We would like to thank FMC BioPolymer USA for product samples of Avicel. This work was supported through the Defense Threat Reduction Agency grant HDTRA1-13-1-0005 to KK and the NIH research grant 1R15AI100001-01A1to KK.

1.细胞的制备和试剂<ol><li>前一天的测定中，板适当的宿主细胞中所讨论的病毒（ 表1和2）在90 - 100％汇合。 </li><li>制备10％的甲醛溶液固定在卫生署2 O.在例子中，混合5.56毫升的36％的股票甲醛14.44毫升蒸馏水2 O（DH 2 O）。 注意:使用适当的安全处理方法和使用通风时的甲醛。 </li><li>制备结晶紫染色:1％结晶紫（CV）在20％乙 ​​醇和卫生署2 O. </li><li>准备2个斑块介质（细胞类型依赖性以2倍浓度; 见表2）。过滤用0.2μm过滤器（如果有）的试剂是不育的。 </li><li>固定覆盖的制剂（ 表3） <ol><li>对于液体覆盖，使微晶纤维素在卫生署2 O.无菌2.4％的溶液为了防止结块，粉末添加到含有水的第一个烧瓶中在迅速搅拌棒混合，倒入微晶纤维素在缓慢和快速地搅拌，在室温（RT）下。当溶液证明太粘稠搅拌棒均匀化，切换到一个烧瓶摇动器&gt; 30分钟，用重的搅拌以确保均匀的同质化。 注:储备溶液可以在较低浓度下进行，但溶液可以分离和将需要被重新混合以确保均匀化。微晶纤维素的工作溶液可用于从0.6-3％。 </li><li>均化后，高压灭菌，在室温封闭溶液和存储。 </li><li>对于琼脂糖和羧甲基纤维素（CMC）的叠加，调制原液中的dh 2 2％CMC或0.6％琼脂糖的O操作。混合与搅拌棒和高压锅或微波炉带入的解决方案。 </li></ol></li></ol> 2.稀释和感染<ol><li>当天电镀后，目视检查开始前的测定中细胞的汇合和生存能力。确保标准的细胞形态一次一〜90％汇合单层存在。 </li><li>执行传染性样本的十倍系列稀释。使用的细胞生长介质用于病毒繁殖作为稀释剂（ 表2）。变化的基础上所讨论的病毒的预期滴度所需稀释度的数量，并且总是使用未感染的对照样品，以独立地确保在菌斑鉴定细胞生存力和援助。 </li><li>从连续稀释，感染细胞45分钟至1小时（ 表1）。用接种物的足够体积，以覆盖细胞，同时保持体积尽可能的低，以最大程度地与单层（ 表4）病毒接触。轻轻摇动板每20分钟，以保证均匀覆盖，防止细胞单层的干燥。 </li><li>在感染后，覆盖直接固定化介质的接种物中使用1阱（ 表2）的一个适当的量:1混合2×斑块媒体和immobiliz的荷兰国际集团选择的叠加（CMC，琼脂糖或微晶纤维素）。轻轻摇动混匀。 <ol><li>对于液体覆盖，将1:1的温热2×斑块媒体和1.2 - 2.4％的RT库存微晶纤维素，得到的0.6-1.2％的Avicel覆盖介质的工作溶液。 </li><li>对于琼脂糖覆盖，使用1:1的混合物加热2×斑块介质中，并加热0.6％琼脂糖的储备溶液，在56℃水浴中进行30分钟，以平衡获得最终琼脂糖/覆盖0.3浓度的温度％。 </li><li>对于CMC，制备2％原液和处理所描述的琼脂糖覆盖。 </li><li>当施加叠加到该单层，总是平衡热琼脂糖或CMC在56℃的水中，以防止损坏的单层。确保解决方案是温暖的，但不烫手，防止细胞死亡，降低病毒滴度。最琼脂糖覆盖将开始凝固低于42°C，迅速开展工作和/或准备小批量，以防止凝固在处理。 </li></ol></li><li>除了覆盖后，孵化板产生明显的斑块，显然是可数的。斑的形成可以利用2-14天，取决于病毒的被分析， 表1中 。 注意:一旦液体覆盖板被放置在孵化器，不要动它们。发病期间的液体覆盖的移动会导致模糊的斑块。琼脂糖和CMC覆盖是半固体的，可以移动或在光学显微镜下周期性地检查以监测斑块的发展。 </li></ol> 3.固定和染色细胞<ol><li>以固定细胞，倒出或者吸出的Avicel覆盖，并用30分钟将10％甲醛溶液至过夜（&lt;1毫升每孔为一个6孔板中）固定细胞。 <ol><li>对于琼脂糖或CMC，直接添加甲醛溶液，以覆盖为1小时至过夜。 注:样品可以保存更长的时间，在固定剂，只要它不不蒸发，干燥，因为这会扭曲的单层。 </li></ol></li><li>染色前和固定后，弃去甲醛去除半固体插头琼脂糖和中央军委无论是自来水或手动用锅铲。冲洗的Avicel斑块与水染色前除去残留的覆盖层/固定剂。 </li><li>对于染色，覆盖细胞与供〜15分钟，结晶紫溶液最小量。岩盘如果必要，以确保均匀覆盖。 </li><li>轻轻洗去结晶紫染色水。一旦固定，染色，干燥，储存斑块无限期以供将来分析。 </li></ol> 4.确定病毒滴度<ol><li>计数噬斑在每个孔中，取平均值为相同的稀释的任何技术重复。对于大板格式，拥有超过5 100多斑更少或更多的折扣井。注意斑块的大小和形态。阴性对照应具有均匀的单层及可以作为一个参考对照。 </li><li>通过取斑的平均数目为一个稀释和总稀释倍数的倒数确定的库存样品的病毒效价。 </li></ol><img alt="式（1）" fo:content-width="3in" src="/files/ftp_upload/52065/52065eq1.jpg" width="300" />注意:作为一个例子，30和32噬斑计数为1×10 -7稀释[31（平均值）/ 10 -7（稀释）×0.4毫升（接种物）]将得到的7.75滴度×10 8 pfu的的重复/毫升。

