我们感谢河野洋平山内和罗伯塔·曼奇尼为我们提供了试剂。 ，欧洲研究委员会（ERC），以及由瑞士国家科学基金会（Sinergia）的AH实验室是由居里夫人初始培训网络（ITN）的支持。

1.缓冲区和股票溶液的制备<ol><li>由470毫克的Tris盐酸，390毫克的MES水合物和580毫克氯化钠溶解在80毫升的DDH 2 O制备的MNT缓冲液（20mM MES，30毫摩尔Tris和100mM NaCl）中调整缓冲至pH 7.4，并将其与的DDH 2 O带来至100ml的最终体积</li><li>通过在80毫升的dh每2 O溶解9.76克MES水合物准备500毫米MES缓冲三个相同的解决方案。通过用浓NaOH滴定调节缓冲剂至pH 5.8，5.4和5.0，分别。带来各缓冲器的100毫升的dh 2 O的最终体积</li><li>通过在80ml各溶解7.88克Tris盐酸的dh 2 O制备的500mM Tris缓冲液两个相同的解决方案，并调节pH值至分别7.4和6.4，通过用浓HCl滴定。使缓冲至100ml与卫生署2 O的最终体积</li><li>用蒸馏水2 O准备50％的甘油溶液，搅拌均匀，直到GLYC埃罗尔是均匀混合。过滤通过一个Steritop滤波器单元（0.22微米孔径）成玻璃瓶的溶液。 </li><li>制备10％NP-40溶液和1M NaCl的卫生署2 O. </li><li>通过在4毫升的DDH 2 O溶解两片准备25倍浓缩的蛋白酶抑制剂</li></ol> 2.甘油梯度的制备<ol><li>制备5毫升洗涤剂缓冲主混合物（300毫摩尔NaCl，2％NP-40，2倍浓缩的蛋白酶抑制剂），用于每一pH条件下的溶液至试验（pH为7.4，6.4，5.8，5.4和5.0）。为了这个目的，混合9毫升1M NaCl的的6毫升10％的NP-40，2.4毫升25×蛋白酶抑制剂的股票，并且9毫升的DDH 2 0。 </li><li>对于每一pH条件下吸管4.4毫升的主混合物到50毫升锥形管中。 </li><li>用于pH值高于5.8添加0.6毫升相应调节pH的500毫摩尔Tris原液到管。用于pH 5.8和更低的添加0.6毫升相应调节pH的500mM的MES原液。 </li><li>制作5毫升含有300mM NaCl的缓冲液，2×蛋白酶抑制剂，60毫摩尔Tris调节至pH 7.4和的DDH 2 O这将作为不含去污剂的控制梯度缓冲液。 </li><li>如果需要，通过分别添加浓HCl或NaOH溶液，精细调节溶液至pH 7.4，6.4，5.8，5.4和5.0的pH值。 </li><li>将5ml的50％甘油原液添加到5ml导致六个不同的25％甘油溶液含洗涤剂和不含去污剂的缓冲混合物。通过用pH试纸检查的pH值。 注意:如果所测量的pH值从所需值显著不同，各自的溶液应该再次制备。 </li><li> 7比:通过在3混合50％甘油的库存与卫生署2 O制备15％甘油溶液中。 </li></ol> 3. IAV的超速离心<ol><li>通过使用5ml的注射器和娘家姓添加3毫升15％甘油溶液到超清晰离心管（13.2毫升14毫米×89毫米）DLE（21g的9厘米长）。在管的内壁不离开滴因为这可能会扰乱梯度的完整性。重复此一共有六个离心管，每个的五个pH条件来测试和一个用于控制的样品。 </li><li>小心地将15％甘油层下3.4毫升25％甘油溶液，通过使用5ml的注射器和长针。小心两层不能混用。重复此为所有六个条件与相应的调节pH的甘油溶液进行测试。 注:II级生物安全柜工作，为下面的步骤。 </li><li>轻轻覆盖用30微升澄清含有IAV（X31，H3N2）在1ml MNT缓冲液稀释的尿囊液的甘油梯度（相当于总病毒蛋白的约20-30微克）对于每个梯度。 </li><li>平衡对立管，将它们放入一个SW41超速离心转子。离心150分钟，55,000 XG和12℃。 </li><li>离心后，小心通过使用干净巴斯德吸管和一抽吸器除去上清液， 即 ，既甘油层。重悬在40微升（1×）的非还原性LDS样品缓冲液沉淀。它到吸管上下数次以完全溶解沉淀是​​重要的。将样品转移到1.5 ml离心管。 </li></ol>沉淀部分和考马斯染色的4 SDS-PAGE <ol><li>加热所有样品在95℃下进行10分钟。此时的样品可以贮存于-20℃，直到它们通过SDS-PAGE进行分析。 </li><li>负载20微升溶解丸粒到一个预制梯度（4-12％）的Bis-Tris微型凝胶在1×MOPS SDS 200伏运行1小时运行缓冲液。 </li><li>使用40％的甲醇和10％冰醋酸在DDH 2 O定影溶液</li><li>孵育在定影溶液的凝胶1小时，并在15厘米的细胞培养皿用胶体考马斯溶液足够体积，同时轻轻染色过夜在室温下摇动。 注:以关闭盘以避免染色溶液的蒸发是很重要的。 </li><li>退色在DDH 2 O.凝胶直至凝胶背景变得清晰更换DDH 2 O的每次15-20分钟。存储在DDH 2 O的凝胶在4°C，直到它被扫描带量化。 </li><li>扫描高分辨率的凝胶，并使用市售的或定制的软件，用于蛋白质谱带强度的定量。减去从靠近相应频带的区域中的背景信号和（在pH 7.4）正常化这些值到不含去污剂的对照样品。 </li></ol>

