We thank F. Chisari for providing Huh-7.5.1 cell line. We would like to thank Justine Ho for editing the manuscript. This work was supported by Cedars-Sinai Medical Center Institutional Programmatic Research Award and National Center for Advancing Translational Sciences, Grant UL1TR000124 to V.A.

Protokolün genel hatları Şekil 1 &#39;de gösterilmiştir. 1.. Hücreler <ol><li> 10-15% fetal sığır serumu (FBS), 10 mM esansiyel olmayan amino asitler, 10 mM Hepes, penisilin (100 birim / ml), streptomisin (100 mg / ml) ve 2 mM L-glutamin içeren tam büyüme ortamı hazırlayın. </li><li> Hepatit C viral replikasyon döngüsünün in vitro analizi için, yukarıdaki takviyeleri ihtiva eden tam bir büyüme ortamı içinde Huh-7.5.1 hücreleri 13 koruyun. </li><li> % 5 CO2 ile 37 ° C &#39;de belirtilen desteklenmiş büyüme ortamı ile Huh-7.5.1 hücrelerinde kültür viral suşları. </li></ol> 2.. Virüs ve Plasmid yapılar <ol><li> HCV RNA&#39;nın tamamlayıcı DNA (cDNA) formunu oluşturmak ve genetik manipülasyon kolaylığı için bir plazmid vektörü içine klonlamak. Not: Japon fulminant hepatit 1 keşfi, JFH-1 (genotip 2a HCV), HCV araştırma için kritik olan ve olduğunu sahiptir izolatöncelikle HCV çoğaltma döngüsü 11-13 incelemek için kullanılır. JFH-1 HCV göre arası ve intragenotypic kimerik virüsler genellikle 14-18 araştırma kullanılır. Sentetik intragenotype 2a kimerik virüs, FNX-HCV, ve bir monosistronik kimerik muhabir gerginlik İnşaat önceden 19 ve yapı detayları bu protokol kapsamı dışındadır tarif edilmiştir. </li><li> O (nükleotidler 1-2878) J6CF ana suş (NCBI erişim no. AF177036), yanı bir 5&#39;NTR, yapısal bölgeleri ve P7, NS2 ve yapısal olmayan bölgelerin bir parçası içerdiği pFNX-HCV intragenotype 2a virüsü kullanarak de JFH-1 virüs suşu (NCBI erişim no. AB047639) &#39;in yapısal olmayan bileşenler olarak. Not: FNX-HCV Jc1 intragenotype 2a kimerik HCV&#39;nin sekansına göre sentezlendi önce 15 bildirdi. </li><li> Bir R takarak (adım 2.2 yukarıda) pFNX-HCV gerginlik dayalı bir monosistronik kimerik muhabir viral yapıyı, pFNX-Rluc, üret5 &#39;ve NTR (nt 388 ve 389 arasında) Çekirdek geni arasındaki enilla lusiferaz gen. Daha sonra, bölünme sinyali olarak çalışacak bir ayak ve ağız hastalığı virüsü 2A (F2A) peptid dizisi kullanılarak lusiferaz geni ve bu yapının çekirdek gen bağlayın. </li><li> PFNX-HCV veya pFNX-Rluc viral bir arka plan kullanarak RNA polimeraz null (Pol-) viral yapı mühendisi. , AAG amino asit tortuları ile, bu viral omurgaların birinin, NS5B polimeraz katalitik kalıntıları, GDD (nt 8615-8623 aa 2759-2761) değiştirin. </li></ol> 3.. In Vitro HCV RNA transkripsiyon <ol><li> Bir intragenotype 2a kimerik virüs FNX-HCV ve HCV-FNX Pol, null viral replikasyon (Şekil 2A) değerlendirilmesi için (Pol-) HCV kullanın. </li><li> Xbal kısıtlama enzimi ile linearize plazmid, viral ve sonra kör uçlar elde etmek için mung fasulye nükleaz ile muamele. Anyon değişim kromatografisi ile sindirilmiş plazmid arındırın. O bütünlüğünü doğrulayınf jel elektroforezi (Şekil 2B) agaroz DNA tabi tutulması ile doğrusallaştırılmış plasmid. </li><li> T7-RNA polimerazı kullanılarak doğrusallaştırılmış viral plazmid uyarlamak. </li><li> Bir RNA saflaştırma kiti kullanılarak yeni sentezlenmiş DNase ile muamele edilmiş RNA arındırın. </li><li> , Agaroz jel elektroforezi (Şekil 2C) ile RNA üretimini kontrol edin. </li><li> Spektrofotometresi ile RNA niceliğini. </li><li> 10 ug çalışma alikotlar içinde -80 ° C &#39;de üretilen RNA saklayın. Not: Aynı anda her viral numune ve kontrol için tüm RNA transkripsiyonu ile her bir deney tasarımı içinde RNA kalitesi varyasyonu en aza indirmek için. </li></ol> 4. HCV RNA Transfeksiyonu ve Örnek Toplama <ol><li> Tripsin kullanılarak Huh-7.5.1 hücreler hasat. </li><li> Hücreler, santrifüj yıkayın ve soğuk düşük serum ortamı ile iki kez süspansiyon tekrar süspansiyon. Ml başına 1 x 10 7 hücre düşük serum medya hücreleri tekrar süspansiyon. </li><li> 10 &amp; # toplam Mix956, bir 0,4 cm&#39;lik bir elektroporasyon küveti içinde yeniden süspansiyon haline getirilmiş hücreler (4 x 10 6 hücre) 400 ul ile transkribe viral RNA g. , 270 V de elektroporasyon ile 100 Ω ve 950 uF hücreleri transfekte. </li><li> % 15 FBS, 10 ml tam büyüme ortamı içinde elektroporasyona hücrelerin yeniden süspanse edin. Not: Huh-7.5.1 hücreleri% 15 Fetal Bovine Serumu kültürlendiğinde Elektroporasyona tabi tutulan hücrelerin artan hayatta kalma görülmektedir. </li><li> 4, 48 ve 96 saat: her ikisi de T-25 şişelerinde (kap basma 1.2 x 10 ~ 6 hücreleri) ve 48 kuyucuklu plaklar içinde, aşağıdaki zaman noktalarında her biri için (oyuk başına 1 x 10 4 hücreleri) hücrelerin Plate. </li><li> Kültür şişelerinden ve levhalardan ölü hücre kalıntılarını uzaklaştırmak üzere,% 10 FBS katkılı taze büyüme ortamı ile 4-8 saat post-transfeksiyon ile Ortamı değiştirin. </li><li> 48, 96 saat zaman noktalarında, ve 4 ° C&#39;de 10 dakika boyunca 1500 rpm&#39;de santrifüj ile hücreler toplandı örnekleri hücre tortularını çıkarmak hücre kültürü süpernatanları hasat º C </li><li> -80 ° C de hücresiz süpernatantlar Mağaza Lyse Western blot ile protein ve RNA analizi için ve belirtilen zaman noktalarında transkripsiyon-nicel PCR ters hücreleri. </li></ol> 5.. HCV genom kopyalarının değerlendirilmesi için Transkripsiyon-kantitatif PCR (RT-qPCR) Ters <ol><li> HCV genomik RNA kopya sayısını belirlemek için iki adımlı bir RT-qPCR gerçekleştirin. </li><li> Ters uyarlamak 1 ters transkriptaz ve 5&#39;NTR (JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3 &#39;) bağlanan HCV duyu şeridi için spesifik bir primer kullanılarak toplam hücresel RNA ug veya temizlik gen Peptidilprolil izomeraz G (PPIG R, için özel bir primer : 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3 &#39;). Ayrıca, HCV sense iplik primeri kullanılarak bilinen genom kopyalarının (10 1 9 standart 10) (Kademe 3 de üretilen) FNX-HCV RNA nın ters transkripsiyonunu. </li><li> (JFH RT spesifik HCV primerler kullanılarak elde edilen transkripsiyonu cDNA 50 ng kullanılarak qPCR yürütmekQF: 5&#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3 &#39;) ve DNA qPCR süper karışımını içeren yeşil boya bağlayıcı. Hem de temizlik gen PPIG için qPCR yapın (primerler PPIG F: 5&#39;-GAAGAGTGCGATCAAGAACCCATGAC-3 &#39;; PPIG R: 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#39;) Not: örnekleri arasında, doğru hücre HCV RNA seviyesini hesaplamak için, cep temizlik geni PPIG kullanımı , normalleşme için ekspresyon seviyesi (Ct çevrimi). Kopya sayısının saptanması için standart olarak FNX-HCV genomunun kopya numarasını kullanın. </li><li> 15 saniye için 95 ° C ve 60: qPCR çalışan HCV RNA kopya sayısını belirlemek için, aşağıdaki koşulları kullanarak &#160; Gerçek zamanlı PCR sistemi kullanılarak 30 saniye (40 devir) için C numara yerine. </li><li> Genom replikasyonu için sonuçları Şekiller 3A ve 3B bakınız. </li></ol> 6.. Western Blot Analizi HCV Protein İfadesi (Şekil 3C) algılama için <ol><li> Hücre lizatları, fr kullanınom viral RNA protein, western blot analizi için 96 saat sonrası transfeksiyon transfekte. </li><li> SDS-PAGE ve poliviniliden difluorür (PVDF) membran transfer kullanılarak hücre lizatı giderin. </li><li> % 5 kaymağı alınmış süt, fosfat tamponlu tuzlu su (PBS) içinde% 0.2 Tween 20 ihtiva eden bir blokaj çözeltisi kullanılarak membran bloke eder. </li><li> Primer fare monoklonal