<ol>
	<li>Cooper, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plaque+assay+of+animal+viruses.">The plaque assay of animal viruses.</a> Adv. Virus Res. 8, 319-378 (1961).</li><li>Dulbecco, R., Vogt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+problems+of+animal+virology+as+studied+by+the+plaque+technique.">Some problems of animal virology as studied by the plaque technique.</a> Cold Spring Harb. Symp. Quant. Biol. 18, 273-279 (1953).</li><li>Hartley, J. W., Rowe, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+cytopathic+and+plaque+assays+for+mouse+hepatitis+viruses.">Tissue culture cytopathic and plaque assays for mouse hepatitis viruses.</a> Proc. Soc. Exp. Biol. Med. Soc. Exp. Biol. Med. N. Y. 113, 403-406 (1963).</li><li>Juarez, D., Long, K. C., Aguilar, P., Kochel, T. J., Halsey, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+plaque+assay+methods+for+alphaviruses.">Assessment of plaque assay methods for alphaviruses.</a> J. Virol. Methods. 187, 185-189 (2013).</li><li>Shurtleff, A., Keuhne, A., Biggins, J., Keeney, A. <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+alternative+overlays+in+plaque+assays.">Use of alternative overlays in plaque assays.</a> , (2011).</li><li>Shurtleff, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+the+Filovirus+Plaque+Assay+for+Use+in+Preclinical+Studies.">Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies.</a> Viruses. 4, 3511-3530 (2012).</li><li>Matrosovich, M., Matrosovich, T., Garten, W., Klenk, H. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+low-viscosity+overlay+medium+for+viral+plaque+assays.">New low-viscosity overlay medium for viral plaque assays.</a> Virol. J. 3, 63 (2006).</li><li>Klebe, R. J., Harriss, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technically+simple+&#8216;non-lethal&#8217;+vital+staining+procedure+for+viral+plaque+and+cell+transformation+assays.">A technically simple &#8216;non-lethal&#8217; vital staining procedure for viral plaque and cell transformation assays.</a> Arch. Virol. 81, 359-362 (1984).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells.">Replication and plaque assay of influenza virus in an established line of canine kidney cells.</a> Appl. Microbiol. 16, 588-594 (1968).</li><li>Abiko, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outbreak+of+Human+Metapneumovirus+Detected+by+Use+of+the+Vero+E6+Cell+Line+in+Isolates+Collected+in+Yamagata,+Japan,+in+2004+and+2005.">Outbreak of Human Metapneumovirus Detected by Use of the Vero E6 Cell Line in Isolates Collected in Yamagata, Japan, in 2004 and 2005.</a> J. Clin. Microbiol. 45, 1912-1919 (2004).</li><li>Baer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+DNA+damage+signaling+upon+Rift+Valley+fever+virus+infection+results+in+cell+cycle+arrest+and+increased+viral+replication.">Induction of DNA damage signaling upon Rift Valley fever virus infection results in cell cycle arrest and increased viral replication.</a> J. Biol. Chem. 287, 7399-7410 (2012).</li><li>Austin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+Activation+following+Rift+Valley+Fever+Virus+Infection+Contributes+to+Cell+Death+and+Viral+Production.">p53 Activation following Rift Valley Fever Virus Infection Contributes to Cell Death and Viral Production.</a> PLoS ONE. 7, (2012).</li><li>B&#246;ttcher-Friebertsh&#228;user, E., Stein, D. A., Klenk, H. -. D., Garten, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+influenza+virus+infection+in+human+airway+cell+cultures+by+an+antisense+peptide-conjugated+morpholino+oligomer+targeting+the+hemagglutinin-activating+protease+TMPRSS2.">Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2.</a> J. Virol. 85, 1554-1562 (2011).</li><li>Youil, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+influenza+virus+replication+in+Vero+and+MDCK+cell+lines.">Comparative study of influenza virus replication in Vero and MDCK cell lines.</a> J. Virol. Methods. 120, 23-31 (2004).</li><li>Tobita, K., Sugiura, A., Enomoto, C., Furuyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plaque+assay+and+primary+isolation+of+influenza+a+viruses+in+an+established+line+of+canine+kidney+cells+(MDCK)+in+the+presence+of+trypsin.">Plaque assay and primary isolation of influenza a viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin.</a> Med. Microbiol. Immunol. (Berl.). 162, 9-14 (1975).</li></ol>

The authors have nothing to disclose and no competing or financial interests in the protocol or products described.

