笔者想在注射浸润的发展和在体外装配检测实验室的几名成员，感谢他们的宝贵建议。这项工作是由从美国加州大学的资助。部分支持这项研究是由国家科学基金会（CBET-1144237）的资助。

1。植物材料<ol><li> 烟草在包壳实验中使用的植物应该是在4叶期（约3-4周老厂）。 </li></ol> 2。植物注射浸润细胞病毒基因组的功能部件的交付和表达<ol><li> 第1天：农杆菌菌株（如高血压105或GV 3101）窝藏pCass-BMV RNA，BMV RNA 2和BMV RNA 3 1,2种植为辅，卡那霉素50毫克/毫升LB琼脂板（选择农杆菌pCASS载体）和利福平100mg/ml（选择）。孵化器在28°C正在开展为期两天。 </li><li> 第3天：从到2毫升含有50毫克/毫升，卡那霉素和利福平100毫克/毫升，1天在28°Ç轨道摇床设定在250转的LB肉汤LB琼脂平板上接种单菌落。 </li><li> 第4天：接种50毫升的LB肉汤补充卡那霉素和100 mg / ml的利福平，10毫米MES的pH为5.6毫克/毫升和100毫米ED 50乙酰丁香酮与文化毫升在250毫升三角瓶，16小时在28°的轨道摇床在250 RPMÇ 。 </li><li> 第5天：文化转移（外径600时达到1.0）橡树岭管或无菌猎鹰管和离心机于5000 rpm 10分钟，在4°C </li><li>溶于10毫升的10 mM MgCl 2的离心沉淀10分钟，于5000 rpm，在4°C，重复这一步，更多的时间，以确保完全去除抗生素。 </li><li>暂停10毫升含有MgCl 2的 10毫米和10毫米的MES（pH值5.6）的缓冲沉淀。 </li><li>衡量每个BMV RNA的RNA的1，2和RNA 3文化的OD 600调整到600外径0.1使用10毫米MgCl 2的和10mm MES（pH值5.6）。 </li><li>混合1毫升每所有三个0.1外径600文化停牌。 </li><li>添加乙酰丁香酮100毫米，搭配绅士LY和混合物不受干扰，在室温下保持3小时。 </li><li>吸引到培养液1毫升。注射器没有针头。 </li><li> 2-3扩大北叶的背面一侧渗入以上文化暂停本生 （5叶期，老厂3-4周），轻轻按下一个半叶的背面表面注射器。 </li><li>这种渗透过程重复到其他叶片。 </li><li>绿房子保持在24°C的渗透植物</li><li>收获渗入3到4天的渗透（dpi）的叶。 </li></ol> 3。 BMV病毒颗粒的纯化<ol><li>收集N。本生留下一个包含所有三个野生型BMV agrotransformants混合物agroinfiltrated。 </li><li>在无菌杵和迫击炮叶片研磨与等量的：BMV提取缓冲液（0.5 M的醋酸钠（W / V）; 0.08 M MgAc，pH值4.5和1/100，这是要在使用前添加β-巯基乙醇的体积）3。为了最大限度地提高病毒产量，建议加酸水洗砂（约0.5-1克），有利于细胞破碎容易磨和高效。 </li><li>通过2-3层薄纱布叶提取物的筛选，收集乱舞。 </li><li>再次研磨的薄纱布上的留成部分与BMV提取液等体积的叶片材料的初始重量。 </li><li>重复过滤程序，用薄纱布。这些步骤应在4°C进行</li><li>滤液转移到离心管中，并添加5分钟，在室温下预冷的氯仿和涡量相等（或震动），直到悬浮液的颜色变成浅绿色。 </li><li>离心15分钟，12,000 RPM乳化的解决方案，在4°C。 </li><li>收集水相，搅拌30分钟，以消除任何残留氯仿磁力搅拌器。 </li><li>暂停转移到无菌超离心管。 </li><li底图1毫升。 10％的蔗糖（在病毒提取液配制）作为缓冲3。 </li><li>在贝克曼超速离心机为3小时，在30000转离心4°C。 </li><li>弃上清，并暂停BMV暂停缓冲量所需的颗粒（准备用无菌蒸馏水悬浮缓冲液稀释BMV提取液的1/10）。 </li><li>从上面的步骤部分纯化的病毒粒子的制备受到150分钟28000转的10-40％蔗糖密度梯度离心，在4°C </li><li>收集病毒带手动或由分馏。病毒带将出现蓝白色的光照下。 </li><li>稀蔗糖溶液含有至少50％的病毒样本与病毒悬浮缓冲。 </li><li> 3个小时在贝克曼超速离心机转速在30000离心稀释病毒含有蔗糖溶液在4°C。 </li><li>最后暂停在VIR所需量的高纯度的病毒颗粒我们暂停缓冲。 </li><li>测量外径260分光光度计（毫克/毫升）的病毒颗粒的浓度。 </li></ol><img alt="式（1）" src="/files/ftp_upload/3645/3645eq1.jpg" />注：基于RNA的含量，为BMV的消光系数是5 4。 <ol start="19"><li>验证纯度的病毒颗粒的透射电镜下观看（图A） </li></ol> 4。