Current support is provided by an SBIR (Small Business Innovation Research) administrative supplement grant from NIDCRR44 DE024456. NIDCR HIV grant that evolved out of NIDCR grant U01 DE017855 for the development of a confirmatory point-of- care diagnostic for HIV. We gratefully acknowledge Silke Weisenburger(PEPperPRINT Heidelberg, Germany) for her technical assistance and kind support. We also thank NYU Langone Medical Center for the LI-COR Odyssey Imaging System.

These data are a part of an ongoing research study conducted at New York University College of Dentistry and were approved by the Institutional Review Board of the New York University School of Medicine, IRB # H10-01894. Clinical samples used in this study were de-identified samples used previously for diagnosis and with permission from the Wadsworth Center of New York State Department of Health, Albany, NY.
1. Installing the ZIKV High-density Peptide Microarray Slide in a specific Incubation Tray
<ol>
	<li>Handle the glass slide by the edges using powder-free gloves. The glass slide dimensions are 75.4 mm by 25.0 mm and 1 mm of thick. Place the slide in the incubation tray with the microarray surface facing up.</li>
	<li>Place the glossy side of the seal facing downwards onto the microarray printed surface. To ensure a good position, overlap the screw holes of the seal and the base plate.</li>
	<li>Place the upper part of the tray onto the seal to create a microarray chamber.</li>
	<li>Secure the slide in the tray by tightening equally the thumbscrews one after another by hand in an alternating pattern starting from the upper left followed by lower right. Place the lid of the incubation tray.</li>
</ol>
2. Background Interaction Detection: Staining the Microarray with Secondary Antibodies
NOTE: Use a pipette to deposit the solutions (standard and blocking buffers, diluted secondary antibodies) in the corner of the microarray chamber.
<ol>
	<li>Incubate the microarray with a standard buffer (1x Phosphate buffered saline (PBS), 0.05% Tween 20, pH 7.4, filtered with a 0.45 &#181;m filter) (total volume of 2,000 &#181;L/microarray) for 15 min at room temperature (RT) on an orbital shaker at 140 rpm.</li>
	<li>Remove the standard buffer by aspirating with a pipette from the corner of the microarray chamber. Fill empty slide holders with blank slides to prevent breaking of the microarray slide.</li>
	<li>Block the microarray with the blocking buffer (total volume of 2,000 &#181;L/microarray) for 60 min at RT on an orbital shaker at 140 rpm.</li>
	<li>Remove the blocking buffer by aspirating with a pipette from the corner of the microarray chamber.</li>
	<li>Incubate the microarray with secondary antibody, anti-human IgM fluorochrome conjugated, diluted 1: 5,000 (total volume of 2,000 &#181;L/microarray) in the staining buffer (10% blocking buffer in standard buffer) for 30 min at RT in the dark on an orbital shaker at 140 rpm.</li>
	<li>Remove the secondary antibody by aspirating with a pipette from the corner of the microarray chamber.</li>
	<li>Wash the microarray 3x, 1 min per wash with standard buffer (total volume of 2,000 &#181;L/microarray at RT on an orbital shaker at 140 rpm. After each wash, remove the standard buffer by aspirating with a pipette from the corner of the microarray chamber.</li>
	<li>Immerse the slide 2x into a freshly prepared dipping buffer (1 mM Tris, pH 7.4), (total volume of 200 mL).</li>
	<li>Dry the microarray carefully, for approximately 1 min, by aspirating excess fluid very carefully from the top to the bottom of the slide without touching the slide surface. Analyze the slide in a microarray scanner reader.</li>
</ol>
3. Exposure of the Microarray to Host Serum
CAUTION: Perform this step under laboratory safety conditions, Biosafety Level 2 (BSL-2) because of the potential infectious nature of the serum specimens. Work within a Class II biological safety cabinet (BSC).
