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) 的 gii. 4 悉尼 (加入: JX459908) 组装在衣. 
		注: 衣在研究中获得了 Atmar (休斯敦, 德克萨斯州贝勒医学院) 的礼遇. </li>
		<li> 稀释衣至50和 #181; 在1x 磷酸盐缓冲盐水 (PBS, pH 7.2) 中, 最大容积为15和 #181; L 在单个上限 PCR 管。确保该管含有至少 600 ng 的衣壳。确保每个治疗配体组合有 600 ng 的衣壳. </li>
		<li> 预热热循环到所需的热处理. 
		注: 为本议定书的目的, 治疗68和 #176; C 为不同的时间 (0-25 min) 被选择了. </li>
		<li> 预热一个额外的热循环到4和 #176; C 用于立即冷却。在所需时间, 在热循环上添加带有衣壳溶液的管子。立即转移到预4和 #176; C 循环加热后5分钟。
		<ol> <li> 包括95和 #176; C 作为变性衣壳控制的5分钟治疗。包括未经处理的 (23 和 #176; C) 衣壳溶液作为一个积极的控制。包括 PBS 与无衣壳作为一个额外的负面控制. </li>
		</ol> </li> <li> 在离心机中短暂旋转, 将所有液滴放到管底, 并将 PBS 中处理过的衣壳溶液稀释到3和 #181;确保至少有200和 #181; 稀释的、经过处理的衣壳 (100 和 #181; l/井). </li>
	</ol> </li> <li> 应用100和 #181; 每个井的衣壳解决方案, 确保每个处理至少有两个井。另外, 包括2控制井与100和 #181; L PBS 为每个配体;这提供了测定的背景吸光度。盖上盖板, 用石蜡膜封住边缘, 以减少蒸发, 然后在4和 #176 的摇箱上过夜; C 或室温下2小时. </li>
	<li> 用0.05% 吐温 20 (PBST) 在 PBS 中制作5% 脱脂牛奶固体 (w/v), 准备阻塞缓冲器. </li>
	<li> 从盘中取出经过处理的衣壳溶液。添加200和 #181; 阻塞缓冲区的 i/o。在室温或夜间孵育2小时, 密封在4和 #176; C. </li>
	<li> 准备化适 M6-2 18 、 29 和化合成 HBGA 血型 A 或化类型 H 的配体绑定解决方案。
	<ol> <li> 将化适稀释到1和 #181; 我在核酸水中。确保200和 #181 有足够的解决方案; 每种治疗的总数量。将所选的化 HBGA 稀释到30和 #181; 在阻塞缓冲区中的 g/mL。确保有足够的200和 #181 的解决方案; 每种治疗的总容积. 
		注: 使用较少的 HBGA, 10 和 #181; HBGA 0.25% 脱脂牛奶中的 PBST 可以替代。化 M6-2 和 HBGA 的浓度已经经过优化和报告. </li>
	</ol> </li> <li> 删除阻塞缓冲区, 并使用200和 #181 清洗该板3次; PBST。用无菌纸巾把盘子倒置, 晾干残余的水分. </li>
	<li> 添加100和 #181; 配体结合液的 L/井, 确保每个热处理-配体组合有2口井。在室温下, 在60转1小时左右的轨道振动筛上孵化盖有盖子的盘子. 
	注: 一定要包括2口井每一个未经处理的衣和完全变性衣为每个配体作为正负控制, 分别。此外, 还包括2和 #34; 没有衣壳和 #34; 每个配体的井为板材背景控制. </li>
	<li> 去掉配体溶液, 用200和 #181 冲洗3次井; PBST。将无菌纸巾上的盘子倒置, 以干燥残留的水分. </li>
	<li> 在 PBS 中添加100和 #181; 0.2 和 #181 的溶液的 L/井; 亲和辣根过氧化物酶。在室温下孵育15分钟, 在轨道振动筛上轻轻摇动. 
	注意: 不同的亲和-辣根过氧化物酶结合物将有不同的浓度。请查阅用户和 #39 的手册, 以确定酶的 ELISA 应用的理想浓度范围, 并确保它是最佳的. </li>
	<li> 移除共轭解。用200和 #181 洗涤3次; 我/好 PBST将无菌纸巾上的盘子倒置, 以干燥残留的水分. </li>
	<li> 添加100和 #181; L/井33和 #39;, 55 和 #39;-tetramethylbenzidine (TMB) 衬底, 并允许颜色发展为 5-15 min. 观察任何颜色的负控制井, 并确保在复制板之间的同一时间持续发展。也要注意使用的平板读写器的吸光度范围. 