对于一个成功的噬斑测定中最关键的因素在于所述协议为特定病毒培养中的问题的优化作为条件可显著变化。重点要考虑到有:主机与病毒有关，合适的病毒生长的条件下，充分稀释范围的细胞相容性，为了明确区分斑块，并正确叠加选择和染色的细胞和病毒中的问题。 而VEEV和RVFV都采用了许多相同的宿主细胞类型中的非常类似的条件下生长，在斑块形态和生长动力学的差异而变化显著。当对空斑测定，其传统上被用作传播和指示细胞系对出血热病毒和甲病毒使用Vero细胞，VEEV通常生长到更高滴度比RVFV和演示增加的复制动力学，在48 HPI 4显影较大的均匀性斑块， 10 - 12。在对比的VEEV，RVFV要求72 HPI和演示斑块，通常要小得多，可变的尺寸。 在反对RVFV和VEEV，流感病毒是高度的宿主细胞特异性，并且很难通过组织培养繁殖。对于流感病毒，病毒进入和融合通过与病毒糖蛋白hamagglutinin（HA），它通过与宿主细胞的α-唾液酸表面受体结合介导进入靶细胞的细胞表面受体的结合通常被启动。 HA是三聚体糖蛋白，它存在于所有的流感病毒的膜包，并且需要切割进亚基HA1和HA2由一个特定的宿主细胞蛋白酶。为了进一步使问题复杂化，这些切割位点可病毒13株之间往往各不相同。作为能够裂解的HA被限制到特定的组织蛋白酶的表达，蛋白酶芳ë常常加到细胞培养基中，以促进病毒融合和进入所选宿主细胞中13。 TPCK胰蛋白酶是一种常用的蛋白酶是，用于在与两个MDCK和Vero细胞的细胞培养物的一个例子:通过病毒的HA 14,15的蛋白水解活化促进多循环复制（在不存在任何胰蛋白酶失活血清的）。 如本研究和其他人，在病毒生命周期的不同可以影响不同类和病毒物种间重叠的选择，盘格式和收集时间，以显著差异。在我们的研究中，琼脂糖覆盖层表现出在较高的滴度比任CMC或液体覆盖两个RVFV和乙型流感病毒更清晰的噬菌斑，补强之间广泛的病毒和细胞类型及其用途。用琼脂糖的低终浓度大大计算机辅助在去除固体塞和简化的染色。 CMC全面展示了Poorest功效的叠加层测试的所有三种病毒，并产生非常小的，模糊的斑块为乙型流感病毒。虽然整体CMC展示了最不理想的特点，其在快速增长的和极其致命的病毒可能会证明是有利的，因为它没有出现减少斑块大小与效价只有最小的减少使用。液体覆盖被证明是最通用的，因为它是非常简单的准备，增加易用性，并且是可比较的所有所选择的病毒的琼脂糖。在一个更高的吞吐量的96孔板格式，应用和除去没有因凝固抑制，与传统的叠加，并提供准确和一致的结果。用液体覆盖时的思考在于不透明颜色，而无法监视斑块的形成作为所述板不能移动，直到集合中的点，限制了这种适应的病毒与先前表征的复制动力学。 <p类="jove_content"&gt;在空斑试验条件稍作修改可以显著改变的结果，而任何新的系统或技术的评估和性能始终是必要的。而最优化和标准化的空斑检测协议不用于每一种情况存在，我们的报告表明的通用性和便于在传统的用途以及新型的液体覆盖对空斑测定，同时提供足够的背景为进一步的用户修改。

