我们要感谢的技术支持和USGS西部生态研究中心提供资金（S. Schwarzbach）和援助（K. Spragens T.格雷厄姆）M. Scullion爱达荷州技术和R.酥。主持的创新技术中心 - 国防和国土安全部（www.idhs.org），国防部和美国空军研究实验室部支持下进行这项研究。动物处理动物护理和使用委员会的美国地质调查局西部生态研究中心和美国地质调查局捆扎实验室鸟儿许可证批准的协议。任何使用本出版物中的贸易，产品或公司名称仅是描述目的，并不意味着美国政府的认可。

1。野生鸟类捕捉，使用水雾网<ol><li>滨鸟捕获，设立一个活跃的，如沼泽，海岸线，滩涂觅食网站的薄雾网。 </li><li>幻灯片特拉梅尔线循环极周围的雾网的一端垂直插入泥极。 </li><li>伸出净，特拉梅尔循环通过插入第二个极点雾网的另一端，垂直插入泥杆，确保特拉梅尔线教。 </li><li>一旦鸟被捕获，提取净的鸟类和返回绑扎站。 </li></ol> 2。泄殖腔拭子采样<ol><li>在拍摄后立即收集泄殖腔拭子</li><li>找到泄殖腔，用指尖推回周围羽毛</li><li>解开一个无菌涤纶或聚酯倾斜棉签，果蒂第一，和滋润更易于在擦拭用无菌病毒运输媒体的一角。润燥后，整个头部的棉签轻轻插入泄殖腔。拭泄殖腔内缓慢捻转的拭子和删除泄殖腔拭子的周长。 </li><li>直接插入到病毒的传输介质样品瓶拭子末端，旋转棉签轴之间的拇指和食指，棉签是在媒体。有一个助手完成拉〜1厘米的小瓶底部棉签回尖端的程序和一把剪刀切轴棉签拭子末端仍然在小瓶和轴不干预第封闭。第小瓶紧紧。与酒精切割棉签轴之间消毒的剪刀。 </li><li>放置在冷冻盒，内含干冰或冰袋小瓶。 </li></ol> 3。口咽部拭子采样<ol start="1"><li>轻轻打开鸟嘴，使鼻​​孔缝可见</li><li>解开一个干净的棉签从包，果蒂第一，滋润提示与病毒的传输介质，更易于在擦拭</li><li>不接触到任何其他表面的提示，插入的嘴和棉签鼻孔开放口的屋顶上，并继续对口咽或咽喉地区</li><li>将直接进入病毒传输介质的小瓶拭子末端。旋转棉签轴之间的拇指和食指，棉签是在媒体。如上所述，助理拉〜1厘米的小瓶底部的棉签回头和切轴棉签一把剪刀，所以拭子末端仍然在小瓶和轴不干预第关闭。第小瓶紧紧。与酒精切割棉签轴之间消毒的剪刀。 </li><li>放置在冷冻盒含有干冰或冰袋小瓶。 </li></ol> 4。禽流处理和发布<ol start="1"><li>返回鸟捕捉和及时的释放（&lt;20分钟）的网站。处理程序应该意识到动物福利的原则，为鸟类在取样过程中的痛苦的迹象（如擦伤，捕捉性肌病和低或热疗）警报。一个基本的急救箱应该包括在设备清单。 </li></ol> 5。测试设施<ol start="1"><li>测试样品可以在任何封闭的空间，如研究拖车或帐篷，与电源（发电机电压调节）运行PCR单位连接的计算机。 </li></ol> 6。 RNA的提取<ol start="1"><li> RNA提取的RNeasy试剂盒稍作修改，以制造商的说明，以下Spackman等。 </li><li>涡街3-5小号棉签中期和350μL转移到2 ml离心管。注:在PCR阳性标本需要rescreened或H5病毒测试需要运行，其余样品置于冰上。 </li><li>添加350μL的RLT缓冲（β- ME）和第5号的旋涡</li><li>的RNA级70％的乙醇添加350μL。 </li><li>在5000 XG离心5分钟。 </li><li>自旋列放置在一个2 ml收集管中加入600μL上清。保留其余样品在试管架。 </li><li>万XG和丢弃流过离心20秒。 </li><li>重复步骤17-18，直到所有的样品已经通过离心柱通过。 </li><li>离心柱放置在一个干净的2ml收集管。 </li><li> RW1缓冲液加入700μL的离心柱和离心25秒10,000 × G.丢弃的流量通过。 </li><li>加入500μl的RPE缓冲区的离心柱和离心25秒10,000 × G.丢弃的流量通过。 </li><li>共有两个视网膜色素上皮缓冲区清洗重复步骤22。 </li><li>离心14000小工的一个额外的2分钟旋转列倒掉收集管中。 </li><li>离心柱放置在一个干净的1.5 ml离心管，加入无核酸酶水50μL。在室温下孵育60秒。 </li><li>洗脱RNA的万XGF 60秒旋转或60秒。将纯化的冰样本。 </li></ol> 7。准备阴性对照<ol start="1"><li>冻干试剂准备使用PCR检测TaqMan探针A型流感的目标1（Asay的ASY - 0109）从爱达荷州技术冻干试剂检测试剂盒的说明。 </li><li>离心3秒阴性对照试剂瓶（红色），以确保颗粒在底部。 </li><li>删除第视觉确保颗粒在底部。 </li><li>加入20μL2 ×重整缓冲液和20μL试剂级水。 </li><li>重温和旋涡的最大速度为5小号。 </li><li>离心3秒，使液体瓶底。 </li><li>目视检查颗粒水化，如果不重复步骤31-32。 </li><li>成的LightCycler毛细管水合混合物加入19μL，重复到第二个毛细管吸管的步骤，以获得一式两份。 </li></ol> 8。准备测试样本<ol start="1"><li>离心3秒未知的试剂瓶（蓝色），以确保颗粒在底部。 </li><li>删除第视觉确保颗粒在底部。 </li><li>加入40μL纯化的RNA。 </li><li>重温和旋涡的最大速度为5小号。 </li><li>离心3秒，使液体瓶底。 </li><li>目视检查颗粒水化，如果不重复步骤38-39。 </li><li>水合混合成的LightCycler毛细管加入19μL，重复到第二个毛细管吸管的步骤，以获得一式两份。 </li></ol> 9。准备阳性对照<ol start="1"><li>离心3秒阳性对照试剂瓶（绿色），以确保颗粒在底部。 </li><li>删除第视觉确保颗粒在底部。 </li><li>加入20μL2 ×重整缓冲液和20μL试剂级水。 </li><li>重温和旋涡的最大速度为5小号。 </li><li>离心3秒，使液体瓶底。 </li><li>目视检查颗粒水化，如果不重复步骤45-46。 </li><li>成的LightCycler毛细管水合混合物加入19μL，重复到第二个毛细管吸管的步骤，以获得一式两份。 </li></ol> 10。离心的毛细管<ol start="1"><li>一旦所有的批处理毛细管已准备装入装有毛细管适配器的微型离心机。 </li><li>离心机在最低设置按钮1秒的脉冲这转移液体，准备进行测试底部。 </li></ol> 11。操作快速<ol start="1"><li>创建一个新文件。标签阳性对照，阴性对照和样品（他们的ID号）。 </li><li>计划1:RNA样品制备举行<ol><li>周期= 1 </li><li>分析模式=无</li><li>目标温度= 40 </li><li>孵化时间为30分钟</li><li>温度变化率= 20 ° C / S（默认） </li><li>二级温度目标= 0（默认） </li><li>步长= 0（默认） </li><li>步骤延迟= 0（默认） </li><li>采集模式=无（默认） </li></ol></li><li>计划2:RNA的变性<ol><li>周期= 1 </li><li>分析模式=无</li><li>目标温度= 94 </li><li>孵化时间= 120秒</li><li>温度变化率= 20 ° C / S（默认） </li><li>二级温度目标= 0（默认） </li><li>步长= 0（默认） </li><li>步骤延迟= 0（默认） </li><li>采集模式=无（默认） </li></ol></li><li>计划3:RNA扩增<ol><li>周期= 45 </li><li>分析模式=自动分析</li><li>目标温度，段1 = 94;段2 = 60 </li><li>孵化时间段1 = 0秒，段2 = 20秒</li><li>温度变化率，赛格1＆2 = 20 ° C /秒（默认） </li><li>二级温度目标，赛格1＆2 = 0（默认） </li><li>步长，赛格1＆2 = 0（默认） </li><li>步骤延迟，赛格1＆2 = 0（默认） </li><li>采集模式，段1 =无;段2 =单</li></ol></li><li>荧光参数<ol><li>显示模式:荧光通道2（CH2 / 1） 收益: <ol><li> CH 1（F1）= 1 </li><li> CH 2（F2）= 8 </li><li> CH 3（F3）= 1 </li></ol></li><li>要显示每个样品的实时荧光数据，选择感兴趣的样本，并点击"显示图形"按钮，在屏幕底部</li></ol></li><li>对于每一个毛细血管，结果包括以下内容: <ul><li> ＃ - 标识的传送带传送带的位置</li><li>样品名称 - 提供样品名称</li><li>控制 - indentifies每个样品的样品类型</li><li>分数 - 计算样品中样品的荧光和背景荧光水平之间的关系</li><li>结果 - 显示样品的总体结果</li></ul></li></ol> 12。代表性的成果: 现状:一个红色的"本"呼叫测试/未知样品表明的目标是确定在该样本和控制是成功的。 未检出:一个绿色"未检出"测试/未知样品的呼吁表示，没有确定的目标是在该样本和测试控制是成功的。 请重复:一个"请重复"的指示，积极或消极的控制失败。 一个成功的快速7200分析的一个例子是如图4所示。阳性对照扩增，并生成检测到的荧光，在约25个周期（X轴）（y轴）。循环阈值（CT）&gt; 35商定的截止大多数禽流感病毒参考实验室。因此，此方法的阳性对照为禽流感病毒的检测范围内接受的周期数（0-35）的荧光信号。相比之下，阴性对照不产生后甚至45个循环的荧光信号。同样，来自西部鹬的十二个采集的样品不产生荧光信号，表明鸟类是禽流感病毒的负面。鼓励在传统实验室的所有阳性标本的后续测试，以确保没有错误产生误报。 <img src="files/ftp_upload/2829/2829fig1.jpg" alt="图1" /> 图1。涉禽捕获使用薄雾网。 <img src="files/ftp_upload/2829/2829fig2.jpg" alt="图2" /> 图2。至少鹬泄殖腔和口咽样品的收集。 <img src="files/ftp_upload/2829/2829fig3.jpg" alt="图3" /> 图3。编程RNA扩增迅速7200。 <img src="files/ftp_upload/2829/2829fig4.jpg" alt="图4" /> 图4。Fluorogram 7200便携式快速RRT - PCR显示阳性和阴性对照，应该会出现在一个成功的实验产生<a href="http://www.jove.com/files/ftp_upload/2829/2829fig4large.jpg">的。点击这里查看完整大小</a>的影像

