该项目由赠款1R03M​​H08127-01和7R03MH08127-02由美国国立卫生研究院对问李的支持，并通过从医学系在UAB的资金影响到Q力。从Molette基金和奥本大学的支持表示赞赏。我们也感谢工作过程中从普林车女士和沃洛Musiienko先生的技术协助。

1。细胞，病毒和抗病毒化合物<ol><li>维护的BSR细胞中，幼仓鼠肾（BHK）细胞10，在Dulbecco改良的Eagle培养基（DMEM）含有5％胎牛血清（FCS）的衍生物，100U/ml青霉素和100微克/毫升链霉素。 </li><li>对于所有三个实验，板细胞的DMEM含1％FCS，100U/ml青霉素和100微克/毫升链霉素，为优化以前5。该培养基被称为对于所有三个实验测定培养基。 </li><li>孵育中所有单元的培养箱中在37℃，含5％CO 2和80-95％的湿度。 </li><li>噬斑纯化和繁殖如前面8中所述类型10 BTV（BTV-10）。稀BTV-10在测定培养基中的每个指定的检测。 </li><li>溶解在DMSO中的所有测试化合物，形成与浓度的股票为10毫米。存储库存在-20°C。 </li><li>使用试验介质为候化合物稀释到所需的浓度ð分析2。 </li></ol> 2。基于CPE的检测使用CTG套件<ol><li>种子的BSR细胞到384孔微孔板（黑色;由16×24格式）通过MicroFlo选择分配器。播种密度为5,000个细胞/孔，和播种量为20μL的抗病毒疗效分析。 </li><li>细胞孵育2-3小时，直至获得细胞粘附于板彻底。 </li><li>添加的抗病毒化合物与10μM的终浓度向每个孔中。完全混合。 </li><li> BTV稀释到所需要的滴度，并加入5微升BTV与内政部表示，每孔。对于控制均匀，加入5微升试验培养基。培养感染的细胞72小时，在37℃，含5％CO 2和80-95％的湿度。 </li><li>在72 HPI，解冻和平衡CTG缓冲区和冻干CTG基板，使用前室温。由根据第混合冻干酶/底物和缓冲试剂重组均质CTG试剂溶液E制造商的指示。 </li><li>平衡的测定板至室温15分钟。 </li><li>通过分配器添加CTG试剂等体积（25微升）到每个孔中。在室温下在黑暗中温育15分钟后，使用多模读取器具有0.1秒的积分时间测量发光信号。 </li></ol> 3。剂量 - 反应分析<ol><li>种子的BSR细胞到384孔微孔板（黑色;格式由16×24），通过一个分配器，以5000个细胞/孔在20微升的抗病毒效力测定法和25μl的细胞毒性测定法，接种量分别接种密度。 </li><li>培养细胞2-3小时，在37℃，含5％CO 2和80-95％的湿度，直至细胞被很好地附着到板上。 </li><li>在384孔板的每个化合物的四分之一面积（相当于一个96孔板），如表1中所示的过程测定。分配8个重复每个浓度在一个单一的检测。通过将病毒仅而不化合物分配的第一列作为不添加化合物和病毒的阳性对照，并且最后一列（ 第 12 届 ）作为阴性对照。 </li><li>化合物稀释到50μm在测定培养基中，并加入到20微升/孔的抗病毒疗效检测的第二栏。使用8通道半自动移液器25μM的浓度混合的化合物的五倍。 </li><li>使用8通道半自动移液器，转移在第 2栏20微升混合物到下一列（第3 次 ），搅拌均匀，形成的12.5μM的浓度存在。