<ol>
	<li>Inactivate serum sample at 56 &#176;C for 30 min and centrifuge at 16,000 rcf for 5 min at 4 &#176;C13. 
	NOTE: Use a pipette to deposit the solutions (staining, standard buffer and diluted serum) in the corner of the microarray chamber.</li>
	<li>Dilute serum sample in staining buffer starting with a 1:1,000 (total volume of 2,000 &#956;L/microarray) dilution. Keep it at 4 &#176;C until use.</li>
	<li>Incubate the microarray with the staining buffer (total volume of 2,000 &#956;L/microarray) for 15 min at RT on an orbital shaker at 140 rpm.</li>
	<li>Remove the staining buffer by aspirating with a pipette from the corner of the microarray chamber.</li>
	<li>Incubate the microarray with the diluted serum sample overnight at 4 &#176;C on an orbital shaker at 140 rpm.</li>
	<li>Remove the diluted serum sample by aspirating with a pipette from the corner of the microarray chamber.</li>
	<li>Wash the microarray 3x, 1 min per wash with standard buffer (total volume of 2,000 &#956;L/microarray at RT on an orbital shaker at 140 rpm. After each wash, remove the standard buffer by aspirating with a pipette from the corner of the microarray chamber.</li>
</ol>
4. Staining with Secondary Antibodies and Labeled Control Antibodies
NOTE: Use a pipette to deposit the solutions (standard buffers, diluted secondary and label antibodies) in the corner of the microarray chamber.
<ol>
	<li>Dilute the secondary antibody in the staining buffer. 
	NOTE: Use anti-human IgM fluorochrome conjugated antibody or anti-human IgG fluorochrome conjugated antibody at a dilution of 1:5,000 (total volume of 2,000 &#181;L/microarray) in staining buffer.</li>
	<li>Mix control label antibody (monoclonal anti-HA fluorochrome conjugated) at a dilution of 1:1,000 (total volume of 2 000 &#181;L/microarray) in staining buffer with secondary antibody previously diluted in staining buffer.</li>
	<li>Incubate with the microarray for 30 min at RT in the dark on an orbital shaker at 140 rpm.</li>
	<li>Remove the mix of diluted secondary and label antibodies by aspirating with a pipette from the corner of the microarray chamber.</li>
	<li>Wash 3x with standard buffer (total volume of 2,000 &#181;L/microarray). Each wash is for 1 min on an orbital shaker at 140 rpm. After each wash, remove the standard buffer by aspirating with a pipette from the corner of the microarray chamber.</li>
</ol>
5. Microarray Scanning
<ol>
	<li>Immerse the slide 2x into freshly prepared dipping buffer (1 mM Tris, pH 7.4) (total volume of 200 mL).</li>
	<li>Dry the microarray carefully, around 1 min, by aspirating very carefully from the top to the bottom of the slide without touching the slide surface.</li>
	<li>Scan the microarray following the instructions of the scanner. Place the slide onto the scanner with the printed surface facing up.</li>
	<li>Create a new project and select the scanning area. Set scanner parameters as: Resolution: 21 &#181;m, Intensity for both 700 and 800 nm channels: 7.0, Scanning quality: medium and Offset: 0.8.</li>
	<li>Acquire and save the scan raw image as a 16-bit grayscale TIFF file format.</li>
</ol>
6. Microarray Analysis Using a Specific Software
<ol>
	<li>Open the raw image (TIFF image). Open the array grid file.</li>
	<li>Align the array grid to the scan image with the computer&#39;s mouse or keyboard arrow keys.</li>
	<li>Select &#34;Quantify Selection&#34; in the specific software, which creates a readout file that contains the signal intensity for each spot, the background value, and the corresponding peptide sequence. 
	NOTE: This output file can also be imported into a spreadsheet program for additional analysis and graphing.</li>
</ol>
7. Microarray Storage
<ol>
	<li>Store the microarray sealed in the dark at 4 &#176;C under oxygen free nitrogen or argon gas.</li>
</ol>