antikoru NS3 ile membran (1 1000 sulandırma) ve beta-aktin (1 5000 sulandırma) inkübe edin. </li><li> Turp peroksidaz (1 5000 sulandırma) konjuge keçi anti-fare IgG&#39;si ilave edin ve kimyasal-ışıma ile tespit. </li></ol> 7. İmmünofloresan Deneyi (IFA) <ol><li> Imüno-flüoresan deneyi için -20 ° C&#39;de 30 dakika boyunca metanol kullanılarak HCV-enfekte edilmiş ve transfekte hücreler düzeltildi. </li><li> PBS ile üç kez yıkanır ve hücre IFA bloklama tamponu ile bloklayın. </li><li> Tavşan poliklonal veya fare monoklonal anti-DSR (lütfen Dr Dasgupta&#39;yı, UCLA tarafından sağlanan), anti-NS5A öncelikle antikor kullanınNA antikoru 1:200 (1 ug / ml) içinde bir seyreltmede J2 ve 5 saat için bir gece boyunca 4 ° C&#39;de inkübe edin º C </li><li> Üç kez birincil antikor ardından hücrelerin PBS ile yıkayın. </li><li> • Keçi anti-tavşan IgG-488, poliklonal antikor veya ikincil bir 1:1000 seyreltmesi (1 ug / ml) keçi anti-fare IgG-594 poliklonal ikincil antikor ilave edin ve oda sıcaklığında 1 saat boyunca inkübe edilir. </li><li> PBS floresan mikroskop (Şekil 3D) kullanarak Hoechst boyası ve görünümü kullanarak üç kez ve leke çekirdekler hücreleri yıkayın. </li></ol> 8. Ölçüm Virüs titresi <ol><li> Levha naif Huh-7.5.1 hücreler yaklaşık 3 x 10 3 hücre / oyuk, 96 oyuklu bir plaka kullanılmıştır. Ertesi gün, büyüme ortamı kullanılarak HCV RNA transfekte edilmiş hücrelerin hasat hücresiz kültür yüzer 10-kat seri dilüsyonları gerçekleştirmek ve Huh-7.5.1 hücreler üzerine, üç kopya halinde inoküle. </li><li> -20 ° C&#39;de 30 dakika boyunca metanol (kullanarak 72 saat sonrası enfeksiyon hücreleri saptamak </suC) ve Bölüm 7&#39;de belirtildiği gibi, HCV NS5A proteini için immunostain ° p&gt;. </li><li> NS5A pozitif odak saymak ve mililitre başına odak noktası oluşturan birimin ortalama sayısı (FFU) hesaplamak için yüksek seyreltme kullanın. HCV titresi için Şekil 4&#39;e bakınız. </li></ol> Viral genom replikasyonu ve enjeksiyon için 9. Renilla Lusıferaz Muhabir Deney <ol><li> Viral genomun replikasyonu, 48 oyuklu plakalarda üç kere levha HCV RNA-elektroporate Huh-7.5.1 hücreleri (1 x 10 4 hücre / çukur) değerlendirilmesi için. </li><li> Lyse 6 saat sonra pasif lizis tamponu 75 ul, 48 saat ve 96 saat post-transfeksiyon ile hücreler. </li><li> -80 ° C, oda sıcaklığı ve mağazada, 15 dakika boyunca yavaşça plakaları sallayın </li><li> , Renilla lusiferaz enzim aktivitesinin ölçülmesi, oda sıcaklığına kadar protein lisat ve Renilla lusiferaz deney reaktif çözülme için. </li><li> , 96 wel oyuk başına protein lizat 20 ul ekle<html> l beyaz ışıldama plakası ve bir luminometre içerisinde plaka yerleştirilir. </li><li> Renilla lusiferaz analiz ayıraçı (coelenterazine + alt-tabaka tamponu) ile kaplanır ve 2 saniye ön okunan bir süre sonra, 100 ul koyun; 10 saniye boyunca yayılan ışık entegre. </li><li> Biyolojik üçlü lusiferaz değerleri her bir numune için ortalama ve standart sapmayı hesaplayın ve istatistik analiz için verileri (Şekil 5) tabi tutulması. </li><li> Enfeksiyon değerlendirmek için, 48-çukurlu plakalarda naif Huh-7.5.1 hücreler üzerine üç kopya olarak belirtilen zaman noktalarında (48 saat ve 96 saat) için HCV RNA-transfekte edilmiş hücrelerden elde edilen hücre içermeyen yüzer 500 ul inokülasyon. </li><li> De sonra 6 saat sonrası enfeksiyon başına taze orta 500 ul ile viral aşının değiştirin. </li><li> Lyse 8,2-8,7 (Şekil 5) adımda tarif edildiği gibi Renilla lusiferaz deneyi için 48 saat post-enfeksiyon ve konuya hücreler. </li></ol>itle &quot;&gt; 10. İstatistiksel Analiz <ol><li> Grafiklerde hata çubukları standart sapmalar (SD) gösterir. P değerleri, eşleştirilmemiş t testi ile hesaplanmıştır. </li></ol>