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The authors have nothing to disclose.

病毒衣壳是亚稳大分子复合物。虽然病毒粒子组装需要病毒基因组的衣壳和冷凝，下一轮感染的启动取决于这个紧凑的衣壳的结构拆卸。病毒已经进化到利用各种细胞机制，以控制涂层脱壳周期，包括细胞受体，伴侣分子，蛋白水解酶，由马达蛋白或解旋酶，以及pH和离子开关26,27提供物理力。在这里，我们描述了基于甘油梯度离心特异性测试的促进IAV芯拆卸因子的效果的体外方法。该技术可以潜在地适于解剖的其它包膜病毒脱壳过程。 通过使用胶体考马斯，主要结构IAV蛋白M1，NP和HA可以清楚地在SDS-PAGE即使相对小的病毒partic检测文件编号。然而，沉淀的衣壳结构的更深入的表征和其组合物将需要后续的分析，例如电子显微镜（如先前提出的28），Western印迹，动态光散射或质谱。该协议的进一步扩展可以包括较低的（25％）甘油特定核心组件的相位和分析的分馏。 下部甘油层到5.0（为X31）最优融合pH值的调整导致了基质层的几乎完全离解（ 图2）16。但是，大约30％的输入的NP信号仍然可以在该pH检测。这可能是由于没有在此设置的蜂窝脱壳因素，如最近鉴定的组蛋白脱乙酰6（HDAC6）18，不完全拆卸发生。在细胞内，从Na +的病毒核心和接触开关的缓慢酸化至K + </suP&gt;素数完全释放vRNP通过脱壳控制的因素的16病毒。我们不能排除，在我们的设置中，非离子型去污剂部分破坏酸暴露和不稳定芯。然而，在芯在中性pH沉降没有影响观察到的类似蛋白质谱带强度表示为相对于非溶解的样品（ 图2A，比较泳道1和2）。虽然，这里给出证明该测定是高度可再现的和（因此是）鲁棒足够用于频带量化，变化的一定范围内可以观察到。 在这个协议中所用的甘油是在实验的结果是至关重要的。如先前报道17，超速离心通过蔗糖梯度动摇的IAV芯，这可能是由于由蔗糖产生一个高渗透力。通过蔗糖梯度IAV的纯化可能会产生同样的效果。因此，我们比较蛋生长X31从CL派生<html> arified尿囊液和蔗糖纯化的股票。 （数据未显示）没有大的差异可能相对于核心稳定性酸性pH的影响被观察到。尽管如此，为了排除渗透活性蔗糖的潜在的副作用，我们建议执行与澄清尿囊液或浓缩的细胞培养上清液在体外脱壳测定。 除了梯度材料的选择，它保持梯度不影响裂解病毒核心的沉降行为，并确保可再现的结果的完整性是重要的。此外，超速离心后的沉淀级分的不完全再悬浮可能导致不同的结果。最后，我们强烈建议在实验的当天理想地调节含去污剂的缓冲溶液的pH为离子浓度的微小变化可能对病毒核心稳定性有强烈影响。 内容"&gt;重要的是要注意，根据特定IAV菌株，不同的拆卸效率可能发生基于各个M1的性能是重要的和NP变体，这可能也适用于突变病毒。因此，对于矩阵层和vRNP pH值的阈值解离已用于测试额外的影响之前的各IAV应变来确定。有关对病毒核心稳定性，我们建议的底部甘油层调节至pH值，使得M1的半最大拆卸实现特定条件的分析，这是可能通过相应地修改所述含去污剂的层来调查特定离子或还原剂的影响。推定蜂窝脱壳因子可直接加入到甘油梯度，在细胞质因素的情况下，在梯度的底部的第三甘油层可以是引入，其调节至中性pH值，包含感兴趣的细胞因子，并且是自由洗涤剂。以这种方式，第Ë三层梯度将更加紧密地模仿病毒通过内吞系统和基因区段的释放通道进入中性细胞质。 综上所述，我们提出了一个强大而简单的体外实验研究IAV启动和脱壳，这对通用的修改的可能性，以解决IAV条目的上下文不同的问题。此外，它可以应用于调查与相关脱壳机制的其它包膜病毒条目的进程。