通过从第 3列的转移20微升混合物到下一列（ 第 4 次 ），以形成另一种2倍稀释为6.25μM的浓度重复此过程。重复此两方面的连续稀释，直到第 11列。吸并添加并混合样稿后丢弃20μl，以11 个列中的混合物OUND。 </li><li>从第2列添加BTV-10的基础上，以0.01的MOI，向每个孔中，以与5微升/孔的体积11列。添加病毒后，在每列的最终化合物浓度应为：20μM2栏，10μM的第3列，并继续以2倍稀释液降至柱11为0.04μM的最终浓度。 </li><li>加入5微升培养基到第1列的单元格只能控制（正）和5μL/孔BTV的柱12作为北京电视台唯一的感染控制（负）。 </li><li>稀释化合物以200μM的细胞毒性测定法的初始浓度。类似地，将25μl化合物/孔的具有8通道的半自动移液器加至柱2和混合5倍至100μM的浓度存在。不要添加BTV。 </li><li>进行2倍系列稀释液通过吸移25μl的从第2列到相邻的第3栏，并一直持续到最后一列（ 第 12 次 ）。在塔12中，混合后，吸出并丢弃25微升米ixture。在第2列中的最终浓度应为100微米和12 个列应为0.2微米。列1是细胞唯一的控制。 </li><li>两种抗病毒疗效和细胞毒性测定法，孵育所述板在37℃，含5％CO 2和80-95％湿度下72小时后处理。使用CTG试剂盒如上述（协议步骤2.5-2.7）测量细胞活力。 </li></ol> 4。 （TOA）测定时，加成<ol><li>种子的BSR细胞在384 - 孔微量培养板1-24柱（黑色;由16×24的格式），通过一个分配器，在5000个细胞/孔，并在接种体积为15微升/孔。 </li><li>对于每个化合物，利用所有24列具有八个复制品的每个时间点在384孔板中（ 表2）的一半。通过加入10μl/孔的测定培养基中分配作为细胞仅控制第1列。标记的列24为BTV感染仅对照（阴性对照）加入5微升/孔的测定培养基中的ð5微升/孔的病毒以0.01的MOI为25微升/孔的终体积。 </li><li>从列2-22在不同时段后感染（HPI）作为抗病毒疗效的评价栏中选择偶数列。在这些列，感染细胞与5微升/孔BTV在0.01 MOI。在不同的HPI，还添加5微升/孔的稀释的化合物至每孔中，以形成25微升/终体积良好。为表示-2和-1 HPI添加化合物的BSR细胞之前BTV感染。对于0 HPI，同时化合物和BTV添加到文化。 </li><li>同时，从柱3-23指定奇数列作为化合物仅控制，该化合物在不同时间点被添加作为指定（-2，-1，0，1，2，4，8，16， 24，48 HPI）。在这些列中，添加5微升/孔的测定培养基和5微升/孔的稀释的化合物，以形成25微升/终体积良好。 </li><li>处理后，培养细胞在37℃，5％CO 2与80-95％的湿度。 </li><li>确定细胞存活率在72 HPI使用的CellTiter格洛套件如前（协议步骤2.5-2.7）描述。 </li></ol> 5。数据分析<ol><li>使用适当的内部软件的基础上通过所述多模读取器获得的发光信号的第一处理的所有数据。确定的平均值（平均），标准偏差（STDEV）每处理以及变异系数（CV），这就需要一个值不大于10％。 </li><li>从上面的软件处理过的数据传送到生物稳定性和图形软件工具。进行非线性回归分析，以确定EC 50和CC 50的值。 </li></ol>