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没有利益冲突的声明。

这里介绍的快速诊断方法，有利于高效，准确的监测禽流感病毒的野鸟样本进行测试。要少得多严格的便携式RRT - PCR标本的存储需求，适合于远程的情况下，维持冷链可能是不切实际的的，如果不可用液氮货主或干冰。此外，我们发现，随着冻干试剂的样品分析，足够直接与实验室为基础的分子分析的最小的知识领域的生物学家。这项技术是时间效率和成本效益。一个经营者，这是可行的运行三?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>的RNeasy微型离心柱</td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td>收集管（1.5 2毫升） </td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td>缓冲区的RLT </td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td>缓冲区RW1 </td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td>缓冲区视网膜色素上皮</td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td>无RNase水</td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td> 14.3中号β-巯基乙醇的解决方案</td><td> Fisher Scientific则</td><td> BP176100 </td><td></td></tr><tr><td> 100％的乙醇</td><td> Fisher Scientific则</td><td> NC9602322 </td><td></td></tr><tr><td>涡精灵2，120V </td><td>科学工业</td><td> SI - 0236 </td><td></td></tr><tr><td> TaqMan探针流感A目标1（水解探针） </td><td>爱达荷州技术</td><td> Asay的- ASY - 0109 </td><td></td></tr><tr><td> LightCycler配套20毫升毛细管</td><td>罗氏应用科学部</td><td> 04929292001 </td><td></td></tr><tr><td> 2毫升管肩微量离心</td><td>爱达荷州技术</td><td></td><td>包括快速套件</td></tr><tr><td>坚固耐用的高级病原鉴定设备（快速）7200 </td><td>爱达荷州技术</td><td></td><td>包括快速套件</td></tr><tr><td>基于Pentium的笔记本电脑与Windows XP专业版</td><td>爱达荷州技术</td><td></td><td>包括快速套件</td></tr><tr><td> LightCycler配套数据分析软件</td><td>爱达荷州技术</td><td></td><td>包括快速套件</td></tr></tbody></table>

