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 reached. Add Tween-20 to 0.005%.</li>
		<li>Prepare the glycan samples. Dilute PAA conjugated glycans, (diLacNAc (Gal&#946;1-4GlcNAc&#946;1-3Gal&#946;1-4GlcNAc&#946;-PAA), 3SLNLN (Neu5Ac&#945;2-3Gal&#946;1-4GlcNAc&#946;1-3Gal&#946;1-4GlcNAc&#946;-PAA) and 6SLNLN (Neu5Ac&#945;2-6Gal&#946;1-4GlcNAc&#946;1-3Gal&#946;1-4GlcNAc&#946;-PAA (Figure 1A)) are to be diluted to 100&#181;g/ml in printing buffer from step1.1.1. Monovalent glycans, diLacNAc (Gal&#946;1-4GlcNAc&#946;1-3Gal&#946;1-4GlcNAc&#946;-(CH2)3NH2, 3SLNLN (Neu5Ac&#945;2-3Gal&#946;1-4GlcNAc&#946;1-3Gal&#946;1-4GlcNAc&#946;--(CH2)3NH2) and 6SLNLN (Neu5Ac&#945;2-6Gal&#946;1-4GlcNAc&#946;1-3Gal&#946;1-4GlcNAc&#946;--(CH2)3NH2)) to 100&#181;M in printing buffer from step 1.1.1. Finally, prepare the amine-functionalized dyes, to use as grid markers/landing lights. Dye marker spots are printed at 1 &#181;M. 
		NOTE: Atto488-NHS dye is used in our prints. However, any amine-functionalized dye, readable by the slide scanner is appropriate for this purpose.</li>
		<li>Transfer 10 &#181;l of each glycan sample to 384-well microtiter plates, used for the printing robot. Store unused glycans in printing buffer solution at -20 &#176;C in sealed tubes. Tansfer 10 &#181;l of the dye marker to the printing plate. &#160;</li>
	</ol>
	</li>
	<li>Printing 
	NOTE: All steps that concern touching or moving the slides must be done with gloves.
	<ol>
		<li>Place arrayer pins in the printing head according to the correct configuration of the layout. For this layout use one pin to print all samples and arrays. Print the glycans in blocks of six, skipping one feature (a feature is defined as single spot of a certain compound), on each side, such that spots of the same sample are not printed exactly beside each other. 
		NOTE: The end user must decide the configuration. &#160;</li>
		<li>Remove slides from bags, open boxes, and remove any dust particles or small pieces of plastic that might be on their surfaces by blowing ultra-high purity Nitrogen gas on each slide. Place the desired number of slides, coated side up, on the arrayer stage.</li>
		<li>Program the arrayer, use the parameters of 27 spots per visit to the source plate, using 3 &#34;pre-spots&#34; onto poly-L-lysine coated slides, to remove excess liquid at the tip of the printing pins
		<ol>
			<li>Turn on MPU (Main Power Unit) and Climate Control Unit. Open Tiling Analysis software on computer.</li>
			<li>Select layout to the print. Select Options &#8594; Pin Tool to be used for printing the array 1 x 1 print tool - 1 single pin. Select the Target tab &#8594; Tool Array Definition &#8594; Print Pattern and add the pitch (spot-to-spot distance), number of rows and columns to accommodate the samples to be printed, and the printing pattern of the samples (for this array an 8 x 8 printing pattern is used at 340 &#181;m pitch for 7 total samples).</li>
			<li>Select Target &#8594; Slide Layout and program the block-to-block distance to allow printing of each replicate array into the 48-well (4 x 12) slide template. Select Source and add the number of source pickups (1 for each sample when using 1 pin) (for this print 7 source pickups will be used to accommodate 6 sample wells and 1 dye marker well). 
