Name: development of a hepatitis b virus reporter system to monitor the early stages of the replication cycle
Uploaded: 2017-02-01T13:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle at JoVE.com

This work was supported in part by the Research Program on Hepatitis from Japan Agency for Medical Research and Development (AMED) and by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

Reporter protein kodlayan rekombinant HBV 1. Üretim <ol><li> HepG2 hücrelerinin hazırlanması <ol><li> Hücre kültürü ortamı hazırlayın (Dulbecco değiştirilmiş Eagle ortamı,% 10 fetal sığır serumu (FBS ile takviye (DMEM)), 100 U / ml penisilin, 100 ug / ml streptomisin ve 100 U / ml esansiyel olmayan amino asitler). </li><li> Kültür ortamında 10 ml Transfeksiyondan bir önceki gün içinde 10 cm&#39;lik bir kollajen kaplı tabak Plaka 4 x 10 6 HepG2 hücreleri. Nemlendirilmiş bir% 5 CO2 inkübatöründe 37 ° C&#39;de, HepG2 hücreleri inkübe edin. Not: yaklaşık olarak 4 x 10 6 HepG2 hücreleri kültür ortamında 10 ml, 10 cm kollajen kaplı tabak içinde plakalanır, bunlar% 70-90 konfluent ertesi gün olacaktır. </li></ol></li><li> transfeksiyon <ol><li> PUC1.2HBV ö epsilon 15 5 ug ve pUC1.2HBV / NL 15 5 ug ile transfekt HepG2 hücreleri transfeksiyon kullanılaraküreticinin talimatlarına göre reaktif. </li><li> Sonraki gün, kültür ortamı çıkarın ve taze kültür ortamı 10 ml. </li><li> Bir hafta sonra transfeksiyon 50 ml tüp rekombinant HBV ihtiva eden kültür ortamı aktarmak 1.3.1 adıma devam edin. transfekte edilmiş hücreleri içeren plaka taze kültür ortamı 10 ml. Not: Rekombinant HBV üretimi 4 hafta süre ile muhafaza edilir. Rekombinant HBV ihtiva eden kültür ortamı 1 ay boyunca 4 ° C &#39;de muhafaza edilebilir. </li></ol></li><li> Rekombinant HBV saflaştırılması <ol><li> santrifüj ile rekombinant HBV (5 dakika boyunca 2300 x g) ihtiva eden kültür ortamından hücre debrisini uzaklaştırmak. </li><li> 0.45 um&#39;lik bir membran filtresinden süpernatant geçirin. </li><li> Kültür ortamına:% 26 PEG / 1.5M NaCI (130 g, NaCl, 49 g, 0.5 M EDTA, pH 8.0, 1 ml, 1 M HEPES pH 5 ml 7.6 500 ml polietilen glikol 6000) bir eşit hacmi ekleme ihtiva eden rekombinantHBV ve hafifçe karıştırın. 4 ° C&#39;de bir gece boyunca inkübe edilir. </li><li> 4 ° C&#39;de 20 dakika boyunca 2300 x g&#39;de santrifüjleyin. </li><li> Süpernatant atılır ve TNE 0.5 ml (10 mM Tris, 50 mM NaCI, 1 mM EDTA) içinde pelet çözülür. </li><li> 5 dakika için 2300 x g&#39;de santrifüjleme ile çöpleri çıkarın. </li><li> TNE içinde 0.8 ml% 20 sükroz üzerinde yük yeniden birleştirici HBV ihtiva TNE 0.5 mi. </li><li> 15 ° C&#39;de 3 saat boyunca 100,000 x g&#39;de santrifüjleyin. </li><li> mümkün olduğunca süpernatant kadar atmak ve pelet kaydedin. kültür ortamının başlangıç ​​40 ml başına serumsuz DMEM, 1 ml içinde süspanse edin. </li><li> 4 ° C&#39;de bir gece boyunca inkübe edilir. </li><li> 0.45 um&#39;lik bir filtre ile filtrelenir. -80 ° C de, 0.5 ml alikotları ve mağaza hazırlayın. Not: orijinal virüs numunenin raportör proteini kirlenme gözlenirse, 2 saat boyunca 100,000 x g&#39;de TNE içinde% 5-30 sükroz yoğunluk gradyanı ile ultra santrifüj ile virüs arındırmak ya da ileri geri CsCI yoğunluk dengelenmiş santrifüj50 saat boyunca 150.000 x g&#39;de m 1.1-1.6 g / ml olmuştur. </li></ol></li></ol> Rekombinant HBV 2. Enfeksiyon <ol><li> % 10 FBS, 100 U / ml penisilin, 100 ug / ml streptomisin, 250 ug / ml G-418 ve 100 U / ml ile takviye edilmiş DMEM içinde HBV enfeksiyonuna duyarlı olan stabil bir şekilde NTCP (HepG2 / NTCP) 15&#39;i ifade eden kültür HepG2 hücreleri nemlendirilmiş bir% 5 CO2 inkübatöründe 37 ° C&#39;de gerekli olmayan amino asitler,. </li><li> Enfeksiyondan bir gün önce, plaka yaklaşık 5 x 10 4 HepG2 / kültür ortamına 0.1 ml, 96-çukurlu kollajen kaplı plaka NTCP hücreleri 14. </li><li> 37 ° C su banyosu içinde çözülme rekombinant HBV şişede kalan buz küçük bir bit olana kadar. </li><li> Aşağıdaki birleştirerek bulaşmasına orta hazırlayın: 1X fosfat tamponlu salin (PBS), dimetil sülfoksit, 2 ul (DMSO),% 40 PEG8000 10 ul rekombinant HBV 10 ul ve taze kültür ortamına 78 ul96 oyuklu bir plakanın her bir kuyucuğu. </li><li> 96 gözlü plakanın bir oyuğuna rekombinant HBV çözeltisi 100 ul ekle. </li><li> Enfeksiyondan bir gün sonra, virüs fraksiyondan kirletici raportör proteinini çıkarmak için, göz başına 300 | il PBS ile enfekte edilmiş hücrelerde 3 kez yıkayın. </li><li> Bir hafta ya da daha az,% 2 DMSO içeren kültür ortamında 200 ul enfekte hücreler inkübe edin. </li></ol> 3. Analiz <ol><li> Bir hafta sonra enfeksiyon, PBS ile enfekte edilmiş hücrelerde 3 kez yıkayın. </li><li> enfeksiyonlu hücrelere liziz tamponunda 50 ul ekle. </li><li> 5 dakika boyunca kültürü plakası sallayın, ve daha sonra 5 dakika boyunca 2000 x g&#39;de santrifüjlenir. </li><li> Luminometre plakasına haberci substratın 50 ul ekle. </li><li> raportör alt-tabakayı içeren bir luminometre plakasına hücre lizatının 20 ul ekle. kısaca vorteks ile karıştırın. </li><li> Luminometreye plaka koyun ve 15 okuma başlatın. </li></ol>

