Name: alternative in vitro methods for the determination of viral capsid structural integrity
Uploaded: 2017-11-16T15:00:00
Description: Watch this Scientific Journal Video about Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity at JoVE.com

这项工作得到了农业和食品研究倡议的支持, 2011-68003-30395 号来自美国农业部、国家粮食和农业研究所通过 NoroCORE 项目进行的竞争性赠款。美国农业部提供了开放获取费用的资金。我们要感谢罗伯特 Atmar (贝勒医学院, 休斯敦, 德克萨斯州) 好心提供我们纯化的衣和瓦莱丽拉帕姆为她的协助与 TEM 图像。我们还要感谢弗兰克 n. 巴里帮助我们开始了实验。

1. Plate-based 结合法评价高阶病毒衣壳结构的损失 注意: 在这里提出的一个非常类似的 ELASA 协议已被发布 29 ,和类似的 ELASA 化验报告以前曾作为研究文章的一部分在别处 17 , 18 , 22 。
<ol> <li> 对人类病毒的热处理 gii 4 悉尼衣 <ol> <li> 获得纯化的人类病毒主要衣壳蛋白 (VP1...

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这里描述的协议和技术提供了一种评估人类病毒衣壳完整性/功能的方法。这些方法可用于获得对病毒灭活治疗效果的机械论洞察力, 并有可能补充更多传统的失活评估方法, 如 RT qPCR 或斑块检测。例如, 仅通过斑块法对化学或物理失活的评价, 就可以衡量病毒失活的程度, 而不是该失活的根本机制。此外, 病毒聚集可能导致低估的效价, 因此高估治疗效果30。这些方法可以通过提供?...