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	<li>Kelser, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meet+dengue's+cousin.">Meet dengue's cousin.</a> Zika. Microbes Infect. 18 (3), 163-166 (2016).</li><li>Musso, D., 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=Detection+of+Zika+virus+in+saliva.">Detection of Zika virus in saliva.</a> J Clin Virol. 68, 53-55 (2015).</li><li>Siqueira, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+Clinical+Manifestations+of+Patients+Infected+with+Zika+Virus.">Oral Clinical Manifestations of Patients Infected with Zika Virus.</a> Oral Health. , (2016).</li><li>Duarte, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+of+Zika+Virus+Infection+in+Pregnant+Women.">Challenges of Zika Virus Infection in Pregnant Women.</a> Rev Bras Ginecol Obstet. 38 (6), 263-265 (2016).</li><li>Buus, 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=High-resolution+mapping+of+linear+antibody+epitopes+using+ultrahigh-density+peptide+microarrays.">High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays.</a> Mol Cell Proteomics. 11 (12), 1790-1800 (2012).</li><li>Kouzmitcheva, G. A., Petrenko, V. A., Smith, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+diagnostic+peptides+for+lyme+disease+through+epitope+discovery.">Identifying diagnostic peptides for lyme disease through epitope discovery.</a> Clin Diagn Lab Immunol. 8 (1), 150-160 (2001).</li><li>Hamby, C. V., Llibre, M., Utpat, S., Wormser, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Peptide+library+screening+to+detect+a+previously+unknown+linear+diagnostic+epitope:+proof+of+principle+by+use+of+lyme+disease+sera.">Use of Peptide library screening to detect a previously unknown linear diagnostic epitope: proof of principle by use of lyme disease sera.</a> Clin Diagn Lab Immunol. 12 (7), 801-807 (2005).</li><li>t Hoen, P. A., 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=Phage+display+screening+without+repetitious+selection+rounds.">Phage display screening without repetitious selection rounds.</a> Anal Biochem. 421 (2), 622-631 (2012).</li><li>Townend, J. E., Tavassoli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traceless+Production+of+Cyclic+Peptide+Libraries+in+E.+coli.">Traceless Production of Cyclic Peptide Libraries in E. coli.</a> ACS Chem Biol. 11 (6), 1624-1630 (2016).</li><li>Turchetto, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+expression+of+animal+venom+toxins+in+Escherichia+coli+to+generate+a+large+library+of+oxidized+disulphide-reticulated+peptides+for+drug+discovery.">High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery.</a> Microbial Cell Factories. 16 (1), 6 (2017).</li><li>Carmona, S. J., Sartor, P. A., Leguizamon, M. S., Campetella, O. E., Aguero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Peptide+Discovery:+Prioritization+of+Pathogen+Diagnostic+Markers+Using+Multiple+Features.">Diagnostic Peptide Discovery: Prioritization of Pathogen Diagnostic Markers Using Multiple Features.</a> Plos One. 7 (12), e50748 (2012).</li><li>Lagatie, O., Van Dorst, B., Stuyver, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+three+immunodominant+motifs+with+atypical+isotype+profile+scattered+over+the+Onchocerca+volvulus+proteome.">Identification of three immunodominant motifs with atypical isotype profile scattered over the Onchocerca volvulus proteome.</a> PLoS Negl Trop Dis. 11 (1), e0005330 (2017).</li><li>Basile, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+ELISA+kit+(YF+MAC-HD)+to+detect+IgM+to+yellow+fever+virus.">Development and validation of an ELISA kit (YF MAC-HD) to detect IgM to yellow fever virus.</a> J Virol Methods. 225, 41-48 (2015).</li><li>Madden, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BLAST+Sequence+Analysis+Tool.">The BLAST Sequence Analysis Tool.</a> The NCBI Handbook. , (2013).</li><li>Services, U. S. D. o. H. a. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories (BMBL). , (2009).</li><li>Pellois, J. 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=Individually+addressable+parallel+peptide+synthesis+on+microchips.">Individually addressable parallel peptide synthesis on microchips.</a> Nat Biotech. 20 (9), 922-926 (2002).</li><li>Stafford, 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=Physical+Characterization+of+the+"Immunosignaturing+Effect".">Physical Characterization of the "Immunosignaturing Effect".</a> Mol Cell Proteomics. 11 (4), (2012).</li><li>Gardner, T. 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The authors have nothing to disclose.