<ol>
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The authors have nothing to disclose.

Bu şekilde, hepatit C virüs çoğaltma döngüsü analiz etmek için bir yöntem tarif eder. HCV insan patojen ve öngörülen biyogüvenlik protokolü kesinlikle takip edilmesi gerekir. Bulaşıcı HCV hücre kültür sistemleri, daha önce 11-13,16,17 tarif edilmiştir. Resimli protokolü takip ederken biz uygulamak birkaç önemli nokta vardır. İlk olarak, alt çalışmalar için olduğu gibi tam uzunlukta viral genomik RNA&#39;nın iyi kalitede olması için büyük önem taşımaktadır. Viral cDNA&#...

Hepatit C virüsü (HCV), siroz ve karaciğer kanserine neden olur. Bu 350,000 kişi yılda 1-3 ölüyor, dünya çapında 170 milyon kişiyi etkilemektedir. HCV 9.6 kb&#39;lik bir genom boyutuna sahip bir pozitif iplikli bir RNA virüsüdür. HCV genomu, proteolitik olarak 10 polipeptitlere çeşitli hücresel ve viral proteazlar tarafından yarılır ~ 3000 amino asit kalıntıları tek bir poliprotein olarak tercüme edilir. HCV cinsi Hepatit C virüs in prototip virüsüdür ve Flaviviridae familyası 4 aittir. Maruz kalması üzerine, HCV bireylerin% 80, kronik enfeksiyon oluşturur. Enfeksiyon tanısı karaciğer bozulmasını önlemek için terapötik müdahaleye izin verebilir çoğunlukla asemptomatik ve zamanında. Güncel Tedavinin yetersiz olduğunu ve hiç aşı 5,6 mevcuttur. Hepatit C etyolojisi ilk 1989, 7 &#39;de tarif edilmiştir. HCV replikasyonu incelenmesi hepatit C aşısı ve tedavi araştırmalar için önemlidir, ancak bu olmuştuuzun süre etkili bir viral kültür sisteminin olmaması nedeniyle engellenmektedir. HCV&#39;nin moleküler klon intrahepatik, aşılamadan 8 üzerine şempanzelerde enfeksiyon olduğu gösterilmiştir. Daha sonra, HCV alt-genomik replikonlar, bir hücre kültür sistemi 9,10 viral genom çoğaltma aşamasını incelemek için izin verilmiş olan, tarif edilmiştir. Bir genotip 2a HCV Discovery JFH-1 (Japon fulminan hepatit-1) izole, hücre kültürü enfekte edebilen HCV çoğaltma araştırma 11-13 için yeni yollar açtı. Genotip 2a gerginlik JFH-1 arası ve içi genotipik kimerik virüs ve genotip 1 HCV bazlı bazlı bulaşıcı kültür sistemleri de 14-18 vardır. Biz başarıyla protein alanları ve sis etkili RNA elemanlarının 19,20 yüksek çözünürlüklü fonksiyonel profil haritasını elde etmek JFH-1 suşu ve HCV intragenotype 2a kimerik virüs kullandık. Buna göre, burada biz olanak rutin kullanılan etkili bir kültür sistemi açıklanmaktadırHCV çoğaltma döngüsü ve konak-patojen etkileşimi çeşitli aşamaları okuyor. Bu viral genomun replikasyonu ve intragenotype 2a HCV ve bir Renilla lusiferaz raportör göre HCV de novo enfeksiyon değerlendirmek virolojik ölçümlerde mevcut.