	注: 根据所使用的配体/试剂的质量, 显影时间可能有所不同. </li>
	<li> 停止反应的发展与100和 #181; L/井1M 磷酸。立即阅读板 450 nm。保存数据. 
	注: 酸的引入通常会使反应剧烈减慢, 但不会完全停止。在读吸光度之前, 不要把停止的盘子留在长时间内. </li>
</ol> 2。Plate-based 测定结果的分析 <ol> <li> 在电子表格程序中保存原始吸光度数据。用特定的处理和配体产生每个井对的平均吸收。使用这些平均值进行所有后续分析. 
	注: 比较两个井的吸收, 以确保没有对水井有显着不同的价值观. </li>
	<li> 分析绝对衣壳完整性 (所有绑定), 减去 #34 的原始吸光度; 没有衣壳和 #34; 从其他所有样品的吸光度值中控制. 
	注: 另外, 由于高阶 (二级、三级、第四系) 结构损失, 可直接分析。而不是减去阴性 (无衣壳) 控制的吸收, 减去完全变性衣壳控制的价值。这消除了所有的绑定信号, 由于非特异绑定到仪器, 以及绑定由于衣壳序列。HBGA 和适 M6-2 显示几乎没有约束力的完全变性衣壳, 所以任何方法产生类似的结果。在选择要使用的分析时, 必须考虑非特异适绑定的假阳性信号. </li>
	<li> 与阴性或变性控制调整的吸收, 划分治疗吸收由他们各自正面 (没有治疗) 控制吸收并且乘100。这提供了被处理的衣壳相对于未经治疗的控制信号的百分比. </li>
	<li> 要估计每个配体的结合信号的程度, 可归因于衣壳序列, 减去和 #34; 没有衣壳和 #34; 从初始吸收的负控制, 并采取变性衣壳的调整吸收。除以正控制信号的调整吸光度, 并乘以100。这给出了明显比例的信号, 由于配体结合到变性衣壳/衣壳序列. </li>
</ol> 3。用于检测热处理后病毒聚集的 dl 注意:下面的步骤可能是特定于所使用的软件和仪器, 但可以适用于类似的设备. 
<ol> <li> 在 dl 仪器软件中创建一个新的大小 SOP 使用: 材料 = 蛋白质, 分散剂 = PBS, 温度 = 25 和 #176; C, 平衡时间 = 0 秒, 细胞类型 = 一次性试管 (小容量, ZEN0040), 测量角度 = 173 和 #176; 反向散射,测量持续时间 = 自动, 测量数 = 3, 测量之间的延迟 = 0 秒, 数据处理: 分析模型 = 一般用途 (正常分辨率)。对所有其他参数使用默认设置. </li>
	<li> 选择软件中的绿色运行箭头, 并命名该示例. </li>
	<li> 按照1.1.1 通过1.1.4 的步骤进行 vlp 的热处理. </li>
	<li> 在 1x PBS 中稀释热处理后的 vlp 1:10, 最终浓度为5和 #181;选)用1和 #181 过滤样品; m 孔径大小, 除去尘埃颗粒;dl 对尘埃非常敏感, 因为大颗粒比小颗粒散射的光要多得多. </li>
	<li> 传输50和 #181; 将稀释的 vlp 成小容量的一次性试管。将试管插入仪器的样品室. </li>
	<li> 开始运行, 并使用 #39; 相关和 #39;, #39; 累积量拟合和 #39;, #39; 专家建议和 #39; 选项卡, 用于评估数据质量. 
	<ol> <li> 在运行过程中, 检查多索引值是否小于 0.3, 表示质量度量. #160; 在和 #8216; 多视图和 #8217; 选项卡上, 确保相关系数在短时间内是恒定的且接近, 但不超过 1,并在以后的时间急剧下降到 0, 颗粒大小的特点。使用 #8216; 专家建议和 #8217; 在测量期间对数据质量进行反馈的选项卡. </li>
		<li> 测量完成后, 再次检查多索引值小于0.3。确保累积量拟合线紧跟数据点并 #8216; 累积值拟合 #8217; 选项卡. </li>
	</ol> </li> <li> 将每个测量所报告的 Z 平均粒子直径的平均值作为每个样本的粒度。在 nm 和 #176 中绘制直径; C. </li>
</ol> 4。实时检测病毒聚合的 dl 注意: 下面的步骤可能是特定于所使用的软件和仪器, 但可以适应和应用于类似的设备. 