			NOTE: The source dialogue should turn GREEN to show that the number of source visits matches the samples to be printed in the print pattern.</li>
			<li>Select Target&#160;&#8594; Adapter Plate and Slide Layout and adjust the slide number (5 slides are printed at a time for this print with 1 pre-spotting slide).</li>
			<li>Release the tray by clicking the T1 button to activate the tray and add the desired number of slides to the tray, paying close attention to the locations of the pre-spotting slide (BLUE) and &#34;real&#34; slides.</li>
			<li>Place plain glass slides on all remaining vacuum ports to block the vacuum and click OK to activate the vacuum. Check that all slides are held in place and click OK to return the tray to the home position.</li>
			<li>Load the printing plate into the plate holder and ensure the rack is set correctly in place. Click GO and allow the printing process to proceed.</li>
		</ol>
		</li>
		<li>Print the remaining 24 spots in replicates of 6 spots per array (4 arrays/source visit).</li>
		<li>Load the 384-well plates into the printer and begin printing. Maintain relative humidity, while printing, throughout the print between 55 and 65%. The printer stops after printing 5 slides. &#160; 
		NOTE: While the microarray in this assay is printed in a specific 8 x 8 pattern, different microarray printing robots will require equipment specific programming to achieve the desired layout. It is up to the end user to determine the most appropriate pattern given their equipments&#8217; specifications.</li>
	</ol>
	</li>
	<li>Humidification/Immobilizing 
	NOTE: Once the slides have finished printing, they undergo a temporary immobilization step, also called humidifying. Humidifying post-print helps to homogenize the sample attachment by re-wetting the spots and ensuring complete blocking.
	<ol>
		<li>Put slides in a 100% humidity chamber immediately after printing for a period of 30 min. Construct the chamber by placing very wet paper towels flat in the bottom of a large glass dish, placing the slides on some sort of rack, such as a test tube rack, print side up, and sealing with plastic wrap to trap in the moisture.</li>
		<li>Number slides with an alcohol/solvent resistant marking pen and store in a desiccation box. Desiccate overnight.</li>
	</ol>
	</li>
	<li>Numbering, Blocking, and Storage
	<ol>
		<li>Prepare the blocking buffer&#160;&#8211; 50 mM ethanolamine in 50 mM borate buffer. First, dissolve boric acid, in ddH2O, to a final concentration of 50 mM in a flask containing a magnetic stir bar. Stir the solution vigorously on a stirring plate until all solid has dissolved. While constantly stirring, add the appropriate amount of ethanolamine from a 16.54 M stock solution to reach the desired 50 mM concentration. Add concentrated sodium hydroxide to adjust to a final pH of 9.2.</li>
		<li>Immerse the printed slides in the blocking buffer solution for 1 hr.</li>
		<li>Following 1 hr, rinse the slides in ddH2O to remove any excess traces of blocking buffer. Dispose blocking buffer in an appropriate waste container.</li>
		<li>Transfer the slides to glass staining holders and spin dry. Dry arrays by placing them in a centrifuge equipped with swinging plate holders, at a speed of 10 x g for 5 min.</li>
		<li>Once the slides are dry, they can be incubated immediately or stored. Storage conditions are -20 &#176;C in a sealed plastic bag. &#160;</li>
	</ol>
	</li>
</ol>
2. Analyzing Glycan Binding Proteins
<ol>
	<li>Prepare a humidified chamber in which the arrays to be hybridized will fit. A simple chamber consists of a Pyrex dish, layered on the bottom with dampened paper towels and a rack, upon which the slides will rest.</li>
	<li>Use biotinylated lectins SNA (Sambuca nigra agglutinin) and ECA (Erythrina cristagalli agglutinin) at 10 &#956;g/ml + 2 &#181;g/ml of Streptavidin-555 dye, in probing buffer (PBS + 0.01% Tween-20). For HA (A/Vietnam/1203/04 H5N1 and A/KY/07 H1N1, in this example), use a starting concentration of 20 &#956;g/ml pre-complexed, as previously described 34. Briefly, make a HA:Mouse-&#945;-Strep:Goat-&#945;-Mouse-Alexa647 mixture in blocking buffer (PBS + 3%BSA + 0.01% Tween-20), at a 4:2:1 molar ratio. 
	NOTE: The array is probed with lectins to ascertain that glycans are immobilized and detectable (Figure 1B).</li>
	<li>Transfer the glycan binding protein-antibody mix solution of 20 &#181;l to a 384-well plate and incubate on ice for 30 min. Serially dilute lectin and HA samples 1:1, in their appropriate buffer, 8 times, by mixing 10 &#181;l of sample with 10 &#181;l of buffer.</li>
	<li>Pipette 8 &#181;l of the sample onto the micro-well surface and incubate in the sealed humidification (100% RH) chamber for 90 min.</li>
	<li>Wash the slides one at a time by holding the edges and dipping them four times in a mixture of PBS and 0.05% Tween, then four times in PBS, and finally four times in deionized H2O. Using the same drying protocol described in the blocking step, spin the arrays dry in a centrifuge for 5 min at 10 x g.</li>
</ol>
3. Scanning and Data Analyses 
NOTE: The glycan microarrays can be scanned at different settings, depending on the microarray scanner&#8217;s manufacturer. By varying the laser power and resolution, the instrument can produce an image with as many signals as possible within its dynamic range (Figure 1C).