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			<td height="21" style="height:21px;width:569px;">Name of Material/ Equipment</td>
			<td style="width:267px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">10-017-CV</td>
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			<td height="21" style="height:21px;width:569px;">Non essential amino acid</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">MT25025CI</td>
			<td></td>
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			<td height="21" style="height:21px;width:569px;">HEPES</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">15630080</td>
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			<td height="21" style="height:21px;width:569px;">Glutamax</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
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			<td height="21" style="height:21px;width:569px;">Opti-MEM Reduced Serum Medium,no Phenol Red</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11058-021</td>
			<td></td>
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			<td height="21" style="height:21px;width:569px;">Huh-7.5.1</td>
			<td style="width:267px;">The Scripps Research Institute</td>
			<td style="width:119px;"></td>
			<td>The cell line was kindly provided by Dr. Francis Chisari to Dr. Arumugaswami under executed MTA between The Scripps Research Institute and Cedars-Sinai Medical Center</td>
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			<td height="23" style="height:23px;width:569px;">Plasmids (pFNX-HCV, pFNX-HCV Pol null, pFNX-Rluc, and pFNX-Rluc Pol null)&#160;</td>
			<td style="width:267px;">Cedars-Sinai Medical Center</td>
			<td style="width:119px;"></td>
			<td>The HCV plasmids were synthesized by Dr. Arumugaswami using overlapping oligo-nucleotides.&#160;</td>
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			<td height="21" style="height:21px;width:569px;">XbaI</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">R0145S</td>
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			<td height="21" style="height:21px;width:569px;">Mung Bean Nuclease</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">M0250S</td>
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			<td height="21" style="height:21px;width:569px;">T7 RiboMAX Express Large Scale RNA Production System</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">P1320</td>
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			<td height="21" style="height:21px;width:569px;">Rneasy Mini Kit</td>
			<td style="width:267px;">Qiagen</td>
			<td style="width:119px;">74104</td>
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			<td height="21" style="height:21px;width:569px;">Nanodrop 2000</td>
			<td style="width:267px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Electroporation Cuvette (4 mm)</td>
			<td style="width:267px;">Bioexpress</td>
			<td>E-5010-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Gene Pulser Xcell Total System</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">165-2660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-dsRNA antibody J2&#160;</td>
			<td>English &#38; Scientific Consulting Kft.</td>
			<td style="width:119px;">10010200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 488</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 594</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVDF membrane package</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">162-0263</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Blotting Grade Blocker Non Fat Dry Milk</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6404XTU</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Tween-20</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6531XTU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [8 G-2]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab65407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [H23]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab13830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td style="width:119px;">115-035-003</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Amersham ECL Prime Western Blotting Detection Reagents&#160;</td>
			<td style="width:267px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2236</td>
			<td></td>
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			<td height="21" style="height:21px;width:569px;">SUPERSCRIPT III RT&#160;</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">18080085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SYBR QPCR SUPERMIX W/ROX</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11744500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ViiA 7 real-time PCR system</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Renilla Luciferase Assay System kit</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">E2810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">RNase-Free DNase</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">M6101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">GloMax-Multi Detection System (Luminometer)</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for analyzing hepatitis c virus replication

