危险品得到了德克萨斯大学西南医学中心资助的学者计划提供的资金支持。作者感谢迈克尔 Whitt (田纳西州大学健康科学中心) 和肖恩. 惠兰 (哈佛医学院) 提供 VSV-DsRed 和 VSV-卢克。作者还感谢加里 Luker (密歇根大学医学院) 为 VACV--FL-GFP 菌株的亲切礼物。

1. 一般毒蛾(LD652) 细胞和病毒培养
<ol><li>
LD652 细胞培养与电镀
	<ol>
<li>培养蛾LD652 细胞, 维持生长培养基 (材料表) 中的细胞单层, 在正常大气下孵化27摄氏度。保持细胞在10厘米组织培养处理的菜肴和通过细胞后, 达到80% 融合。</li>
		<li>对板, 去除黏附的 LD652 细胞从板材通过吹打媒介反复地到单层 (这些细胞不要求 trypsinization 去除), 并且稀释样品在生长媒介到大约密度 1 x 105细胞/毫升。吸管1毫升稀释培养/井的24井板。种子至少三复制井/治疗类型为每个时间点 (最大八治疗/板材)。</li>
		<li>允许单元格至少满足1小时。</li>
	</ol>
</li>
	<li>
痘苗病毒制剂
	<ol>
<li>为了准备 VACV 的储存, 在 HeLa 细胞中放大病毒20或非洲绿猴肾细胞 (BSC-1 或 BSC-40 细胞)20,21,22。 注: VACV 应变西部储备 (VACV) 在这里描述的化验工作很好。</li>
		<li>滴定 VACV 菌株通过斑块检测 BSC-1 或 BSC-40 细胞20,22。 注: 所有显示的 VACV 多重感染 (语言) 这里描述的 LD652 细胞感染是基于从 BSC-40 斑块化验得出的效价。</li>
	</ol>
</li>
	<li>
水泡口炎病毒制剂
	<ol>
<li>为了准备 VSV 种群, 在 BHK 细胞中放大病毒23。</li>
		<li>滴定 VSV BSC-40 或 BHK 细胞膜上的斑块测定13,23。 注意: 这里描述的所有 VSV mois 》杂志 LD652 细胞感染是基于从 BSC-40 斑块化验得出的滴度。</li>
	</ol>
</li>
</ol>2. 基于荧光的 VSV 的联合感染和活细胞成像抢救方法
<ol><li>
LD652 细胞的播种与感染
	<ol>
<li>板 4万 LD652 细胞/井8井室。</li>
		<li>对于基于荧光的活细胞成像, 使用 VSV 菌株编码荧光蛋白。在这里提出的实验使用 VSV-DsRed17, 一种重组 VSV 菌株, 表达自由 DsRed 蛋白不融合到任何其他 VSV 蛋白。 注: VSV 感染 LD652 细胞不病变 96 hpi, 因此, 在评估 VSV 复制在这个时间框架内, 细胞死亡不是一个混杂的因素。</li>
		<li>在无血清的培养基中制备 VSV DsRed 和 VACV-GFP-绿色荧光蛋白 (可以持续森林;材料表)分别在1和25的教学语言中。 注: VACV-gfp 是一种重组 VACV 菌株, 表达了卢克和 GFP24之间的融合。使用25或更大的 VACV 菌株的语言确保所有 LD652 细胞感染13。如果使用不同的 coinfecting 病毒, 该语言将必须由用户的经验判断。每种感染治疗应建立在重复的水井, 包括模拟感染的治疗, 其中只有可持续森林可持续的添加到细胞。</li>
		<li>在27摄氏度的0.2 毫升接种中孵化细胞2小时。</li>
		<li>用0.5 毫升/良好的生长培养基冲洗细胞。</li>
		<li>在生长培养基中孵化25微米细胞活性染料 (材料表) 中的细胞, 45 分钟27摄氏度。</li>
		<li>吸入介质, 用0.5 毫升/井冲洗井 1x, 去除过量染料。</li>
		<li>保持细胞在0.3 毫升/井的生长培养基。</li>
	</ol>
</li>
	<li>
活细胞图像捕获
	<ol>
<li>提前30分钟打开共聚焦显微镜, 并装上8井腔盘。 注: LD652 细胞可以在室温下成像, 从20–25°c, 但为最佳条件, 房间的温度应调整为27摄氏度。</li>
		<li>设置适当的激发/发射设置, 每个荧光标记蛋白的成像, 以及相对比图像设置。</li>
		<li>调整每个通道的激光强度。 注: 这可能需要运行一个试点实验, 以确定在整个时间过程中观察到的荧光信号强度范围, 将在最后的实验中使用。</li>
		<li>使用10X 目标, 捕获每个井的相对比度和荧光图像每 1–5 h 为传染的期间到一个期望的时间点 (e. g, 48–72 hpi)。 注意: 在每个井中捕获多个视野是很重要的, 以便准确估计整个文化中的感染率。</li>
	</ol>
</li>
	<li>
图像分析
	<ol>
<li>使用图像分析软件对适当的荧光通道进行自动图像分析 (e., 405 毫微米, 488 nm, 568 nm)。</li>
		<li>要计算受感染细胞的百分比, 请将表示 VSV 感染 (e., DsRed 阳性细胞为 VSV DsRed 感染) 的荧光细胞总数除以细胞活性染料标记的存活细胞总数所有治疗的视野。</li>
	</ol>
</li>
</ol>3. LD652 细胞中基于发光的 VSV 抢救方法的一般病毒感染协议
<ol><li>
接种的制备
	<ol>
