我们感谢伯特Dibbits技术援助。动物育种和基因组，瓦赫宁根大学，荷兰，慷慨地在他们的实验室主办。 ，瑞典，Ottenby鸟天文台的工作人员表示感谢，诱捕和取样的绿头鸭，特别是马格努斯Hellström，马库斯 - 丹尼尔森，马格努松克里斯蒂娜安德森。我们感谢Jonatan Qvist桑妮斯文森，并在Ottenby拍摄的每阿克塞尔Gjöres，和马诺Camon在实验室内进行拍摄。丹尼尔Bengtson提供本出版物的视频部分美丽的鸭照片。使用，进一步加强疾病预防控制中心公共卫生图片库（PHIL <a href="http://phil.cdc.gov/phil/">;</a>材料http://phil.cdc.gov/phil/）:电子显微镜（280号;厄斯金帕尔默博士）和插图（第11823;道格拉斯约旦）的流感病毒。财政支持KNJV（荷兰皇家猎人协会），荷兰农业部，Faunafonds的STICHTING Eik信托（在荷兰），瑞典研究理事会（赠款。2007-20774）和欧共体创办Newflubird项目。 RNA提取的化学品，RNA公司Ambion公司，公司的一个厚礼。这是第245号从Ottenby鸟天文台的贡献。

