Name: a miniaturized glycan microarray assay for assessing avidity and specificity of influenza a virus hemagglutinins
Uploaded: 2016-05-29T14:00:00
Description: Watch this Scientific Journal Video about A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins at JoVE.com

This work was supported in part by the Scripps Microarray Core Facility, and a contract from the Centers for Disease Control (J.C.P.). RPdV is a recipient of a Rubicon and VENI grant from the Netherlands Organization for Scientific Research (NWO). The Consortium for Functional Glycomics (http://www.functionalglycomics.org/) funded by NIGMS grant GM62116 (J.C.P.) provided several glycans used in this study. This is publication 29113 from The Scripps Research Institute.

NOTE: All steps are carried out at room temperature unless otherwise indicated.&#160;
1. Array Construction
<ol>
	<li>Glycan Preparation and Plate Setup
	<ol>
		<li>Prepare a stock of printing buffer. First make 500 ml of a 150 mM stock solution of dibasic sodium phosphate. Also make 50 ml of a 150 mM stock solution of monobasic sodium phosphate solution. In a flask, using a magnetic stir bar and pH meter, slowly titrate the dibasic sodium phosphate solution with the monobasic solution until a pH of 8.5 is reach...

<ol>
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Assessing IAV receptor specificity is an important step in analyzing pandemic potential of avian viruses. Sialic acid recognition by the virus is linked to several biological properties such as binding to and release from the cell. Knowledge of which amino-acid mutations are necessary for avian viruses to achieve &#945;2-6 binding and cross the species barrier enables pandemic preparedness. Several assays are used to determine receptor specificity; however, all have their drawbacks, including only measuring avidity and n...

Wild waterfowl are the natural reservoir for IAV, but IAV is able to cross the species barrier to poultry and mammals, including, humans. Avian IAVs recognize &#945;2-3 linked sialic acids (avian-type receptors), whereas human viruses bind &#945;2-6 linked sialic acids (human-type receptors). To be able to efficiently replicate and transmit between humans an avian IAV needs to bind to human-type receptors 1.
IAVs are divided based on serology that characterizes the antigenicity of their hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins. HA binds to sialic acids, whereas NA is the receptor-destroying enzyme at the other end of the viral lifecycle and cleaves sialic acid 2. All human infecting viruses, including H1N1, H2N2 and H3N2, have an avian origin 3. Over the last two of decades several avian to human crossovers have occurred, with H5N1, H7N7, and H7N9 being the most well-known; however, other subtypes have infected humans more sporadically (H6N1, H7N1, H7N2, H9N2, H10N7, H10N8) 4. Fortunately, it seems that none of these viruses have been able to fully adapt to human-type &#945;2-6-linked sialic acid receptors 5-8. Adaptation of avian or other zoonotic viruses to accommodate replication and transmission in human hosts could have a devastating effect on human health. Therefore, prior knowledge of how these viruses are evolving to bind human-type receptors will help world-wide surveillance of emerging influenza viruses.
Determination of receptor preference was first elucidated using erythrocytes of different species and remains a favored assay among flu researchers 9-12. The demonstration that avian viruses recognize &#945;2-3 sialic acid and human viruses &#945;2-6 linked sialic acid was originally based on an assay using hemagglutination of erythrocytes enzymatically engineered to contain each of the linkages 13,14. Although the readout is hemagglutination, a standard assay for virologists, the underlying glycan structures are not defined, only the terminal linkage. Additionally, the limited availability of the sialyltransferases, used to re-sialylate the cells, have limited the use of this assay 15-18. Subsequently, other methods of determining receptor-binding preferences were introduced using sialylated glycan structures linked to poly-acrylamide (PAA) or poly-glutamate (PGA) structures in plate-based assays 19,20. Several variations are possible in coating either the glycans or viruses to microtiter plates, each of which results in a robust, reliable and very sensitive ELISA-type assay 21-23. Alternatively, biotin-linked glycans can replace PAA/PGA and can be conjugated to streptavidin-coated plates 2,24. Although some specific sera may be required, ELISAs are standard and several glycans linked to PAA are readily commercially and non-commercially available (Consortium for Functional Glycomics (http://www.functionalglycomics.org)).
Glycan microarray technologies have emerged as an invaluable tool to determine receptor specificity, as multiple different glycans are spotted, and binding to a wide array of different structures can be assessed within a single assay 25-29. The binding of IAV to these structures provides a better understanding of the glycan structures that IAV preferentially recognizes 30-33. Glycan microarrays require small amounts of sample volume to perform a binding assay and only uses minute amounts of glycan per spot (2 nl). However, these arrays are typically used only to evaluate specificity of glycans receptors. Analysis of multiple viruses, or hemagglutinin proteins, at multiple concentration ranges can be prohibitive due to the number of slides required. Furthermore, to date, no relative avidity assay has been developed using glycan array techniques.
To combine the low sample requirement afforded by glycan microarray techniques and the sensitivity of PAA-linked glycans in ELISA-based assays, we sought to develop a multi-well glycan array that would allow for high-throughput analysis with similar or better resolution as compared to the traditional ELISA-based assay. Simultaneously, we wanted to minimize the amount of biologicals and reporter chemicals consumed. The ultimate result is a miniaturized avidity assay, specifically developed for monitoring IAV specificity and is equally applicable to assess other glycan binding proteins. Using a glass slide separated into 48 micro-wells by a Teflon mask, 6 different glycans are spotted in 6 replicates per well. The microarray platform affords the same trends in receptor binding seen in the macro ELISA format with several advantages. These include (I) printing of compound in 6 replicates, using minimal sample, versus the coating of several rows in a plate, using 100 &#181;l per well; (II) multiple different compounds analyzed simultaneously in a single well, including controls; (III) a massive decrease in incubation volume and; (IV) a larger dynamic range using a fluorescent readout. A single slide can be calculated to be equivalent to 18x 96-well plates.
With the following protocol, any lab capable of fabricating and analyzing spotted microarrays should be able to manufacture this miniaturized ELISA format.

