Geçerli destek NIDCRR44 DE024456 bir SBIR (küçük iş yenilik araştırma) yönetim ek grant tarafından sağlanır. NIDCR dışında gelişti NIDCR HIV verme doğrulayıcı bir bakım noktası kontrol kalkınma HIV U01 DE017855 verin. Biz minnetle kabul Silke Weisenburger(PEPperPRINT Heidelberg, Germany) onun teknik yardım ve tür destek için. Ayrıca NYU Langone Tıp Merkezi LI-COR Odyssey görüntüleme sistemi için teşekkür ediyoruz.

Bu veri, New York University College, diş hekimliği bir devam eden araştırma parçasıdır ve New York Üniversitesi Tıp Fakültesi, IRB kurumsal inceleme kurulca onaylanmış # H10-01894. Bu çalışmada kullanılan klinik örnekler daha önce kullanılan tanı ve izinle üzerinden Wadsworth Center, New York Dışişleri Bakanlığı Sağlık, Albany, NY de-tespit edildi.
1. ZIKV yüksek yoğunluklu peptid Mikroarray slayt yükleme içinde belirli bir kuluçka tepsi
<ol><li>Cam slayt pudrasız eldiven kullanarak kenarları tarafından ele. Cam slayt 75.4 mm 25.0 mm ve 1 mm kalın boyutlardır. Slaydı yukarı dönük olacak şekilde Mikroarray yüzeyli kuluçka tepsisine yerleştirin.</li>
	<li>Aşağıya doğru Mikroarray yazdırılan yüzeye karşı karşıya mühür parlak yanına koyun. İyi bir pozisyon sağlamak için vida deliklerini taban plakası ve mühür üst üste.</li>
	<li>Üst kısmındaki tepsiyi Mikroarray odası oluşturmak için mühür üzerine yerleştirin.</li>
	<li>Slayt içinde belgili tanımlık tepsi eşit taktıktan birbiri ardına sağ alt tarafından takip sol üst başlayarak bir alternatif model elle sıkılaştırma tarafından güvenli. Kuluçka tepsisi kapağını yerleştirin.</li>
</ol>2. arka plan etkileşim algılama: Mikroarray ikincil antikorlar ile boyama
Not: bir pipet (Standart ve engelleme arabellekleri, seyreltilmiş ikincil antikorlar) çözümleri Mikroarray odası köşede yatırmak için kullanabilir.
<ol><li>Standart arabelleği (0,45 µm filtre ile filtre 1 fosfat tamponlu tuz (PBS), %0.05 ara 20, pH 7.4, x) ile Mikroarray kuluçkaya (toplam hacmi 2.000 µL/Mikroarray), oda sıcaklığında (RT) üzerinde bir orbital Çalkalayıcı 140 devirde 15 dakika.</li>
	<li>Standart arabellek Mikroarray odası köşesinden bir pipet ile alıyorum tarafından kaldırın. Boş slayt sahipleri Mikroarray slayt kırma önlemek için boş slaytlar ile doldurun.</li>
	<li>RT üzerinde bir orbital Çalkalayıcı 140 RPM, 60 dk için engelleme arabellek (toplam hacmi 2.000 µL/Mikroarray) ile Mikroarray engelleyin.</li>
	<li>Engelleme arabellek Mikroarray odası köşesinden bir pipet ile alıyorum tarafından kaldırın.</li>
	<li>İkincil antikor, Birleşik Anti-insan IgM fluorochrome ile Mikroarray kuluçkaya, RT, 30 dk içinde bir orbital Çalkalayıcı 140, karanlık için (%10 standart arabellek arabellek engelleme) seyreltilmiş 1: boyama içinde 5.000 (toplam hacmi 2.000 µL/Mikroarray) tampon d/dak.</li>
	<li>İkincil antikor Mikroarray odası köşesinden bir pipet ile alıyorum tarafından kaldırın.</li>
	<li>3 Mikroarray yıkama x, 1 min başına yıkama ile standart arabellek (2.000 µL/Mikroarray RT bir orbital Çalkalayıcı 140 devirde tarih itibariyle toplam hacmi. Her yıkama sonra standart arabellek Mikroarray odası köşesinden bir pipet ile alıyorum tarafından kaldırın.</li>
	<li>Slayt 2 sokmak içine taze hazırlanmış daldırma arabellek (1 mM Tris, pH 7,4) x, (toplam hacmi 200 mL).</li>
	<li>Mikroarray dikkatle, yaklaşık 1 dakika için slayt altına çok dikkatli bir şekilde üstten aşırı sıvı slayt yüzeyine dokunmadan alıyorum tarafından kuru. Slayt içinde a microarray tarayıcı okuyucu analiz.</li>
</ol>3. Serum barındırması Mikroarray pozlama
Dikkat: Seviye 2 (BSL-2) serum örnekleri potansiyel bulaşıcı doğası nedeniyle laboratuvar güvenlik koşulları altında bu adımı gerçekleştirin. Bir sınıf II biyolojik güvenlik içinde dolap (BSC) çalışır.