<ol>
	<li>Hemati, B., 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=Bluetongue+virus+targets+conventional+dendritic+cells+in+skin+lymph.">Bluetongue virus targets conventional dendritic cells in skin lymph.</a> J. Virol. 83, 8789-8799 (2009).</li><li>Gu, L., 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=Novel+Virostatic+Agents+against+Bluetongue+Virus.">Novel Virostatic Agents against Bluetongue Virus.</a> PLoS ONE. 7, e43341 (2012).</li><li>Meiswinkel, 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=The+2006+outbreak+of+bluetongue+in+northern+Europe--the+entomological+perspective.">The 2006 outbreak of bluetongue in northern Europe--the entomological perspective.</a> Prev. Vet. Med. 87, 55-63 (2008).</li><li>Szmaragd, 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=Mortality+and+case+fatality+during+the+recurrence+of+BTV-8+in+northern+Europe+in+2007.">Mortality and case fatality during the recurrence of BTV-8 in northern Europe in 2007.</a> Vet. Rec. 161, 571-572 (2007).</li><li>Li, Q., Maddox, C., Rasmussen, L., Hobrath, J. V., White, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+development+and+high+throughput+antiviral+drug+screening+against+Bluetongue+virus.">Assay development and high throughput antiviral drug screening against Bluetongue virus.</a> Antiviral Research. 83, 267-273 (2009).</li><li>Noah, J. W., 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=A+cell-based+luminescence+assay+is+effective+for+high-throughput+screening+of+potential+influenza+antivirals.">A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals.</a> Antiviral Res. 73, 50-59 (2007).</li><li>Che, P., Wang, L., Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development,+optimization+and+validation+of+an+assay+for+high+throughput+antiviral+drug+screening+against+Dengue+virus.">The development, optimization and validation of an assay for high throughput antiviral drug screening against Dengue virus.</a> Int. J. Clin. Exp. Med. 2, 363-373 (2009).</li><li>Li, Q., Li, H., Blitvich, B. J., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Aedes+albopictus+inhibitor+of+apoptosis+1+gene+protects+vertebrate+cells+from+bluetongue+virus-induced+apoptosis.">The Aedes albopictus inhibitor of apoptosis 1 gene protects vertebrate cells from bluetongue virus-induced apoptosis.</a> Insect Mol. Biol. 16, 93-105 (2007).</li><li>Petty, R. D., Sutherland, L. A., Hunter, E. M., Cree, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+MTT+and+ATP-based+assays+for+the+measurement+of+viable+cell+number.">Comparison of MTT and ATP-based assays for the measurement of viable cell number.</a> J. Biolumin. Chemilumin. 10, 29-34 (1995).</li><li>Buchholz, U. J., Finke, S., Conzelmann, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+bovine+respiratory+syncytial+virus+(BRSV)+from+cDNA:+BRSV+NS2+is+not+essential+for+virus+replication+in+tissue+culture,+and+the+human+RSV+leader+region+acts+as+a+functional+BRSV+genome+promoter.">Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.</a> J. Virol. 73, 251-259 (1999).</li><li>Phillips, T., Jenkinson, L., McCrae, C., Thong, B., Unitt, J. <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+high-throughput+human+rhinovirus+infectivity+cell-based+assay+for+identifying+antiviral+compounds.">Development of a high-throughput human rhinovirus infectivity cell-based assay for identifying antiviral compounds.</a> J. Virol. Methods. 173, 182-188 (2011).</li><li>Harvey, T. J., 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=Tetracycline-inducible+packaging+cell+line+for+production+of+flavivirus+replicon+particles.">Tetracycline-inducible packaging cell line for production of flavivirus replicon particles.</a> J. Virol. 78, 531-538 (2004).</li><li>Puig-Basagoiti, F., 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=High-throughput+assays+using+a+luciferase-expressing+replicon,+virus-like+particles,+and+full-length+virus+for+West+Nile+virus+drug+discovery.">High-throughput assays using a luciferase-expressing replicon, virus-like particles, and full-length virus for West Nile virus drug discovery.</a> Antimicrobial Agents and Chemotherapy. 49, 4980-4988 (2005).</li><li>Puig-Basagoiti, F., 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=Triaryl+pyrazoline+compound+inhibits+flavivirus+RNA+replication.">Triaryl pyrazoline compound inhibits flavivirus RNA replication.</a> Antimicrob. Agents Chemother. 50, 1320-1329 (2006).</li><li>Duan, M., 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=In+vitro+and+in+vivo+protection+against+the+highly+pathogenic+H5N1+influenza+virus+by+an+antisense+phosphorothioate+oligonucleotide.">In vitro and in vivo protection against the highly pathogenic H5N1 influenza virus by an antisense phosphorothioate oligonucleotide.</a> Antivir. Ther. 13, 109-114 (2008).</li><li>Ray, D., Shi, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+flavivirus+antiviral+drug+discovery+and+vaccine+development.">Recent advances in flavivirus antiviral drug discovery and vaccine development.</a> Recent Pat. Antiinfect. Drug Discov. 1, 45-55 (2006).</li><li>Severson, W. E., 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=High-throughput+screening+of+a+100,000-compound+library+for+inhibitors+of+influenza+A+virus+(H3N2).">High-throughput screening of a 100,000-compound library for inhibitors of influenza A virus (H3N2).</a> J. Biomol. Screen. 13, 879-887 (2008).</li><li>Severson, W. E., 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=Development+and+validation+of+a+high-throughput+screen+for+inhibitors+of+SARS+CoV+and+its+application+in+screening+of+a+100,000-compound+library.">Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library.</a> J. Biomol. Screen. 12, 33-40 (2007).</li><li>Bolken, T. 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=Identification+and+characterization+of+potent+small+molecule+inhibitor+of+hemorrhagic+fever+New+World+arenaviruses.">Identification and characterization of potent small molecule inhibitor of hemorrhagic fever New World arenaviruses.</a> Antivir. Res. 69, 86-97 (2006).</li><li>Van Loock, M., 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=A+novel+high-throughput+cellular+screening+assay+for+the+discovery+of+HIV-1+integrase+inhibitors.">A novel high-throughput cellular screening assay for the discovery of HIV-1 integrase inhibitors.</a> J. Virol. Methods. 179, 396-401 (2012).</li><li>Kampmann, T., 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=In+silico+screening+of+small+molecule+libraries+using+the+dengue+virus+envelope+E+protein+has+identified+compounds+with+antiviral+activity+against+multiple+flaviviruses.">In silico screening of small molecule libraries using the dengue virus envelope E protein has identified compounds with antiviral activity against multiple flaviviruses.</a> Antiviral Res. 84, 234-241 (2009).</li><li>Kirchmair, J., 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=Development+of+anti-viral+agents+using+molecular+modeling+and+virtual+screening+techniques.">Development of anti-viral agents using molecular modeling and virtual screening techniques.</a> Infect. Disord. Drug Targets. 11, 64-93 (2011).</li></ol>

