Name: a simple flow cytometry based assay to determine in vitro antibody dependent enhancement of dengue virus using zika virus convalescent serum
Uploaded: 2018-04-10T14:00:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum at JoVE.com

Açıklanan proje JJM için Uniformed Hizmetleri Üniversitesi Sağlık Bilimleri Enstitüsü'nden fon tarafından desteklenmiştir. Görüş ya da burada yer alan iddialar yazarların özel olanları ve resmi veya yansıtıcı olarak görüşlerini Savunma Bakanlığı, Uniformed Hizmetleri Üniversitesi Sağlık Bilimleri Enstitüsü veya başka bir Ajansı ABD olmak yorumlanacaktır değildir Hükümet.
WGV tüm deneylerin gerçekleştirilen ve veri analiz; JJM tasarlamış ve çalışma denetlenir; WGW ve JJM kağıt yazdı.

Burada açıklanan protokol göstermek için kullanılan serum numuneleri ilişki değerlendirme ve Akreditasyon laboratuvar hayvan bakımı için ev ve bakım için uygun olarak yerel, eyalet ve federal ilkelerinde edildi rhesus makak elde International (AAALAC)-akredite tesis. Tüm hayvan deneyleri gözden geçirilmiş ve kurumsal hayvan bakım ve kullanım Komitesi tarafından onaylanmış ve örnekleri paylaşım protokolü bir doku ile elde.
1. gün 1
Not: bir BSL-2 laboratuvar doku kültürü için kullanılan steril laminar akış Biyogüvenlik kabini içinde aşağıda açıklanan tüm adımları gerçekleştirin.
<ol><li>Serum numuneleri, oda sıcaklığında erimek ve 100 µL serum örnekleri için steril bir tüp aktarın. Isı 30 dk 56° c su banyosu veya sıcaklığı ayarlanabilir bir thermomixer için devre dışı. 1:1-soğuk RPMI-%10 (10 fetal sığır serum ile RPMI) kullanarak 1:1,000 için arasında serum örneği 10 kat seri dilutions olun.</li>
	<li>Seri olarak seyreltilmiş serum örneği 10 µL steril bir 96-şey V-alt plaka her şey için transfer. Sadece ve serum örneği ve RPMI-10 sadece RVPs ve serum örneği olmadan olmadan denetim Wells, RPMI-10 RVPs ile iki kümeleri içerir. Her serum örneği ve triplicates RPMI-10 denetimleri ayarlayın.</li>
	<li>Dang-1, 2, 3 ve 4 RVPs-80 ° C dondurucu ve tezcan çıkarın 37 ° C su banyosu giriyordu. Hemen buz çözdürülen RVPs aktarın. Yaklaşık 170 µL RVPs her serum örneği için elde etmek (RVPs x 3 Wells her serum seyreltme için 10 µL (1:1, 1:10, 1: 100, 1:1, 000) ve RVPs x 3 Wells beher-in RPMI-10 RVP ile 10 µL sadece kuyu kontrolü).</li>
	<li>RVPs 10 µL her de serum örneği içeren 96-şey V-alt plaka ve RPMI-10 RVPs (serum) denetim wells ile içine pipet. RVPs RPMI-10 yalnızca için (hiçbir serum örneği ve hiçbir RVP) kuyuları eklemeyin. İyice yukarı ve aşağı pipetting tarafından Mix 5-10 kat. 10 µL soğuk eklemek RPMI-10 RVPs soğuk her RPMI 10 yerine (hiçbir serum örneği ve hiçbir RVP) kuyu kontrol sadece.</li>
	<li>96-şey V-alt plaka için bir kuluçka aktarmak ve plaka varlığında % 5 CO237 ° C'de 1 saat kuluçkaya. 96-şey V-alt plaka kuluçka iken, % 70 etanol ile kabine Biyogüvenlik yüzeyini temizlemek ve UV ışığı 15dk için çalıştırın.</li>
	<li>İyi bir steril 5 mL damlalıklı kullanarak hücreleri karıştırın ve hücre 5 mL steril 15 mL konik tüp transfer K562 hücrelerinin tamamen Konfluent bir T75 şişesi kuluçka makinesi çıkarın. Not: K562 hücre RPMI-10 içinde muhafaza ve 1 x 106/mL subcultured.</li>
	<li>Hücreleri steril 15 mL konik tüp sizden 10 µL hücreleri kaldırarak saymak ve 10 µL tryphan mavi ile karıştırın. 15 mL konik tüp hücrelerde toplam sayısını belirlemek için bir hemasitometre kullanarak hücreleri saymak.</li>
	<li>15 mL konik ~ 1200 x g hücreleri ile 10 dakika santrifüj kapasitesi, süpernatant dikkatle boşaltmak ve sıcak RPMI-10 saat 80.000 hücreler/30 µL medya (2,66 106 hücre /mL x) bir konsantrasyon hücreleri resuspend.</li>