<ol> <li> 在 dl 仪器软件中创建一个新的大小 SOP 使用: 材料 = 蛋白质, 分散剂 = PBS, 温度 = 根据需要, 平衡时间 = 0 秒, 细胞类型 = 石英试管, 测量角度 = 173 和 #176; 反向散射, 测量持续时间 = 自动,测量数 = 50 (可根据聚集速率增加或减少), 测量之间的延迟 = 0 秒, 数据处理: 分析模型 = 多个窄模式 (高分辨率)。对所有其他参数使用默认设置. </li>
	<li> 选择软件中的运行箭头打开并命名一个新的样本, 并允许仪器达到温度平衡. </li>
	<li> 将 vlp 在1X 和 #160 中挂起;P bs 在离心管中, 浓度为5和 #181; g/毫升和 200-500 和 #181 之间的体积; l 涡悬浮. </li>
	<li> 自旋向下 5-十年代在一个台式离心机的 vip 悬挂 de-gas。这有助于防止在热处理时气泡形成, 影响尺寸测量. </li>
	<li> 将 vip 悬挂转换为小体积石英试管-避免气泡和吸管慢慢地对试管壁。仪器达到温度平衡后, 将试管插入样品室. </li>
	<li> 等待十年代的样品温度平衡, 然后开始运行。在运行开始时启动计时器。在第一个测量结束时停止计时器。把这段时间作为第一个时间点。从软件记录的测量时间获取后续时间点. </li>
	<li> 启动运行, 并监视相关、分发适应和专家建议以评估数据质量. 
	注: 当样品在聚合过程中被多时, 这些测量结果不一定少于0.3。
	<ol> <li> 确保相关系数不变且接近, 但在短时间内不超过 1, 并且在一个或多个后期急剧下降, 这是粒度的特征. </li>
		<li> 确保分布适应线紧跟数据点. </li>
	</ol> </li> <li> 分析大小数据。
	<ol> <li> 确定每个测量时间点和初始时间点之间的时间差。测量工期并非完全相同. </li>
		<li> 使用强度分布, 记录每个测量/时间点的最大区域的峰值大小. </li>
		<li> 在最小值中绘制峰值直径与时间的图. </li>
	</ol> </li> </ol> 5。tem 样品制备 <ol> <li> 获得为 tem 设计的碳支撑膜 (镍) 网格. 
	注: 咨询与核心设施的推荐试剂及其处理使用的设施显微镜。一定要在装有干燥剂或真空室的盒子里储存网格, 以尽量减少暴露在湿气中. </li>
	<li> 撕裂大约5厘米 x 5 厘米的滤纸。获得玻璃培养皿和适当的自闭式反向镊子, 设计用于显微镜. </li>
	<li> 切割约2.5 厘米 x 5 厘米的石蜡片。放在台式上. </li>
	<li> 热处理病毒 GII. 4 悉尼衣 <ol> <li> 按照上述步骤 1.1.1-1.1.5 中所述的热处理程序 (除: 使用 10 mM HEPES (pH 7.4) 进行治疗而不是 PBS, 不要进一步稀释衣治疗后 (保持50和 #181 的解决方案, 克/毫升)。吸管整个〜15和 #181; 在石蜡膜上除去经治疗的衣壳溶液. </li>
	</ol> </li> <li> 使用镊子抓取网格边缘, 允许它们在边缘上关闭以容纳网格, 并将网格碳颠倒放在处理后的解决方案的顶部。让孵化10分钟, 使衣壳附着在网格上. 
	<ol> <li> 根据衣壳制剂的纯度, 如果需要, 在10-15 和 #181 的形式下添加冲洗 10 mM 的 HEPES 溶液; 在经过处理的衣壳液滴后, L 液滴放在线上. </li>
		<li> 10 分钟后, 用水滴拾取网格。将网格垂直放置在一张滤纸上, 使其远离水滴。选取 10-15 和 #181 的液滴; L HEPES 缓冲器与电网, 举行三十年代, 然后灯芯远离另一块滤纸洗. </li>
	</ol> </li> <li> 将15和 #181; 在空白区域的石蜡膜上放置2% 铀醋酸液滴。
	<ol> <li> 用网格提取铀乙酸的液滴, 并在四十五年代保存. 铀乙酸酯, 一定要除去大部分的溶液。将网格碳侧放在一块滤纸上, 小心网格不 #34; 粘 #34; 对镊子上的湿气。将过滤纸与网格放在一个开放的玻璃培养皿中. 
		注意: 不要使用塑料培养皿, 因为网格会被静电吸引, 并成为粘在盘子里. </li>
	</ol> </li> <li> 对所有处理进行重复。将培养皿半与网格干燥过夜, 在观察 TEM 之前晾干. </li>
	<li> 查阅相关机构的显微镜设备, 观察准备好的网格。一些设施允许使用者对他们的设施和 #39 设备的操作进行培训。有关特定显微镜的设置和观察的具体细节是 beyond 本文的范围. </li>
</ol>