1。鸭捕获和泄殖腔抽汲<ol><li>他们通过吸引与诱惑鸭或食品陷阱涉足鸭属阿纳斯 （或其他鸟类）在一个笼子里，并放入个人卡板箱。运输包装盒中的时间应保持在最低限度，例如，通过安装在短距离内的陷阱一个场站。在每个研究物流的情况下可能会有所不同。要求，需要为每个新设置的意见和相关的动物伦理委员会的批准。另外，还猎人拍摄鸟类进行采样。 </li><li>它的翅膀紧紧地抱着它的身体，以鸭出的方块。品尝新鲜的下降，这将是目前在大多数情况下，直接从盒子的底部。它们捡起来，用无菌棉签和应用流体用Whatman FTA卡。如果没有，进行泄殖腔擦拭。 </li><li>在它的后面打开的鸭子。这允许访问泄殖腔和方便采样。泄殖腔位于腹部，靠近基地的尾巴下面是一个突出的结构。一个有经验的人可以在同一时间持有和样品的鸟，否则第二个人可以协助。 </li><li>小心地插入无菌塑料粘胶棉签（Copan公司，意大利），约1厘米，使泄殖腔的温柔漩涡。卷棉签从泄殖腔在用Whatman FTA卡表面的流体。已进行取样后立即释放动物是可取的。 </li><li>在室温下干燥至少1小时的FTA卡，然后分别存储每个样本的纸袋（如信封）。在室温下储存可能，可能与潮湿的气候中的​​硅珠。发送和接收机构的生物安全人员可以允许通过普通邮件发送FTA卡，因为病原体成为与FTA卡表面接触后无效。 </li></ol> 2。病毒RNA提取<ol><li>提取的FTA卡包括粪便/泄殖腔流体材料。打孔三个光碟与哈里斯2毫米打孔和地方所有成核糖核酸免费96well板块以及的。彼此之间新的样品，仔细清洁的打孔机，用酒精和一个金精密擦拭（Kimtech）。始终使用阳性和阴性对照。阳性对照，可以由新鲜适用的FTA卡的实验室应变，或一个自然的样本，这是了解产生积极的结果。作为阴性对照，一拳从一个空的自由贸易协定卡光盘。此外，留下另一位后来在实验中的PCR控制的自由（与水为模板）。 </li><li>加入70μlRNA的快速提取液（Ambion公司）和热封板（AbGene热封口机）。与确定的速度不会导致溢出（这取决于所用的板振动筛模型;提前测试），在室温下孵育5分钟一盘振动筛。 </li><li>根据制造商的协议MagMAX - 96病毒RNA提取试剂盒（Ambion公司），携带病毒RNA分离。简介:准备裂解/约束力的解决办法（套件），每孔加入130μl。转移50μL提取FTA卡材料，每口井。 1分钟摇板。 </li><li>加入20μL准备珠混合（套件），每个样品和混合吹打向上和向下。确定速度摇动5分钟。 RNA分子结合的磁珠。 </li><li>移动板磁性的96站捕捉到的珠。根据磁独立使用的模型，这可能需要超过5分钟。当溶液变成了彻底清除，删除和丢弃上清液。然后删除从磁立场板。 </li><li>洗磁珠两次洗涤液1和洗涤液2（套件）的两倍。对于每个4洗涤步骤每个样品加入150μl准备洗涤液，摇匀确定速度为1分钟，移动板磁立场，捕捉珠约5分钟（或直到解决的办法是彻底清除），删除和弃去上清液。从磁立场板，然后删除，添加洗涤液，并进行清洗步骤，如前面。在最后的洗涤步骤，可能和空气的干板振动筛2分钟，在室温下珠中删除尽可能多的洗涤液。 </li><li>洗脱缓冲液中加入50μL（套件），从珠子释放RNA和摇板振动筛上的4分钟。如前所述捕捉珠。上清现在包含分离出的RNA，这是为下游应用做好准备。 </li></ol> 3。 RT - PCR检测禽流感病毒基质基因开展25μL反应一步访问RT - PCR检测系统的逆转录聚合酶链反应（RT - PCR检测）（Promega公司）（从克劳斯等调整 18和Fouchier 等 22。 ） <table border="1"><tbody><tr><td>核酸免费水</td><td> 1.5μl </td></tr><tr><td> AMV / Tfl5缓冲区</td&gt; <td> 5μL </td></tr><tr><td> dNTPs </td><td> 0.5μL </td></tr><tr><td>底漆M52C 22（10μmol） </td><td> 2.5μl </td></tr><tr><td>底漆M253R 22（10μmol） </td><td> 2.5μl </td></tr><tr><td> 硫酸镁 （25毫米） </td><td> 7μl </td></tr><tr><td> AMV反转录酶（5u/μl） </td><td> 0.5μL </td></tr><tr><td> TFL聚合酶（5u/μl） </td><td> 0.5μL </td></tr><tr><td> RNA样品</td><td> 5μL </td></tr></tbody></table>在Biometra T1的热循环PCR的条件是:最初的45分钟反转录在45 ° C，2分钟预变性94 ° C和40个循环:94℃，1分钟，56℃1分钟， 68℃2分钟。增加7分钟伸长率在68 ° C的结论放大。 4。 