可行的病毒样本进行准确的分离和量化一直是病毒学正在进行的研究目标。直到斑块分析1952年问世的一种手段，以定性和定量计算出动物病毒滴度最早是1,2。这个技术首先适于与从噬菌体测定法，已预先用来计算在植物生物学1,2-库存噬菌体的效价修饰。而对于病毒定量的替代手段，至今已开发并适于，例如免疫测定，荧光和透射电子显微镜，可调电阻脉冲感测（TRPS），流式细胞术，重组报告系统，并定量逆转录聚合酶链反应（定量RT-PCR）的，这些方法不能识别和定量复制能力的病毒体1,3。而在技术和技术的进步不断完善和改变景观; plaquË试验继续代表在确定病毒浓度感染裂解病毒体1,4的黄金标准。 在一个噬斑分析，宿主细胞的汇合单层的感染未知浓度已连续稀释到一个可数的范围，典型地为5-100的病毒粒子之间的裂解病毒。受感染的细胞单层上覆盖在固定化覆盖介质，以防止病毒感染由通过液体介质的过程中病毒增殖或者机械或对流流动乱扩散。而固体或半固体覆盖，如琼脂糖，甲基纤维素或羧甲基纤维素（CMC），传统上一直使用的，液体的覆盖已成为具有新颖的液体覆盖的发展越来越有吸引力的替代，如微晶纤维素5- 7。利用液体与传统覆盖斑块检测有几个优势叠加可以APPL灭蝇灯在室温下，并施加和去除是显著容易。由于液体覆盖不需要暖，细腻，不耐热的病毒也可能被证明更容易牌匾。 初次感染和应用固定化叠加，个别斑块，或细胞死亡的区域之后，将开始发展为病毒感染和复制被限制到周围的单层。感染的细胞将继续复制裂解感染周期，进一步传播感染，导致越来越明显和离散斑。根据所使用的病毒的生长动力学和宿主细胞，可见斑块通常会在2-14天形成。细胞单层然后可以用标准的明视野显微镜，以便容易地识别斑块用肉眼进行计数，或者更典型地固定和counterstained通过中性红或晶体暴力。有各种各样的噬菌斑计数渍可用，每个发行日EIR特定的优点和缺点。结晶紫的加入量通常在收集点和覆盖层的固定/拆卸后，提供了一个快速的和独特计数器污点其允许非常小的噬斑中识别时的混合形态存在。中性红具有早期应用和与覆盖恒定接触的优点，允许在现场监测显影斑块形成，具有未知病毒或复制动力学工作时，这是特别有用的。然而，我们发现用中性红时染色通常不是作为不同的。 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）提供了几个优点，作为黄色染料染色活细胞的深蓝色和病毒噬斑可以不除去叠加的作为算用中性红。通过MTT，得到活细胞和死细胞之间的高对比度也允许小斑检测在较早的时间点在感染后虽然存储仍然需要去除覆盖8。结晶紫可以在水和醇中的溶液被简单地制成，并且提供了用于混合斑块形态灵敏度的高度，我们选择了它作为优选的和简化的计数器染色剂用于协议证明，因为我们正在利用几个家族的井病毒特征。 固定和染色被感染细胞单层后，噬菌斑计数，以滴定病毒原料样品中的噬斑形成单位（pfu）每毫升条款。日志下降应当注意连续稀释之间，并且根据板的大小，间5-100菌斑计数，以用作参照阴性对照。据统计样本将减少10％，每100斑块时，比较样品重复3计算各不相同。用空斑试验，以确定病毒滴度的优点在于它们的定量感染性病毒颗粒的实际数目瓦特能力ithin样品。作为多个病毒粒子有可能感染的单个的细胞中，单元与病毒体的术语是在斑块滴定1,2-使用。 斑块形态会发生很大不同下生长条件和病毒的物种之间。斑块大小，透明度，边框定义和分配都应该注意的，因为它们可以提供关于所讨论的病毒的生长和毒力因子的有价值的信息。 基本的空斑测定原理也可以被调整和修改在许多不同的方式，如在使用焦点形成测定（游离脂肪酸）的。游离脂肪酸不依赖于细胞裂解和复染以检测斑块形成，而是采用免疫染色技术来通过标记的抗体直接检测细胞内的病毒蛋白。增加的敏感性，降低的温育时间在感染后，最重要的是给定量的非裂解性病毒都是不同的能力使用时游离脂肪酸的优势。而被广泛使用的，关键限制因素在游离脂肪酸与传统的空斑测定出在需要适当的抗体并能够只用探针病毒蛋白亚单位与实际传染性病毒体4。 本研究的目的，我们将限制我们的讨论古典牌匾检测，并描述了使用传统固体和半固体覆盖（琼脂糖和CMC），随着新型液体微晶纤维素覆盖。

<table><tbody><tr><td>6-well CellStar plate for tissue culture</td><td>Greiner Bio-One</td><td>658 160</td><td></td></tr><tr><td>12-well CellStar plate for tissue culture</td><td>Greiner Bio-One</td><td>665 180</td><td></td></tr><tr><td>96-well CellStar plate for tissue culture</td><td>Greiner Bio-One</td><td>655 180</td><td></td></tr><tr><td>96-2ml deep well plate</td><td>USA Scientific</td><td>C15046314</td><td>For serial dilutions</td></tr><tr><td>DMEM</td><td>Quality Biologicals</td><td>112 013 101</td><td>Cell Media/Diluent</td></tr><tr><td>2x EMEM for plaques</td><td>Quality Biologicals</td><td>115 073 101</td><td>Warm to 37 &amp;#176;C</td></tr><tr><td>MEM 100x&amp;nbsp;</td><td>Cellgro</td><td>25 025 Cl</td><td></td></tr><tr><td>Sodium Pyruvate&amp;nbsp;</td><td>Cellgro</td><td>25 000 Cl</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140 122</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030 081</td><td></td></tr><tr><td>HyClone FBS&amp;nbsp;</td><td>Thermo Scientific</td><td>SH30910.03</td><td>Heat inactivate before use at 62 &amp;#176;C for 30 min</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldritch</td><td>A7030-50G</td><td></td></tr><tr><td>Trypsin TPCK</td><td>Sigma Aldritch</td><td>T1426-50mg</td><td>Make aliquots/Avoid freeze thawing</td></tr><tr><td>HEPES&amp;nbsp;</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>DEAE-Dextran</td><td>Sigma Aldritch</td><td>D9885-10G</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma Aldritch</td><td>C3886-25G</td><td></td></tr><tr><td>Formaldehyde Solution</td><td>Sigma Aldritch</td><td>F8775-500ml</td><td></td></tr><tr><td>Ethanol denatured Reagent Grade</td><td>Sigma Aldritch</td><td>362808-1L</td><td></td></tr><tr><td>Avicel RC-591 NF</td><td>FMC BioPolymer USA</td><td>RC-591 NF</td><td>Shake vigorously when reconsitiuting&amp;nbsp; for &gt;30 min to homogenize</td></tr><tr><td>UltraPure Agarose&amp;nbsp;</td><td>Invitrogen</td><td>16500-100</td><td></td></tr><tr><td>Carboxymethyl cellulose, Sodium Salt Medium Viscosity&amp;nbsp;</td><td>Sigma Aldrich</td><td>C4888</td><td></td></tr></tbody></table>