	<li>96-şey V-alt plaka İnkübatör (Adım 1.6) kaldırmak, 96-şey V-alt plaka her şey için K562 hücre 30 µL aktarmak ve karıştırın iyice yukarı ve aşağı pipetting 5-10 kat. 96-şey V-alt plaka için bir kuluçka aktarmak ve plaka varlığında % 5 CO237 ° C'de 1 saat için kuluçkaya.</li>
	<li>1s için kuluçka sonra 96-şey V-alt plaka kuluçka ve santrifüj ~ 1200 x g, 5 dakikalığına kaldırın. Santrifüjü sonra plaka % 10 çamaşır suyu içeren bir kap içine baş aşağı çevirerek kuyuları medyadan dikkatle boşaltmak.</li>
	<li>Her şey hücrelerde 125 sıcak RPMI-10 µL içinde resuspending tarafından yıkama bir pipet ile iyice karıştırın ve ~ 1200 x g 5 dk. Decant medya için plaka plaka % 10 çamaşır suyu içeren bir kap içine baş aşağı çevirerek santrifüj kapasitesi. Bu yıkama iki kez tekrarlayın.</li>
	<li>Çamaşır sonra RPMI-10 her şey, damlalıklı ile yukarı ve aşağı karışımı sıcak 100 µL ekleyin ve %5 CO2huzurunda 37 ° C'de 48 h plaka kuluçkaya.</li>
</ol>2. gün 3
<ol><li>100 µL hücre ve medya/iyi kuluçka makinesi üzerinden içeren 96-şey V-alt plaka çıkarın ve bir biyogüvenlik kabini taşımak. Bir çok kanallı pipet, karışımı kullanarak içeriği her şey ve hücreleri transfer ve medyaya 96 iyi U-alt plaka veya 5 mL polipropilen borular için önceden etiketli.</li>
	<li>96-şey V-alt plaka %1 100 µL ile her kuyuya durulama Paraformaldehyde (PFA) 1 x fosfat tamponlu tuz (PBS) ve 96 de U-alt plaka veya etiketli 5 mL polipropilen borular içinde anılan sıraya göre wells transfer adım 2.1 bir son vermeye konsantrasyonu % 0,5 PFA/iyi veya tüp. İyice bir çok kanallı pipet kullanarak karıştırmak, alüminyum folyo ile plaka kapak ve kuluçka hücreleri düzeltmek 30 dakika boyunca oturmak izin.</li>
	<li>Akış Sitometresi ( Tablo reçetesi Örneğin bakınız) hazırlamak için yan ve floresan ayarların yanı sıra ileri dağılım kalibre günahı K562 hücreleri çalıştırarak. GFP hücrelerden yayılan tek Floresans sadece floresan kanal gerekli FL1, olduğu. Kazanmak ~ 30, 000 – 50.000 hücreleri her örnek. Not: Herhangi bir Akış Sitometresi tek bir floresan okuma yeteneğine sahip olarak RVPs ile enfekte hücreleri sadece GFP varlığı nedeniyle yeşil Floresans yayarlar ve diğer bir floresan kanal ihtiyaç vardır veri alma için kullanılabilir.</li>
</ol>3. veri analizi
<ol><li>
Herhangi bir Akış Sitometresi Analizi yazılım kullanarak Akış Sitometresi alınan verileri analiz (tablo malzemeleri görmek).
	<ol>
<li>Küme ilk kapısı, vs FSC-tek hücre eklemek veya auto-floresan birini analiz12den çıkartmak için H (ileri dağılım-yükseklik) FSC-A (ileri dağılım üzerinde – alan) temel. O zaman, singlet Geçitli hücreler SSC-A göre kapı (yan dağılım-alan) vs FSC-A herhangi bir autofluorescent enkaz dışlamak için.</li>
		<li>SSC-A vs GFP ifade tabanlı için SSC-A vs FSC-A Geçitli hücreler analiz GFP-A. GFP + hücrelerin etrafında bir kapı ayarlayarak GFP + hücreler her seyreltme serum ve denetim örnekleri için yüzdesini belirlemek.</li>
	</ol>
</li>
	<li>3 tarafından hücrelerinin toplam yüzdesi, GFP + onaylatılacak Wells bölerek GFP + hücreler her serum Numune dilüsyonu ve denetim wells için ortalama yüzdesini belirleyin.</li>
	<li>Kat-geliştirme enfeksiyon hücrelerinin GFP + serum numuneleri hücrelerinin GFP + RPMI-10 RVPs (serum örneği) denetim wells ile ortalama yüzde bölünen her seyreltme için ortalama yüzde bölerek hesaplayın.</li>
	<li>Kat-geliştirme (y ekseni) seyreltme karşı (x ekseni) grafik ve istatistiksel analiz Tukey'nın post-hoc testi birden çok karşılaştırmalar için ardından ANOVA kullanarak.</li>
</ol>