<ol><li>Serum örneği için 30 dk 56 ° C'de ve 16.000 rcf, santrifüj 4 ° C135 min için devre dışı bırakabilirsiniz. Not: bir pipet (boyama, standart tampon ve seyreltik serum) çözümleri Mikroarray odası köşede yatırmak için kullanabilir.</li>
	<li>Serum örneği boyama arabelleği 1:1,000 (toplam hacmi 2.000 μL/Mikroarray) seyreltme ile başlayan sulandırmak. 4 ° C'de kullanımı kadar devam et.</li>
	<li>RT bir orbital Çalkalayıcı 140 devirde tarih itibariyle 15 dakika Mikroarray boyama arabelleği (toplam hacmi 2.000 μL/Mikroarray) ile kuluçkaya.</li>
	<li>Boyama arabellek Mikroarray odası köşesinden bir pipet ile alıyorum tarafından kaldırın.</li>
	<li>Gecede 4 ° C'de bir orbital Çalkalayıcı 140 devirde üzerinde seyreltik serum örneği ile Mikroarray kuluçkaya.</li>
	<li>Seyreltik serum örneği tarafından Mikroarray odası köşesinden bir pipet ile alıyorum kaldırın.</li>
	<li>3 Mikroarray yıkama x, 1 min başına yıkama ile standart arabellek (2.000 μL/Mikroarray RT bir orbital Çalkalayıcı 140 devirde tarih itibariyle toplam hacmi. Her yıkama sonra standart arabellek Mikroarray odası köşesinden bir pipet ile alıyorum tarafından kaldırın.</li>
</ol>4. ikincil antikorlar ve etiketli denetim antikorlar ile boyama
Not: bir pipet (Standart arabellekleri, seyreltilmiş ikincil ve etiket antikorlar) çözümleri Mikroarray odası köşede yatırmak için kullanabilir.
<ol><li>İkincil antikor boyama arabellekte sulandırmak. Not: Kullanım Anti-insan IgM fluorochrome antikor veya anti-insan IgG fluorochrome konjuge antikor 1:5,000 (toplam hacmi 2.000 µL/Mikroarray) Boyama arabellekte bir seyreltme, Birleşik.</li>
	<li>Mix kontrol etiket antikor (monoklonal anti-HA fluorochrome Birleşik), daha önce tampon boyama seyreltilmiş ikincil antikor ile tampon boyama içinde 1:1,000 (toplam hacmi 2 000 µL/Mikroarray) seyreltme.</li>
	<li>RT, 30 dk içinde karanlık bir orbital Çalkalayıcı 140 devirde Mikroarray ile kuluçkaya.</li>
	<li>Seyreltilmiş ikincil ve etiket antikorlar tarafından Mikroarray odası köşesinden bir pipet ile alıyorum kaldırın.</li>
	<li>3 x standart arabellek (toplam hacmi 2.000 µL/Mikroarray) ile yıkayın. Her yıkama 1dk bir orbital Çalkalayıcı 140 devirde üzerinde içindir. Her yıkama sonra standart arabellek Mikroarray odası köşesinden bir pipet ile alıyorum tarafından kaldırın.</li>
</ol>5. Mikroarray tarama
<ol><li>Slayt 2 sokmak x içine taze hazırlanmış daldırma arabellek (1 mM Tris, pH 7,4) (toplam hacmi 200 mL).</li>
	<li>Kuru Mikroarray dikkatle, çok dikkatli bir şekilde en baştan slayt köküne slayt yüzeyine dokunmadan alıyorum tarafından yaklaşık 1 dk,.</li>
	<li>Tarayıcının talimatları Mikroarray inceden inceye gözden geçirmek. Slayt tarayıcı üzerine yazdırılan yüzeye yukarı dönük olacak şekilde yerleştirin.</li>
	<li>Yeni bir proje oluşturmak ve tarama alanını seçin. Tarayıcı parametreleri olarak belirleme: çözünürlük: 21 µm, 700 ve 800 nm kanallar için yoğunluğu: 7.0, tarama kalitesi: Orta ve uzaklık: 0.8.</li>
	<li>Elde etmek ve tarama raw görüntü bir 16-bit gri tonlamalı TIFF dosya biçiminde kaydedin.</li>
</ol>6. Mikroarray belirli bir yazılım kullanarak çözümleme
<ol><li>Ham görüntü (TIFF İmaj) açın. Dizi kılavuz dosyasını açın.</li>
	<li>Bilgisayar fare veya klavye ok tuşları ile tarama görüntü dizi kılavuza hizalayın.</li>
	<li>"Ölçmek seçimi" sinyal yoğunluğu her nokta için arka plan değeri ve karşılık gelen peptit dizisi içeren bir okuma dosyası oluşturur belirli yazılımında seçin. Not: Bu çıkış dosyası Ayrıca ek çözümleme ve grafik için bir elektronik tablo programına aktarılabilir.</li>
</ol>7. Mikroarray depolama
<ol><li>Oksijen serbest azot veya argon gazı altında 4 ° C'de karanlıkta mühürlü Mikroarray saklayın.</li>
</ol>