AI阳性样品的筛选和净化的目标片段从凝胶<ol><li>负载2μL5倍上样染料（BioRad公司）和1％琼脂糖凝胶电泳，用溴化乙锭的1％（每100毫升凝胶2.5μl）预检染色（罗氏）6μl水混合的PCR产物2μL。 </li><li>一个DNA大小的标准（BioRad公司的EZ负荷100 bp的阶梯）一起运行在120V 1小时凝胶，凝胶系统与可视化。见在图1中的例子。 </li><li>选择样品扩增片段与预期大小范围（200个基点和300基点（目的片段为244 bp的），之间。6μl5倍加载染料装入这些候选人的整个PCR反应体积（〜23μl剩下2） ％溴化乙锭染色的琼脂糖凝胶电泳和运行2个小时。 </li><li>广场上的紫外线透射（Bioblock科学）的凝胶和目视检查。参见图2中的一个例子。正确的大小，向香港海关乐队用手术刀和个人1.5 ml反应管置于凝胶。片段从凝胶净化，Zymoclean凝胶DNA回收试剂盒（Zymo研究）的实例。 </li></ol> 5。 PCR产物的测序和鉴定<ol><li>开展Sanger测序目标片段，例如在ABI 3730毛细管音序器与ABI大染料3.1化学（美国应用生物系统公司）。准备测序反应中含有加入10μl卷10-20吴凝胶纯化模板cDNA，1.75μl5倍稀释缓冲液，0.5μL大染料V3.1预混料，1μL正向引物（M52C 20，10毫米），和ddH2O。骑自行车的条件是:初始变性1分钟，25次:10小号在96℃，在45℃和4时60分5 S ° C。净化，并准备根据您的内部协议的测序反应。 </li><li>确定对核苷酸的数据库，如GenBank23，如基于Web的工具，如在为国家生物技术信息（NCBI，中心的爆炸<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">造成</a>的cDNA序列http://blast.ncbi.nlm.nih.gov/Blast.cgi）。 </li></ol> 6。代表性的成果: 野鸭（阿纳斯platyrhynchos）进行抽样Ottenby鸟天文台于2007年12月。从每凫一个FTA卡上的样本是在本议定书中所述。 FTA卡在船后，两年中在-20 ° C冷冻保存。克劳斯等人在隔离测试实验室相同的FTA卡样品18作为阳性对照，以及它的9个十倍串行稀释。两个阴性对照一）提取从一个空的自由贸易协定卡，以测试是否有结转的冲床，II）RT - PCR反应，核酸免费水为模板，进行测试，如果污染发生时，或在PCR反应的准备。 84样本进行了分析。图1中可以找到一个从这些84个样本的PCR产物的凝胶图片。从天然样品中的多种非特异性条带，可以观察到由于粪便中的各种微生物污染。然而，长244 bp的目标片段的引物对。整个PCR反应体积的一个子集的样品生产约正确的尺寸范围（200 bp和300 bp的之间）的片段，凝胶（图1）装上。乐队从凝胶中切的画像补充图1中可以找到。除了阳性对照，这些样本中的两个（69899和69912）新的协议。一个对NCBI核苷酸数据库的BLAST搜索AI基质基因（图3）透露他们的身份，而所有其他乐队类似细菌的序列最密切的，或者没有产生一个可读序列。 <img alt="图1" src="/files/ftp_upload/2832/2832fig1.jpg" /> 图1。 2μLPCR阳性对照产品的凝胶图片的PCR产物进行初步筛选。erial稀释的阳性对照，两个阴性对照和84泄殖腔样本被装。凝胶面板，底部面板中的48个样品中48个样品。红色箭头指示为100 bp的DNA大小标准使用的凝胶车道。图中，红色圆圈显示在预期的尺寸范围内带。蓝色圆圈表示一个非特异性扩增（左上）或低于100大小的BP（右下）的引物二聚体人工制品。 <img alt="图2" src="/files/ftp_upload/2832/2832fig2.jpg" /> 图2。与乐队之间的200个基点和300基点（bp的目标片段244），在正确的尺寸范围内的碎片样本的选择。样本选择。绿色箭头指示的阳性对照，两个红色箭头表示被确认为禽流感病毒的积极通过比较他们的cDNA序列在NCBI核苷酸数据库的样本。 <img alt="图3" src="/files/ftp_upload/2832/2832fig3.jpg" /> 图3。代表在NCBI BLAST搜索的屏幕截图。切除片段所获得的cDNA序列核苷酸数据库在NCBI网站查询。样本正确识别AI矩阵基因片段。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/2832/2832fig3large.jpg">若要查看这个图片的大小的一个完整的版本，请点击这里</a> 。 <img alt="补充图1" src="/files/ftp_upload/2832/2832supfig1.jpg" /> 补充图S1。从凝胶切除选定的样品带。这张照片是从图1中描绘后，候选人之间的200 bp和300 bp的带凝胶采取切出。