<li>30分钟前的感染, 解冻的股票 VSV-卢克16 (重组 VSV 菌株, 表达自由萤火虫荧光素酶蛋白不融合到任何其他 VSV 蛋白)。如果在 VACV 复合感染中对 VSV-卢克的抢救进行化验, 也会使 VACV-西铁病毒在冰上解冻。 注: 其他病毒 (除 VACV) 可能在复合感染期间抢救 VSV-卢克, 但这将必须由每个用户经验主义地确定。</li>
		<li>在 VACV 的存在或不存在的情况下, 通过稀释 VSV-卢克来制备接种, 以便在增加总接种体积0.2 毫升/井时, 分别达到10和25的教学效果。</li>
	</ol>
</li>
	<li>
细胞感染
	<ol>
<li>仔细吸入成熟的 LD652 培养基, 以避免干扰单层和接种0.2 毫升病毒/好。这一次被定义为 0 hpi。增加无菌可持续森林管理, 以作为 "模拟感染" 的阴性对照治疗。</li>
		<li>在接种中孵化细胞2小时, 在27摄氏度。</li>
		<li>在 2 hpi, 取出接种的吸入, 并替换为1毫升/良好 LD652 生长介质。</li>
		<li>允许感染继续 24–72 hpi。</li>
	</ol>
</li>
</ol>4. 荧光素酶测定法
<ol><li>
细胞裂解物的制备
	<ol>
<li>小心地吸入上清, 并添加1毫升的 Dulbecco 磷酸盐缓冲盐水 (DPBS)/好。</li>
		<li>使用1毫升注射器的柱塞, 将细胞刮成 DPBS。</li>
		<li>将细胞转移到离心管, 离心机在 400 x g 20 分钟4摄氏度。</li>
		<li>同时, 在2O 无菌 RLB 中, 制备5x 报告裂解缓冲液的1x 稀释剂。</li>
		<li>离心后, 在不干扰细胞颗粒的情况下吸入 DPBS。</li>
		<li>并用重悬每个颗粒在150µL 1x RLB。</li>
		<li>冷冻-解冻样品1x 使用5分钟孵化在-80 °c 冰箱后, 一个快速解冻的室温水浴。将裂解物存储在-80 摄氏度, 直到准备分析。</li>
	</ol>
</li>
	<li>
荧光素酶测定
	<ol>
<li>在冰上解冻裂解物。</li>
		<li>吸管 20 ul 的裂解成一个坚实的黑色或白色的96井微板块。</li>
		<li>添加 100 ul 的荧光素酶检测试剂每一个井。</li>
		<li>立即测量光强度使用紫外线 (适当的设置为特定 luminometers 将必须由用户经验主义地确定)。</li>
	</ol>
</li>
	<li>
荧光素酶检测分析
	<ol>
<li>对 lu 信号进行规范化处理, 从控制处理获得的 lu 读数 (代表性结果)。在至少进行了三项独立实验之后, 通过适当的统计检验, 可以分析每个治疗/对照组获得的平均 LU。</li>
	</ol>
</li>
</ol>5. 应用免疫印迹
<ol><li>使用适当的试剂和仪器, 用裂解物提取用于 SDS 页和后续免疫印迹法的卢克化验。</li>
</ol>6. LD652 细胞培养 VSV 的效价
<ol><li>根据步骤 1.1, 板 LD652 细胞。</li>
	<li>病毒根据步骤3感染细胞。</li>
	<li>在所需的时间点, 收集上清液到无菌离心管。</li>
	<li>颗粒细胞使用 1000 x g 10 分钟在4摄氏度和转移上清液到新的管。</li>
	<li>贮存上清液在-80 摄氏度或进行滴定通过斑块检测 BSC-40 细胞。 注: 如果滴定 VSV 来自含有 VACV 的 LD652 细胞培养物, 建议在2苷内将100微克/毫升胞嘧啶 AraC (BSC-40) 添加到 hpi 培养基中, 以防止残留 VACV 颗粒在 BSC-40 膜13上形成斑块。AraC 是一种病毒 dna 聚合酶抑制剂, 阻止 VACV DNA 复制25 , 但不影响 VSV 复制。</li>
</ol>7. LD652 细胞中基于发光的 VSV 抢救方法的变异: rna 干扰和质粒转染实验
<ol><li>
病毒或寄主转录 rna 干扰介导的 dsRNA 的制备
	<ol>
<li>设计基因特异引物尾在 5 ' 末端与 T7 启动子序列 (TAATACGACTCACTATAGGG) 瞄准 coinfecting 病毒 (例如, VACV) 或L. 蛾mRNA 的兴趣。这些引物应产生 400–600 bp 的 PCR 产物, 用于有效的 rna 介导的击倒。见金门等。13用于在 LD652 细胞中通过 dsRNA 介导的干扰来击倒 VACV 或L. 蛾记录的引物序列。 注: l. 蛾mRNA 记录可以从出版的 l. 蛾转录数据库26、27、28中确定。</li>
		<li>通过rt-pcr 生成 DNA 模板 (cDNA 合成: 1 周期50°c 30 分钟;PCR:40 周期94°c 为十五年代, 50 °c 三十年代和68°c 为四十五年代每个)。</li>
		<li>使用 pcr 纯化试剂盒净化 pcr 产品。</li>
		<li>以纯化 PCR 产品为模板, 用体外转录纯化试剂盒对 dsRNA进行转录纯化。</li>
	</ol>
</li>
	<li>
dsRNA 或质粒 DNA 转染 LD652 细胞的研究
	<ol>
<li>种子 1 x 105细胞/井24井板并且让细胞安定至少 1 h。</li>
		<li>为每个井被转染, 稀释 4 ul 再染试剂入 100 ul 的可持续森林。在室温下孵育高达15分钟。可以对此进行缩放, 以便为所有 transfections 执行主组合。</li>