We designed a protocol utilizing a high-density peptide microarray containing the entire Zika virus protein sequence (French Polynesian strain). The microarray was manufactured by printing 3,423 different overlapping linear peptides. Each peptide was 15 amino acids and varied by only one residue from its nearest neighbor in the sequence (i.e., 14 residue overlap). Although shorter overlaps can be printed, epitope mapping is more precise with longer overlaps. Each peptide was printed in duplicate to increase reliability.
It is essential to start the process with pre-staining the microarray with the secondary antibody to detect possible background interactions and differentiate them from the sample specific signal. Using a specific blocking buffer is also important as other blocking buffers like Bovine Serum Albumin (BSA) or dried milk powder can result in reduced signal intensity. A validated serum sample for ZIKV IgM antibodies was first incubated with the microarray at 1:1,000 but no signal was detected. Subsequent incubation at a lower dilution of 1:500 resulted in a low intensity signal coming from peptides that reacted with IgM antibodies in the sample. The best results were obtained when the Zika validated serum sample was diluted at 1:250. We did not retest the serum sample at a higher concentration to avoid an increase of the background signal. Careful handling of the microarray is essential to avoid scratching the microarray surface. Therefore, the solutions such us diluted serum and standard buffer are added to and removed from the corner of the microarray chamber. Also, important for a successful result is the slide specific incubation tray provided by the company that manufactured the microarray, which is designed to allow working with minimal sample volumes. The incubation tray dimensions are 13.0 cm by 9.0 cm and 3 cm of thick. There are 3 slides designated cavities in the incubation tray that allow assaying 3 different slides simultaneously. However, in this experiment we used only 1 slide. Blank microscope slides were placed in the other two cavities to balance the tray and prevent breaking of the array slide. The incubation tray consists of 4 parts: a base plate where the slides are placed in designated cavities; a sealer made up of silicon to separate each slide from the others; an upper part that contains thumbscrews to join the sealer with the base plate and create the microarray chambers for adding solutions for the assay such as washing buffer or serum sample. The sealer and upper part avoid contamination or mixing of solutions between slides. The last section component is a lid to minimize the risk of evaporation or environmental contamination of samples. An orbital shaker is preferred to get optimal wetting and staining conditions.
Depending on when the virus infected the host the resulting specific Zika IgM antibody concentration in sera can be high or low. Therefore, it was anticipated that using a serum sample with a higher IgM concentration would increase the relative fluorescence signal intensity. After IgM detection, the same serum sample was incubated at a dilution of 1:250 with the microarray, but an anti-human IgG was used as the secondary antibody. Alternatively, different antibody classes can be detected simultaneously within the same microarray using a mixture of secondary antibodies each conjugated with different fluorescence dyes. The same microarray can be stained again with a more concentrated serum sample if the signal intensity is low. If stored properly the microarray is stable for several months and can possibly be used again.
Quantification of spot intensities and peptide annotations was performed using&#160;proprietary software from the company that manufactured the microarray that also provides a &#34;.psf&#34; file with the template of the array format. The psf file is essentially a grid that is aligned to the raw scanner image using the proprietary software. A software algorithm analyzes the relative fluorescence intensities of each spot into raw (intensity value of the spot&#39;s signal), background (estimated value of the signal caused by non-specific binding) and foreground (subtracted the background from the raw value) signal. It also calculates the foreground median values and aggregate foreground median corresponding to the mean of the two median spot values of each peptide in duplicate. In addition, the software identifies consensus motifs/epitope within overlapping peptides. Alternatively, signal intensities can be quantified with the microarray scanner software and then using the template of the array format, the peptide sequence can be identified.
Following this protocol, several promising peptide candidates were identified based on the fluorescence intensity. Subsequent BLAST14 analysis was used to identify residues with potential cross-reactivity to other flavivirus. A total of 14 identified peptides are present only in ZIKV as identified in 3 proteome databases. A second series of microarrays is planned that will contain the selected peptides from the first array. This will allow testing multiple validated specimens as well as samples from people infected only with flavivirus&#39;s other than ZIKV to confirm the specificity. For screening, we will adapt an ELISA assay format, coating the plates with the selected peptides and testing the binding to ZIKV specific antibodies in human serum and saliva samples.
ZIKV handling requires working in Biosafety Level 2 (BSL-2) facility15. As we worked with a serum sample validated for ZIKV infection we worked in BSL-2 facility performing all the sample manipulation within a Class II (or higher) biological safety cabinet (BSC). This is why we inactivated the virus heating the serum sample at 56 &#176;C for 30 min before incubating the sample with the microarray13. Following heat inactivation, it is also recommended continue working in a BSL-2 facility with appropriate good laboratory safety practice to avoid the chance of infection.
The microarray that we designed contains only linear peptides and consequentially conformational epitopes that might be valuable as diagnostics may have been missed. This limitation can be addressed by using a high-density microarray containing cyclic constrained peptides that mimic looped epitope structures16. This protocol can rapidly be adapted to other pathogens because once the protein sequence is known a new microarray can be designed for discovery of potential diagnostic peptides. It can also be adapted for other applications such as biomarker discovery17, antigen and epitope discovery for vaccine development or therapeutic targets18,19.