作者宣称，他们有没有竞争的财务权益。

抗病毒的命中初始识别，用于抗病毒药物发现和开发的关键步骤之一是开发强大的测定法，其中包括选择一个量化指标，开发一种简单的协议，从而获得足够的信号和低于10％的CV。大多数生化或基于细胞的筛选被设计成由于在筛分过程所需的再现性和潜在的大量分子筛选的提供基于最稳健的，简单和便宜的测定法，化学的起点。该CPE为基础的检测被指定为满足这些要求，以确定从一个大的复合图...

BTV是一个原型双链RNA病毒属中的环状病毒 ， 呼肠弧病毒家族。 BTV是家畜中最重要的疾病，包括绵羊，山羊，牛等家畜之一，与世界各地的1,2 $ 3十亿/年亏损。异国情调的BTV血清型是在上市的重要动物病原体“美国农业部的严重后果家畜病原体。”重新出现的BTV的最近，已经引起疾病的牛大爆发和羊在整个北欧3,4几个国家。作为其经济意义的结果，作为一个模型系统，BTV已经成为广泛的分子，遗传和结构研究的主题，并且几种疫苗已经开发出来。然而，由于缺乏适当的检测对抗病毒药物的发现，有没有可用的对抗BTV抗病毒药物。 在最近的高通量筛选（HTS）活动使用BTV作为模型系统，我们ðeveloped，优化和验证一个基于CPE的检测，以确定对虫媒病毒5潜在的广谱抗病毒药物。基于CPE的测定是，已在抗病毒的药物发现对许多病毒诱导快速和可观察到的CPE /凋亡5-7被用于一个公认的测定。在我们的系统，北京电视台后感染，CPE是显而易见的脊椎动物细胞，包括海拉，BSR和HEK 293T 8。 BTV-诱 ​​导的CPE可以监测和量化使用各种细胞活力的检测方法，包括的CellTiter格洛细胞活力试剂盒（试剂盒CTG）9。该试剂盒确定基于对细胞内ATP的定量在培养的活细胞的数目呈现，这标志着代谢活跃的活细胞的存在。在优化条件下，这里提出的基于CPE的检测表明其可行性与“混合和计量”一步到位的协议，并与稳定的荧光信号的灵活性。同时，有毒化合物热度庆安细胞活力将被排除在这个基于CPE的检测。在基于CPE的检测表明其稳定性和抗病毒的药物发现对抗BTV的可靠性，并且已经用于筛选NIH的分子库的小分子库（MLSMR），从而导致潜在的抗病毒铅化合物（次六个新集群的识别）5。 当一个潜在的抗病毒化合物中已使用基于CPE测定法确定，这将需要经受10浓度的剂量-反应实验来确定抗病毒活性和细胞毒性2的范围内。的抗病毒效力，表示为50％抑制浓度（IC 50）或50％有效浓度（EC 50），是一种药物，其抑制基线和最大之间病毒诱导的CPE的中途的浓度。抗病毒药物的细胞毒性， 即 50％的细胞毒性浓度（CC 50），是浓度的药物诱导的基线值和最大值之间的细胞毒性的50％。选择性指数（SI），记为50％的SI（SI 50）从CC 50 / IC 50，它确定对病毒诱导的CPE抗病毒的特异性来计算。该IC 50（或EC 50），CC 50和SI 50值是至关重要的措施，以确定抗病毒化合物是否是有效的和有选择性的进行进一步的药物开发。 当一种抗病毒药表明没有明显的毒性在体外 ，还防止了病毒诱导的CPE和生产性病毒的生命周期，它以表征其农业部2是重要的。我们通过开展ToA的检测，以确定病毒的生命周期是受抗病毒的可能（S）步实施这种表征。通常，抗病毒化合物加入到细胞中在不同的时间前或后病毒感染。如果抗病毒药物加入到被感染的细胞中张贴到其目标STEP感染的过程中，它比较时向其中添加之前的步骤中的人会导致较低的活性。因此，ToA的研究是一种用于确定化合物的抗病毒效力，其潜在目标的关键，无论是在病毒的生命周期或参与病毒生命周期的宿主机器。 对于所有三个实验，细胞存活率用CTG套件下列制造商的指示5确定。该检测系统输出，可以使用不同的内部软件进行分析，充分的发光信号。每个测定的至少一式三份用8副本进行验证和执行。对于所有获得的数据，三个参数，包括平均值（AVE），标准偏差（STDEV），和共同有效的变化（CV）进行分析，以确定检测的鲁棒性。一次测定的鲁棒性进行了测定，将数据进行进一步的分析，以及使用各种生物统计学和图形化绘制C工具2。