在体外组装衣壳蛋白亚基的制备<ol><li>准备1X病毒分离缓冲液（0.5 M的氯化钙 ;的50 mM Tris盐酸，pH值7.5，1.0毫米EDTA;数码地面1.0毫米，0.5毫米PMSF）1升5。 </li><li>准备透析膜，根据Sambrook 等 6。 </li><li>免除需要将透析袋纯化病毒的浓度。 </li><li>放入包含1X病毒分离缓冲区烧杯中含有的病毒透析袋。 </li><li>透析24小时内，在4°Çwhilé搅拌。 </li><li>在12000 XG 15分钟收集24小时后，离心透析袋的解决方案，在4°C。这将BMV RNA的分离游离的病毒粒子。 </li><li>收集上清贝克曼离心机35000转和3个小时在4°C至颗粒任何未游离的病毒粒子的离心机。 </li><li>收集上清。 </li><li>在这个阶段，当务之急是从任何污染的病毒粒子RNA的分离衣壳蛋白亚基。要做到这一点，从第8步上清应受到另一轮重组衣壳蛋白亚基超过晚上不添加任何核糖核酸（RNA 1X装配缓冲，见下文），由高速离心（30,000 rpm的3小时在4°c）颗粒任何组装病毒颗粒。这个重组的步骤后，上清含外壳蛋白亚基将无任何残留污染的RNA。然而，以确保这一点，它是最好执行附加在V体外重组与利用RNA 1X装配缓冲唯一的外壳蛋白亚基。经过一夜重组，准备必须是自由组装病毒颗粒（验证用TEM）。 </li><li>在外径254和280 nm测量或其他方法，如Bradford法确定的衣壳蛋白亚基的浓度。 </li><li>执行12-15％的SDS-PAGE印迹，以确定分离衣壳蛋白亚基的完整性。 </li></ol> 5。含有病毒颗粒的RNA 在体外装配<ol><li>准备RNA被重新组装成病毒颗粒的成绩单，并计算浓度7。 </li><li>混合衣壳蛋白亚基和转录RNA的比例为1:5（重量/重量）5。 </li><li>免除上述混合物透析袋，并妥善保护，以避免任何泄漏。 </li><li>准备1000毫升。 RNA的，1X装配缓冲液（50 mM氯化钠的50 mM Tris-盐酸，pH值7.2 10毫米氯化钾;氯化镁2 5毫米，1.0毫米的数码地面电视）5。 </li><li>将透析袋，含有对1X装配缓冲反应混合物倒入烧杯，搅拌。 </li><li>在4°C透析轻轻搅拌24小时内重新组装反应。 </li><li> 24小时后收集透析袋的混合物，并添加1.5毫升RNA装配缓冲。 </li><li>通过这种混合物通过Centricon-100柱离心30分钟，在低转速（2000 XG）。 </li><li>洗一次列在4°C，30分钟装配缓冲1.5毫升。 </li><li>重复两次以上的洗涤步骤。 </li><li>最后，通过离心洗脱重新组装的病毒粒子，在10,000 g，4°C下5分钟。 </li><li>估计在外径260纳米的病毒粒子浓度。 </li></ol> 6。制造光学病毒鬼（OVGs） <ol><li>吲哚菁绿（ICG）8日至所需的浓度比（吲哚本报告中使用的绿色和衣壳蛋白的重量比为1:10）衣壳蛋白的纯化<html> ð调匀吸取。 </li><li>透析衣壳蛋白对OVG组装缓冲器（1 M氯化钠和1 mM的数码地面电视，pH4.8，50毫米醋酸钠; 1毫米EDTA）和ICG解决方案，在4°C 24小时。 </li><li> 24小时后，收集透析袋（12 kDa的孔径）和离心机的解决方案，1小时9万转4℃（图B） </li><li>去除上清，涡旋或在4°C，允许它本身暂停BMV暂停缓冲隔夜暂停BMV暂停缓冲沉淀</li><li>验证通过TEM（图C）OVGs形态。 </li><li>测量吸光度，从240纳米到950纳米，使用分光光度计（卡里50，瓦里安公司）;证实ICG的存在特征吸收峰大约700 nm和790 nm的8。 （图D） </li><li> ICG的典型〜700 nm和800 nm的荧光峰，可以看到后OVG解决方案620 nm激发荧光光谱仪（图D）（3的Fluorolog，乔宾-Yvon公司）。 </li></ol>_title“&gt; 7代表结果。 <img alt="图A" src="/files/ftp_upload/3645/3645figA.jpg" /> 负染BMV纯化病毒从北 图A. TEM图像本生的植物agroinfiltrated与所有三个野生型的BMV agroconstructs（比例尺= 100纳米）的混合物。 <img alt="图B" src="/files/ftp_upload/3645/3645figB.jpg" /> 图B颗粒在体外组装，高速离心后，ICG的含OVGs。 <img alt="图C" src="/files/ftp_upload/3645/3645figC.jpg" /> 图C. TEM图像（比例尺= 100纳米）的负染ICG的含OVGs。 <img alt="图D" src="/files/ftp_upload/3645/3645figD.jpg" /> （底部） 图四吸光度（上）和荧光光谱OVGs。激发波长为用于OVG排放瓦特为620纳米。