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The authors declare that they have no competing financial interests.

HBV genomu çekirdeği, polimeraz, yüzey ve X ORF (Şekil 1) dahil olmak üzere dört ana açık okuma çerçevelerini (ORF) sahiptir. Bu dört HBV ORF transkripsiyon sıkıca dört rehberleri tarafından düzenlenir: prekor / çekirdek geliştirici arttırıcı II içeren, S1 promotör, S2 promotör ve X yükseltici zenginleştirici 18 I içeren. 3.5 kb ve 3.4 kb mRNA&#39;ların sırasıyla prekor ve Core proteinler çevrilir. Büyük zarf proteini (L) en büyük alt-genomik mR...

Hepatit B virüsü (HBV) ile kronik enfeksiyon kronik karaciğer hastalıklarında 1 için önemli bir risk faktörüdür. Mevcut tedavi stratejileri interferon uyarımlı gen fonksiyonlarının 2 ile HBV pol fonksiyonu ve / veya enfekte olmuş bireylerde bağışıklık yanıtlarını aktive tip I interferon verilmesini de dolaylı bastırıcı HBV proliferasyonunu inhibe nükleotid analoglan dayanmasına rağmen, bu tedaviler HBV ortadan kaldırmaz DNA tamamen 3. Ayrıca, bir anti-pol maddeler karşı dirençli olan HBVlerin çıkışı endişe 4 taşımaktadır. şirketinden insan bağışıklık eksikliği virüsü (HIV) veya hepatit C virüsü (HCV), yaşam döngüsünün farklı adımlarını hedefleme kombine anti-viral ajanlar uygulanması başarılı bastırmak ya da virüs (ler) ortadan kaldırmak için gösterilmiştir. Bu fikrin, H farklı aşamalarında doğrudan etki anti-HBV ajan (lar) gelişimine benzerBV yaşam döngüsü gelecek HBV terapileri kurulması için önemlidir. Genel olarak, hedef virüs in vitro kültür sistemi basit bir kurulması anti-virüs maddelerinin geliştirilmesini kolaylaştırır. Bununla birlikte, anti-HBV maddeleri taramak için in vitro kültür sistemlerinin geliştirilmesi için en az iki engeller bulunmaktadır. İlk HBV enfeksiyonu / proliferasyonu için uygun bir in vitro hücre kültürü sisteminin olmamasıdır. Kurulan hücre hatlarında yayılır, HIV ve HCV gibi diğer virüsler, aksine, bunun nedeni, dar konukçu aralığının içeren deney sınırlamaları in vitro HBV yetiştirmek için zordur. Bu HBV enfeksiyonunun, 5, 6 karşı hassas olan, insan hepatoma hücre hattı HepaRG, belirli hücre kültür sistemlerinin kullanılması, 7, bu problemlerin üstesinden gelmek için geliştirilmiştir. Ayrıca, ürokinaz ty izole PXB hücreleri,PE plazminojen aktivatör transjenik / primer insan hepatosit (Ph) ile aşılanır SCID fareleri, HBV enfeksiyonu ve çoğaltma 8 duyarlı olduğu gösterilmiştir. Bununla birlikte, HepaRG HBV replikasyon seviyesi HBV enfeksiyonu / çoğaltma seviyelerinin tutarsız ve &#39;tekrarı sonuçlara neden olabilir kültürden sonra hücre farklılaşması durum, bağlıdır. PXB yaygın HBV enfeksiyonu deneyleri için kullanılan ama durumu ile sınırlıdır. Bir tetrasiklin uyanlabilir HBV sentezleme hücre soyu, HepAD38, aynı zamanda yaygın HBV replikasyonunu incelemek için kullanılmıştır, ama bu sistem, sadece, transkripsiyon sonrası olup, HBV enfeksiyonu 9 giriş adımında değerlendirme sağlar. Son zamanlarda, HBV için işlevsel bir reseptör olarak, sodyum