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Flaviviruses DENV ve ZIKV gibi önemli kanıt homoloji birbirleri ile13,14cross-react antikor üreten her iki onların yapısal ve yapısal olmayan proteinlerde paylaşmak. Bu cross-reactive antikor yanıt-e doğru geliştirmek gösterilmiştir enfeksiyonu ile ilgili vivo içinde ve vitro Contegra serotip veya diğer flaviviruses1,2. Çapraz enfeksiyon geliştirmek için potansiyel hasta...

Antikor bağımlı geliştirme (ADE) enfeksiyon sayede virüs serotip tarafından indüklenen kısmen cross-reactive antikor yanıt artırır artan viral çoğaltma ve viremi yol virüs, başka bir serotip alımını bir süreçtir. ADE kapsamlı dang virüs (DENV) enfeksiyonları dört büyük serotipleri yaygın nerede belgelemiştir. Hastaların alt kümede, ADE Dang kanamalı ateşi (DHF) ile ilişkilidir. Biz son zamanlarda Zika virüs (ZIKV) enfeksiyon DENV ADE içinde vitro neden oldu ve büyük olasılıkla DENV viremi vivo içinde1 iyileştirmesini katkıda DENV cross-reactive antikor yanıt düzeyi önemli ölçüde yüksek indüklenen göstermiştir , 2. antikor bağımlı geliştirme deneyleri vardır ilgili virüs ile ikincil enfeksiyon geliştirmek ve flavivirus enfeksiyonlarının patogenezinde içine değerli bilgiler sağlamak ve bilgilendirmek için antikorlar kapasitesini değerlendirmek için değerli bir araç aşıların geliştirilmesi.
Açıklanan tahlil DENV RVPs enfeksiyon için normalde söyleyebileceğin K562 hücreleri ile birlikte burada kullanır. RVPs tek bir çoğaltma3turdan sonra ifade edilir bir alt genomik yeşil flüoresan protein (GFP) replicon kodlamak yapısal olarak sağlam çoğaltma beceriksiz DENV viral parçacıkların vardır. Bu nedenle, RVPs ile enfekte olma hücreleri yeşil bulabilsem ve Akış Sitometresi veya mikroskobu kullanılarak kolayca algılanabilir. Bu tahlil kullanılan RVPs ticari kaynaklardan elde edilmiştir. Onlar ancak, olabilir diğer virüslere karşı oluşturulan ve bu el yazması açıklanan tahlil kullanılır. Bu arada, K562 hücreler antikorlar Fc bölgesine bağlamak ve alt antikor4,5konsantrasyonları nötralize huzurunda bulaşabilir bir FcγIII reseptör ifade lösemi hücre satırı vardır.
ADE deneyleri kapsamlı bir şekilde kullanıldığını şiddetli Dang ve vitro ADE mekanizmaları betimlemek için risk faktörleri araştıran çalışmalarda6,7,8. Burada açıklanan ADE tahlil vitro enfeksiyon RVPs kullanarak geliştirmek ve Akış Sitometresi ile karşılaştırıldığında ya gerektiren kullanılmakta, diğer deneyleri plak şekillendirme belirlenmesi için serum kapasitesini belirlemek için hızlı ve kolay kullanılabilir ikisi de zaman alıcı ve emek hücreleri6,7,8,9,10,11, birimleri (pfu) Vero hücreleri veya bir antikor boyama enfekte yoğun.