甲型流感病毒（IAV）是包膜病毒属于正粘病毒科的。它的基因编码的一个分段，负义单链RNA基因组。在人类中，IAV引起呼吸道感染，发生的季节性疫情和承担全球性流行病1的潜力。在结合于宿主细胞表面2上的唾液酸残基，IAV是通过网格蛋白依赖的内吞作用和网格蛋白独立的途径3-8内化。在胞吞液泡的酸性环境（pH值&lt;5.5）触发在IAV穗糖蛋白血凝素（HA），这导致在病毒融合和已故体膜9的一个主要的构象变化。一旦IAV衣壳（这里也被称为病毒"核心"）已经从晚期内涵体（LES），它在胞质溶胶中，随后通过病毒核糖核蛋白（vRNPs）转运到细胞核未涂覆逃脱 - 的部位O˚F病毒复制和转录10-13。之前的HA的酸活化的病毒经历在内吞系统，素数为随后的拆卸14-16芯pH的逐渐减少。在此"启动"步骤中的病毒膜M2离子通道介导的质子和K +10,14,16的流入。该病毒内部的离子浓度的变化扰乱了相互作用的基质蛋白M1和八个vRNP束建立，并促进IAV脱壳在以下膜融合10,14-17细胞质中。 起动步骤的直接和定量的分析已经阻碍了一个事实，即内涵体难以接近实验。入门过程，而且高度非同步的。此外，终点测定法，如释放的病毒RNA或感染性测量的定量R​​T-PCR，没有提供关于病毒capsi的生化状态的详细的图片ð在进入任何给定的一步。同时通过siRNA或药物治疗的内体扰动已显著贡献IAV进入18-20的理解，微调是困难的，容易出现非特异性副作用，在严格控制的内吞体成熟方案。 为了避免这些问题，我们已经适于基于使用速度梯度离心17的以前开发体外协议。相对于其他的尝试21,22，23,24，其中大多数是基于蛋白水解裂解和清洁剂处理，然后进行EM分析的组合，这种方法使一个容易量化的结果。离心通过不同的梯度层允许沉降颗粒被暴露，并与以受控的方式变化的情况作出反应。在所提出的协议，IAV从澄清尿囊液的或纯化的病毒颗粒衍生的被沉淀成两层甘油梯度，其中所述底层包含非离子型洗涤剂NP-40（ 图1）。作为病毒进入第二，含去污剂的层，病毒脂质包膜和包膜糖蛋白轻轻溶解并留下。核心，八个vRNP束的组成和由一个基质层包围，沉积物作为一个稳定的结构进沉淀部分。病毒核心蛋白，例如M1和vRNP相关核蛋白（NP），可以在通过SDS-PAGE和考马斯染色的沉淀来识别。特别是，考虑市售梯度凝胶的优点，并与高度敏感的胶体考马斯25染色，使高精度，即使是少量的病毒核心相关蛋白的检测。 此设置的基础用于测试是否不同的条件，例如pH，盐浓度，和推定脱壳因素上的核心部件和芯STABIL的沉淀行为的影响<html>性。到这个目的，在甘油梯度只有较低的，含去污剂的层是通过将因子或感兴趣的条件进行修改。该技术已在研究的不同的pH值和盐浓度的IAV芯16的完整性的影响特别有价值的。 IAV脱壳的中间步骤可以在随后vRNP离解在pH 5.5和下部16,17（ 图1）温和的酸性pH值（&lt;6.5）进行监测，包括在基质层的离解。后者步骤是由高K +浓度的甘油层的存在进一步增强，反映了晚内吞环境16。因此，当病毒和芯通过梯度沉降他们经历了改变环境中核内体，模仿的条件。结果是在体外病毒核心的逐步解体，补充细胞生物学实验得出的结果。 T"&gt;这里介绍的方法，使快速和IAV的高度重复性分析（X31和A / WSN / 33）和IBV（B /李/ 40）脱壳由酸性pH值触发增钾+16,17以及拆卸的在碱性pH曝光17副粘病毒核心。可以想象，该方法可以适于其它包膜病毒进入细胞过程中深入了解病毒衣壳的结构和衣壳拆卸的生化特性。