		<li>稀释多达1微克的 dsRNA 或总质粒 DNA 与 100 ul 的可持续森林研究为每个井被转染。在室温下孵育高达15分钟。 注: 我们以前发现, 转染1微克的 dsRNA/105 LD652 细胞足以用于 > 80% 击倒的病毒或主机成绩单13, 但 dsRNA 剂量需要修改实验设置/条件将不得不有经验的确定。</li>
		<li>结合转染试剂和 dsRNA/质粒 DNA 稀释与1:1 比值 (例如, 100 ul 稀释 dsRNA 与 100 ul 稀释转染试剂) 和孵化20分钟室温。</li>
		<li>同时, 用1毫升/井的可持续森林洗涤水井, 然后在每个井中添加 500 ul 的可持续森林。</li>
		<li>慢慢添加转染试剂 dsRNA (或质粒 DNA) 混合物滴入每个井。</li>
		<li>用细胞孵化5小时的转染试剂。<ol>
<li>对于涉及 coinfecting 病毒编码的转录记录的干扰实验 (如VACV), 在5小时转染潜伏期后, 用含有 VSV-卢克的病毒接种取代转染试剂 (有或无 VACV 或另一种 coinfecting 病毒) (步骤 3)。随后, 将包含 VSV 的病毒接种替换为完全生长介质 2 hpi。卢克化验 (步骤 4) 然后可以执行在提取裂解物在不同的时间点, 以确定是否 coinfecting 病毒记录的击倒导致 VSV-卢克救援的损失。</li>
			<li>对于蛾转录的 rna 干扰实验, 用完整的生长介质替换转染组合, 并允许在 VSV (步骤 4) 感染前24小时进行击倒。卢克化验 (步骤 4) 然后可以执行在提取裂解物在不同的时间点, 以确定如果蛾记录的被击倒促进 VSV-卢克抢救。</li>
			<li>对于 transfections 的质粒表达载体的免疫调节剂, 用 VSV-卢克 (步骤 4) 取代转染试剂, 允许在感染前24小时进行蛋白表达。卢克化验 (步骤 4) 然后可以执行在提取裂解物在不同的时间点, 以确定是否候选免疫调节蛋白的表达促进 VSV-卢克抢救。 注: 在这里描述的转染条件使用质粒 DNA 的1微克/井, 导致 40–60%13的转染效率。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

<ol>
	<li>Nomaguchi, M., Fujita, M., Miyazaki, Y., Adachi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+tropism.">Viral tropism.</a> Frontiers in Microbiology. 3, 281 (2012).</li><li>Werden, S. J., 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+role+of+cell+signaling+in+poxvirus+tropism:+the+case+of+the+M-T5+host+range+protein+of+myxoma+virus.">The role of cell signaling in poxvirus tropism: the case of the M-T5 host range protein of myxoma virus.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1784 (1), 228-237 (2008).</li><li>McFadden, G., Mohamed, M. R., Rahman, M. M., Bartee, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+determinants+of+viral+tropism.">Cytokine determinants of viral tropism.</a> Nature Reviews: Immunology. 9 (9), 645-655 (2009).</li><li>Silva, J. 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=Second-generation+shRNA+libraries+covering+the+mouse+and+human+genomes.">Second-generation shRNA libraries covering the mouse and human genomes.</a> Nature Genetics. 37 (11), 1281-1288 (2005).</li><li>Moffat, 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=A+lentiviral+RNAi+library+for+human+and+mouse+genes+applied+to+an+arrayed+viral+high-content+screen.">A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.