人类病毒负责全球相当大的公共卫生负担, 每年造成大约6亿8500万疾病和21.2万人死亡1, 耗资数十亿美元的2,3。今天, 病毒检测和量化涉及基因组放大和/或 ligand-based 平台。前者通常是首选, 因为它比较敏感, 提供了定量信息, 并且通常具有较低的误报结果的风险4。最常见的病毒检测和定量的基因组扩增技术是逆转录酶定量聚合链式反应 (RT-qPCR)。因为它的目标和放大人类病毒基因组的一小部分, RT qPCR 单独是不能区分传染性和非传染性的粒子, 作为自由基因组 RNA, 受损衣含有基因组, 和衣致命突变/截断的基因组仍可通过 RT qPCR 放大。虽然最近已经报告了两个新的体外培养技术为人类病毒56 , 但它们仍然依靠 RT qPCR 进行病毒定量化, 而且还不是常规临床/环境的可行方法。测试人类的病毒。因此, RT-qPCR 仍然是临床/环境测试的黄金标准。
其他的体外方法是为了更好地估计病毒颗粒的传染性状态而开发的。这些方法一般可以归类为那些处理病毒衣壳完整性和评估基因组完整性的人。前者的做法更受欢迎。一个常用的方法是核糖核酸预处理前提取病毒基因组 RNA, 但这并不解释的病毒微粒, 保持完好, 但缺乏能力, 以约束宿主受体/因素, 以及病毒粒子, 有致命的突变或已截断或稍有损坏的基因组7。衣壳的完整性也可以根据病毒对人类病毒 co-receptor/因素的能力进行评估, 这些碳水化合物被称为组织血型抗原 (HBGAs)。HBGAs 是存在于宿主肠上皮细胞作为糖蛋白或脂 (在多个其他组织中) 的一部分, 并可能在体液中分泌, 如唾液。在其表面含有 HBGA 样物质的肠道细菌也被证明绑定人类病毒8,9,10, 这样的交互可能会促进病毒感染5。使用 HBGA 结合的基本概念是, 只有能够结合假定受体/共同的病毒粒子才能够感染细胞。多项研究表明, bead-based HBGA 结合在先核酸扩增是一种有前途的传染性判别方法11,12,13,14, 15,16。关于 HBGAs 的一个主要挑战是, 没有一个单一的 HBGA 类型可以约束所有人类病毒基因型。为了解决这一问题, 猪胃粘液蛋白, 其中含有 HBGAs, 已被用于取代纯化的 HBGA 碳水化合物的利益, 易于合成, 一致性, 降低成本。
摩尔et al.的最新出版物17引入了一套用于估计人类病毒衣壳完整性的新技术。代替 HBGAs, 摩尔et al.17研究了广泛反应的核酸适 (M6-2)18和广泛反应的单克隆抗体 (NS14)19到 heat-treated GII 的绑定. 4 悉尼病毒衣 (病毒样颗粒或 vlp), 并比较这对合成 HBGAs 的约束力。核酸适是短 (〜 20-80 nt) 单链核酸 (ssDNA 或 RNA), 折叠成独特的三维结构作为其序列的结果, 并绑定一个目标。因为它们是核酸, 所以成本更低;易于化学合成、纯化和改性;和稳定的热量。一些报告的适产生反对病毒存在, 其中一些表现出广泛的反应, 各种菌株18,20,21,22。例如, 摩尔et al.17表明, 适 M6-2 绑定到纯化、组装的人类病毒衣 (vlp), 并在依赖于病毒衣壳的 HBGA 中表现出类似的行为, 以维持更高的顺序 (例如,二级和第三级) 蛋白质结构发生绑定。另一方面, 在衣完全变性后, 有相当比例的病毒 vip 结合抗体 NS14 残留。摩尔et al.研究中病毒结合的评价17使用了类似于 ELISA 的简单的 plate-based 方法, 但适用于替代抗体 (因此该方法被称为 ELASA)。这种高通量的实验方法被用来评估不同的处理对病毒衣壳的影响, 这对了解病毒灭活的机制, 在接触物理或化学压力源时有价值然而, 这种方法的一个缺点是较低的灵敏度和缺乏耐受性的高级别的基质相关污染物, 导致非特异性的约束力适。
摩尔et al.17使用了另一种方法, 即动态光散射 (dl) 来监测病毒粒子在热处理时的聚集情况。dl 通常用于评估纳米粒子悬浮液的大小, 并已扩展到蛋白质23,24和病毒25,26,27。粒子在溶液中散射的光的强度随粒子大小的函数而波动, 从而允许计算扩散系数, 然后使用已确定的公式进行粒子直径。dl 技术可以区分分散粒子的水动力直径到纳米尺度, 它允许检测分散的病毒、单个衣壳蛋白或聚, 以及基于大小27的病毒聚合体。病毒聚集, 以增加的粒子大小为代表, 表明了衣壳完整性的损失。由于衣壳变性, 其结构被破坏, 疏水性残留物就会暴露, 导致微粒粘在一起形成聚合体。dl 可用于在指定处理后测量粒子大小或实时聚合的动力学17,28。这种方法的优点是可以在溶液中观察衣壳的行为, 但需要高浓度的纯化衣壳, 这可能并不完全代表该病毒在其自然状态。
摩尔et al.使用的最终方法17是透射电子显微镜 (TEM)。虽然这种方法缺乏敏感性, 不产生定量数据, 但它允许可视化不同处理对病毒衣壳结构的影响。虽然尚未理想的临床/环境设置, 这些方法的组合使用是有价值的了解人类病毒灭活。本文的目的是为 plate-based 结合测定 (ELASA)、dl 和 TEM 制备方法提供 in-depth 的协议, 用于研究热处理对衣壳中病毒衣壳的影响。在摩尔et al.中提供的完整性17。