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我们什么都没有透露。

这里介绍的注射浸润方法，可以广泛适用于广泛的植物病毒。这种方法的一个共同特征是同步交付多个agroconstruct相同的细胞，通常经常使用的植物病毒机械接种相关的一个主要缺点。 在体内和体外装配使用雀麦花叶病毒模型的研究，可以有效地进行，由以下几个小技巧。（i）对于N 的成功渗透本生离开，没有水的浸润前24小时的植物;（二）农杆菌悬浮培养扩散到细胞?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> MES钠盐</td><td> Sigma-Aldrich公司</td><td> M2993 </td></tr><tr><td>吲哚青绿</td><td> Sigma-Aldrich公司</td><td> I2633 </td></tr><tr><td>贝克曼超速离心机</td><td>贝克曼</td><td>型号：L8-70M </td></tr><tr><td> centricon-100列</td><td> Millipore公司</td><td> YM-100 </td></tr><tr><td>分光光度计</td><td>卡里50，瓦里安公司</td><td>零件编号 10068900 </td></tr><tr><td>分光光度计</td><td>乔宾-Yvon公司的Fluorolog 3。 </td><td>零件编号FL3-21 </td></tr></tbody></table>

simple and robust in vivo and in vitro approach for studying virus assembly

在与正义RNA基因致病人类，动物和植物，后代包壳进入成熟和稳定的病毒粒子的病毒是在一个特定的主机的基数在建立感染阶段。因此，包壳的研究解密的信息，调节病毒组装的机制，形成传染性病毒颗粒的诀窍。在制定遏制病毒的传播和疾病控制的新方法，这些信息是至关重要的。病毒包壳，可以在体内和体外研究。基因在体内的包壳，是一个高度管制的选择性的过程，涉及大分子的相互作用和亚条块分割。因此，研究领导剖析事件，其中包括在体内的病毒包壳，将提供的基本知识，了解如何病毒增殖和组装。最近在体外包壳已开发为在IMA生物医学领域的研究ging和治疗中的应用。非笼罩植物病毒纳入企业在体外带负电荷的外来物质的包壳中遥遥领先。雀麦草花叶病毒（BMV），非笼罩多元的RNA病毒致病的植物，已被用来作为一个模型系统研究基因在体内和体外包装。在烟草植物的包壳检测， 农杆菌介导的瞬时表达，是指作为注射浸润，高效和强大的技术相同的单元是一个用于同步多个组件的交付和表达。在这种方法中， 农杆菌携带二进制进行的desiredviral基因cDNA的载体细胞悬浮到间隙withina的使用没有超过1毫升一次性注射器（针）的复杂的叶渗透。的T-DNA转移到植物细胞中的DNA插入这个过程的结果;插入瞬时仍然在细胞核，然后由主机聚合酶II转录，导致瞬时表达。由此产生的转录（封顶，加尾），然后出口到细胞质中的翻译。约24至48小时的潜伏期后，渗透叶子的部分，可以为microscopyor生化分析采样。注射浸润允许细胞大量（数百到数千），同时要转。在体外包壳，BMV纯化病毒衣壳蛋白分离，对分离缓冲含有钙，RNA和非游离病毒颗粒通过离心去除氯透析。贫基因衣壳蛋白亚基重组所需的病毒基因组元件或如吲哚菁染料的非病毒成分。

Watch this Scientific Journal Video about Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly at JoVE.com

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

同步提供多个病毒成分对植物细胞通过一种简单，高效和可靠的方法<em&gt;农杆菌</em&gt;介导的瞬时表达描述。这种方法是适合就读复制，包壳其次<em&gt;在体外</em&gt;到非病毒部分基因组的重组耗尽适用于生物医学应用光学病毒鬼。

简单而强大在体内和在体外方法研究病毒大会

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

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Immunology and Infection

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

12.1K 次观看.  University of California, Riverside. 同步提供多个病毒成分对植物细胞通过一种简单，高效和可靠的方法农杆菌介导的瞬时表达描述。这种方法是适合就读复制，包壳其次在体外到非病毒部分基因组的重组耗尽适用于生物医学应用光学病毒鬼。

视频: Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

科研

免疫与感染

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

一种新型<em&gt;在体外</em&gt;模型研究人类全血细胞间的相互作用

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

体外方法比较标的结合和CDC诱导治疗性抗体之间：应用在Biosimilarity分析

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

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

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

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

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...