<table>
	<tbody>
		<tr>
			<td>NEXTERION&#174; Slide H MPX-48&#160;</td>
			<td>Schott</td>
			<td>1091525</td>
			<td>Microwell slides</td>
		</tr>
		<tr>
			<td>ProScanArray Plus&#160;</td>
			<td>PerkinElmer</td>
			<td>discontinued</td>
			<td>confocal microarray scanner</td>
		</tr>
		<tr>
			<td>Innoscan 1100AL Scanner/Mapix Software</td>
			<td>Innopsys</td>
			<td>-</td>
			<td>confocal microarray scanner</td>
		</tr>
		<tr>
			<td>MicroGrid II&#160;</td>
			<td>Digilab</td>
			<td>-</td>
			<td>microaray printer</td>
		</tr>
		<tr>
			<td>SNA</td>
			<td>Vector Labs</td>
			<td>B-1305&#160;</td>
			<td>Plant Lectin</td>
		</tr>
		<tr>
			<td>ECA</td>
			<td>Vector Labs</td>
			<td>B-1145&#160;</td>
			<td>Plant Lectin</td>
		</tr>
		<tr>
			<td>Anti-Strep Antibody</td>
			<td>IBA</td>
			<td>2-1509-001&#160;</td>
			<td>Anitbody for HA binding</td>
		</tr>
		<tr>
			<td>Anti-Mouse Alexa-647</td>
			<td>Life</td>
			<td>A-21235</td>
			<td>Anitbody for HA binding</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P2287&#160;</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>di-basic Sodium Phosphate</td>
			<td>Sigma</td>
			<td>255793</td>
			<td>printing buffer component</td>
		</tr>
		<tr>
			<td>mono-basic Sodium phosphate</td>
			<td>Sigma</td>
			<td>229903&#160;</td>
			<td>printing buffer component</td>
		</tr>
		<tr>
			<td>poly-l-lysine solution</td>
			<td>Sigma</td>
			<td>P8920&#160;</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma</td>
			<td>221465</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sigma</td>
			<td>493546</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>incubation/washing buffer</td>
		</tr>
		<tr>
			<td>SMP4B pins</td>
			<td>Telechem</td>
			<td>SMP4B</td>
			<td>printing pin</td>
		</tr>
		<tr>
			<td>Compressed Nitrogen (Grade5)</td>
			<td>Praxair</td>
			<td>UN1066</td>
			<td>general dusting/drying tool</td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma</td>
			<td>B6768-500G</td>
			<td>Slide blocking reagent</td>
		</tr>
		<tr>
			<td>ethanolamine</td>
			<td>Sigma</td>
			<td>E9508-500ML</td>
			<td>Slide blocking reagent</td>
		</tr>
		<tr>
			<td>Atto 488</td>
			<td>AttoTec</td>
			<td>AD 488-91</td>
			<td>Gridmarker on array</td>
		</tr>
		<tr>
			<td>PAA-LNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA368</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>PAA-3SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA362</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>PAA-6SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA343</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>LNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te98</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>3SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te175</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>6SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te176</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>384-well microtiter plate</td>
			<td>Matrix TechCorp</td>
			<td>4361</td>
			<td>Printing plate</td>
		</tr>
		<tr>
			<td>VWR lab marker</td>
			<td>VWR</td>
			<td>52877-150</td>
			<td>Slide Numbering</td>
		</tr>
		<tr>
			<td>Wheaton slide staining dish</td>
			<td>Sigma</td>
			<td>Z103969-1EA</td>
			<td>Blocking and Drying</td>
		</tr>
	</tbody>
</table>

