &rarr;10&deg;C (hold)</li>
	<li>Clean the Rd C sample with the DNA Clean and Concentrator-5 Kit (Zymo Research) per the manufacturer's instructions. Elute in 10 &mu;L.</li>
	<li>Add 1 uL of 1M bicarbonate (Sigma) and 1 uL Cy5 (GE Healthcare) to the Rd C sample.</li>
	<li>Incubate x 1 hour in the dark.</li>
	<li>Clean the Cy3-labeled sample with the DNA Clean and Concentrator-5 Kit (Zymo Research) per the manufacturer's instructions. Elute in 12 &mu;L.</li>
</ol>
5. Hybridization to Virochip Microarray
<ol>
	<li>Optional: use 1.5 &mu;L to check cDNA concentration and the amount of dye incorporated on a Nanodrop spectrophotometer (for normalization of samples)</li>
	<li>In a PCR strip tube add 1 &mu;L 25X fragmentation buffer, 5 &mu;L 5X blocking buffer, 9.5 &mu;L (or amount to normalize) of Cy3-labeled sample, and water to a total volume of 25 &mu;L (Agilent)</li>
	<li>Add 25 &mu;L of 2X GEx hybridization buffer (Agilent). Spin down briefly to remove bubbles.</li>
	<li>Load 40 &mu;L of the sample onto gasket slide.</li>
	<li>Place Agilent-printed Virochip array (University of California, San Francisco / Agilent) (side marked "Agilent" down) on top of gasket slide and fasten screw tightly.</li>
	<li>Hybridize overnight in 65&deg;C oven, speed setting at 10 rpm.</li>
	<li>To wash arrays, preheat buffer 2 (Agilent) to 37&deg;C. Take apart gasket slide / array sandwich underwater in buffer 1 (Agilent). Wash in buffer 1 x 1 min. Wash in preheated buffer 2 x 1 min. Slowly remove slide from buffer 2, being careful to avoid bubbles (use kimwipe to wick away extra moisture being careful to avoid directly touching active side of array)</li>
	<li>Place slide into slide holder and insert into array scanner.</li>
</ol>
6. Virochip Scanning and Analysis
<ol>
	<li>Scan Cy3-labeled array at 5 &mu;m or 2 &mu;m according to the instrument's standard scanning protocol (Figure 2B).</li>
	<li>Analyze Virochip microarrays by visual inspection, cluster analysis, or the automated Virochip analysis program, E-Predict11. By each of these three methods, influenza virus is readily detected in the nasal swab sample (from a child with influenza-like illness) (Figure 2C)</li>
</ol>
7. Representative Results:
When the protocol is done correctly, a smear should be seen on agarose gel electrophoresis after the Rd B step (Figure 2A). The Virochip microarray on the Agilent platform will be difficult to analyze visually (Figure 2B), but if a virus is present it should be readily identified by visual inspection, cluster analysis, and / or E-Predict (Figure 1C). Samples can be spiked with measured quantities of nucleic acid from a known virus (e.g. tobacco mosaic virus; MS2 bacteriophage) to serve as a positive control for the assay.
<img src="/files/ftp_upload/2536/2536fig1.jpg" alt="Figure 1" /> 
 Figure 1. Schematic of Virochip microarray processing and analysis. Extracted nucleic acid from clinical samples are randomly amplified, labeled with fluorescent dyes, and hybridized to the Virochip microarray. Microarrays are scanned at 2 &mu;m resolution and analyzed using a variety of computational tools, including E-Predict.
<img src="/files/ftp_upload/2536/2536fig2.jpg" alt="Figure 2" /> 
 Figure 2. Steps in the Virochip assay. After amplification by random PCR, a smear of 200 - 1000 bp can be visualized by gel electrophoresis (A). (B) Three Virochip microarrays out of the 8 arrays / glass slide are shown, with a small region of one microarray blown-up in the inset on the bottom right corner. (C) Automated microarray viral analysis using E-Predict revealing the presence of influenza A virus in the clinical sample. The high similarity score (s=0.55) corresponds to a p-value that is close to zero, thus making this an extremely significant prediction.

