这项工作是由FWO-VLAANDEREN，在IOF项目IOF10 / StarTT / 027和根特大学特别研究基金BOF12 / GOA / 014的支持。

注意：下面的转染和共免疫沉淀的协议被建立用于一个9厘米培养皿格式。其他格式也是可能的缩放协议之后。 1.播种人胚肾（HEK）293T细胞<ol><li>转染每9厘米培养皿1.2×10 6个细胞的12ml Dulbecco改性Eagle培养基（DMEM）中的补充有10％胎牛血清，2mM的L-谷氨酰胺，0.4mM的丙酮酸钠之前种子的HEK293T细胞的一天， 0.1mM非必需氨基酸，100U / ml青霉素和0.1mg / ml链霉素。 </li><li>生长的细胞16小时，在37℃和5％的CO 2。 </li><li>目视检查的形态和细胞的生存力与转染前一个倒置光学显微镜。细胞需要是亚汇合以获得最佳的转染效率。 </li></ol> 2.磷酸钙HEK293T细胞的转染注意：使用0.5-1 pCAXL-NP或空pCAXL质粒结合1-3微克每9cm培养皿pCAXL-MX1的微克。使用在所有样品中总质粒DNA的量相等;调节用空质粒如果必要的。 <ol><li>准备转染后的缓冲区： <ol><li>制备的Tris-EDTA（TE）为1.0毫摩尔Tris盐酸pH为8.0和0.1mM EDTA的pH为8.0的浓度。 </li><li>制备的BS / HEPES用25mM的HEPES浓度（5.96克/升，4-（2-羟乙基）-1-哌嗪乙磺酸），274 mM氯化钠（16克/升），10毫米氯化钾（0.74克/升）， 1.5mM的NaHPO 4·12H 2 O（0.5克/升）和11.1毫葡萄糖（2克/升）。调节pH至7.05。 </li><li>制备的CaCl 2 / HEPES用1.25M的氯化钙2·2H 2 O（183.8克/ L）和125毫米的HEPES（29.79克/ L）的浓度。调节pH至7.05，用NaOH。 </li></ol></li><li>使用前暖转缓冲区在37℃。 </li><li>通过稀释在600微升TE中的质粒DNA制备质粒样品。在6孔板的孔中制备这些混合物。 </li><li>添加150微升的CaCl 2 / HEPES的在一个逐滴的方式向质粒样品和吹打3次上下混合。 </li><li>准备通过逐滴加入质粒溶液的转染溶液（TE + DNA +氯化钙2 / HEPES; 750微升）以750微升的BS / HEPES缓冲液中新鲜6孔板提供的。均匀分布的质粒溶液在整个井含BS / HEPES缓冲液。 </li><li>摇板振荡器上的转染溶液90秒以1,000rpm。 </li><li>为5分钟，在室温下孵育该混合物。 </li><li>加的转染溶液（1.5毫升）逐滴向细胞。使用一个微量吸P1000滴在细胞中的转染溶液。之分散在整个9厘米培养皿摇板非常轻柔。 </li><li>孵育所述细胞在37℃和5％CO 2的6小时。然后通过抽吸去除中，立即更换无线第12毫升新鲜，预热的培养基。轻轻的新鲜培养基添加到细胞中，以防止细胞脱离。对于这一点，保持吸管的尖端靠在井的侧面，轻轻推出的介质。 </li><li>孵育细胞另外16小时，在37℃和5％的CO 2。 </li></ol> 3.免疫共沉淀注：转染后执行免疫共沉淀24小时。 <ol><li>制备低盐裂解缓冲液和高盐洗涤缓冲液中。 <ol><li>通过称量NEM的量和在无水乙醇中溶解制备的2M N-乙基马来酰亚胺（NEM）的储液。准备NEM原液使用前新鲜。 注意：NEM很毒，准备和使用这种原液在通风橱中。 </li><li>准备以50mM的Tris-HCl pH为8，150 mM氯化钠，5mM的乙二胺四乙酸（EDTA），1％NP40和蛋白酶抑制剂混合物的浓度低的盐裂解缓冲液（溶解1表吨在50ml裂解缓冲液）。添加NEM至25mM的（ 即，稀释1:80）的终浓度。添加蛋白酶抑制剂和NEM后，保持在冰上。 注：请务必在使用前刚加入蛋白酶抑制剂和NEM。 </li><li>准备以50mM的Tris-HCl pH为8，500 mM氯化钠，5mM的EDTA和1％NP40的浓度高盐洗涤缓冲液。请注意，高盐洗涤缓冲液中不包含的NEM。 </li></ol></li><li>配制细胞裂解液。 <ol><li>除去培养基并用2ml冰冷的磷酸盐缓冲盐水（PBS）洗细胞。轻轻地添加洗涤液，因为HEK293T细胞分离容易。 </li><li>取出PBS和每9厘米培养皿中加入600微升冰冷的低盐裂解液。 </li><li>孵育板20分钟在冰上。确保该板保持水平，以保证在板表面用裂解缓冲液完全覆盖。轻轻摇动板每5分钟。 </li><li>收集细胞裂解物在一个1.5ml微量管和离心3分钟，在4℃和16000×g离心以沉淀不溶性级分。 </li><li>传送可溶部分， 即细胞裂解物，以一个新的1.5 ml离心管，并保持在冰上。立即继续进行共免疫沉淀协议，以防止相互作用蛋白的解离。执行下列所有尽可能在冰上或在4℃下的步骤，以限制蛋白质水解活性在裂解物。 </li></ol></li><li>代的免疫复合物。 注：在这一步骤中，感兴趣的蛋白结合的合适的抗体。研究NP-MX1的互动，使用鼠标抗NP单克隆抗体。 <ol><li>对于每个样品，混合135微升溶胞产物与2微升抗NP单克隆抗体和113微升低盐裂解缓冲液（250微升总体积）的。存储剩余的溶胞产物在-20℃下用于进一步分析，如免疫印迹，来记录推测INTERA的表达水平ction伙伴中的转染细胞。 注意：可替换地，测量裂解物的蛋白质浓度（ 例如，使用Bradford试剂），并使用总蛋白的固定量， 例如，400微克，对于每个溶胞产物。 </li><li>孵育在一个转向轮在4℃下将抗体溶解物混合3小时。此步骤可以扩展到一个保温过夜。 </li></ol></li><li>制备的G蛋白珠。 注意：G蛋白珠运输和储存在20％乙醇中保存。胎边浆料通常由50％珠和这些珠需要洗涤它们用于免疫沉淀的免疫复合物之前。 <ol><li>使用50微升珠， 即，将100μl珠浆料，每个样品。洗所需的在1管中的共免疫沉淀测定法所有的样品珠的量。切1毫升移液管尖的尖端，以缓解珠浆料移液。 </li><li>离心G蛋白珠浆在8,000×克和4℃持续30秒。除去乙醇溶液，并添加低盐裂解缓冲液的等体积。离心机的G蛋白珠浆在8000×g离心，4℃，30秒，轻轻除去上清液。重复此洗涤步骤3次。 注意：用于洗涤珠粒不需要含有蛋白酶抑制剂或NEM的低盐裂解缓冲液。 </li><li>估计的G蛋白珠的体积并加入低盐裂解缓冲液的等体积以使一个新的50％珠浆料在低盐裂解缓冲液。 </li><li>对于每个样品，转移100μl的珠浆液在一个新的1.5 ml离心管，并在冰上储存直至使用。小心分割前悬浮珠浆料，因为这些珠快速沉降到管的底部。 </li></ol></li><li>免疫的免疫复合物通过G蛋白珠和其洗脱。 <ol><li>使用G蛋白珠进行免疫沉淀，离心所有管30秒，在8000×g离心，4℃和前通过目测有珠子存在所有样品中等量验证。如有必要，再次调节的珠的量在一些样品和离心机。弃上清。要小心，不要打扰沉淀的G蛋白珠。 </li><li>短暂离心所述免疫复合物（ 即 ，裂解物用抗体，250微升），30秒，在8000×g离心，4℃，收集在试管底部的完整样品。转移的免疫复合物的G蛋白珠（50微升）。 </li><li>孵育在转向轮60分钟，在4℃。不孵育这些免疫复合物长于75分钟，用珠，以减少蛋白质的非特异性结合到所述G蛋白珠。 </li><li>离心G蛋白珠（与结合免疫复合物），持续30秒，在8000×g离心，4℃并除去上清液。要小心，不要打扰沉淀的G蛋白珠。可选：保存这个上清在4℃或-20℃，供以后分析，例如，为了估计未结合的蛋白质的量。 </li><li>约5分钟，900微升高盐裂解液洗的G蛋白珠。确保该珠粒在最佳洗涤的洗涤缓冲液完全重悬。离心G蛋白珠进行30秒，在8000×g离心，4℃，并丢弃上清液。重复此洗涤步骤4次。要小心，不要打扰沉淀的G蛋白珠以避免免疫沉淀的物质损失。 </li><li>在最后一次洗涤步骤后，加入50微升2×Laemmli样品缓冲液至珠并加热该悬浮液10分钟，在95℃下洗脱（共）免疫沉淀的蛋白质。 <ol><li>制备出10毫升6×Laemmli缓冲液用1克十二烷基硫酸钠，3.5毫升甘油，3.5毫升的1M的Tris-HCl pH为6.8和420微升β巯基乙醇的。通过添加蒸馏水调节至10ml的总体积。稀释在蒸馏水的3倍，得到2×Laemmli缓冲液。 注意：＃946;巯基乙醇是有毒的，准备和使用Laemmli缓冲液在通风橱中。 </li></ol></li><li>加热后，离心G蛋白珠进行30秒，在8000×g离心并储存样品于4℃（短期）或-20℃（长期）。 </li></ol></li></ol> 4.分析的（共）免疫沉淀的蛋白<ol><li>可视化存在于细胞裂解物和共免疫沉淀的洗脱物进行SDS-PAGE 20和蛋白质印迹21,22的蛋白质。通常情况下加载一半的Laemmli洗脱液的凝胶。要小心，不要采取样品进行凝胶装载时打扰沉淀的G蛋白珠。 MX1和NP表达，揭示具有抗MX1和抗NP抗体，分别为14。检测用HRP基于化学发光和X射线胶片显影的频带。 </li></ol>