taurokolat cotransporting polipeptidin tanımlanması (NTCP) değişken bir HBV kültürü sisteminin 10 geliştirilmesine imkan tanımıştır. Gerçekte, bu tür Huh7 olarak duyarlı olmayan hepatokarsinoma hücrelerinin NTCP ekspresyonu veHepG2 HBV enfeksiyonunun 10 sağlar ve bu nedenle, HBV hassas hücre hatlarının seçimi deney sınırlamaları birçok çözme genişletilmiştir. İkinci bir sorun, HBV enfeksiyonu ve çoğaltma değerlendirmek için basit bir deney sisteminin olmamasıdır. HBV enfeksiyonu değerlendirilmesi genellikle HBV DNA, RNA ve proteinler analiz ederek yapılır. Ancak, bu virüs belirteçlerinin ölçümü genellikle masraflı ve zaman basit değil zaman tüketen vardır. Bu nedenle, basit bir deney sisteminin geliştirilmesi, bir raportör gen kullanılarak, HBV deney sistemleri ile ortaya çıkan problemlerin üstesinden gelinebilir. Bununla birlikte, bir HBV kapsid içinde paketlenebilir genom boyutu sınırlı olduğundan, - en az 3.7 kb 11 - Bir raportör genin boyutu mümkün olduğu kadar kısa olması gerekir. Ayrıca, viral replikasyon için gerekli olan genom boyunca dağılmış birden çok cis elemanlarının varlığı bölgesi için uygun pozisyonlar sınırlar<html> genomuna haberci genin yerleştirilmesine. Çeşitli raporlar, HBV genomunda 11, 12, 13 içine, HIV-1 Tat, yeşil floresan protein ve DsRed dahil olmak üzere yabancı genleri, eklemek için çalıştılar. Bununla birlikte, bu yeniden birleştirici HBVlerin HBV enfeksiyonu / çoğaltma ya da HBV enfeksiyonu / çoğaltma etkileyen faktörler yüksek verimli tarama taranması için kullanışlı değildir. Bu esas olarak bir araya getiren virüslerin, düşük verimlilik ve yetersiz virüs üretiminin neden raportör gen ekspresyonunun düşük yoğunluktadır. Bu sorunları aşmak için, virüs üretiminin yüksek verim ile bir muhabir HBV inşa. Bu virüs girişinden transkripsiyonu için, HBV replikasyon döngüsünün erken aşamalarında izlenmesi için son derece duyarlıdır. Bu, küçük bir (171 amino asit) lüminesan raportör tasarlanmış olduğu için bu elde etmek için, NanoLuc (NL), bir işaretleyici gen olarak seçilmiştirclass = &quot;xref&quot;&gt; 14. Ayrıca, NL ateşböceği ya Renilla lusiferaz daha parlak 150 kat yaklaşık ve lüminesan Reaksiyon yanlış isabet oranı yüksek verimli tarama düşük olacağını düşündürmektedir ATP&#39;ye bağımsızdır. Rekombinant HBV üretim verimi yaklaşık 1/5 ebeveyn HBV bölgesinin ve önceki HBV yeniden birleştirici virüs rapor seviyelere benzer; anti-HBV maddelerinin kitlesel taranması için kullanılabilir ancak, NL parlaklığı virüsü verimliliği ilgili sorunların üstesinden gelmektedir. Birincil hepatositleri kullanılarak anti-HBV maddelerinin taranması, HepaRG, HepAD38 ve NTCP-kalıt hepatositler, geleneksel yöntem (ler) anti-HBV maddelerinin taranması için yararlı olabilir. Ancak, burada açıklanan sistem, kolay kullanım, yüksek hassasiyet ve tarama için düşük maliyetli gibi çeşitli avantajlara sahiptir. Bu avantajlar, geliştirmek ve terapötik amaçlar için yeni HBV maddelerin belirlenmesi için kapsamlı incelemeler için uygundur.

