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
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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.

1.生产重组乙型肝炎病毒的编码蛋白质记者<ol><li> HepG2细胞的制备 <ol><li>制备细胞培养基（Dulbecco改良的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），100U / ml青霉素，100微克/毫升链霉素和100单位/毫升的非必需氨基酸）。 </li><li>板4×10 6个 HepG2细胞中在10ml培养基的转染前一天10厘米胶原包被的培养皿。孵育HepG2细胞于37℃在湿润的5％CO 2培养箱。 注意:当大约4×10 6个 HepG2细胞在10cm的胶原包被的培养皿铺板在10ml培养基中，它们将是70-90％汇合的第二天。 </li></ol></li><li> 转 <ol><li>转染HepG2细胞pUC1.2HBV增量小量15的5微克和pUC1.2HBV / NL 15 5微克使用染试剂根据制造商的说明。 </li><li>次日，除去培养基并加入10毫升的新鲜培养基。 </li><li>一周转染后，将含有重组乙肝培养基转移到50ml管，并进行到步骤1.3.1。 10毫升新鲜培养基添加到含转染的细胞的板。 注:重组乙肝病毒的产量保持4周。含有重组乙肝病毒的培养基可以储存在4℃下1个月。 </li></ol></li><li> 重组乙型肝炎病毒的分离纯化 <ol><li>从含有通过离心重组乙肝病毒（2300 xg离心5分钟）的培养基中除去细胞碎片。 </li><li>通过0.45μm膜过滤器传递上清液。 </li><li>的26％的PEG / 1.5 M氯化钠（聚乙二醇6000 130克，氯化钠49克，1 50ml的0.5M EDTA pH 8.0的和5ml的1M HEPES pH为7.6在500毫升）中加入等体积的培养基含重组HBV，轻轻混匀。在4℃孵育过夜。 </li><li>离心2300 XG在4℃下20分钟。 </li><li>弃去上清液并溶解沉淀在0.5ml TNE（10毫摩尔Tris，50mM NaCl中，1mM EDTA）中。 </li><li> 2,300 xg离心除去离心碎片5分钟。 </li><li>负载0.5毫升含重组乙肝TNE的拖到TNE0.8毫升20％的蔗糖。 </li><li>离心机在15℃100000×g下3小时。 </li><li>丢弃尽可能多上清尽可能的，并保存沉淀。悬浮它在1毫升每40 ml起始培养基的无血清DMEM中。 </li><li>在4℃孵育过夜。 </li><li>通过0.45微米的过滤器过滤。在-80℃制备各取一份0.5ml和存储。 注意:如果发现原来的病毒样本的记者蛋白污染，10万XG在TNE 5-30％的蔗糖密度梯度超离心纯化病毒2小时，或来回氯化铯密度离心平衡米1.1-1.6克/毫升15万XG为50小时。 </li></ol></li></ol> 2.重组乙肝病毒感染的<ol><li>培养HepG2细胞稳定表达NTCP（HepG2细胞/ NTCP）15易受HBV感染的DMEM补充有10％FBS，100U / ml青霉素，100微克/毫升链霉素，250微克/毫升的G-418和100U / ml的非必需氨基酸，在37℃在湿润的5％CO 2培养箱。 </li><li>感染前一天，板约5×10 4的HepG2 / NTCP细胞14进入在0.1ml培养基中的96孔胶原包被板。 </li><li>在37℃水浴解冻HBV重组，直到有剩余的小瓶冰小一点。 </li><li>由以下结合制备用于感染的培养基:在1×磷酸盐缓冲盐水（PBS），2微升二甲基亚砜（DMSO）中，加入10μl40％PEG8000的重组乙肝病毒的10微升和78微升新鲜培养基每一个96孔板的孔。 </li><li>加入100微升的重组乙肝病毒溶液的96孔板的一个孔中。 </li><li>感染后一天，洗净感染的细胞3次，用每孔300μl的PBS中，以从病毒级分除去的污染报告蛋白。 </li><li>孵育感染细胞在200μl含有2％DMS​​O的一周或更少的培养基。 </li></ol> 3.分析<ol><li>感染后一周，洗感染的细胞3次，用PBS。 </li><li>加入50μl裂解缓冲液的受感染的细胞。 </li><li>摇动培养板进行5分钟，然后在2000 xg离心离心5分钟。 </li><li>加入50微升记者基板进入光度计板。 </li><li>加入20μl的细胞裂解物与含记者基板光度计板。涡旋简要混合。 </li><li>放置在光度计板，并开始阅读15。 </li></ol>

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The authors declare that they have no competing financial interests.

HBV基因组有四个主要的开放阅读框（ORFs），包括核心，聚合酶，表面和X的ORF（ 图1）。这四个HBV ORF的转录紧紧四个启动子调控:含有增强II的前核心/核心启动子，含有增强我18启动S1，S2启动子和X子。 3.5 kb和3.4 kb的mRNA的分别翻译成前C区和核心蛋白。大包膜蛋白（L）的从最大的亚基因组mRNA的（2.5kb的）产生的，而中间（M）和小的表面蛋白（S）是从短转录（2.1kb的）?...