<table>
	<tbody>
		<tr>
			<td>Plate-Based Binding Assay for Evaluating Loss of Higher Order Norovirus Capsid Structure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well, medium-binding polystyrene EIA plate</td>
			<td>Fisher (Costar)</td>
			<td>07-200-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Lid for 96-well plate</td>
			<td>Thermo Fisher</td>
			<td>3098</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin film (Parafilm)</td>
			<td>Thermo Fisher</td>
			<td>PM999</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS (pH 7.2)</td>
			<td>Life Technologies</td>
			<td>20012-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS (pH 7.2) + 0.05% Polysorbate 20</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified, assembled human norovirus GII.4 capsids</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Individual 0.2 ml PCR tubes</td>
			<td>Genesee Scientific</td>
			<td>22-154G</td>
			<td></td>
		</tr>
		<tr>
			<td>2 thermocyclers</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop mini centrifuge</td>
			<td>Fisher Scientific</td>
			<td>12-006-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk solids</td>
			<td>Thermo Fisher</td>
			<td>LP0031B</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic histo-blood group type A disaccharide, biotinylated</td>
			<td>Glycotech</td>
			<td>01-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated aptamer M6-2 (stock concentration 100 &#181;M in nuclease-free water): 5&#39;-/5Biosg/AGTATACGTATTACCTGCAGCTGGGAAGAGGTCCGGTAAATGCAGGGTCAGCCCGGAGAGCGATATCTCGGAGATCTTGC -3&#8217;</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-horseradish peroxidase conjugate (ELISA grade), We use Life Technologies SNN2004</td>
			<td>Life Technologies</td>
			<td>SNN2004</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#8217;,5,5&#8217;-tetramethylbenzidine (TMB) substrate, room temperature</td>
			<td>Thermo Fisher</td>
			<td>50-76-00</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M phosphoric acid</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader capable of reading 450 nm</td>
			<td>Tecan</td>
			<td>Infinite m200 Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Analysis of Plate-Based Assay Results</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel or similar program</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dynamic Light Scattering Experiments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer ZSP, or similar particle size analysis instrument</td>
			<td>Malvern Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientifc</td>
			<td>02-215-365</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz cuvette</td>
			<td>Hellma</td>
			<td>104-002-10-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Small volume disposable cuvette (to reduce VLP use)</td>
			<td>Malvern Instruments</td>
			<td>ZEN0040</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy Sample Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel electron microscopy grids with carbon support film</td>
			<td>Ladd Research</td>
			<td>10880-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Self-closing reverse electron microscopy tweezers</td>
			<td>Ted Pella</td>
			<td>5372-NM</td>
			<td></td>
		</tr>
		<tr>
			<td>Qualitative filter paper</td>
			<td>Sigma-Aldrich (Whatman)</td>
			<td>WHA1002055</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass petri dishes</td>
			<td>Sigma-Aldrich</td>
			<td>BR455743</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mM stock HEPES solution (pH 7.2)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>2% uranyl acetate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

alternative in vitro methods for the determination of viral capsid structural integrity

人类病毒在全世界造成了相当大的公共卫生和经济损失。新兴的体外培养的进展尚不适用于常规的病毒检测。目前的检测和量化技术, 主要依靠核酸扩增, 不区分传染性的非传染性病毒颗粒。这篇文章的目的是介绍最近在一起使用的技术进展的具体细节, 以便获得关于病毒微粒传染性状况的进一步信息。一种方法是评估病毒 ssDNA 适与衣的结合。适具有易合成、改性、价格低廉、稳定等优点。另一种技术, 动态光散射 (dl), 具有观察衣壳行为在溶液中的优势。电子显微镜允许可视化的结构完整性的病毒衣。尽管前景看好, 但每种技术都存在一些缺陷, 如对样品基质中的正电荷分子的非特异性适结合, 对 dl 的纯化衣壳的要求, 电子显微镜的灵敏度较差。然而, 当这些技术结合使用时, 所产生的数据提供了关于病毒衣壳完整性的更全面的信息, 可以用来推断传染性, 这些信息对于准确评估灭活方法或病毒检测的解释。本文提供了使用这些方法来区分传染性的人病毒粒子的协议。

人类病毒的结构完整性 GII. 4 悉尼衣 (vlp) 使用了摩尔et al.提出的几种新方法进行了评估17. 首先, 进行了一项实验, 将衣的完整性作为其绑定一个假定的受体/共同 (HBGA) 或 ssDNA 适 (M6-2) 的能力的功能进行了比较 (图 1)。显示的结果以前是由摩尔et al.提供的17, 并证明了适 M6-2-capsid 绑定行为与病毒 HBGA 结合,...

Watch this Scientific Journal Video about Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity at JoVE.com

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

利用病毒基因组扩增的常规检测方法由于不能区分传染性的非传染性微粒而受到限制。本文的目的是提供的其他方法, 以帮助区分传染性病毒颗粒使用适结合, 动态光散射和透射电子显微镜详细的协议。

替代体外测定病毒衣壳结构完整性的方法

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Research

JoVE Journal

Immunology and Infection

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

HIV - 1衣壳大会通过低温电子显微镜的结构和迭代螺旋实时空间重构

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

科研

免疫与感染

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

病毒浓度的测定通过空斑试验：用传统的和新型覆盖系统

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

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

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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 ...