<ol>
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作者没有什么可透露的。

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>&lt;strong&gt;Plate-Based Binding Assay for Evaluating Loss of Higher Order Norovirus Capsid Structure&lt;/strong&gt;</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 &amp;micro;M in nuclease-free water): 5&#039;-/5Biosg/AGTATACGTATTACCTGCAGCTGGGAAGAGGTCCGGTAAATGCAGGGTCAGCCCGGAGAGCGATATCTCGGAGATCTTGC -3&amp;rsquo;</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&amp;rsquo;,5,5&amp;rsquo;-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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Analysis of Plate-Based Assay Results&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Dynamic Light Scattering Experiments&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Transmission Electron Microscopy Sample Preparation&lt;/strong&gt;</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

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

alternative-vitro-methods-for-determination-viral-capsid-structural

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

8.0K 次观看.  North Carolina State University. 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。

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

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

科研

免疫与感染

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

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

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

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

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

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 ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

生物学

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

高灵敏检测方法的沙粒，细胞附着的测量

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

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 ...

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.

Here we provide a detailed procedure for large-scale production of research-grade AAV vectors using adherent HEK 293 cells grown in cell stacks and affinity chromatography purification. This protocol consistently yields &#62;1 x 1013 vector genomes/mL, providing vector quantities appropriate for large animal studies.

在细胞堆中生产腺相关病毒载体，用于大型动物模型的临床前研究

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. ...

Plaquing of Herpes Simplex Viruses

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.

单纯疱疹病毒的扁平化

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can ...