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM medium</td>
			<td>Gibco</td>
			<td>1134218</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>1000185</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gbico</td>
			<td>1049769</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>CellTiter-Glo (CTG) kit</td>
			<td>Promega</td>
			<td>TB288</td>
			<td>For cell viability measurement</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Fisher</td>
			<td>S25309B</td>
			<td>Diluted from 95%</td>
		</tr>
		<tr>
			<td>Antiviral huashilcompounds</td>
			<td>NIH MLSMR and de novo synthesis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BTV-10</td>
			<td>ATCC</td>
			<td>VR-187</td>
			<td></td>
		</tr>
		<tr>
			<td>BSR cell</td>
			<td>Developed in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy-II multi-mode microplate reader</td>
			<td>BioTek</td>
			<td></td>
			<td>For luminescent signal reading</td>
		</tr>
		<tr>
			<td>MicroFlo select dispenser</td>
			<td>BioTek</td>
			<td></td>
			<td>Adding cells, virus, and reagents</td>
		</tr>
		<tr>
			<td>384-well flat-bottom microplate</td>
			<td>CORNING</td>
			<td>28908031</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Gen. 5 software</td>
			<td>BioTek</td>
			<td></td>
			<td>For analysis of reading outputs from Synergy-II multi-mode microplate reader</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad</td>
			<td>Version 5</td>
			<td>For biostatic analysis and plot</td>
		</tr>
	</tbody>
</table>

assays for the identification of novel antivirals against bluetongue virus

为了找出对BTV潜在的抗病毒药物，我们已经开发，优化和验证这里提出三个实验。在基于CPE测定法的开发是为了评估在第一测定化合物是否表现出任何抗病毒活性，并已用于筛选大量化合物库。同时，抗病毒药物的毒性也可以使用基于CPE的测定法进行评价。剂量反应实验的目的是确定疗效所选抗病毒的范围内， 即 50％抑制浓度（IC 50）或有效浓度（EC 50），以及它的范围的细胞毒性（CC 50）。 TOA的分析受聘为农业部初步研究，以确定在BTV病毒的生命周期或宿主细胞机制的可能影响本小说抗病毒药物的作用机制。这些测定是在细胞培养系统的抗病毒功效的评价是至关重要的，并已被用于我们最近的研究导致ing到了一些对BTV新的抗病毒药物的鉴定。

1。化合物的抗病毒疗效基于细胞的CPE测定法的开发，优化和使用该发光基CTG试剂盒，以确定新的抗病毒药物对抗BTV如先前描述的2,5验证体外 。在10剂量响应测定法来反映抗病毒疗效通过测量基础上，在活细胞中5,11呈现细胞ATP的定量在培养代谢活细胞的数目的确定铅化合物和细胞毒性。在我们以前的报告中，一些潜在的抗病毒化合物进行了评价，包括通?...

Watch this Scientific Journal Video about Assays for the Identification of Novel Antivirals against Bluetongue Virus at JoVE.com

Assays for the Identification of Novel Antivirals against Bluetongue Virus

三个实验，包括细胞病变效应（CPE）为基础的试验中，剂量反应实验和时间的 - 加法（TOA）测定法已被开发，优化，验证和用于识别对蓝舌病毒的新的抗病毒药物（BTV），以及以确定可能的机制， - 行动（MOA）为新发现的抗病毒药物。

为检验新型抗病毒药物的鉴定对蓝舌病病毒

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

assays-for-identification-novel-antivirals-against-bluetongue

Research

JoVE Journal

Immunology and Infection

13.9K 次观看.  University of Alabama at Birmingham. 三个实验，包括细胞病变效应（CPE）为基础的试验中，剂量反应实验和时间的 - 加法（TOA）测定法已被开发，优化，验证和用于识别对蓝舌病毒的新的抗病毒药物（BTV），以及以确定可能的机制， - 行动（MOA）为新发现的抗病毒药物。 

视频: Assays for the Identification of Novel Antivirals against Bluetongue Virus

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

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

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 ...

科研

免疫与感染

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

免疫监视人乳铁蛋白的细胞摄取及其相关抗病毒活性抗丙型肝炎病毒

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

早期的病毒进入测定的鉴定和抗病毒化合物的评价

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

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 ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群<em&gt;体外</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

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 ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

猪全血表达编码和非编码 rna 类的鉴定

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...