<table><tbody><tr><td>DENV 1-4 RVP</td><td>Integral Molecular</td><td>RVP-501</td><td></td></tr><tr><td>K562 cells</td><td>ATCC</td><td>CCL-243</td><td></td></tr><tr><td>RPMI</td><td>Corning</td><td>10-040-CV</td><td></td></tr><tr><td>FBS</td><td>GE lifesceinces</td><td>SH 30910.03</td><td>10% FBS in RPMI-10</td></tr><tr><td>Penicillin-Streptomycin</td><td>MP biomedicals</td><td>1670049</td><td>100 IU Pen/mL, 100 ug Strep/mL in RPMI-10</td></tr><tr><td>HEPES</td><td>Cellegro</td><td>25-060-Cl</td><td>0.025M in RPMI-10</td></tr><tr><td>MEM Non-essential Amino Acid Solution (100&amp;times;)</td><td>Sigma</td><td>M7145</td><td>1x in RPMI-10</td></tr><tr><td>Sodium Pyruvate</td><td>Cellegro</td><td>25-000-Cl</td><td>1mM in RPMI-10</td></tr><tr><td>L-glutamine</td><td>Fisher Scientific</td><td>MT25005CI&amp;nbsp;</td><td></td></tr><tr><td>Sterile V-bottom plates</td><td>Thomas Scientific</td><td>333-8001-01V</td><td></td></tr><tr><td>BD Falcon Polypropylene 5 ml FACS tubes</td><td>VWR</td><td>60819-728</td><td></td></tr><tr><td>Non-sterile U-bottom plates</td><td>Falcon</td><td>Ref 353910</td><td>A high throughput alternative to FACS tubes</td></tr><tr><td>5mL, sterile, serological pipette</td><td>Denville</td><td>P7127</td><td></td></tr><tr><td>200uL sterile pipette tips</td><td>Denville</td><td>P3020 CPS</td><td></td></tr><tr><td>20uL sterile pipette tips</td><td>Denville</td><td>P1121</td><td></td></tr><tr><td>50-200uL multichannel pipette</td><td>Denville</td><td>P3975-8-B</td><td></td></tr><tr><td>5-50uL multichannel pipette</td><td>Denville</td><td>P3975-9-B</td><td></td></tr><tr><td>20% formadehyde</td><td>Tousimis</td><td>&amp;nbsp;#1008B</td><td></td></tr><tr><td>Water Bath</td><td>ThermoFisher</td><td>TSCOL35</td><td></td></tr><tr><td>thermomixer</td><td>Eppendorf</td><td>2231000387</td><td>An alternative to a waterbath for heat inactivation</td></tr><tr><td>CO2 Incubator</td><td>ThermoFisher</td><td>13-998-213</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>E7023</td><td></td></tr><tr><td>Liquid Bleach</td><td>Fisher Scientific</td><td>NC9724348</td><td></td></tr><tr><td>Flowjo 9.8</td><td>TreeStar, Inc.</td><td></td><td>Flow cytometry analysis software</td></tr><tr><td>BD FACSDiva 6.1.2</td><td>Becton Dikinson</td><td></td><td></td></tr><tr><td>BD LSR II flow cytometer</td><td>Becton Dikinson</td><td></td><td></td></tr><tr><td>Liquid Bleach</td><td>Fisher Scientific</td><td>NC9724348</td><td></td></tr></tbody></table>

a simple flow cytometry based assay to determine in vitro antibody dependent enhancement of dengue virus using zika virus convalescent serum