<table border="0" cellpadding="0" cellspacing="0" width="1103">
	
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">cOmplete&#8482;, EDTA-free protease inhibitor tablets</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:116px;">11873580001</td>
			<td style="width:473px;">The stock solution can be stored at 2 to 8 &#176;C for 1 to 2 weeks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glacial acetic acid</td>
			<td>Merck Millipore</td>
			<td>100063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol anhydrous BioChemica</td>
			<td>AppliChem</td>
			<td>A1123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td>Merck Millipore</td>
			<td>100317</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Long injection needle (21 GA, 9 cm, bevel or blunt-end)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M8250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Merck Millipore</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td>Now commercially available as IGEPAL&#174; CA-630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE&#174; 4-12% Bis-Tris mini gels, 10 wells, 1.0 mm</td>
			<td>Life Technologies</td>
			<td>NP0321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE&#174; LDS sample buffer (4X)</td>
			<td>Life Technologies</td>
			<td>NP0008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE&#174; MOPS SDS running buffer (20X)</td>
			<td>Life Technologies</td>
			<td>NP0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH indicator strips, pH 4.0 - 7.0</td>
			<td>Merck Millipore</td>
			<td>109542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QC Colloidal Coomassie Stain</td>
			<td>BIO RAD</td>
			<td>1610803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Merck Millipore</td>
			<td>106406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodiumhydroxide</td>
			<td>Merck Millipore</td>
			<td>106498</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Steritop&#8482; filter unit</td>
			<td>Merck Millipore</td>
			<td>SCGPT05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW41 Ti, ultracentrifuge rotor set</td>
			<td>Beckman Coulter</td>
			<td>331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thinwall, Ultra-clear centrifuge tubes, 13.2 ml, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris hydrochloride&#160;</td>
			<td>AppliChem</td>
			<td>A1087</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid</td>
			<td>Virapur</td>
			<td></td>
			<td>Freshly thawed on 4&#176;C</td>
		</tr>
	</tbody>
</table>

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.