<ol>
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作者宣称，他们有没有竞争的财务权益。

研究抗病毒药蛋白及其病毒靶之间的相互作用是了解这些蛋白质的抗病毒机制的细节非常重要的。这可以给新的见解如何病毒及其宿主共同进化，并依据新的抗病毒策略的发展。此处所描述的优化的共免疫沉淀协议允许研究小鼠MX1蛋白及其病毒靶，流感的NP蛋白质之间的相互作用。这个协议的最重要的方面是，在裂解缓冲液中加入NEM的，作为对NP-MX1相互作用是检测不到的，在缺少NEM（ 图3）<...

粘病毒性（MX）蛋白质是抗病毒病原体的先天免疫防御的重要组成部分。这些蛋白质是由I型和III型干扰素诱导的大dynamin上样GTP酶。相应的Mx基因是存在于几乎所有的脊椎动物中的一个或多个拷贝和它们的基因产物抑制多种病毒，包括正粘病毒科 （ 例如 ，流感病毒）， 弹状 （ 例如 ，水泡性口炎病毒）， 布尼亚病毒科 （ 如 ，拉克罗斯病毒）和逆转录病毒 （ 例如，人免疫缺陷病毒-1）1-4。目前还不清楚这些蛋白质如何认识这种病毒浩如烟海，没有任何明显的共享主序列基序在这些病毒。的Mx蛋白的相互作用与病毒指标分析，可能涉及高阶配合其他宿主细胞的因素，将有助于了解的分子机制吨帽子进化的病毒和其宿主之间的军备竞赛。 哺乳动物的Mx蛋白和病毒靶之间的相互作用已经研究最广泛的用于人类的MxA。人类的MxA能抑制多种病毒，包括正粘病毒A型流感和Thogoto病毒的复制。 MxA蛋白结合的Thogoto病毒核蛋白复合物（vRNPs），从而防止它们的核条目，这导致感染5的块。此MxA蛋白和Thogoto病毒vRNPs之间相互作用已被证明具有共沉淀和免疫共沉淀实验6-9。怎样的Mx蛋白质阻碍A型流感病毒是不太清楚。一个主要的问题是，它不是简单的证明的MX蛋白和流感基因产物之间的相互作用。其中一份报告证实人类的MxA和NP蛋白质之间的相互作用在A型流感病毒感染的细胞10。这种相互作用可能仅通过共immunopr显示ecipitation如果细胞已用裂解之前交联试剂硫代（琥珀酰亚胺丙酸酯），这表明该相互作用是暂时和/或弱。最近的研究已经表明，不同甲型流感毒株的差分的Mx灵敏度由NP蛋白11,12的原点来确定。本着这一精神，甲型流感病毒可以从一定程度控制的Mx通过突变的具体残留在NP蛋白13逃跑。这表明，甲型流感病毒对主机的Mx的主要目标是NP蛋白质，最有可能NP组装vRNP复合物。然而，这些较近期的研究中证明了流感的NP或vRNPs，要么人类的MxA或鼠标MX1之间的相互作用。 最近我们发现，对于第一次，流感NP和鼠标MX1蛋白具有优化免疫共沉淀协议14，其在此处详细描述之间的相互作用。在一般情况下，共同-i的mmunoprecipitation是最常用的生化方法来调查蛋白质 - 蛋白质相互作用之一。这种技术通常优于其他技术， 例如，酵母双杂交，因为它允许研究蛋白质-蛋白质相互作用在其天然环境中。共免疫沉淀，可以进行上内源性表达的蛋白质，如果可用的抗体针对感兴趣的蛋白。备选地，感兴趣的蛋白质可以表达的通过转染或感染的细胞和亲和标记可被使用。除了上述的优点，所描述的共免疫沉淀协议允许弱和/或瞬态蛋白质相互作用的检测。在这个优化的协议的主要成分是添加N-乙基马来酰亚胺（NEM）的细胞裂解缓冲液中。 NEM是一种烷基化试剂，与游离硫醇基团，例如存在于半胱氨酸反应，在pH为6.5-7.5，以形成稳定的硫代酯（ 图1）。在较高的pH值，NEM也可以与氨基反应或进行水解15。 NEM通常用于阻断游离硫醇基团，以防止二硫键的形成或抑制酶活性。例如，NEM常被用来阻止desumoylating酶，它们是半胱氨酸蛋白酶。在所描述的免疫共沉淀的协议，NEM最初包含在裂解缓冲液，因为它已被报道，流感蛋白的蛋白修饰可以影响病毒蛋白16之间的相互作用。出乎意料的是，在加入NEM的证明是关键文件通过共免疫沉淀流感NP和鼠标MX1之间的相互作用。目前还不清楚为什么加NEM是至关重要检测NP-MX1互动。可能的交互过瞬态和/或弱。 NEM可以稳定的相互作用， 例如，通过保留MX1的特定构象，病毒蛋白或甚至一个未知第三COMPO新界东北堆填区。 NEM这种稳定作用已被观察到之前， 例如，对于核苷酸还原酶M1和其抑制剂吉西他滨（F2dC）17之间的相互作用。 MX1和NP都含有多个半胱氨酸残基可通过NEM进行修改。例如，最近的一项研究雷尼等人证明，无茎的MxA-变种包含三个溶剂暴露半胱氨酸残基可被碘乙酰胺进行修改。突变这些残基对丝氨酸没有影响的MxA的酶活性，但阻止二硫键介导的聚集18。由于这些半胱氨酸保守MX1，这表明在MX1类似的半胱氨酸可以通过NEM并因此影响其构象或溶解性进行修改。此外，NEM还可能影响MX1，这对于MX1的抗流感活性所必需的GTP酶活性，并由此稳定MX1和NP之间的相互作用。然而，NEM上的GTP酶ACTI有直接的影响MX1的VITY是不可能的，因为NEM还需要检测流感NP和GTP酶的MX1蛋白14的无活性突变体之间的相互作用。很明显，需要更多的研究来解开NEM对NP-MX1的互动效果。 总之，所描述的共免疫沉淀协议允许研究抗病毒药MX1蛋白及其病毒靶，流感的NP蛋白质之间的相互作用。该协议也可以用来研究依赖于特定蛋白质的构象的稳定化等弱或瞬时相互作用。依赖于特定的构象的蛋白质-蛋白质相互作用之前已经描述的， 例如，对于钙结合蛋白如钙调蛋白19。最后，NEM的有益作用，也可以在该检测蛋白质 - 蛋白质相互作用，例如共沉淀测定法等方法使用。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM high glucose</td>
			<td>Gibco</td>