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.

Printing, Scanning &#38; Data analyses
To ensure proper printing, it is vital to have the correct alignment of the spotted grid within the teflon mask, which delineates each array on the slide. During printing, due to the nature of the teflon coating, spots cannot be seen by the naked eye on the MPX slides. Attention is paid then to the appearance of the pre-spots on the poly-L-Lysine coated slides. Directly after printing, each...

Watch this Scientific Journal Video about A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins at JoVE.com

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

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 ...

The overall goal of this miniaturized glycan array is to analyze the specificity and avidity of influenza A virus hemagglutinins. This assay can help to answer key questions regarding influenza A virus receptor binding and avidity. The main advantage to this technique is that within a single assay we can address specificity as well as relative binding avidity, normally not achieved within typical glycan array and plate-based assays.This protocol begins with preparations of the glycan samples. The base solution for the glycans is a stock printing buffer which, when made, should be slowly titrated to a pH of 8.5 and then given an addition of surfactant. Using the prepared printing buffer, dilute the PAA-conjugated glycans to 100 micrograms per milliliter.Then, dilute the monovalent glycans to 100 micromolar also in the printing buffer. Finally, make working stocks of the amine-functionalized dyes at one micromolar. Now load ten microliters of each glycan sample into a well of a 384-well microtiter plate.This is enough solution to print five slides. Also transfer ten microliters of the dye marker to one well of the printing plate. Prepare to print the arrays by first positioning the arrayer pins and printing head according to the layout.In this case, one pin can print all the samples. The arrays of glycans are printed in blocks of six surrounded by an empty spot on all sides. Put on gloves to handle the slides.Unpack them and blow any debris off their surfaces with ultra high purity nitrogen gas. Next, position the slides coated-side up on the arrayer stage. Then, program the arrayer.In this case, program 27 spots per visit to the source plate. Three pre spots placed on the non-array area of a poly-L-lysine coated slide are used to remove excess liquid at the tip of the pin. The other 24 spots per pin visit are used to place six spots in four replicate arrays.Details are provided in the text protocol. Now, load the multi-well plates into the printer and start printing the arrays. While printing, make sure that the humidity stays between 55 and 65%Pay attention to the appearance of the pre spots on the poly-L-lysine coated slides.After printing, visually inspect each slide. Check for correct alignment of the spots within the teflon borders, which is vital, and check for the correct number of spotted features. To help homogenize the attachment of the dots, put the printed slides in a 100%humidity chamber with their printed side up immediately after they are made.Use wet paper towels to line a container and a makeshift rack. Wrap the chamber in plastic to trap in the moisture. After half an hour, remove the slides.Then, number the slides with a solvent-resistant marker and store them in a desiccation box overnight. The next day, prepare a fresh stock of blocking buffer with vigorous stirring. Adjust the pH of 9.2 using concentrated sodium hydroxide.Then, incubate the slides in the buffer for an hour. To remove the block, rinse the slides in double-distilled water. Then, transfer the slides to glass staining holders and load the holders into a swinging plate holder to spin the slides dry at 10 G's for five minutes.Once dry, if needed store the slides at minus 20 degrees celsius in sealed plastic bags. In