viral concentration determination through plaque assays: using traditional and novel overlay systems

空斑试验仍然是最准确的方法，感染性病毒体和病毒物质，通过在细胞培养离散的斑（感染单位和蜂窝死区）的数量的直接定量之一。在这里，我们将演示如何执行基本斑块检测，以及不同的重叠和技术如何影响斑块的形成和生产。典型的固体或半固体覆盖基材，如琼脂糖或羧甲基纤维素，已被用来限制病毒的传播，通过所述液体生长培养基防止滥感染。固定覆盖限制蜂窝感染的直接围绕单层，从而允许离散可数的病灶和随后的斑块形成的形成。克服固有的使用传统覆盖的困难，一种新型的液体覆盖利用微晶纤维素和羧甲基纤维素钠已被越来越多地用作一个替换标准中空斑试验。液体覆盖斑块检测可以在任何标准的6或12孔板格式，可以容易地进行按传统工艺，不需要特殊的设备。由于其液体状态，并且随后的易于应用和去除的，微培养板格式可以备选地使用作为一种快速，准确和高吞吐量的替代较大规模的病毒滴定。使用非加热的粘稠液体聚合物提供了机会，以简化工作，节省试剂，孵化空间，并增加在传统或遏制高的实验室，因为没有试剂，加热或玻璃器皿中使用时的操作安全性是必需的。液体覆盖也可能被证明比传统的覆盖某些热不稳定的病毒更为敏感。

