Name: zika virus specific diagnostic epitope discovery
Uploaded: 2017-12-12T13:00:00
Description: Watch this Scientific Journal Video about Zika Virus Specific Diagnostic Epitope Discovery at JoVE.com

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 Tr...

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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 reli...

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 (<strong class="xfi...

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 ...

The overall goal of this experiment is to identify Zika virus specific peptides for use in a diagnostic assay to detect Zika virus antibodies. This is critical, as the virus disappears from the blood in approximately 10 days, while antibodies persist for years. This method allows us to identify antigens and epitopes utilizing a microarray of the entire Zika proteome to identify peptides that capture either IgM or IgG antibodies in a sample of serum from an infected individual.The main advantage in detecting both early antibodies, IgM, and later antibodies, IgG, is to provide the clinician a timeline of the infection. And this is particularly important in pregnant women. While this protocol provides insight into the cause of Zika virus infection, it can be applied to other pathogens, using a microarray specific for that pathogen if its protein sequence is known.Handle the glass slide by the edges using powder-free gloves. The glass slide dimensions are 75.4 millimeters by 25.0 millimeters and 1 millimeter thick. Place the slide in the incubation tray with the microarray surface facing up.Fill the empty slide holders with blank slides to prevent the microarray slide from breaking. 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.Place the upper part of the tray onto the seal to create a microarray chamber. Secure the slide in the tray by equally tightening the thumb screws one after another by hand in an alternating pattern. Start from the upper left and proceed to the lower right.Place the lid on the incubation tray. Careful handling of the microarray is essential to avoid scratching the microarray surface. Solutions are added to and then removed from the corner of the microarray chamber.Drying the microarray is also important to provide a good scan image. Incubate the microarray with 2, 000 microliters of standard buffer per microarray for 15 minutes at room temperature on an orbital shaker at 140 rpm. Remove the standard buffer by aspirating with a pipette from the corner of the microarray chamber, then block the microarray with a blocking buffer for 60 minutes at room temperature on an orbital shaker at 140 rpm.Remove the blocking buffer by aspirating from the corner of the microarray chamber with a pipette. Add anti-human IgM fluorochrome conjugated secondary antibody diluted 1:5, 000 in the staining buffer to the microarray. Then incubate the microarray for 30 minutes at room temperature in the dark on an orbital shaker at 140 rpm.Use a pipette to remove the secondary antibody by aspirating from the corner of the microarray chamber. Wash the microarray for one minute with 2000 microliters with standard buffer per microarray at room temperature on an orbital shaker at 140 rpm. Remove the standard buffer using the demonstrated technique and repeat the wash two times.Next, immerse the slide twice into 200 milliliters of a freshly prepared dipping buffer. Dry the microarray carefully for approximately one minute 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 as demonstrated later in this video.Inactivate the serum sample at 56 degrees Celsius for 30 minutes. Then centrifuge the sample at 16, 000 RCF for five minutes at four degrees Celsius. Keep the sample at four degrees Celsius until use.Then incubate the microarray with the staining buffer for 15 minutes at room temperature on an orbital shaker at 140 rpm. Remove the staining buffer by aspirating from the corner of the microarray chamber with a pipette. Dilute the serum sample and staining buffer starting with a 1:1, 000 dilution.Incubate the diluted sample with the microarray overnight at four degrees Celsius on an orbital shaker at 140 rpm. Remove the diluted serum sample using the demonstrated technique. Then wash the microarray three times as before.Dilute the secondary antibody in the staining buffer. Use anti-human IgM fluorochrome conjugated antibody or anti-human IgG fluorochrome conjugated antibody at a dilution of 1:5, 000 in staining buffer. Then mix the control-labeled antibody at a dilution of 1:1000 in staining buffer with the diluted secondary antibody.Incubate with the microarray for 30 minutes at room temperature in the dark and on an orbital shaker at 140 rpm. Remove the mix of diluted secondary and labeled antibodies by aspirating from the corner of the microarray chamber with a pipette. Then wash the microarray three times with standard buffer.Each wash is for one minute on an orbital shaker at 140 rpm. Finally, remove the washing buffer from the microarray chamber as before. To perform microarray scanning, immerse the slide twice into 200 milliliters of freshly prepared dipping buffer.Dry the microarray for around one minute by very carefully aspirating from the top to the bottom of the slide without touching the slide surface. Scan the microarray following the instructions of the scanner. Place the slide onto the scanner with the printed surface facing up.Create a new project and select the scanning area. Set scanner parameters with a resolution of 21 microns, an intensity of 7.0 for both the 700 and 800 nanometer channels, a scanning quality of medium, and an offset of 0.8. Acquire and save the scan draw image as a 16-bit grayscale TIF file format.To analyze the data, open the raw image. Then open the array grid file. Align the array grid to the scan image with the computer's mouse or keyboard arrow keys.Select quantify selection 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. Finally, store the microarray sealed in the dark at four degrees Celsius under oxygen-free nitrogen or argon gas. Shown here is the raw fluorescence image of staining the microarray with anti-human IgG after incubating with a specific Zika virus serum sample diluted 1:250.HA control peptides frame the microarray. Peptides that react strongly to IgG are shown in the zoom panel. Similarly, shown here is a raw fluorescence image of staining the microarray with anti-human IgM after incubating with a specific Zika virus serum sample diluted 1:250.Again, the HA control peptides frame the microarray. Peptides that react strongly to IgM are shown in the zoom panel. Shown here is a representative Zika virus epitope consensus sequence reacting with IgM in the host serum sample peptides.Peptides from the epitope consensus sequence are highlighted in red, and the peptide with the highest fluorescence intensity is marked in bold. After watching this video, you should have a good understanding of how to process a high-density peptide microarray. Once mastered, this technique can be done in 24 hours if it is performed properly.While attempting this procedure, it's important to remember to carefully handle the microarray to avoid scratching the microarray surface. Following this procedure, other methods like ELISA or a surface plasmon resonance binding assay can be performed to determine if the identified peptides bind specifically to Zika virus antibodies and not to other Flavivirus antibodies. After its development, this technique will pave the way for researchers in the field of diagnostics for the discovery of diagnostic peptides using serum and saliva specimens.Don't forget that working with infectious agents as Zika virus can be extremely hazardous, and precautions such as heat-inactivating the serum sample and working in Biosafety Level 2 facilities should always be taken while performing this procedure.

zika-virus-specific-diagnostic-epitope-discovery

Research

JoVE Journal

Immunology and Infection

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 ...

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 ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...