Zika virus (ZIKV) diagnosis based on clinical symptoms is challenging because it shares vectors, geographic distribution, and symptoms with Dengue and Chikungunya virus infection1. Given the risk for adverse pregnancy outcomes in women infected with ZIKV during pregnancy, it is important to distinguish between the 3 viruses. Although the current molecular diagnostic tests are specific, they are only useful in blood or saliva during the relatively short period of acute infection2,3. Serological assays are essential for diagnosis outside this initial period of infection4.
The development of a ZIKV specific serological assay is challenging for two reasons: first, the Zika antigens that the human immune system responds to are not currently known; and second, conserved flaviviruses amino acid sequences induce antibody cross-reactivity. Our objective was to discover unique ZIKV specific peptides to be used in diagnostics. Different approaches have been developed to screen peptide libraries covering entire proteins including phage, bacterial, and yeast surface display5,6,7,8,9,10. Our strategy was to use a high-density peptide microarray that permits rapid and inexpensive high-throughput serological screenings11,12 and subsequently the identified peptides can be used to improve current serological assays for detection of ZIKV infection.
This protocol enables the discovery of Zika virus specific diagnostic peptides using a high-density peptide microarray (Figure 1). The high-density peptide microarray was produced using the peptide laser printing technology. The entire ZIKV protein sequence consisting of 3,423 amino acid residues based on the French Polynesian strain (GenBank: KJ776791.2), was printed on a standard glass slide in blocks of 15 linear residues with an overlap of 14 amino acid residues in duplicate for a total of 6,846 peptide spots. In addition to the entire Zika protein sequence peptides, the microarray utilizes Influenza hemagglutinin (HA) peptides for internal controls.
A Zika validated positive serum sample obtained from Wadsworth Center (Albany, NY), was used to identify specific Immunoglobulin M (IgM) and Immunoglobulin G (IgG) reactive peptides. After incubating with the sample overnight, the microarray was stained with a secondary fluorochrome conjugated antibody (anti-human IgM or anti-human IgG), and analyzed on a microarray scanner. Quantification of spot intensities and peptide annotation was performed with a specific software provided by the same company that manufactured the microarray.