<ol>
	<li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+and+genotyping+of+viral+pathogens.">Microarray-based detection and genotyping of viral pathogens.</a> Proc Natl Acad Sci U S A. 99, 15687-15692 (2002).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+a+critical+respiratory+illness+caused+by+human+metapneumovirus+by+use+of+a+pan-virus+microarray.">Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.</a> J Clin Microbiol. 45, 2340-2343 (2007).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+detection+of+human+parainfluenzavirus+4+infection+associated+with+respiratory+failure+in+an+immunocompetent+adult.">Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.</a> Clin Infect Dis. 43, e71-e76 (2006).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+DNA+microarrays+for+detection+of+viruses+in+acute+respiratory+tract+infections+in+children.">Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.</a> J Pediatr. 153, 76-83 (2008).</li><li>Kistler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-viral+screening+of+respiratory+tract+infections+in+adults+with+and+without+asthma+reveals+unexpected+human+coronavirus+and+human+rhinovirus+diversity.">Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity.</a> J Infect Dis. 196, 817-825 (2007).</li><li>Rota, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+coronavirus+associated+with+severe+acute+respiratory+syndrome.">Characterization of a novel coronavirus associated with severe acute respiratory syndrome.</a> Science. 300, 1394-1399 (2003).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+discovery+and+sequence+recovery+using+DNA+microarrays.">Viral discovery and sequence recovery using DNA microarrays.</a> PLoS Biol. 1, e2-e2 (2003).</li><li>Urisman, A. <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+a+novel+Gammaretrovirus+in+prostate+tumors+of+patients+homozygous+for+R462Q+RNASEL+variant.">Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.</a> PLoS Pathog. 2, 25-25 (2006).</li><li>Kistler, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+divergent+avian+bornaviruses+from+cases+of+proventricular+dilatation+disease:+identification+of+a+candidate+etiologic+agent.">Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.</a> Virol J. 5, e25-e25 (2008).</li><li>Chiu, C. Y. <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+cardioviruses+related+to+Theiler's+murine+encephalomyelitis+virus+in+human+infections.">Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections.</a> Proc Natl Acad Sci U S A. 105, 14124-14129 (2008).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Predict:+a+computational+strategy+for+species+identification+based+on+observed+DNA+microarray+hybridization+patterns.">E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.</a> Genome Biol. 6, R78-R78 (2005).</li></ol>

No conflicts of interest declared.

The Virochip protocol as described here is complex and requires a meticulous and skilled research technician. Correct reagent concentrations and conditions for PCR, labeling, and hybridization are critical. The Virochip protocol can be easily modified to accommodate analysis of diseased tissues by use of a tissue extraction method such as TRIzol (Invitrogen) for nucleic acid extraction. Probes can be added or deleted as necessary to obtain the desired spectrum and breadth of coverage; for instance, probes for detection of nonviral targets such as bacteria and fungi can be designed and added "on-the-fly". Some applications of the Virochip assay currently under development include broad-spectrum viral diagnostics in the clinical laboratory, outbreak investigation, purity screening of drugs and vaccines, and novel viral pathogen discovery.

<table border="1">
	<tbody>
		<tr>
			<th>Name of reagent / material</th>
			<th>Name of reagent / material</th>
		</tr>
		<tr>
			<td>PrimerA</td>
			<td>5'-GTTTCCCAGTCACGATA-(N9)-3'</td>
		</tr>
		<tr>
			<td>Primer B</td>
			<td>5'-GTTTCCCAGTCACGATA-3'</td>
		</tr>
		<tr>
			<td rowspan="5">RT Master Mix (5 &mu;L)</td>
			<td>2 &mu;L 5X RT buffer</td>
		</tr>
		<tr>
			<td>1 &mu;L 12.5 mM dNTP (Invitrogen)</td>
		</tr>
		<tr>
			<td>1 &mu;L water</td>
		</tr>
		<tr>
			<td>0.5 &mu;L 0.1M DTT</td>
		</tr>
		<tr>
			<td>0.5 &mu;L SSIII RT (Invitrogen)</td>
		</tr>
		<tr>
			<td rowspan="3">Sequenase Mix (5 &mu;L)</td>
			<td>1 &mu;L 5X Sequenase buffer</td>
		</tr>
		<tr>
			<td>3.8 &mu;L water</td>
		</tr>
		<tr>
			<td>0.15 &mu;L Sequenase (USB Corporation)</td>
		</tr>
		<tr>
			<td rowspan="5">Rd B PCR Master Mix (45 &mu;L)</td>
			<td>5 &mu;L 10X PCR buffer</td>
		</tr>
		<tr>
			<td>1 &mu;L 12.5 mM dNTP (Invitrogen)</td>
		</tr>
		<tr>
			<td>1 &mu;L 100 pmol / &mu;L primer B</td>
		</tr>
		<tr>
			<td>1 &mu;L KlenTaq LA (Sigma)</td>
		</tr>
		<tr>
			<td>37 &mu;L water</td>
		</tr>
		<tr>
			<td rowspan="5">Rd C PCR Master Mix (45 &mu;L)</td>
			<td>5 uL 10X PCR buffer</td>
		</tr>
		<tr>
			<td>1 &mu;L 12.5 mM dNTP (Invitrogen)</td>
		</tr>
		<tr>
			<td>1 &mu;L 100 pmol / &mu;L primer B</td>
		</tr>
		<tr>
			<td>1 &mu;L KlenTaq LA (Sigma)</td>
		</tr>
		<tr>
			<td>37 &mu;L water</td>
		</tr>
		<tr>
			<td rowspan="4">Hybridization buffer (25 &mu;L)</td>
			<td>1 &mu;L 25X fragmentation buffer (Agilent)</td>
		</tr>
		<tr>
			<td>5 &mu;L 5X blocking buffer (Agilent)</td>
		</tr>
		<tr>
			<td>9.5 &mu;L (or amount to normalize) of Cy3-labeled sample</td>
		</tr>
		<tr>
			<td>Add water to total volume of 25 &mu;L</td>
		</tr>
		<tr>
			<td>Agilent Virochip microarray</td>
			<td>license pending; available from Chiu laboratory, UCSF</td>
		</tr>
	</tbody>
</table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

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Immunology and Infection

18.7K Views.  University of California, San Francisco.  The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Video: Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...