			<td>52100-047</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Ethylmaleimide</td>
			<td>Sigma</td>
			<td>E-3876</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma</td>
			<td>I-30212</td>
			<td>also known as NP40</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11 873 580 001</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-NP monoclonal antibody</td>
			<td>NIH Biodefense and Emerging Infections Research Resources Repository</td>
			<td>NR-4282</td>
			<td>ascites blend of clones A1 and A3</td>
		</tr>
		<tr>
			<td>anti-RNP polyclonal serum</td>
			<td>NIH Biodefense and Emerging Infections Research Resources Repository</td>
			<td>NR-3133</td>
			<td>directed against A/Scotland/840/74 (H3N2)</td>
		</tr>
		<tr>
			<td>Protein G Sepharose 4FF</td>
			<td>GE Healthcare</td>
			<td>17-0618-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyperfilm ECL 18 x 24 cm</td>
			<td>GE Healthcare</td>
			<td>28-9068-36</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Western Blotting Substrate</td>
			<td>Pierce</td>
			<td>32106</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

N-乙基马来酰亚胺是可用于不可逆地修饰的游离巯基， 例如 ，以抑制半胱氨酸蛋白酶（见图1）的有机化合物。 抗病毒MX1蛋白抑制A型流感病毒的复制与病毒核蛋白的相互作用。这里描述的优化免疫共沉淀协议允许研究这个NP-MX1互动。转染HEK293T细胞与抗病毒MX1蛋白在流感NP蛋白的存在或不存在的表达载体。接着，将NP蛋白是从总细胞裂解物被拉?...

Watch this Scientific Journal Video about Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein at JoVE.com

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

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 ...

co-immunoprecipitation-mouse-mx1-protein-with-influenza-virus

Research

JoVE Journal

Immunology and Infection

17.6K 次观看.  VIB. 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.

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

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 ...

科研

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 ...

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

在野生鸟类禽流感病毒快速诊断：使用便携式的RRT - PCR和在外地的冻干试剂

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

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, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

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 ...

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 ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

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 ...

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.