<table><tbody><tr><td>Nano-Glo Luciferase Assay Regent</td><td>Promega</td><td>N1110</td><td></td></tr><tr><td>Penicillin-Streptomycin Mixed Solution</td><td>Nacalai tesque</td><td>09367-34&amp;nbsp;</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution</td><td>Thermo Fisher Scientific</td><td>11140050</td><td></td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>11995065</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>100 mm/collagen-coated dish</td><td>Iwaki</td><td>4020-010</td><td></td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>L3000001&amp;nbsp;</td><td></td></tr><tr><td>Polyethylene glycol (PEG) 6000&amp;nbsp;</td><td>Sigma-Aldrich</td><td>81255</td><td></td></tr><tr><td>Polyethylene glycol (PEG) 8000&amp;nbsp;</td><td>Sigma-Aldrich</td><td>89510</td><td></td></tr><tr><td>NaCl</td><td>Nacalai tesque</td><td>31319-45&amp;nbsp;</td><td></td></tr><tr><td>0.5 mol/L EDTA Solution</td><td>Nacalai tesque</td><td>06894-14</td><td></td></tr><tr><td>Tris-HCl</td><td>Nacalai tesque</td><td>35434-21</td><td></td></tr><tr><td>Millex-HP, 0.45 &amp;mu;m, polyethethersulfone, filter</td><td>Merck Millipore</td><td>SLHP033RS</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Collagen coated 96-well plate</td><td>Corning</td><td>NO3585</td><td></td></tr><tr><td>Passive Lysis 5x Buffer</td><td>Promega</td><td>E1941</td><td></td></tr><tr><td>GloMax 96 Microplate Luminometer</td><td>Promega</td><td>E6501</td><td></td></tr><tr><td>Sucrose</td><td>Nacalai tesque</td><td>30403-55</td><td></td></tr><tr><td>Luminometer plate</td><td>Greiner bio-one</td><td>655075</td><td></td></tr><tr><td>HepG2-NTCP1-myc-clone22</td><td>-</td><td>-</td><td>Reference 15</td></tr><tr><td>pUC1.2HBV delta epsilon</td><td>-</td><td>-</td><td>Reference 15</td></tr><tr><td>pUC1.2HBV/NL</td><td>-</td><td>-</td><td>Reference 15</td></tr><tr><td>50 ml tube</td><td>Violamo</td><td>1-3500-02</td><td></td></tr><tr><td>Anti-Myc antibody</td><td>Sigma-Aldrich</td><td>C3956</td><td></td></tr><tr><td>HBIG</td><td>Japan Blood Products Organization</td><td>-</td><td></td></tr><tr><td>IFN-&amp;beta;</td><td>Mochida Pharmaceutical</td><td>14987224005413</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3393</td><td></td></tr></tbody></table>

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.