Antikor bağımlı geliştirme enfeksiyon Dang viral patogenezinde önemli bir rol oynamaktadır göstermiştir. Antikorlar veya serum enfeksiyon söyleyebileceğin hücre hatlarında geliştirmek kapasitesini ölçmek geleneksel deneyleri üzerinde kullanarak viral çıkış plak deneyleri tarafından takip medya enfeksiyon ölçmek için güveniyorlar. Son zamanlarda, bu deneyleri dang virüs (DENV) enfeksiyonu fluorescently etiketli antikorları kullanarak hücre hatlarında inceledik. Her iki bu yaklaşım bu teknikler yaygın kullanımı kısıtlamak kısıtlamalar bulunmaktadır. Burada, biz yeşil flüoresan protein ve K562 hücreleri antikor bağımlı geliştirme (ADE) DENV enfeksiyon rhesus elde edilen serum kullanarak incelemek için hızlı dang virüs muhabir viral parçacıkların (RVPs) kullanarak bir basit vitro tahlil tarif maymunların Zika virüs (ZIKV) ile enfeksiyon sonra 16 hafta. Bu teknik güvenilirdir, hücreleri en az manipülasyonu içerir, canlı çoğaltma yetkili virüs kullanımı gerektirmez ve Akış Sitometresi kullanarak bir nicel okuma almak için yüksek işlem hacmi biçimde gerçekleştirilebilir. Ayrıca, bu tahlil kolayca antikor bağımlı geliştirme (ADE) sarı humma virüsü (YFV), Japon Equine Ensefalit virüsü (JEEV), Batı Nil virüsü (WNV) burada RVPs kullanılabilir vb gibi diğer flavivirus enfeksiyonların incelemek için adapte edilebilir. Tahlil ayarlama kolaylığı, veri analizi ve sonuçları yorumlama çoğu laboratuvar ayarları için son derece uysal yapar.

Sera 4 rhesus makak ZIKV ile enfeksiyon sonra toplanan 16 hafta vardı ve potansiyellerini DENV-1 geliştirmek test üzerinden, 2, 3 ve 4 enfeksiyon K562 hücrelerdeki. Hayvanlar tepe viremi ~ 1 105 kopya ZIKV RNA/mL plazma x 7 gün sonra enfeksiyon tarafından algılamayı sınırın altındaki edildi düzeyleri reddetti enfeksiyon sonra 3 gün vardı. Hiçbir viremi 16 hafta sonrası enfeksiyon, plazma algılandı. O zaman, RVP tahlil gerçekleştirilmiş ve toplanan verileri...

Watch this Scientific Journal Video about A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum at JoVE.com

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

Biz enfeksiyon antikor bağımlı geliştirme tarafından Zika virüs nekahat serum dang virüs muhabir Viral parçacıkların kullanarak ölçmek için basit ve kolay bir iletişim kuralı tanımlamak.

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 ...

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Universidad de Cartagena UDEC

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JoVE Journal

Immunology and Infection

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

8.7K Görüntüleme.  Uniformed Services University. Biz enfeksiyon antikor bağımlı geliştirme tarafından Zika virüs nekahat serum dang virüs muhabir Viral parçacıkların kullanarak ölçmek için basit ve kolay bir iletişim kuralı tanımlamak.

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

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Çoğaltılmış izotermal amplifikasyon Zika, Chikungunya ve dang 1 tespit etmek için teşhis platformu dayalı

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biyokimya

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 ...

İmmünoloji ve Enfeksiyon

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.

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.

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 ...

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 ...

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 ...

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.