空斑试验的准确评估病毒滴度的能力，依赖于许多因素:适当的宿主细胞的选择，适当的培养基和生长条件的细胞和病毒活力，固定化的病毒传播，并精确地确定所述病毒潜伏期的，以便有足够的时间对不同的和可数斑块的形成。 在这项研究中，来自三个有代表性的家庭的病毒被选为展示差异:跨越不同类型的样品叠加选择，潜伏期和斑块形态。委内瑞拉马脑炎（VEEV）被选定为（+），单链RNA病毒的模型，这会导致在马物种和人中显著疾病和代表披膜病毒科家族。乙型流感毒株台湾，一个分段（ - ）单链RNA病毒，主要感染人类，代表正粘病毒科。裂谷热病毒（RVFV），A（ - ）单链RNA节肢动物出生的病毒主要是感染节肢动物，反刍动物和人类中，被选定为布尼亚病毒科的代表。 为RVFV（ 图1），滴度从RVFV的重组减毒活MP12菌株在12孔板形式利用的CMC，琼脂糖，或微晶纤维素其中孵育侧由端72小时感染后叠加的储备液测定（HPI）。代表板块呈现稀释从10 -4〜10 -7能够在面板A.斑块使用CMC可以看到和琼脂糖覆盖呈小，清晰，鲜明的斑块有一个明确的圆形边框。斑有液体覆盖小幅更加丰富，相比于琼脂糖和CMC斑块较大，并提供了一​​个不太明显的边界。病毒滴度比较，B组与所有叠加的表演可比。 为了获得用于MP12随着更大的样本大小，以确定reproduc叠加中更清晰的视觉对比ibility，一个6孔板中也造模，一式三份（ 图2）。在6孔板形式中，使用CMC覆盖的呈小斑比任琼脂糖或液体覆盖，这是可比较的尺寸彼此。而病毒效价分别为所有三个叠加层（图D）之间的类似，形成于琼脂糖和液体覆盖斑块，由于其增加的大小被证明更容易计算。 与RVFV，VEEV滴度和斑块形态明显不同（ 图3A）的不同覆盖中的对比。形成CMC覆盖斑用12孔板格式时，在斑块的大小和灵敏度（B组）的代价表现出了明确和独特的形态。在对比的CMC，使用琼脂糖和液体的叠加导致显著较大的斑块，这表明较低的病毒抑制和增加的敏感性的VEEV复制。这是以前确认时只比较琼脂糖与CMC鸭纸张发表雷斯等人 4。而琼脂糖和液体的覆盖产生较大的斑比CMC中，斑块已定义不明确的边界，并且难于计数在12孔格式中，用液体覆盖层提供的最大边界扩散。当噬菌斑了试用于6孔板（ 图4），较大的6孔形式而言取反的那是困难的，在12孔格式来区分，用琼脂糖证明优于CMC的叠加公开大斑块和液体覆盖的问题斑块定义和灵敏（ 图4D）。 相比于RVFV或plaquing时，如外部蛋白酶的要求VEEV，流感提供了几个独特的挑战。流感病毒以覆盖不同的选择灵敏度也得到了很好的证明，在过去的显著改变人们已经注意到，在修改未成年人作为不同品牌琼脂糖都可以连接使用9。 有趣的乙型流感台应变，使用CMC的作为叠加导致了显着更小的斑块，是难以计数，并证明难以可靠评分（ 图5A）。利用琼脂糖覆盖层提供了最好的斑块（图C），并产生了一个更暗的背景染色（可能是由于增加的单层生存力），并且显示更清晰和更清晰的噬菌斑在直接比较中使用的液体覆盖（面板乙）。 液态聚合物在固体和半固体覆盖，如琼脂糖和CMC的一个明显的优势，在于方便拆卸和应用。半固体覆盖需要加热，以及处理和删除凝固时能证明问题。为了利用这些优势，并确定在高通量的方式进行RVFV利用微晶纤维素的实用性，在96孔板形式进行了试用在不同覆盖concentrati组件（ 图6）。为RVFV的MP-12，稀释液以一式四份在两个0.6和1.2％终浓度的微晶纤维素进行。叠加应用和去除证明简单，其中重复或浓度注意到之间没有明显的差异，表明再现性程度高。当得分，斑块是截然不同的和可数肉眼，展示利用液体覆盖在高通量的方式为RVFV的可行性。 <img alt="图1" fo:content-width="6.5in" width="650" src="/files/ftp_upload/52065/52065fig1highres.jpg" /> 图1:采用12孔板RVFV斑块覆盖比较 Veros铺板以2.5×10 5个细胞在12孔板中，并使用MP12的同一系列稀释的起始样品感染用200μl。感染1.5毫升覆盖0.3％琼脂糖，0.6％微晶纤维素，或1％的CMC（最终℃后oncentrations）中，为了直接比较的叠加层被施加在面板A.斑块显示出计数并滴定在B组。 <img alt="图2" src="/files/ftp_upload/52065/52065fig2highres.jpg" /> 图2:采用6孔板RVFV斑块覆盖比较 Veros铺板以5×10 5细胞在6孔板中，并使用MP12的同一系列稀释的起始样品感染了400微升。三毫升覆盖0.3％琼脂糖，0.6％微晶纤维素，或1％的CMC，为了直接比较的重叠施加这表现在图A，B和C独立的实验，进行了相同的描述为面板的AC，以斑块数和滴定的D组（N = 3）。 <img alt="图3" src="/files/ftp_upload/52065/52065fig3highres.jpg" /> 图3:&lt;利用12孔板强&gt; VEEV斑块覆盖的比较。Veros铺板以2.5×10 5个细胞在12孔板中，并感染了用VEEV TC-83疫苗株的同一系列稀释的起始样品加入200μl。感染后1.5毫升覆盖0.3％琼脂糖，0.6％微晶纤维素，或1％的CMC，为了直接比较的重叠施加这表现在图A的噬菌斑计数，并进行滴定在图B. <img alt="图4" src="/files/ftp_upload/52065/52065fig4highres.jpg" /> 图4:采用6孔板 V EEV斑块覆盖比较 Veros铺板以5×10 5细胞在6孔板中，并感染了用VEEV TC-83的同系列稀释的起始样品加入400μl。感染后，将3ml的覆盖0.3％琼脂糖，0.6％微晶纤维素，或1％的CMC，为了直接比较的重叠施加作为证明在图A，B和C分离实验进行了相同的描述图A - C，有斑块数和滴定的D组（N = 3）。 <img alt="图5" src="/files/ftp_upload/52065/52065fig5highres.jpg" /> 图5:流感斑块覆盖的比较性MDCK细胞以5×10 5细胞在6孔板中，并感染了400微升接种物使用B型流感台的同一系列稀释的起始样本。无胎牛血清（FBS），用在生长培养基或叠加，如胎牛血清可以通过抑制某些蛋白酶需要哪些病毒融合抑制流感传播。 TPCK胰蛋白酶加入到所有应用之前的叠加层，以促进病毒融合和进入与宿主细胞。感染后，将3ml的覆盖0.3％琼脂糖，0.6％微晶纤维素，或1％的CMC，以直接比较施加这表现在A组，B和C.独立实验的覆盖进行了相同的描述在图A - C，有斑块数和滴定的D组（N = 3）。虽然平均被带到了CMC斑块他们证明难以可靠计，因为他们表现出弥漫性边界和非常小的斑块大小。 <img alt="图6" fo:content-width="6.5in" width="650" src="/files/ftp_upload/52065/52065fig6highres.jpg" /> 图6:高通量斑块覆盖 96孔平板Veros的镀以每孔3×10 4个细胞，被感染50μl的接种物的使用RVFV MP12的同一系列稀释的起始样品1小时，一式四份。为覆盖，0.6和1.2％终浓度的微晶纤维素进行了试用，以确定利用液体覆盖在一个高通量的方式，图A和B的可行性和重现。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> RVFV </td><td> VEEV </td><td> B型流感</td></tr><tr><td>电池类型</td><td>维罗</td><td>维罗</td><td> MDCK </td></tr><tr><td>感染期</td><td> 1小时</td><td> 1小时</td><td> 45分钟</td></tr><tr><td>孵化时间</td><td> 3天</td><td> 2天</td><td> 3天</td></tr></tbody></table> 表1:空斑试验接种条件和细胞类型 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> RVFV </td><td> VEEV </td><td> B型流感</td></tr><tr><td>电池类型</td><td>维罗</td><td>维罗</td><td> MDCK </td></tr><tr><td>成长型</td><td> DMEM 1 </td><td> DMEM 1 </td><td> DMEM 2 </td></tr><tr><td>斑块媒体</td><td> 2xEMEM 一 </td><td> 2xEMEM 一 </td><td> 2xEMEM B </td></tr></tbody></table> 1. Dulbecco改良的Eagle培养基补充有10％胎牛血清，1％L-谷氨酰胺，1％Penicllin /链霉素。 2. Dulbecco改良的Eagle培养基补充有1％L-谷氨酰胺，1％Penicllin /链霉素，0.2％牛血清白蛋白，0.025％HEPES，DEAE葡聚糖为50ug / ml的。 答2倍最低必需培养基（500毫升），补充了5％FBS的（25毫升），1％的最小必需氨基酸（5毫升），1％丙酮酸钠（5毫升），1％L-谷氨酰胺（5毫升）中， 2％青霉素/链霉素（10毫升）中。 补充有0.2％牛血清白蛋白，1％最低必需氨基酸（5毫升）中，为50ug / ml时，0.025％HEPES，DEAE葡聚糖，胰蛋白酶TPCK B. 2个最低必需培养基（500毫升）* *之前要准备，加上每25微升1您将使用毫升2微克/毫升股票TPCK胰蛋白酶到等分混合琼脂糖的斑块。 表2:斑块和病毒/细胞生长的媒体 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> RVFV </td><td> VEEV </td><td> B型流感</td></tr><tr><td>电池类型</td><td>维罗</td><td>维罗</td><td> MDCK </td></tr><tr><td>感染期</td><td> 1小时</td><td> 1小时</td><td> 45分钟</td></tr><tr><td>孵化时间</td><td> 3天</td><td> 2天</td><td> 3天</td></tr></tbody></table>叠加解决方案，不会过期，只要不育维​​持时所作。 表3:重叠股票 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 6孔</td><td> 12好</td><td> 96 </td></tr><tr><td> ＃个细胞/孔</td><td> 5×10 5个</td><td> 2.5×10 5个</td><td> 3×10 4个</td></tr><tr><td>接种物的体积（微升） </td><td> 400 </td><td> 200 </td><td> 50 </td></tr><tr><td>覆盖层的体积（ml） </td><td> 3 </td><td> 1.5 </td><td> 0.100 </td></tr></tbody></table> 表4:板块格式