<ol>
	<li>Use a fluorescent slide scanner and scan the arrays immediately after incubation with the glycan binding proteins. Take care to ensure the slide is correctly inserted into the scanning instrument.
	<ol>
		<li>Open scanning software. Click start to open the program. Connect to scanner: Scanner &#8594; Connect or green button in the Scanner navigation panel menu.</li>
		<li>Open autoloader door of the scanner. Place slides in slots in autoloader in specific order. Close autoloader door. Click &#34;read loader&#34; in the Tools navigation panel to auto detect slides in the scanner.</li>
		<li>Set scan parameter: Scanner &#8594; Scan parameters appropriate to the experiment (e.g. 635 laser power high, detection gain 50%). Scan slides: Scanner&#160;&#8594; scan. Select path by creating a folder to save the scan images and name individual scans. Do this prior to the actual scanning. Repeat for each individual slide. Click OK to start scanning.</li>
	</ol>
	</li>
	<li>Use the scanner software to set up the instrument and scan the slides. Set scanner to iteratively scan at low laser power (5 mW) with increasing gain settings of 25%, 50% and 75%. &#160; 
	NOTE: The settings can be tested and optimized, but keep in mind that each scan can possibly lower the fluorescence output of the dyes due to photobleaching.
	<ol>
		<li>Open data acquisition and analysis software such as Mapix software. Click start to open the program. Open scan image: File&#160;&#8594; open image. Open GAL file: Analyze&#160;&#8594; open grid and select the appropriate GAL file.</li>
		<li>Enter blocks mode: Image &#8594; blocks mode to move the grid. Use the grid markers to set the grid to the appropriate location on the slide. Adjust individual blocks as necessary to match the printed array.</li>
		<li>Enter spots mode: Image &#8594; spots mode to adjust location and size of individual spots within the block. Once all blocks and spots are adjusted and in place, measure signal intensities by Analyze&#160;&#8594; photometric calculations. Save the output file.</li>
	</ol>
	</li>
	<li>Once the slides have finished scanning, images are analyzed for binding signals with the image-processing program installed (Figure 1D-G). For image analysis, load a GAL (Array List) file over the scanned TIFF image. 
	NOTE: The GAL file contains both the spot layout pattern and identities of each spot location. GAL files for arrays are created by the printer software using a tab-delimited text file that contains the identities of the samples and their location in the 384-well source plate.</li>
	<li>Use the grid marker/landing lights printed on the microarrays to properly place the GAL grid. Individual spots may need to be moved individually, but a well-printed array will generally allow blocks within the grid to be placed with ease. Following grid placement, collect spot intensities by the software and saved as a tab-delimited text file (.txt). &#160;
	<ol>
		<li>Using spreadsheet program, open the output file from the scanner. Open the appropriate macro file from the folder location. Click View &#8594; Macro &#8594; RunMacro. &#160; 
		NOTE: The pre-written will copy the raw output data, remove unnecessary rows and columns, sort the data by sample, calculate the average mean signal minus background values and plot the resultant values in to an spread-sheet worksheet containing the graphs for each of the six samples tested on the array.</li>
	</ol>
	</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+microarrays+for+decoding+the+glycome.">Glycan microarrays for decoding the glycome.</a> Annu Rev Biochem. 80, 797-823 (2011).</li><li>Song, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sialylated+glycan+microarray+reveals+novel+interactions+of+modified+sialic+acids+with+proteins+and+viruses.">A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses.</a> J Biol Chem. 286 (36), 31610-31622 (2011).</li><li>Gulati, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+H3N2+Influenza+Viruses+Isolated+from+1968+To+2012+Show+Varying+Preference+for+Receptor+Substructures+with+No+Apparent+Consequences+for+Disease+or+Spread.">Human H3N2 Influenza Viruses Isolated from 1968 To 2012 Show Varying Preference for Receptor Substructures with No Apparent Consequences for Disease or Spread.</a> PLoS One. 8 (6), (2013).</li><li>Stevens, J., Blixt, O., Paulson, J. C., Wilson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+microarray+technologies:+tools+to+survey+host+specificity+of+influenza+viruses.">Glycan microarray technologies: tools to survey host specificity of influenza viruses.</a> Nat Rev Microbiol. 4 (11), 857-864 (2006).