Farede Birden Fazla Organdan Zika Virüsünün İzolasyon ve Nicelikselleştirilmesi

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

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Zika Virüsü Kullanan Aedes aegypti Sivrisineklerinde Vektör Yetkinlik Analizleri

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Virus Propagation and Cell-Based Colorimetric Quantification

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and potentially Guillain-Barr&#233; syndrome in adults. Research on ZIKV to understand the disease mechanisms is important to facilitate vaccine and treatment development. The method of quantifying viruses is crucial and fundamental in the field of virology. The focus forming assay (FFA) is a virus quantification assay that detects the viral antigen with antibodies and identifies the infection foci of cells using the peroxidase immunostaining technique. The current study describes the virus propagation and quantification protocol using both 24-well and 96-well (high throughput) formats. Compared with other similar studies, this protocol has further described foci size optimization, which can serve as a guide to expand the use of this assay for other viruses. Firstly, ZIKV propagation is performed in Vero cells for 3 days. The culture supernatant containing ZIKV is harvested and quantitated using the FFA. Briefly, the virus culture is inoculated onto Vero cells and incubated for 2-3 days. Foci formation is then determined after optimized staining processes, including cell fixation, permeabilization, blocking, antibody binding, and incubation with peroxidase substrate. The stained virus foci are visualized using a stereo microscope (manual counting in 24-well format) or software analyzer (automated counting in 96-well format). The FFA provides reproducible, relatively fast results (3-4 days) and is suitable to be used for different viruses, including non-plaque-forming viruses. Subsequently, this protocol is useful for the study of ZIKV infection and could be used to detect other clinically important viruses.

The present protocol describes the propagation of Zika virus (ZIKV) in Vero African green monkey kidney cells and the quantification of ZIKV using cell-based colorimetric immunodetection methods in 24-well and 96-well (high throughput) formats.

Virüs Yayılımı ve Hücre Bazlı Kolorimetrik Miktar Tayini

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and ...

Cell-Based Electrical Impedance Platform to Evaluate Zika Virus Inhibitors in Real Time

Cell-based electrical impedance (CEI)&#160;technology measures changes in impedance caused by a growing or manipulated adherent cell monolayer on culture plate wells embedded with electrodes. The technology can be used to monitor the consequences of Zika virus (ZIKV) infection and adherent cell replication in real time, as this virus is highly cytopathogenic. It is a straightforward assay that does not require the use of labels or invasive methods and has the benefit of providing real-time data. The kinetics of ZIKV infection are highly dependent on the employed cell line, virus strain, and multiplicity of infection (MOI), which cannot be easily studied with conventional endpoint assays. Furthermore, the CEI assay can also be used for the evaluation and characterization of antiviral compounds, which can also have dynamic inhibitory properties over the course of infection. This methods article gives a detailed explanation of the practical execution of the CEI assay and its potential applications in ZIKV research and antiviral research in general.

Here, we demonstrate the use of cell-based electrical impedance (CEI) as a very easy and straightforward method to study Zika virus infection and replication in human cells in real time. Furthermore, the CEI assay is useful for the evaluation of antiviral compounds.

Zika Virüsü İnhibitörlerini Gerçek Zamanlı Olarak Değerlendirmek için Hücre Tabanlı Elektriksel Empedans Platformu

Cell-based electrical impedance (CEI)&#160;technology measures changes in impedance caused by a growing or manipulated adherent cell monolayer on culture ...

An In Vitro Assay for Screening Interferons as Potent Inhibitors of Zika Virus Growth

Source: Contreras, D. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54767">Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons.</a> J. Vis. Exp. (2016)
The video demonstrates an in vitro assay for screening interferons with antiviral activity against the Zika virus, a human pathogen. Within the host cell, the Zika virus undergoes multiplication upon internalization, infecting neighboring cells and creating distinct zones or viral plaques. Type I interferon, acting as an antiviral agent, hinders virus multiplication, thereby preventing the formation of viral plaques in the host cell monolayer.

Zika Virüsü Büyümesinin Güçlü İnhibitörleri Olarak İnterferonları Taramak için İn Vitro Test

1. Screening Cytokine Library against Zika Virus Infection

	Seed naive Vero cells at 1 x 105 cells per well using a 12-well plate. Once the cells are ...

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

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