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Yazarlar ifşa gerek yok.

Biz tüm Zika virüs protein (Fransız Polinezya zorlanma) sıra içeren bir yüksek yoğunluklu peptid Mikroarray kullanan bir iletişim kuralı tasarlanmıştır. Mikrodizi yazdırma 3,423 farklı örtüşen doğrusal peptidler tarafından üretildi. Her peptid 15 amino asitler yapıldı ve onun en yakın komşu sırada (Yani, 14 kalıntı örtüşme) üzerinden tek bir kalıntı tarafından çeşitli. Daha kısa örtüştüğü yazdırılabilmesi için epitope eşleme ile uzun örtüştüğü daha kesin olsa ...

Vektörel çizimler, dağılım ve belirtiler Dang Chikungunya virüsü enfeksiyonu ve1ile paylaşır çünkü Zika virüs (ZIKV) tanı klinik belirtiler dayalı meydan okuyor. Olumsuz gebelik çıktıları gebelik sırasında ZIKV ile enfekte kadınlarda için risk göz önüne alındığında, 3 virüs arasında ayırt etmek önemlidir. Geçerli moleküler tanı testleri belirli olmasına rağmen onlar sadece akut enfeksiyon2,3nispeten kısa dönemde kan ya da tükürük yararlıdır. Serolojik deneyleri enfeksiyon4bu başlangıç dönemi dışında tanı için gereklidir.
ZIKV belirli serolojik tahlil gelişimi iki nedenden dolayı zordur: ilk olarak, insan bağışıklık sistemi yanıt Zika antijenleri şu anda bilinmemektedir; ve ikincisi, korunmuş flaviviruses amino asit dizileri antikor olan teşvik. Hedefimiz işlem Tanı'daki kullanılmak üzere benzersiz ZIKV belirli peptidler keşfetmek oldu. FAJ, bakteriyel, dahil olmak üzere tüm proteinler kapsayan ekran peptid kitaplıkları için farklı yaklaşımlar geliştirilmiştir ve Maya yüzey görüntülemek5,6,7,8,9, 10. Hızlı ve ucuz yüksek üretilen iş serolojik gösterimleri11,12 izin veren bir yüksek yoğunluklu peptid Mikroarray kullanmak bizim strateji olmuştur ve daha sonra belirlenen peptidler geçerli serolojik geliştirmek için kullanılabilir deneyleri ZIKV enfeksiyon algılanması için.
Bu iletişim kuralı bir yüksek yoğunluklu peptid Mikroarray (şekil 1) kullanarak Zika virüs belirli tanılama peptidler keşfi sağlar. Yüksek yoğunluklu peptid Mikroarray peptid lazer baskı teknolojisi kullanılarak üretilmiştir. 3,423 amino asit kalıntıları eklenmesi ile oluşan tüm ZIKV protein sıra tabanlı Fransız Polinezya strain (GenBank: KJ776791.2), 14 amino asit kalıntılarında için yinelenen bir çakışma ile 15 doğrusal kalıntılarının bloklar halinde standart cam slayt üzerinde yazılı bir Toplam 6,846 peptid noktalar. Tüm Zika protein sıra peptidler yanı sıra Mikroarray grip hemagglutinin kullanır (HA) peptidler iç kontrol için.
Bir Zika olumlu Wadsworth Merkezi'nden (Albany, NY), elde edilen serum örneği belirli immünglobulin M (IgM) ve immünoglobulin G (IgG) Reaktif peptidler tanımlamak için kullanılan geçerliliği. Örnek ile gecede kuluçka sonra Mikroarray bir ikincil fluorochrome konjuge antikoru (Anti-insan IgM veya anti-insan IgG) ile lekeli ve Mikroarray tarayıcı üzerinde analiz. Miktar spot yoğunluklarda ve peptid ek açıklama Mikroarray üretilen aynı şirket tarafından sağlanan özel bir yazılım ile gerçekleştirildi.