</li><li>de Vries, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+hemagglutinin+protein+of+the+new+pandemic+H1N1+influenza+virus:+maintaining+optimal+receptor+binding+by+compensatory+substitutions.">Evolution of the hemagglutinin protein of the new pandemic H1N1 influenza virus: maintaining optimal receptor binding by compensatory substitutions.</a> J Virol. 87 (24), 13868-13877 (2013).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+receptor+binding+preferences+of+recombinant+human+A(H3N2)+virus+hemagglutinins.">Structure and receptor binding preferences of recombinant human A(H3N2) virus hemagglutinins.</a> Virology. 477, 18-31 (2015).</li><li>de Vries, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influenza+A+virus+hemagglutinin+glycosylation+state+affects+receptor-binding+specificity.">The influenza A virus hemagglutinin glycosylation state affects receptor-binding specificity.</a> Virology. 403 (1), 17-25 (2010).</li></ol>

The authors have nothing to disclose

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 not specificity and vice versa.
Here we describe a novel miniaturized tool based on the widely used and accepted ELISA, which uses PAA-linked terminal fragments of more complex glycans. Using 4 well-defined lectins, 2 of plant and 2 of influenza A virus origin, we show that we maintain specificity and relative avidity.
When we calculate the amount of reduction of chemicals and biological used by miniaturizing the ELISA onto a glycan array chip, we reach strikingly large differences. First, the biologicals; we use 10x less assay volume and use 6 test compounds in 6 replicates; this can be calculated to be the equivalent of 36x rows in 96-well plates. The result is that we use 360x less biologicals. Critical steps include proper printing and the risk of high backgrounds using unpurified glycan binding agents or poor detecting antibodies.
The compounds used in this assay, although still available, are not easily made by chemo-enzymatic synthesis. Using microarray-printing technology, each spot is only 2 nl, compared to 100&#160;&#181;l per well of a microtiter plate. Printing a single slide containing 48 wells and 6 compounds, in 6 replicates, we consume approximately 1,152 nl. The same coating procedure in 96-well plates results in coating a total of 1,842 wells with 100 &#181;l of compound in the same concentration. Therefore, printing the compound compared to the plate assay, we attain a striking reduction of 1,500x. Therefore, in total, biologicals are reduced 360-fold and glycan consumption by 1,500-fold. Using robotic liquid handlers, we envision that most steps could be automated, allowing the entire assay be used as a high-throughput screen.
Although printing microarrays and subsequent image analyses do require special machinery and tools, these instruments are not rare and are present at many universities and institutes. Printing is direct and does not require chemical alterations. Therefore, we believe that, with this minimal amount of compounds and simple printing technique, this assay could be more generally applicable. As it reduces the incubation volume, the costs of antibodies and precious biologicals, this technique will make it feasible for labs with limited resources to assess specificity of IAV or other glycan binding proteins with high fidelity while maintaining lower costs.

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 slide should be checked by eye for the presence of each spotted compound that is visible due to the buffer&#39;s salts dried in the spot. Arrays are inspected for correct alignment of spots within the Teflon borders and correct number of spotted features.
Slides are scanned in an iterative process of lower to higher scan power to avoid photo bleaching and allowing the comparison of higher avidity to lower avidity binding proteins, which have higher and lower output signals, respectively. Following scanning, the fluorescent intensity of each spot in the output images are measured using an imaging program. Each array is overlayed with a grid file that matches the number of features printed on the array surface (Figure 1D-G). Using printed fluorescent dyes, the borders of the printed glycans are defined and a mask that is integrated with the identity of the printed sample lassoes each spot. The imaging mask lasso will record all aspects of the imaged spot (size, signal intensity, coordinates in the grid, etc.) and output this information to a tab-delimited text file. Using tabulation software (spreadsheet), the samples are sorted by identity and the mean signal intensity minus background is averaged over 4 of the 6 spots for each sample, leaving out the highest and lowest signal as outliers. A line graph of average mean signal minus background is plotted against the eight protein concentrations applied to the array and contains one line for each sample. The final output graph is smoothed using a non-linear regression calculation (Figure 1I).