Hepatit C Virüsü (HCV) dünya nüfusunun% 3 etkiler ve kronik hepatit, siroz ve hepatosellüler karsinom gibi ciddi karaciğer rahatsızlıkları olur. HCV, Flaviviridae ailesine ait zarflı bir RNA virüsüdür. Mevcut tedavi, tam etkili değildir ve istenmeyen yan etkilere neden olur. Hiçbir HCV aşısı yoktur. Bu nedenle, devam eden bir çaba bir aşı ve daha iyi bir tedavi geliştirmek için gereklidir. HCV bir hücre kültür sistemi viral girişi, genom replikasyonu, paketleme, ve çıkış da dahil olmak üzere HCV gelişmesinin çeşitli aşamalarında çalışmaları için önemlidir. Sunulan mevcut prosedüründe, bir vahşi tip intragenotype 2a kimerik virüs, FNX-HCV ve virüs çoğaltma incelemek için bir Renilla lusiferaz haberci genini taşıyan bir rekombinant virüs FNX-Rluc kullanılır. Bir insan hepatoma hücre hattı (Huh-7 göre) in vitro transfeksiyonu için kullanılmıştır HCV genomik RNA transkripsiyonu. Hücresiz kültür üst sıvıları, protein lizatlan ve tla m HCV RNA büyümeyi değerlendirmek için post-transfeksiyon işaret çeşitli zaman hasat edilmiştir. HCV genomu, çoğaltma durum nicel RT-PCR ve HCV varlığının görüntülenmesi iki-şeritli RNA tarafından değerlendirildi. HCV protein ekspresyonu HCV NS3 ve NS5A proteinleri için spesifik antikorların kullanıldığı Western blot ve imüno deneyleri ile doğrulanmıştır. Kültür yüzer ve viral titresi olarak bulaşıcı parçacıklar serbest HCV RNA transfekte edilmiş hücreler ölçülmüştür. Lusiferaz deneyleri, raportör HCV&#39;nin replikasyon seviyesi ve enfeksiyon riskini değerlendirmek için kullanılmıştır. Sonuç olarak, HCV replikasyon döngüsünün farklı aşamalarında karakterize etmek için çeşitli virolojik ölçümlerde mevcut.

Hepatit C virüs, bir RNA virüsüdür. Bu nedenle, genetik manipülasyon amaçla, HCV genomik cDNA bakteriyel bir plasmid vektörüne klonlanmıştır. A T7 RNA polimeraz promotörü sekans, HCV genomunun 5 &#39;ucuna hemen önce başlandı. HCV analiz akışı bir genel hatları Şekil 1 &#39;de sunulmuştur. Tam 3&#39; ucuna sahip HCV genomik RNA oluşturmak için, plazmit ihtiva eden HCV genomu, Xba I kısıtlama enzimi ile oluşturulan tek sarmallı çıkıntı mung fasulye nükleaz sindir...

Watch this Scientific Journal Video about A Protocol for Analyzing Hepatitis C Virus Replication at JoVE.com

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatit C Virüsü Çoğaltma Analizi için Protokol

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

a-protocol-for-analyzing-hepatitis-c-virus-replication

Research

JoVE Journal

Immunology and Infection

23.9K Görüntüleme.  Cedars-Sinai Medical Center. Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle. 

Video: A Protocol for Analyzing Hepatitis C Virus Replication

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Yılları arasında sinerjistik Araştırma Bioluminescent Görüntüleme kullanma Streptococcus pneumoniae Bebek Fare ve İnfluenza A Virüsü

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

İmmünoloji ve Enfeksiyon

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Model Herpesvirüs Litik çoğaltma Diseksiyon Host-virüs Etkileşim

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes Çalışmaları A Primer Nöron Kültür Sistem Virüs gecikmesi ve Yeniden Simplex

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Fenotipinin yüksek verim Deneyi<em&gt; Salmonella enterica</em&gt; Typhimurium Derneği, Invasion, ve Makrofagların Çoğaltma

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Hepatit C Virüsü Karşı İnsan laktoferrinin Hücresel Alımı ve İlişkili Antiviral Aktivite Monitör İmmünofloresan

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

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

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A Virüsü hemaglutininler aviditesi ve özgünlüğünün değerlendirilmesi için Minyatür Glycan Mikroarray Testi

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Bir Hepatit B Virüsü Muhabir Sisteminin Geliştirilmesi Çoğaltma Döngüsü Erken Aşamaları Monitör

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

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 ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

"Karaciğer-on-a-Chip" kültürleri birincil tetkikine ve Kupffer hücreleri için Hepatit B virüs enfeksiyonu

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

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 ...