Şekil 1 genomu HBV genomu, transkribe edilmiş RNAlar, virüs proteinleri ve kodlama bölgesinin bir şemasını göstermektedir. genomunda raportör geni ve konumu da gösterilir. Şekil 2, raportör genini ihtiva eden HBV raportör plasmidi ve yardımcı plazmit gösterir. PUC1.2HBV / NL raportör pUC1.2HBV 16 prekor mRNA transkripsiyon başlatma sitesinden nükleotid pozisyonları 223-811 silme ve raportör geni sokulması ile...

Watch this Scientific Journal Video about Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle at JoVE.com

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

Burada HBV yaşam döngüsünün erken aşamalarında izlemek için yeni geliştirilmiş hepatit B virüsü (HBV) muhabiri sistemini açıklar. Vitro sistemde BASİTLEŞTİRİLMİŞ yüksek verimli bir strateji kullanılarak anti-HBV maddelerinin taranması yardımcı olacaktır.

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), ...

development-hepatitis-b-virus-reporter-system-to-monitor-early-stages

Research

JoVE Journal

Immunology and Infection

13.1K Görüntüleme.  National Center for Global Health and Medicine.  Burada HBV yaşam döngüsünün erken aşamalarında izlemek için yeni geliştirilmiş hepatit B virüsü (HBV) muhabiri sistemini açıklar. Vitro sistemde BASİTLEŞTİRİLMİŞ yüksek verimli bir strateji kullanılarak anti-HBV maddelerinin taranması yardımcı olacaktır. 

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

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

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 ...

İmmünoloji ve Enfeksiyon

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Gen İfade Tüm Dönemlerinde Viral Fonksiyonu sayısallaştırabilmek için vaksiniya Reporter Virüsler

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Erken Viral Giriş Tespiti için Tahliller ve Antiviral Bileşiklerin Değerlendirilmesi

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Terapötik Hedef Olarak Polipeptitin Sodyum Taurokolat Kotransportasyonu Yoluyla Hepatit B Virüsü Girişini İnceleyen Yetkin Bir Hepatosit Modeli

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Biyokimya

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Algılama düşük kopya sayısı Vitro hepatit B enfeksiyonu tarafından kurulan entegre Viral DNA

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

"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.

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 ...

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

Yüksek Titer Hepatit E Virüs Stokları Üretmek için Bir Hücre Kültürü Modeli

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

Hepatik Olmayan 293T-NE-3NR Hücrelerinde Hepatit B Virüs Enfeksiyonunun Modelilmesi

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

İnsan Serumunda Hepatit B Virüsü DNA'sının Tespiti ve Miktar Tayini

Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal cirrhosis and liver cancer. Accurate detection and quantification of HBV DNA in the blood are essential for diagnosing and monitoring HBV infection. The most common method for detecting HBV DNA is real-time PCR, which can be used to detect the virus and assess the viral load to monitor the response to antiviral therapy. Here, we describe a detailed protocol for the detection and quantification of HBV DNA in human serum or plasma using an IVD-marked real-time PCR-based kit. The kit uses primers and probes that target the highly conserved core region of the HBV genome and can accurately quantify all HBV genotypes (A, B, C, D, E, F, G, H, I, and J). The kit also includes an endogenous internal control to monitor possible PCR inhibition. This assay runs for 40 cycles, and its cutoff is 38 Ct. For the quantification of HBV DNA in clinical samples, a set of 5 quantification standards is provided with the kit. The standards contain known concentrations of HBV-specific DNA that are calibrated against the 4th WHO International Standard for HBV DNA for the nucleic acid test (NIBSC code 10/266). The standards are used to validate the functionality of the HBV-specific DNA amplification and to generate a standard curve, allowing the quantification of HBV DNA in a sample. HBV DNA as low as 2.5 IU/mL was detected using the PCR kit. The high sensitivity and reproducibility of the kit make it a powerful tool in clinical laboratories, aiding healthcare professionals in effectively diagnosing and managing HBV infections.

Yazar Spotlight: Hepatit B Virüsü Tespitindeki Gelişmeler ve Zorluklar 

Real-time polymerase chain reaction (PCR)-based detection and quantification of hepatitis B virus (HBV) DNA is a sensitive and accurate method for diagnosing and monitoring HBV infection. Here, we present a protocol for HBV DNA detection and the viral load measurement of a sample.