乙型肝炎病毒（HBV）慢性感染是慢性肝病1的主要危险因素。虽然目前的治疗策略是基于抑制通过干扰素刺激基因功能2的HBV POL功能和/或激活在感染个体的免疫反应的I型干扰素的施用以及间接抑制HBV的增殖的核苷酸类似物，这些治疗不能消除HBV的DNA完全3。而且，对防聚合酶剂耐药HBVS的出现是关注4。合并的抗病毒剂直接针对人免疫缺陷病毒（HIV）或丙型肝炎病毒（HCV）的生命周期的不同步骤的管理被证明成功地抑制或根除病毒（ES）。类似这样的想法，即直接作用于H的不同阶段抗HBV剂的开发BV的生命周期是建立未来的乙肝治疗方法很重要。 在一般情况下，建立在目标病毒的体外培养系统中的简单的便于防病毒代理的发展。然而，有至少两个障碍的体外培养系统的发展，以筛选抗HBV剂。首先是缺乏一个方便的体外细胞培养HBV感染/扩散制度。不像其他的病毒，如HIV和HCV，其在建立的细胞系增殖，它是很难培养的HBV 体外因为实验的限制，包括一个窄的宿主范围的。使用具体的细胞培养系统，例如人肝癌细胞系HepaRG，这是易受HBV感染，5，6，7已经开发了克服了这些问题。此外，PXB的细胞，从尿激酶TY孤立PE纤溶酶原激活物的转基因/原发性人肝细胞（PHH）接种的SCID小鼠，被证明是容易受到HBV感染和复制8。然而，在HepaRG HBV复制的水平依赖于培养后的细胞的分化状态，这会导致HBV感染/复制水平的不一致和不可再现的结果。 PXB通常用于HBV感染实验，但是由它的可用性的限制。四环素诱导型HBV的表达细胞株，HepAD38，也被广泛用于研究HBV复制，但这种系统只允许转录后，而不是在HBV感染9的进入步骤评价。最近，牛磺胆酸钠cotransporting多肽的识别（NTCP）用作HBV的功能性受体已经允许可变的HBV培养系统10的开发。事实上，在非易感肝癌细胞如的Huh7 NTCP表达和肝癌使HBV感染10，因此，乙肝病毒易感细胞系的选择也不断扩大，解决许多实验的限制。第二个问题是缺少一种简单的测定系统来评估HBV感染和复制的。 HBV感染的评价通常是通过分析HBV DNA，RNA和蛋白质进行。但是，这些病毒标志物的定量是费时，常常是昂贵和不总是简单的。因此，一个简单的测定系统的发展，诸如使用报告基因，可能克服与HBV的测定系统相关的问题。 但是，因为可被包装成一个HBV衣壳基因组大小被限制-小于3.7 kb的11 -报告基因的大小应该尽可能地短。此外，分散在整个基因组中的多个顺式元件，其是病毒复制所必需的存在，限制了可用于在位置<html>报道基因到基因组中的插入可以。若干报告已经试图插入外源基因，包括HIV-1的Tat，绿色荧光蛋白，和红色荧光蛋白，成HBV基因组11，12，13。然而，这些重组HBVS不是用于筛选HBV感染/复制，或用于影响HBV感染/复制因子的高通量筛选是有用的。这主要是因为重组病毒的生产效率低而造成低效病毒产生报道基因表达的降低强度的。 为了克服这些问题，我们构建了记者乙肝病毒生产的高产率。这种病毒是用于监测HBV复制循环的早期阶段，从入口到转录高度敏感。为了实现这一目标，NanoLuc（NL）被选择作为标记基因，因为它是一个小的（171个氨基酸）工程改造发光报告类="外部参照"&gt; 14。另外，NL是约150倍萤火虫或Renilla荧光素酶亮，和发光反应是ATP的独立的，这表明假命中率将是低的为高通量筛选。重组乙肝病毒的生产效率为约1/5的父的HBV，和类似报告以前的HBV的重组病毒的水平;然而，NL的亮度克服病毒生产率的问题，因此它可以被用于抗HBV剂的大量筛选。 使用原代肝细胞的抗HBV剂的筛查，HepaRG，HepAD38和NTCP转导的肝细胞可能是抗HBV剂通过常规方法（多个）的筛选是有用的。然而，在这里所描述的系统具有各种优点，例如简单的处理，灵敏度高，且成本低的筛选。这些优势是适用于高通量分析制定和确定新HBV药物达到治疗目的。

<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.

图1显示了HBV基因组，转录的RNA，病毒蛋白和它们的编码区的基因组的示意图。在基因组中的报告基因和它的位置也被指示。 图2示出了包含报告基因的HBV报告质粒和辅助质粒。所述pUC1.2HBV / NL记者从pUC1.2HBV 16的前C mRNA的转录起始位点删除核苷酸位置223-811，然后插入报告基因构建。通过定点诱变PCR产生与包装信号序列的点突变的pU...

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

这里，我们描述了新开发的乙型肝炎病毒（HBV）报告系统来监视HBV生命周期的早期阶段。 该体外系统的简化将在使用高通量策略抗HBV剂的筛选帮助。

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

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 次观看.  National Center for Global Health and Medicine. 这里，我们描述了新开发的乙型肝炎病毒（HBV）报告系统来监视HBV生命周期的早期阶段。 该体外系统的简化将在使用高通量策略抗HBV剂的筛选帮助。 

视频: 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.

一个协议，用于分析丙型肝炎病毒复制

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

科研

免疫与感染

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.

牛痘病毒记者量化病毒功能在基因表达的各个阶段

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.

早期的病毒进入测定的鉴定和抗病毒化合物的评价

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).

一种通过牛磺胆酸钠共转运多肽作为治疗靶点检查乙型肝炎病毒进入的感受态肝细胞模型

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

生物化学

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.

乙型肝炎体外感染形成的低拷贝数综合病毒 dna 的检测

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.

乙型肝炎病毒感染原代肝细胞和库珀细胞的 "肝片上" 培养

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.

生产高滴度性乙型肝炎病毒库的细胞培养模型

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).

非肝细胞293T-NE-3NRS细胞中乙型肝炎病毒感染的建模

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

人血清中乙型肝炎病毒 DNA 的检测和定量

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.

作者聚焦:乙型肝炎病毒检测的进步和挑战 

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.