Watch this Scientific Journal Video about Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems at JoVE.com

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

病毒浓度的测定通过空斑试验:用传统的和新型覆盖系统

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

viral-concentration-determination-through-plaque-assays-using

Research

JoVE Journal

Immunology and Infection

111.9K 次观看.  George Mason University. Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

视频: Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

对小鼠诺沃克类病毒的空斑法

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

科研

免疫与感染

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

无菌实验室技术:电镀方法

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

生物学

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

重组呼吸道合胞病毒的生成、扩增和滴定

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Plaquing of Herpes Simplex Viruses

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

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

单纯疱疹病毒的扁平化

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

Virus Propagation and Cell-Based Colorimetric Quantification

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and potentially Guillain-Barr&#233; syndrome in adults. Research on ZIKV to understand the disease mechanisms is important to facilitate vaccine and treatment development. The method of quantifying viruses is crucial and fundamental in the field of virology. The focus forming assay (FFA) is a virus quantification assay that detects the viral antigen with antibodies and identifies the infection foci of cells using the peroxidase immunostaining technique. The current study describes the virus propagation and quantification protocol using both 24-well and 96-well (high throughput) formats. Compared with other similar studies, this protocol has further described foci size optimization, which can serve as a guide to expand the use of this assay for other viruses. Firstly, ZIKV propagation is performed in Vero cells for 3 days. The culture supernatant containing ZIKV is harvested and quantitated using the FFA. Briefly, the virus culture is inoculated onto Vero cells and incubated for 2-3 days. Foci formation is then determined after optimized staining processes, including cell fixation, permeabilization, blocking, antibody binding, and incubation with peroxidase substrate. The stained virus foci are visualized using a stereo microscope (manual counting in 24-well format) or software analyzer (automated counting in 96-well format). The FFA provides reproducible, relatively fast results (3-4 days) and is suitable to be used for different viruses, including non-plaque-forming viruses. Subsequently, this protocol is useful for the study of ZIKV infection and could be used to detect other clinically important viruses.

The present protocol describes the propagation of Zika virus (ZIKV) in Vero African green monkey kidney cells and the quantification of ZIKV using cell-based colorimetric immunodetection methods in 24-well and 96-well (high throughput) formats.