<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

Yüksek yoğunluklu peptid microarrays tek standart mikroskobu slaytta altı binden fazla peptidler tarama sağlar. Bu yöntem ilaç bulma, terapötik hedef tanımlama ve geliştirme için uygulanabilir teşhis. Burada, belirli Zika virüs (ZIKV) tanı peptidler yüksek yoğunluklu peptid Mikroarray kullanarak keşfetmek için bir protokol mevcut. ZIKV enfeksiyon 3,423 benzersiz 15 doğrusal amino asit (aa) kalıntıları bir 14-aa kalıntısı örtüşme ile tercüme tüm ZIKV protein içeren bir yüksek yoğunluklu peptid Mikroarray ile inkübe için doğrulanmış bir insan serum örneği içinde yinelenen baskılı. Aynı dizi içinde farklı ikincil antikorlar ile boyama, immünglobulin M (IgM) ve immünoglobulin G (IgG) antikor serum içinde mevcut bağlayabilirsiniz peptidler tespit ettik. Bu peptidler daha fazla doğrulama denemeleri için seçildi. Bu protokol için tasarım, işlemek ve yüksek yoğunluklu peptid Mikroarray çözümlemek için takip strateji tanımlarlar.

Açıklanan protokolü kullanılarak elde edilen sonuçları Şekil 2 ' de gösterilen ve şekil 3. Hiçbir arka plan etkileşimleri Mikroarray ikincil Anti-insan IgM (veri gösterilmez) ile önceden boyama zaman kaydedilmiştir. Anti-bir insanla IgM boyama eşlenik yukarıda arka plan yeşil Floresans yoğunluklarını, IgM ile Bu peptidler (Şekil 2) ana bilgisayar serum örneğind...

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

Zika Virus Specific Diagnostic Epitope Discovery

Bu protokol için bir yüksek yoğunluklu peptid Mikroarray kullanarak Zika virüs belirli tanılama peptidler keşfetmek için bir teknik açıklar. Bu iletişim kuralı readly ortaya çıkan diğer bulaşıcı hastalıklar için adapte edilebilir.

Zika virüs belirli tanılama Epitope bulma

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

zika-virus-specific-diagnostic-epitope-discovery

Research

JoVE Journal

Immunology and Infection

7.7K Görüntüleme.  New York University College of Dentistry. Bu protokol için bir yüksek yoğunluklu peptid Mikroarray kullanarak Zika virüs belirli tanılama peptidler keşfetmek için bir teknik açıklar. Bu iletişim kuralı readly ortaya çıkan diğer bulaşıcı hastalıklar için adapte edilebilir.

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.

Peptid: Epitop-spesifik T-hücrelerinin MHC tetramer bazlı Zenginleştirme

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 ...

İmmünoloji ve Enfeksiyon

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.

Bluetongue Virüs karşı Roman Antiviraller belirlenmesi için Tahliller

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.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

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 Virüs Bulaşıcı hücre kültürü sistemine ve<em&gt; İn Vitro</emİnterferonların&gt; profilaktik etkisi

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.

Yeni Bir Dünyadan Rekombinant Virüsün Kurtarılması ve Karakterizasyonu Zika Virüsü Bulaşıcı Klon

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.

Basit Akış Sitometresi Vitro antikor bağımlı Enhancement-in dang virüs Zika virüs nekahat Serum kullanarak belirlemek için tahlil dayalı

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.

Virüslü Ifnar1- / - farelerin Immunoprivileged organlarında Zika virüs özgü T hücreli yanıt-e doğru değerlendirilmesi

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.

Kodlama ve RNA sınıfları kodlamayan teşhis kodu içinde domuz tam kan ifade.

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.

Antikor-bağımlı donanýmýn birincil insan hücrelerinde Zika şunun miktar

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.

Bir bakteriyel yapay kromozom cDNA klon rekombinant Zika virüs kurtarma

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