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GenBank.">GenBank.</a> Nucleic Acids Res. 38, 46-51 (2009).</li><li>Spackman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+real-time+reverse+transcriptase+PCR+assay+for+type+A+influenza+virus+and+the+avian+H5+and+H7+hemagglutinin+subtypes.">Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes.</a> J. Clin. Microbiol. 40, 3256-3260 (2002).</li><li>Muthukrishnan, M., Singanallur, N. B., Ralla, K., Villuppanoor, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+FTA+cards+as+a+laboratory+and+field+sampling+device+for+the+detection+of+foot-and-mouth+disease+virus+and+serotyping+by+RT-PCR+and+real-time+RT-PCR.">Evaluation of FTA cards as a laboratory and field sampling device for the detection of foot-and-mouth disease virus and serotyping by RT-PCR and real-time RT-PCR.</a> J. Virol. Methods. 151, 311-316 (2008).</li><li>Perozo, F., Villegas, P., Estevez, C., Alvarado, I., Purvis, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+FTA+filter+paper+for+the+molecular+detection+of+Newcastle+disease+virus.">Use of FTA filter paper for the molecular detection of Newcastle disease virus.</a> Avian Pathol. 35, 93-98 (2006).</li></ol>

诱捕，处理和采样鸟类进行了国家立法和由瑞典农业和研究动物伦理委员会局的批准。

这里描述的协议提供了一个辅助方法，以屏幕为禽流感病毒的存在的粪便或类似的样品。这是特别设计，使样品采集的快速和容易。这使得它较少受过训练的人员，如猎人或野生动物管理人员，可能有助于对禽流感监测。无冷却链需要应用，虽然冻结的样品，建议在可能的情况下。室温几天，例如在运输过程中的实验室，是没有问题的FTA卡上的RNA分子，只要卡保持干燥。其他非冷却储存系统，目前正在研究社会评价，如酒精 15或胍 16日，需要特殊的运输安排，因为其危险性质。相比之下，FTA卡法不需要有害物质的航运。然而，在这方面的另一个有趣的存储介质是RNAlater™是没有危险的，无论是17。样品分析，可以在任何标准的分子实验室进行。没有特殊的设备比其他常规的PCR反应和毛细管测序是必要的。所有步骤都可以进行无生物安全级别，因为已经在样本收集潜在的病原体灭活FTA卡的抗菌和抗病毒活性。 在我们的试验与野生鸟类标本的交叉污染和随后的假阳性样品，没有观察到。然而，RNA和PCR时，它始终是最好要特别注意清洁前和后的PCR步骤的工作场所和单独的房间。在通风橱中工作，减少在实验室中的气溶胶污染的风险。移液需要进行过滤嘴。 从先前的研究同时从我们知道的84个样本中有6个是由传统的实时RT - PCR法24和 ​​实时RT - PCR检测的Ct值是已知的相同的鸭的样本。我们的Ct值&lt;30（显示高浓度的病毒RNA）的方法从鸭干检测的阳性标本。其他4个样本，与传统的协议的积极的Ct值&gt; 30（不集中）和不能被检测到我们的协议。 只有具有比较高的病毒滴度的样品呈阳性，在我们的实验和方法的灵敏度，很可能是故障检测所有阳性标本的来源。此外，在目前的研究的样本量非常低，该方法可以完全开发和评估，如果进行更多的控制和严格的实验。但是，如果这些样本将被从一个偏远地区收集，传统的分析不会已经有可能在所有。此外，这些结果源于试点实验，需要进一步优化。定期-20℃冷冻样品采集后的一两年的存储期间也可能影响病毒RNA的质量。这种可能的RNA降解是一个重要的问题，处理与灭活病毒的房间温度存储时。替代液体储存上述样品遭受显着降低，从而影响分析不再延伸的病毒基因组的 16 。虽然没有在我们的研究测试，FTA卡已被证明是适合保存在其他RNA系统是非常相似的禽流感病毒，25，26的，完整的RNA分子。

<table border="1"><tbody><tr><th>试剂或仪器名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>塑料人造丝干棉签</td><td>意大利Copan公司， </td><td> 167KS01 </td><td></td></tr><tr><td> FTA卡</td><td>瓦特曼</td><td></td><td>几个选项</td></tr><tr><td>哈里斯的微型一拳2mm与垫</td><td>瓦特曼</td><td> 1154409 </td><td></td></tr><tr><td>核糖核酸酶自由96well板</td><td>格雷纳生物的一</td><td> 652290 </td><td>或类似</td></tr><tr><td>金精密湿巾</td><td> Kimtech </td><td> 75512 </td><td></td></tr><tr><td> RNA快速提取解决方案</td><td> Ambion公司</td><td> AM9775 </td><td></td></tr><tr><td> MagMAX - 96病毒分离试剂盒</td><td> Ambion公司</td><td> AM1836 </td><td></td></tr><tr><td>单步进入RT - PCR检测系统</td><td> Promega公司</td><td> A1250 </td><td>或类似</td></tr><tr><td>琼脂糖MP </td><td>罗氏公司</td><td> 1388991001 </td><td>多用途琼脂糖</td></tr><tr><td> 5X样缓冲液</td><td> BioRad公司</td><td></td><td>交付DNA梯状</td></tr><tr><td>简易负载100 bp的阶梯</td><td> BioRad公司</td><td> 170-8353 </td><td>或类似</td></tr><tr><td>溴化乙锭的1％ </td><td> Fluka公司</td><td> 46067 </td><td>或类似</td></tr><tr><td> 1.5毫升反应管</td><td> Eppendorf公司</td><td></td><td>或类似</td></tr><tr><td> Zymoclean凝胶DNA回收试剂盒</td><td> Zymo研究</td><td> D4001 </td><td>或类似</td></tr><tr><td> ABI大染料3.1化学</td><td>美国应用生物系统公司</td><td></td><td>或类似</td></tr></tbody></table>

avian influenza surveillance with fta  cards: field methods, biosafety, and transportation issues solved

感染禽流感病毒（AIVs）的许多哺乳动物，包括人类在内的 1 。这些AIVs是多种多样的，在其自然宿主，窝藏几乎所有可能的病毒亚型。人类的流感大流行最初来源于AIVs 3。许多致命的H5N1疫情在近年的人类感染病例的报道。最近，一个新的禽流感病毒相关的应变席卷整个人类的人口，造成"猪流感疫情"4 。虽然人类的贸易和运输活动，似乎是负责的高致病性菌株 5的传播，扩散也可以部分归因于野生鸟类6， 7。然而，所有禽流感病毒株的实际水库的野生鸟类。 在反应和家禽业面临严重的商业损失，已实施了大型监视计划在全球范围内收集信息，对生态AIVs安装预警系统，以检测某些高致病性菌株8-12。传统的病毒学方法要求病毒和分析前是完好的耕地。深冷冻机和沉重的生物安全程序到位，在运输过程中必须严格的冷链。长期监测，因此通常仅限于几个场站，装备精良的实验室。偏远地区无法进行采样，除非实施后勤手续繁琐。这些问题已经被公认为13，14和使用替代的储存和运输策略调查（醇类或胍）15-17 。最近，克劳斯等 18介绍的方法收集，储存和运输的禽流感病毒样本，基于一个特殊的滤纸。 FTA卡19保存在干燥的存储基础20 RNA和渲染病原体接触后21日无效。这项研究表明，FTA卡可以用来检测禽流感病毒的RNA在反转录PCR，可能是导致基因测序，病毒的基因，并确定。 在克劳斯等人 18的研究在实验室分离出禽流感病毒，并单独处理样品。在这里介绍的扩展，从Ottenby鸟天文台（SE瑞典）鸭陷阱野生鸟类的粪便样本进行了测试，直接说明了野战条件下的方法的实用性。捉鸭和泄殖腔拭子样品的采集是证明。目前的协议，包括从单管处理96井设计的工作流程缩放。虽然比传统方法敏感，FTA卡的方法提供了一个优良的大型监控计划的补充。它可以在世界任何地方的样本收集和分析，而不需要保持阴凉链或有害试剂，如酒精或胍，航运方面的安全法规。

Watch this Scientific Journal Video about Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved at JoVE.com

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

维护，检测和序列禽流感病毒的RNA的方法进行了验证和扩展，从鸟类的自然粪便样本。这种技术消除了保持冷静的链和处理传染性病毒的必要性和可应用在一个96孔高通量设置。

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 ...

avian-influenza-surveillance-with-fta-cards-field-methods-biosafety

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

19.2K 次观看.  Wageningen University. 维护，检测和序列禽流感病毒的RNA的方法进行了验证和扩展，从鸟类的自然粪便样本。这种技术消除了保持冷静的链和处理传染性病毒的必要性和可应用在一个96孔高通量设置。

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

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 ...

科研

免疫与感染

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, ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

造型埃博拉病毒的生命周期在生物安全二级条件下的病毒样颗粒包含Tetracistronic小基因组

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

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 ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

禽流感监测与FTA卡:实地方法，生物安全，和运输问题解决了

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