病毒传播和基于细胞的比色定量

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Plaque Assay: A Method to Determine Viral Titer as Plaque Forming Units (PFU)

Source: Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Viruses that infect prokaryotic organisms, called bacteriophages or simply phages, were identified in the early 20th century by Twort (1) and d&#39;H&#233;relle (2) independently. Phages have since been widely recognized for their therapeutic value (3) and their influence on human (4), as well as global, ecosystems (5). Current concerns have fueled a renewed interest in the use of phages as an alternative to modern antibiotics in treatment of infectious disease (6). Essentially all phage research relies on the ability to purify and quantify viruses, also known as a viral titer. Initially described in 1952, this was the purpose of the plaque assay (7). Decades and multiple technological advancements later, the plaque assay remains one of the most reliable methods for determination of viral titer (8).
Bacteriophages subsist by injecting their genetic material into host cells, hijacking the machineries for production of new phage particles, and eventually causing the host to release numerous progeny virions through cell lysis. Because of their minute size, bacteriophages cannot be observed using solely light microscopy; therefore, scanning electron microscopy is required (Figure 1). Additionally, phages cannot be cultivated on nutritional agar plates like bacteria, as they need host cells to prey on.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10514/10514fig1.jpg" /> 
Figure 1: The morphology of a bacteriophage, here exemplified by an E. coli phage, can be studied using scanning electron microscopy. Most bacteriophages belong to Caudovirales (tailed bacteriophages). This particular phage has a very short tail structure and an icosahedral head, placing it in the family of Podoviruses.
The plaque assay (Figure 2) is based on incorporation of host cells, preferentially in log-phase growth, into the medium. This creates a dense, turbid layer of bacteria able to sustain viral growth. An isolated phage can subsequently infect, replicate within, and lyse one cell. With each lysed cell, multiple adjacent ones become immediately infected. Several cycles in, a clear zone (a plaque) can be observed in the otherwise turbid plate (Figure 2B/Figure 3A), indicating the presence of what was initially a single bacteriophage particle. The number of plaque forming units per volume (i.e. PFU/mL) of a sample, can thus be determined from the number of plaques generated.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10514/10514fig2.jpg" /> 
Figure 2: Testing for plaque forming units (PFU) is a common method for determining the number of bacteriophages in a sample. (A) The base of a sterile Petri dish is covered with an appropriate solid nutrient medium, followed by a mixture of soft media, susceptible host cells and a dilution of the original bacteriophage sample. Note that the phage-suspension could, in some cases, also be evenly spread-out across the surface of already solidified soft agar. (B) Growth of the host bacteria forms a lawn of cells in the top agar layer. Bacteriophage replication generates clear zones, or plaques, caused by host cell lysis.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10514/10514fig3.jpg" /> 
Figure 3: Results from PFU-testing show multiple plaques generated by bacteriophages. Due to lysis of susceptible host cells, plaques can be seen as clearing zones in the bacterial lawn, either with (A) full clearance, or (B) partial re-growth caused by generation of resistant bacteria (or possibly by temperate phages in the lysogenic cycle).
Certain temperate phages can adopt what is referred to as a lysogenic lifecycle, in addition to the formerly described lytic growth. In lysogeny, the virus assumes a latent state through incorporation of its genetic material into the genome of the host cell (9), often conferring resistance to further phage infections. This is sometimes revealed through a slight clouding of the plaque (Figure 3B). It is worth noting however, that plaques can also appear blurred due to re-growth of bacteria that have evolved resistance to the phage independent of previous phage infections.
Viruses can attach, or adsorb, to only a limited range of host bacteria (10). Host ranges are further limited by intracellular anti-viral strategies such as the CRISPR-Cas system (11). The resistance/sensitivity towards specific phages displayed by bacterial subgroups has historically been used to categorize bacterial strains into different phage types (Figure 4). Although the effectiveness of this method has now been surpassed by novel sequencing techniques, phage typing can still provide valuable information about bacteria-phage interactions, for instance, facilitating the design of a phage cocktail for clinical usage.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/10514/10514fig4.jpg" /> 
Figure 4: Phage sensitivity of different bacterial strains. Soft agar plates with Cutibacterium acnes strain (A) AD27 and (B) AD35, were spotted with 21 different C. acnes bacteriophages. Only phage 11 were able to infect and kill AD27 while strain AD35 showed sensitivity towards all phages. This technique, termed phage typing, can be used to divide bacterial species and strains into different subgroups based on phage susceptibility.

Determining Viral Titer as Plaque Forming Units PFU

斑块测定:确定病毒性皮石为斑块形成单位（PFU）的方法

Source: Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 ...

教育

JoVE Science Education

高级生物学

微生物学

Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay

Source: Yadavalli, T., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/62195">Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals.</a> J. Vis. Exp. (2021)
This video demonstrates the method to perform a plaque assay using a virus obtained from infected porcine cornea. The porcine cornea is used to test the antiviral efficacy of experimental drugs.

通过噬菌斑测定确定实验药物的抗病毒疗效

1. Virus infection quantification
NOTE: Virus titers from porcine corneas should be evaluated every day to analyze the effect of drug treatment.

	To ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

用于筛选抗病毒化合物的病毒附着测定

NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...