如已经讨论的，在核内体内吸需要呈现IAV芯脱壳胜任。芯的质子化削弱M1和vRNPs的相互作用（病毒RNA，NP组成，和聚合酶复合PB1 / PB2 / PA）。当传入病毒暴露于pH为在早期内涵体（EES）6.5（或更低），并继续进行，直到在的LE在pH5.0周围的病毒熔丝此过程开始。 以模仿，pH降低，甘油梯度的底层以恒定的盐浓度被调节至pH 7.4和5.0之间的值。从澄清的尿囊液衍生X31进行速度梯度离心分析通过SDS-PAGE（ 图2A，B）。不用洗衣粉中存在的梯度（ 图2A，lane1）完整的病毒粒子沉淀，通过的特征模式反映代表HA（HA1和HA2; 63 kDa）的乐队在考马斯，NP（56 kDa的）和M1（27 kDa的）染色凝胶。由于凝胶在非还原条件​​下运行，蛋白水解切割HA1和HA2仍然通过二硫键连接并运行在大约64 kDa的。在还原条件下对应于HA1和HA2频带似乎非常接近的NP和M1频带，分别使得难以忠实解释和确定被完全从病毒核心（ 图2B）衍生的带强度。 在加入NP-40的底部（25％）甘油层病毒包膜包括HA，NA，和M2分别为溶解和超速离心（ 图1; 图2，泳道2）中所述病毒核心单独沉淀成粒料。用pH低于6.5开始，M1被逐渐从沉淀级分丢失，达到pH值5.8和5.4（ 图之间的最小图2A，B）。 pH低于5.8 vRNPs解离，从而从沉淀物丢失。以前，已经显示，事实上，整个vRNPs被释放到上层，因为它们的释放与PB2的从沉淀级分16中的损失相关。测定蛋白条带强度，显示为vRNP束，分别（ 图2）的M 1层和离解的拆卸两个不同的pH值的阈值。额外条带可以在凝胶，这可能对应于聚合酶蛋白PB1（87 kDa的），PB2（86 kDa的），PA（83 kDa的），和M2（11 kDa）的上被观察到。由于IAV病毒体内部的低拷贝数，我们不能可靠地确定他们的身份和他们因此不考虑为定量。 <img alt="图1" src="/files/ftp_upload/53909/53909fig1.jpg" /> 图1:IAV的示意图 <eM&gt; 体外 核心拆装检测 。简言之，将纯化的病毒颗粒装载在两步甘油梯度。底部层含有洗涤剂NP-40和而上部，不含去污剂的层被保持在恒定pH和盐浓度被调节至所需的pH和盐浓度。根据在较低甘油层的情况下，完整的病毒核沉降至底部（1）或解离并留在甘油层（2）。中性pH导致M1与vRNPs组成的完整病毒核心的沉淀。条件脱壳有利，如酸性pH（5.0），结果在病毒核心部件的离解，因此，从沉淀部分损失。继超速，甘油上清液被删除，颗粒通过SDS-PAGE分析。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53909/53909fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </<html> p&gt; <img alt="图2" src="/files/ftp_upload/53909/53909fig2.jpg" /> 图 2: 在 体外 酸化 IAV核心拆装 （A）在较低的甘油层不同的pH值条件下X31内核的体外拆卸。作为对照，NP-40从底部甘油层（第一泳道）被删去。样品通过SDS-PAGE在非还原条件​​，随后进行考马斯染色下分离。病毒蛋白条带在右边标明。 （B）在（A）中示出的病毒蛋白条带的强度的光密度定量。为M1和NP蛋白条带强度进行标准化为那些在pH7.4不使用NP-40。数据表示为重复实验±标准偏差（SD）的装置。从斯托弗等人 ，2014年16改编。ge.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation at JoVE.com

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 ...

in-vitro-disassembly-influenza-virus-capsids-gradient

Nanyang Technological University

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JoVE Journal

Immunology and Infection

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

8.9K 次观看.  ETH Zurich. 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.

视频: In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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免疫与感染

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

通过流式细胞仪测量附件和流感的内在病毒在A549细胞

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.