rapid diagnosis of avian influenza virus in wild birds: use of a portable rrt-pcr and freeze-dried reagents in the field

野生鸟类有牵连的H5N1亚型高致病性禽流感（HPAI）的蔓延，促使监控候鸟迁徙的飞行路线。野生鸟类禽流感病毒（AIV）的采样往往是在偏远地区进行，但结果往往是因为需要运输的样本，以分子检测实验室配备的延误。实时逆转录聚合酶链反应（RRT - PCR）是一种分子的技术，提供了最准确和最敏感的禽流感病毒的诊断方法之一。 RRT - PCR所需的协议以前严格的实验室目前正在适应的领域。冷冻干燥（冻干）的试剂，不需要冷链，湿试剂水平的灵敏度的发展带来了现场的远程测试，以一个实际的目标。 在这里，我们目前在使用单位RRT - PCR（加固高级病原鉴定设备或快速，爱达荷州技术，盐湖城，UT），采用冻干试剂（A型流感的目标1的TaqMan的野生鸟类禽流感病毒快速诊断方法; Asay的ASY - 0109，爱达荷州技术）。该试剂含有适当浓度的单管所有必要的组件进行测试:引物，探针，酶，缓冲液和内部阳性对照，消除与保管不当或处理湿试剂相关的错误。便携式装置执行针对矩阵基因产生的结果，并在2-3小时的屏幕为A型流感。遗传亚型H5和H7型引物也可能设置为目标的血凝素基因。 该系统是适合收集的野生鸟类，迁徙滨鸟种在这里展示了使用泄殖腔和口咽的样品，在北加州拍摄西部鹬（Calidrus马利） 。动物处理动物护理和使用委员会的美国地质调查局西部生态研究中心和美国地质调查局捆扎实验室鸟儿许可证批准的协议。这项技术的主要优势是加快野生鸟类的诊断，增加包含在偏远地区爆发的机会。现场诊断也将证明有用的鉴定和研究野生种群的感染者。收集主机生物学（免疫和生理反应，感染）和空间生态（受感染的鸟类的迁徙表现）信息的机会将提供野生鸟类在何种程度上可以作为禽流感病毒的载体，在长距离的见解。

Watch this Scientific Journal Video about Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field at JoVE.com

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

这项研究描述了在使用一台便携式的RRT - PCR系统的野生鸟类禽流感的诊断。该方法采用冻干试剂的屏幕野生鸟类在非实验室环境中，典型的爆发情况下的优势。利用分子工具提供了快速诊断的准确和灵敏的替代品。

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 ...

rapid-diagnosis-avian-influenza-virus-wild-birds-use-portable-rrt-pcr

Research

JoVE Journal

Immunology and Infection

15.6K 次观看.  USGS Western Ecological Research Center. 这项研究描述了在使用一台便携式的RRT - PCR系统的野生鸟类禽流感的诊断。该方法采用冻干试剂的屏幕野生鸟类在非实验室环境中，典型的爆发情况下的优势。利用分子工具提供了快速诊断的准确和灵敏的替代品。

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

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 ...

科研

免疫与感染

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

禽流感监测与FTA卡:实地方法，生物安全，和运输问题解决了

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

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 ...

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

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

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

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

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

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

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

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

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

在甲型流感病毒的衣壳的体外拆卸梯度离心

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

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

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.