<table>
	<tbody>
		<tr>
			<td>PEPperCHIP Custom Peptide Microarray</td>
			<td>PEPperPRINT</td>
			<td>PPC.001.001</td>
			<td>Custom peptide micarray: our microarray contains the entire Zika virus protein sequence</td>
		</tr>
		<tr>
			<td>PEPperCHIP incubation tray 3/1</td>
			<td>PEPperPRINT</td>
			<td>PPC.004.001</td>
			<td></td>
		</tr>
		<tr>
			<td>PEPperCHIP Staining Kit (680 nm) (anti-HA, DyLight labeling)</td>
			<td>PEPperPRINT</td>
			<td>PPC.037.002</td>
			<td></td>
		</tr>
		<tr>
			<td>PepSLide Analyzer</td>
			<td>PEPperPRINT</td>
			<td>PSA.004.001</td>
			<td>14-days free License for Windows</td>
		</tr>
		<tr>
			<td>Rockland Blocking buffer</td>
			<td>Rockland</td>
			<td>MB-070</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human IgM (mu chain) DyLight 800</td>
			<td>Rockland</td>
			<td>609-145-007</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human IgG Fc DyLight 680</td>
			<td>Thermo Scientific</td>
			<td>SA5-10138</td>
			<td></td>
		</tr>
		<tr>
			<td>LI-COR Odyssey Imaging System</td>
			<td>LI-COR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker device</td>
			<td>IKA</td>
			<td>MTS 2/4 digital microtiter shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Illustrator</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deltagraph</td>
			<td>Redrocks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

zika virus specific diagnostic epitope discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

The results obtained using the protocol described are shown in Figure 2 and Figure 3. No background interactions were noted when pre-staining the microarray with secondary anti-human IgM (data not shown). Staining with an anti-human IgM conjugate resulted in several areas above background green fluorescence intensities, indicating the binding of these peptides with IgM in the host serum sample (Figure 2). IgG detection (red fluorescence signal) revealed only a few peptides (Figure 2).
The specific software provided by the same company that manufactured the microarray analyzes the microarray after scanning. The readout file created contains the signal intensity for each spot, the background value, and the corresponding peptide sequence. The spot fluorescence intensities of each peptide with a strong response to IgM were visualized by plotting the green foreground median values obtained after subtracting the background from the raw value (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56784/56784fig1.jpg" /> 
Figure 1: Schematic Showing the Analysis Process for the High-density Peptide Microarray. <a href="https://cloudflare.jove.com/files/ftp_upload/56784/56784fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56784/56784fig2.jpg" /> 
Figure 2: Peptide Microarray Scanned Images. Raw fluorescence images of staining the peptide microarray after incubating with a ZIKV serum sample diluted 1:250. The IgG reactivity is depicted in red (left), and the IgM reactivity is shown in green (right). HA control peptides frame the peptide microarray. Zoom panels show group of peptides with high fluorescence intensities, indicating a strong response to IgM or IgG. <a href="https://cloudflare.jove.com/files/ftp_upload/56784/56784fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56784/56784fig3.jpg" /> 
Figure 3: Representative Zika Virus Epitope Consensus Sequence Reacting with IgM in the Host Serum Sample. Green foreground median values from overlapping peptides were plotted on a graph. Peptides from the epitope consensus sequence are highlighted in red. The peptide with the highest fluorescence intensity is marked in bold. Vector based graphic design software was used to generate this figure. <a href="https://cloudflare.jove.com/files/ftp_upload/56784/56784fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Zika Virus Specific Diagnostic Epitope Discovery at JoVE.com

Zika Virus Specific Diagnostic Epitope Discovery

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

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Nanyang Technological University

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Immunology and Infection

7.7K Views.  New York University College of Dentistry. In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Video: Zika Virus Specific Diagnostic Epitope Discovery

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

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

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

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

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

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

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

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

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

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

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

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

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

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

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

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

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

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

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

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

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

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial&#160;propagation.

The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter.

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines ...