我们感谢SIGA技术（俄勒冈州Corvallis）提供ST-246。 DKR是由美国国立卫生研究院的练金在免疫学，以波士顿大学（5T32AI 7309）的支持。这项工作是由P41 086180部分支持，美国国立卫生研究院RO1AI1096159-01，和RO3（以JHC）。

1，电池板<ol><li>从板分离HeLa细胞在生长培养基（DMEM，2mM的L-过剩，10％FBS）中稀释至约2.0×10 5个细胞/ ml。分配100μl/孔在黑色壁，透明平底96孔板（20000个细胞/孔）。 </li><li>孵育细胞24小时，直至汇合在37℃培养箱+，5％CO 2。 </li></ol> 2，感染细胞<ol><li>稀释病毒和感染细胞。 <ol><li>解冻TRPV（三人间病毒;早期金星，中级的mCherry，晚TagBFP）和PLV（启动子的维纳斯）荧光病毒和分解利用超声处理5分钟。或者，粗病毒储备可以1:1的比例混合，用0.25毫克/毫升胰蛋白酶在感染媒介并在37℃下进行30分钟。 </li><li>稀释原油库存病毒在37℃感染培养基（DMEM，2毫米L-供过于求，2％FBS）。对于高MOI感染（10 PFU /细胞），病毒稀释股票1.0×10 7 PFU / ml的假设5×10 4细胞/孔和50μl的接种物体积。在感染3重复孔的每个治疗TRPV和另外3重复孔与PLV考虑到具体到每个处理背景荧光相同的治疗方案。 </li><li>通过加入50μl/孔稀释病毒感染媒介传染井。这被定义为时间= 0小时感染后（0 HPI）。 </li></ol></li><li>淡化理想的治疗化合物转化为感染媒介。对于所有的治疗单倍母液稀释应作出并应用到复制井（六96孔如 350μl总体积各50微升）。 <ol><li>稀释试验化合物为感染媒介。 </li><li>稀车辆控制溶剂（ 如 PBS，DMSO）为感染媒介。注意:类似的最终浓度车辆用控制溶剂的应该被用作施加试验化合物（ 例如:如果你的IBT股票被用DMSO中，并在最终的concentratio使用1微升的NS /毫升，然后用DMSO独自在1微升/毫升作为车辆控制）。 </li><li>稀释对照化合物为感染媒介。推荐控制的化合物包括:3.6μM（1微克/毫升）1-β-D-阿糖呋喃胞嘧啶（阿糖胞苷），50μM（11.7微克/毫升）的靛红β-缩氨硫脲（IBT），60微米（50微克/毫升）利福平或5微米（1.9微克/毫升）ST-246 8,15,16。看到预期的效果代表性的成果。注意:使该稀释的两倍（2×）的所需终浓度，因为这是在以1:1的比例添加到该卷已在井中。 </li></ol></li><li>立即加入病毒后，加入含有所需的治疗和控制的稀释步骤2.2 50微升传染媒介。注意:为了早期表达前测定为抑制，化合物（S）可以被直接加入到稀释的病毒接种物（步骤2.1.2）或感染之前对宿主细胞（步骤2.1.3之前）。 </li><li>孵育6-24小时在37＆＃176，C培养箱+，5％CO 2。 </li></ol> 3，修复细胞<ol><li>通过加入100微升8％多聚甲醛到感染介质已经在每个孔中固定的细胞。在室温下孵育为避光15分钟。注:建议到2倍的PFA（8％）直接添加到传染媒介，以防止可能发生的感染牛痘的气雾，如果高滴度的媒体直接倒到垃圾盘前固定。 </li><li>通过翻转板进入废菜除去固定液。 </li><li>加入100μl室温的PBS。 </li><li>与光学透明粘结膜封板。注:板可存放在4℃下，如果不立即读取。请务必阅读，以防止冷凝扭曲分光光度计测量前返回封板至室温。 </li></ol> 4，量化病毒生长<ol><li>测量终点的​​荧光在515:53​​0金星，587:610的的mCherry，和415:457 f或TagBFP（励磁:在nm发射波长）。四个测量应每口井使用的每个通道优化增益设置进行平均。注意:适当的发射波长可能不同，具体取决于您的平板阅读器的具体型号和滤波特性。这可以通过经验进行发射波长扫描，以确定该点那里是为每个通道TRPV与PLV之间的最大差额进行确定。 </li><li>归一化的原始数据，以方便实验之间的比较。 <ol><li>对于每个信道，确定复制TRPV和PLV孔的平均值。 </li><li>减去平均TRPV感染的实验测量每个治疗的平均PLV感染的背景测量。 </li><li>通过将每个背景值中减去由车辆只值以及规范化的数据。 </li><li>一次实验被重复多次，方差单向分析（单向ANOVA）检验和多Çomparison检测后可以进行，以确定是否任何的处理反映从每个通道的车辆唯一的治疗在统计学显著差异。 </li></ol></li></ol> 5，替代协议:动力板测定<ol><li>通过另外的方法执行的所有步骤（步骤2.3）。 </li><li>封板，在酶标仪室粘结膜和地方平衡至37°C。注:必须特别注意​​采取保持一致板在37°C，以防止过度的细胞应激和冷凝。对于动力学分析，这是特别重要的是使用一个缓冲介质（碳酸氢钠和HEPES），以保持适当的pH值不添加5％的CO 2。 </li><li>手动设置酶标仪获得，以防止稍后的时间点饱和。注:准确的增益优化可以通过执行类似条件下的端点检测得到。 </li><li>收购每小时读数8-24 HPI和使用规范化西米征地拆迁过程中的端点检测（步骤4.2）。 </li><li>个别时间点可以使用类似的方法如先前所描述的端点测定统计显着性进行分析。 </li></ol>

<ol>
	<li>Yen, J., Golan, R., Rubins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+infection+&amp;+temporal+analysis+of+virus+gene+expression:+part+1.">Vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1.</a> J Vis Exp. 26 (26), (2009).</li><li>Hansen, S. G., Cope, T. A., Hruby, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BiZyme:+a+novel+fusion+protein-mediating+selection+of+vaccinia+virus+recombinants+by+fluorescence+and+antibiotic+resistance.">BiZyme: a novel fusion protein-mediating selection of vaccinia virus recombinants by fluorescence and antibiotic resistance.</a> BioTechniques. 32 (5), 1182-1187 (2002).</li><li>Popov, S., Mirshahidi, S., Essono, S., Song, R., Wang, X., Ruprecht, R. M. <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+recombinant+vaccinia+viruses+via+green+fluorescent+protein+selection.">Generation of recombinant vaccinia viruses via green fluorescent protein selection.</a> DNA and Cell Biology. 28 (3), 103-108 (2009).</li><li>Bartee, E., Mohamed, M. R., Lopez, M. C., Baker, H. V., McFadden, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+addition+of+tumor+necrosis+factor+plus+beta+interferon+induces+a+novel+synergistic+antiviral+state+against+poxviruses+in+primary+human+fibroblasts.">The addition of tumor necrosis factor plus beta interferon induces a novel synergistic antiviral state against poxviruses in primary human fibroblasts.</a> Journal of Virology. 83 (2), 498-511 (2009).</li><li>Ward, B. M., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+intracellular+movement+of+vaccinia+virus+virions+containing+a+green+fluorescent+protein-B5R+membrane+protein+chimera.">Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera.</a> Journal of Virology. 75 (10), 4802-4813 (2001).</li><li>Johnson, M. C., Damon, I. K., Karem, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+high-throughput+vaccinia+virus+neutralization+assay+for+testing+smallpox+vaccine+efficacy+based+on+detection+of+green+fluorescent+protein.">A rapid, high-throughput vaccinia virus neutralization assay for testing smallpox vaccine efficacy based on detection of green fluorescent protein.</a> Journal of Virological Methods. 150 (1-2), 14-20 (2008).</li><li>Dower, K., Rubins, K. H., Hensley, L. E., Connor, J. H. <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+Vaccinia+reporter+viruses+for+rapid,+high+content+analysis+of+viral+function+at+all+stages+of+gene+expression.">Development of Vaccinia reporter viruses for rapid, high content analysis of viral function at all stages of gene expression.</a> Antiviral Research. 91 (1), 72-80 (2011).</li><li>Dower, K., 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+of+a+pyridopyrimidinone+inhibitor+of+orthopoxviruses+from+a+diversity-oriented+synthesis+library.">Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library.</a> Journal of Virology. 86 (5), 2632-2640 (2012).</li><li>De Clercq, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Potential+of+the+Acyclic+Nucleoside+Phosphonates+Cidofovir,+Adefovir,+and+Tenofovir+in+Treatment+of+DNA+Virus+and+Retrovirus+Infections.">Clinical Potential of the Acyclic Nucleoside Phosphonates Cidofovir, Adefovir, and Tenofovir in Treatment of DNA Virus and Retrovirus Infections.</a> Clinical Microbiology Reviews. 16 (4), 569-596 (2003).</li><li>Prichard, M. N., Kern, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthopoxvirus+Targets+for+the+Development+of+New+Antiviral+Agents.">Orthopoxvirus Targets for the Development of New Antiviral Agents.</a> Antiviral Research. 10, (2012).</li><li>Fenner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+successful+eradication+campaign.+Global+eradication+of+smallpox.">A successful eradication campaign. Global eradication of smallpox.</a> Reviews of Infectious Diseases. 4 (5), 916-930 (1982).</li><li>Breman, J. G., Henderson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+dilemmas--monkeypox,+smallpox,+and+biologic+terrorism.+The+New+England.">Poxvirus dilemmas--monkeypox, smallpox, and biologic terrorism. The New England.</a> Journal of Medicine. 339 (8), 556-559 (1998).</li><li>Baker, R. O., Bray, M., Huggins, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+antiviral+therapeutics+for+smallpox,+monkeypox+and+other+orthopoxvirus+infections.">Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections.</a> Antiviral Research. 57, 1-2 (2003).</li><li>Rimoin, A. 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=Major+increase+in+human+monkeypox+incidence+30+years+after+smallpox+vaccination+campaigns+cease+in+the+Democratic+Republic+of+Congo.">Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo.</a> Proceedings of the National Academy of Sciences of the U S A. 107 (37), 16262-167 (2010).</li><li>Sodeik, B., Griffiths, G., Ericsson, M., Moss, B., Doms, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+vaccinia+virus:+effects+of+rifampin+on+the+intracellular+distribution+of+viral+protein+p65.">Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.</a> Journal of Virology. 68 (2), 1103-1114 (1994).</li><li>Grosenbach, D. W., Jordan, R., Hruby, D. 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+the+small-molecule+antiviral+ST-246+as+a+smallpox+therapeutic.">Development of the small-molecule antiviral ST-246 as a smallpox therapeutic.</a> Future Virology. 6 (5), 653-671 (2011).</li><li>Broyles, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+transcription.">Vaccinia virus transcription.</a> Journal of General Virology. 84 (9), 2293-2303 (2003).</li><li>Condit, R. C., Niles, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+viral+transcription+elongation+and+termination+during+vaccinia+virus+infection.">Regulation of viral transcription elongation and termination during vaccinia virus infection.</a> Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1577 (2), 325-336 (2002).</li><li>D&#237;az-Guerra, M., Rivas, C., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+expression+of+the+2-5A+synthetase/RNase+L+system+results+in+inhibition+of+vaccinia+virus+replication.">Inducible expression of the 2-5A synthetase/RNase L system results in inhibition of vaccinia virus replication.</a> Virology. 227 (1), 220-228 (1997).</li><li>Cohrs, R. J., Condit, R. C., Pacha, R. F., Thompson, C. L., Sharma, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+ppp(A2&#8217;p)nA-dependent+RNase+by+a+temperature-sensitive+mutant+of+vaccinia+virus.">Modulation of ppp(A2&#8217;p)nA-dependent RNase by a temperature-sensitive mutant of vaccinia virus.</a> Journal of Virology. 63 (2), 948-951 (1989).</li><li>S&#225;nchez-Puig, J. M., S&#225;nchez, L., Roy, G., Blasco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+different+leukocyte+cell+types+to+Vaccinia+virus+infection.">Susceptibility of different leukocyte cell types to Vaccinia virus infection.</a> Virology Journal. 1 (1), (2004).</li><li>Bengali, Z., Satheshkumar, P. S., Yang, Z., Weisberg, A. S., Paran, N., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+S2+cells+are+non-permissive+for+vaccinia+virus+DNA+replication+following+entry+via+low+pH-dependent+endocytosis+and+early+transcription.">Drosophila S2 cells are non-permissive for vaccinia virus DNA replication following entry via low pH-dependent endocytosis and early transcription.</a> PLoS One. 6 (2), (2011).</li></ol>

作者什么都没有透露，没有专利正在申请中的病毒或筛选方法。

这里，我们描述了一种多级牛痘记者病毒（TRPV），它提供病毒复制的重现性，实时反馈测量的实际使用情况。通过使用良好定义的痘病毒的抑制剂，我们能够显示，TRPV与动作的每个抑制剂的理解机制相一致的方式进行响应。而TRPV病毒提供了有关病毒阶段级数最全面的信息，两阶段（IREV，LREV）和单级（EV，IV LV）的病毒，也可以类似地使用，同时考虑最优的蛋白质折叠的优点，稳定和优异的信号?...

传统上，病毒表达是利用分子生物学技术（ 例如，RNA印迹法，蛋白质印迹法，微阵列杂交， 等等 ）1研究。虽然这些方法能够提供详细的信息相对于个别分类的mRNA或蛋白质的表达的变化，它们通常不适合于实时性和高通量处理。采用基于荧光的记者替代方法与痘病毒工作时，先前已被应用;然而，他们的开发和使用已经出于不同的目的。一些这样的方法被设计为选择的重组病毒2,3。这些技术表达绿色荧光蛋白在适当的成立和从重组痘苗病毒克隆外源DNA的表达。类似地，一些牛痘株稳定表达可溶性EGFP或下一个本机牛痘启动子表达GFP标记的蛋白已被广泛使用。这些都是典型售货机，ically用于定量病毒进入及复制过程中的基于抗体的中和测定法，化学抑制，或抗病毒效力4-6比较。尽管这些病毒已经被证明非常有用，他们在向因单一暧昧早/晚病毒启动子及其用法提供了有关抑制的点的详细信息的能力是有限的。以前的方法也取得了利用EGFP蛋白的次优的信号 - 噪声特性。 由于痘病毒复制和缺乏可用以测定在病毒基因表达的实时变动对现有工具的复杂性，我们开发了一套单级和多级记者病毒7,8。如在以前的出版物中描述的，这些病毒表达一种，两种，或从天然痘苗病毒启动子3在光谱上不同的荧光蛋白在早期，在感染中间，或最后阶段。这些病毒可能是我们编为病毒复制进度使用荧光显微镜的指标和它们同样适合于高通量的基于平板和阅读器测定。这些病毒很容易在地方的野生型病毒的使用，具有类似的动力学成长，达到相当于滴度7。在策划和创作的这些病毒，很在意拍摄于选择具有高信背景特征具有超强的折叠效率的荧光基团，以方便可靠的定量分析与快速反馈的变化，表达（金星的mCherry和TagBFP）。此外，病毒启动子的组合被选择能产生高保真的，明确的阶段特异性表达，提供有关的全方位早期（C11R启动子）的信息，中间体（G8R启动子）和晚期（F17R启动子）的基因表达，而相比之下，暧昧早/晚推动者。 这些病毒可以作为用于英伟的工具stigating原型痘病毒，牛痘病毒的生命周期。现在还是有很多不知道的宿主 - 病毒相互作用，尽管其悠久的研究历史。牛痘是复杂的，生产超过200独特的蛋白质，其中有许多是免疫调节和主机对立。一旦感染病毒，牛痘病毒立即开始早期mRNA的转录。这是通过被在病毒粒子的包装上的病毒基因组中加载并在暂停状态保持到随后的感染RNA聚合酶和转录因子的促进。这种早期的表现主要生产所需的抑制宿主的免疫系统（mRNA的脱帽，dsRNA的封存和诱饵受体蛋白以及细胞凋亡的抑制剂，应激反应和收费，白细胞介素，和NF-κB信号）和基因组复制的蛋白质。早期表达也产生必要的中间表达的转录因子。中间表达式包括后期转录因子的表达。这表达级联导致生产结构和酶的病毒蛋白质在感染的晚期阶段，这是必要的成熟牛痘病毒体的完整组件。 我们组的荧光记者病毒可以快速进展痘病毒生物学的理解作出。一个在病毒学领域中最常见的和费时的方法是在生长测定。这通常涉及到感染细胞，通过裂解感染的细胞颁布一系列的处理，收获病毒，并通过噬菌斑测定法量化所得到的病毒滴度。使用此处描述的记者病毒允许病毒生长的实时测量，可以很容易检测和并行执行大量的处理之间进行比较。我们预计这组试剂将用于各种协议确定响应药物治疗，RNA干扰K的病毒基因表达的改变nockdown，或宿主范围的限制。 此方法也允许的规模比以前提供更大的高内涵分析，允许高通量的抗病毒的药物屏幕对于感兴趣专门定义的目标的阶段。虽然打击痘病毒感染许多潜在的治疗方法已经确定，唯一被FDA批准的治疗有效治疗痘病毒感染是无环核苷膦酸酯，西多福韦和牛痘免疫球蛋白9,10治疗。尽管消灭天花，1977年11，痘病毒仍然是一个显著威胁人类健康12。对天花病毒的广泛接种疫苗的终止，导致易感性增加其他痘病毒13。例如，中非以前接种疫苗保护的地区在猴痘病毒感染14激增。显著的关注也一直提出了关于潜在的易感性天花病毒的有意释放。由于目前可用的有限的疗法，目前迫切需要对新疗法的发展。这些记者病毒允许快速和高通量小分子抑制剂筛选抑制病毒复制的特定阶段。针对病毒表达阶段抑制目前不是目标抑制剂的鉴定将有利于联合疗法的发展与提高的效力。 要通过观察变化，每个阶段的基因表达进行搜集了关于牛痘细胞生物学。衰减通过化学或感染的宿主细胞的遗传操作通常以降低病毒滴度表示。然而，通过比较改变的病毒表达级联的每一级，可以得到怎样的病毒适应度的影响更完整的理解由一个特定的治疗主编。这些数据已经显示出与传统的病毒滴度输出良好的相关性，但提供更详细的机理信息，以及高吞吐量能力7。

<table border="0" cellpadding="0" cellspacing="0" width="1249">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:747px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:104px;">11995-065</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fetal Bovine Serum - Optima, Heat Inactivated (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">200 mM L-Glutamine, (L-glut)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3712</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Trypsin, 2X, Sterile, Irradiated</td>
			<td>Worthington Biochemical Corp.</td>
			<td>TRLVMF</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl Sulfoxide, Cell Culture (DMSO)</td>
			<td>American Bioanalytical</td>
			<td>AB03091</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1-&#946;-D-Arabinofuranosylcytosine&#160; (AraC), MW= 280 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>C6645</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">isatin &#946; -thiosemicarbazone (IBT), MW= 234 g/mol</td>
			<td>Fisher Scientific</td>
			<td>NC9075202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Rifampicin, MW= 823 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">ST-246, MW= 376 g/mol</td>
			<td>SIGA Labs, Corvallis, OR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">8% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TempPlate RT optically clear film</td>
			<td>USA Scientific</td>
			<td>2978-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

vaccinia reporter viruses for quantifying viral function at all stages of gene expression

痘病毒是一个家族的双链DNA病毒，包括有源人类病原体如猴痘，软疣contagiousum和Contagalo病毒。该系列还包括天花病毒，天花。由于痘病毒复制的复杂性，许多问题仍然对他们的基因表达的策略。在这篇文章中，我们描述的重组痘苗病毒，使之在高通量形式病毒基因表达的单一和多级实时测量的概念和用法。这是通过使用光谱不同的荧光蛋白作为报告的每个病毒复制的三个阶段启用。这些病毒提供了高的信号噪声比，同时保持阶段特异性表达模式，使板为基础的检测和病毒繁殖和复制的显微观察。这些工具具有用途抗病毒药发现，病毒 - 宿主相互作用的研究，和进化的生物迟缓。

因为这些病毒的典型用法的示例，三重荧光记者病毒被用来抑制的几个定义良好的痘病毒抑制剂的点进行比较。 将HeLa细胞接种在组织培养物处理的黑色壁透明平底96孔板中并孵育过夜。汇合的单层细胞感染在感染使用任一三重记者病毒的多重10（MOI）（TRPV， 表1）或启动子的金星（PLV）所详述的板图（ 图1）上。加入含有治疗感染媒体取得0.1％DMSO，3....

Watch this Scientific Journal Video about Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression at JoVE.com

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

我们描述了一种荧光报告牛痘病毒，使病毒的感染性和基因表达的实时测量通过的光谱不同的荧光报告基团的阶段特异性表达的用法。我们详细的板基础的方法准确地确定在哪个病毒复制受到影响响应小分子抑制阶段。

牛痘病毒记者量化病毒功能在基因表达的各个阶段

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

vaccinia-reporter-viruses-for-quantifying-viral-function-at-all

Research

JoVE Journal

Immunology and Infection

11.3K 次观看.  Boston University School of Medicine. 我们描述了一种荧光报告牛痘病毒，使病毒的感染性和基因表达的实时测量通过的光谱不同的荧光报告基团的阶段特异性表达的用法。我们详细的板基础的方法准确地确定在哪个病毒复制受到影响响应小分子抑制阶段。 

视频: Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

科研

免疫与感染

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

有效生成荧光标记疫苗病毒蛋白的方法

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

小鼠胎肝文化系统解剖靶基因功能的终端红细胞生成的早期和晚期

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

功能金报告系统测量特定分枝杆菌误调速率

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...