preparation, make another humidification chamber as before. To detect the immobilized glycans, prepare SNA and ECA biotinylated lectins at 10 micrograms per milliliter in probing buffer, and add two micrograms per milliliter of streptavidin 555 to each dilution.To stain with the hemagglutinins, make a 20 microgram per milliliter solution of pre complex antibody in blocking buffer at a four-to-two-to-one molar ratio as described in the text protocol. Transfer 20 microliters of the prepared solutions into wells of a 384-well plate. Next, serially dilute the lectins and hemagglutinins one-to-one in their appropriate buffers.Make eight dilutions, filling each well with 10 microliters of sample and 10 microliters of buffer. Now, incubate the plate on ice for 30 minutes. Now add eight microliters of each dilution to one of the 48 microwells on a printed slide.Then, incubate the prepared slide in the humidification chamber for 90 minutes. After 90 minutes, wash the slide while holding it from the edge;dip it in PBS-T four times, then dip it in PBS four times and finally, dip it in deionized water four times. Now, spin-dry the slide as before.After spin-drying, immediately scan the slide. Be sure to carefully load the slide into the scanner in the correct orientation. Using a low laser power of five milliwatts iteratively scan the slides, gradually increasing the gain from 25 to 75%While optimizing the scan settings keep in mind that the fluorophores will bleach out with repeated scans.Analyze the saved image for binding signals using the associated software. An array list can be loaded which has the spotting pattern. Overlay it on the scan to help navigate the placement of the grid markers.Then, use the software to analyze the spot intensities and save the results as a tab delimited text file to export into a spreadsheet or other statistical package. Analyze the data by calculating the mean signal intensity minus the background. Take an average of four of the six replicate spots for each sample, leaving out the highest signal and the lowest signal.Then, produce a line graph of the average mean signal minus the background against the eight protein concentrations. Smooth the resulting line using nonlinear regression. The PAA array was used to assess receptor binding specificity of influenza A virus hemagglutinins.The array included non-sialylated controls in the same microwells. Plant lectins with known specificities for the printed compounds were used as positive controls. The ECA lectin bound to terminal galactose beta 1-4 linked to anacetylglucosamine and did not bind to the same compound if the terminal galactose was capped with sialic acid.To test alpha 2-6 linked sialic acid, the SNA lectin was used. As expected, SNA only bound to alpha 2-6 linked sialic acids, but with notable differences in affinity for PAA linked versus non-PAA linked dye lacNAc repeats. Next, an H5 hemagglutinin derived from a Vietnam/1203/04 virus which only recognizes alpha 2-3 linked sialic acids was tested.The binding profile of the H5 hemagglutinin showed the expected specificity with a higher avidity for the PAA linked structures. Finally, the H1 hemagglutinin from a human seasonal H1N1 strain was tested. As expected, this hemagglutinin only bound to alpha 2-6 linked sialic acid-containing structures.Once mastered, this technique can reveal the binding specificities and relative avidities of influenza viruses within a single assay. This technique will greatly enhance the efficiency of influenza binding assays as well as reducing the consumption of precious biologicals.

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Research

JoVE Journal

Immunology and Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

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.

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.

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 ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.