Glycan binding proteins
The PAA-Array is used to assess receptor-binding specificity of influenza A virus hemagglutinins. An additional feature of our array, not present in the analogous plate assay, is the inclusion of non-sialylated controls in the same micro-well. To assess that printed compounds are present after printing, commonly used plant lectins with known specificity were used. ECA binds to terminal galactose linked &#946;1-4 to N-acetyl-glucosamine (Gal&#946;1-4GlcNAc or LacNAc) and will not bind if the terminal galactose is capped with sialic acid. ECA only detects the non-sialylated glycans on our miniaturized glycan array (Figure 2A). The lack of binding to any of the sialylated glycan samples is also indicative that these are fully capped with terminal sialic acid. For &#945;2-6 linked sialic acid, SNA was used as a lectin control (Figure 2B). As expected, SNA only binds to &#945;2-6 linked sialic acids, but with notable differences in affinity for PAA-linked non-PAA-linked di-LacNAc repeats. This difference reflects the sensitivity of PAA-linked glycans and allows the discrimination of proteins that may bind with low avidity. For &#945;2-3 linked sialic acid, an H5 hemagglutinin derived from A/Vietnam/1203/04 virus that is known to recognize &#945;2-3 linked sialic acids only was used 6. The binding profile of the H5 HA indeed shows specificity for &#945;2-3 linked sialic acids, with a higher avidity for the PAA-linked structures (Figure 2C). Finally, the H1 hemagglutinin from a human seasonal H1N1 strain was used that, as expected, only bound to &#945;2-6 linked sialic acid containing structures (Figure 2D).
<img alt="Figure 1" src="/files/ftp_upload/53847/53847fig1.jpg" /> 
Figure 1: Printing, scanning and image analyzes. (A) PAA-conjugated 6SLNLN is shown as a representative glycan structure that is printed on the glass slides. (B) Incubating a glycan binding protein on the multi-well glycan array is shown with an 8 &#181;l volume that creates a droplet on the array surface. (C) Following incubation of the glycan binding proteins on the array and scanning in a confocal fluorescent slide scanner, a representative image is obtained. (D) The image is overlayed with a grid and, using grid markers for proper alignment, the single spots can be analyzed. (E) A close up of a complete set of 8 arrays, in which a single glycan binding protein was analyzed using eight 1:1 dilutions. (F) A single array, in a single well is represented; the array is demarked by the grid markers in the top right (3 spots) and bottom left (2 spots), this glycan binding protein binds a single compound on the array as a replicate of six is clearly visible. (G) The grid is shown with lassos encircling specific individual spots. (H) Imaging software calculates signal values from the image file and outputs a tab-delimited data file. (I) The data can be tabulated in spreadsheet or stastical software to create a representative output graph. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53847/53847fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53847/53847fig2.jpg" /> 
Figure 2: Output of plant lectins (ECA, SNA) and IAV hemagglutinins with different specificities (H5 from A/Vietnam/1203/04, H1 from A/Kentucky/07). (A) ECA binds terminal LacNAc or Galb1-4GlcNAc specifically and detects the presence of the non-sialylated compounds, used as controls for IAV hemagglutinins. (B) SNA recognizes only glycans bearing a terminal &#945;2-6 sialic acid. (C) The recombinant hemagglutinin of the H5N1 (A/Vietnam/1203/04) strain binds to &#945;2-3 sialic acids and provides an avian type receptor binding profile. (D) The recombinant hemagglutinin of a human seasonal H1N1 (A/Kentucky/07) binds to human-type receptors only. Fluorescent signal intensity was measured for each spot, and mean intensity minus mean background was calculated using spreadsheet program. For each glycan, the mean signal intensity is calculated from 6 replicates spots. The highest and lowest signals of the 6 replicates are removed and the remaining 4 replicates are used to calculate the mean signal, standard deviation (SD), and standard error measurement (SEM). The graphs represent the averaged mean signal minus background for each sample and error bars are the SEM value. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53847/53847fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.9K Views.  The Scripps Research Institute. 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.

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

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.