</a> Cell. 124 (6), 1283-1298 (2006).</li><li>Ma, D., Liu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Editing+and+Its+Applications+in+Model+Organisms.">Genome Editing and Its Applications in Model Organisms.</a> Genomics Proteomics Bioinformatics. 13 (6), 336-344 (2015).</li><li>Yin, H., Kauffman, K. J., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+technologies+for+genome+editing.">Delivery technologies for genome editing.</a> Nature Reviews: Drug Discovery. 16 (6), 387-399 (2017).</li><li>Hao, 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=Drosophila+RNAi+screen+identifies+host+genes+important+for+influenza+virus+replication.">Drosophila RNAi screen identifies host genes important for influenza virus replication.</a> Nature. 454 (7206), 890-893 (2008).</li><li>Houzet, L., Jeang, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+screening+using+RNA+interference+to+study+host+factors+in+viral+replication+and+pathogenesis.">Genome-wide screening using RNA interference to study host factors in viral replication and pathogenesis.</a> Experimental Biology and Medicine. 236 (8), 962-967 (2011).</li><li>Marceau, C. 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=Genetic+dissection+of+Flaviviridae+host+factors+through+genome-scale+CRISPR+screens.">Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.</a> Nature. 535 (7610), 159-163 (2016).</li><li>Savidis, G., 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+Zika+Virus+and+Dengue+Virus+Dependency+Factors+using+Functional+Genomics.">Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics.</a> Cell Reports. 16 (1), 232-246 (2016).</li><li>Panda, D., Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+genomic+screening:+elucidating+virus-host+interactions.">Cell-based genomic screening: elucidating virus-host interactions.</a> Current Opinion in Virology. 2 (6), 784-792 (2012).</li><li>Gammon, D. 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=A+single+vertebrate+DNA+virus+protein+disarms+invertebrate+immunity+to+RNA+virus+infection.">A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection.</a> Elife. 3, (2014).</li><li>Letchworth, G. J., Rodriguez, L. L., Del cbarrera, ., J, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis.">Vesicular stomatitis.</a> Veterinary Journal. 157 (3), 239-260 (1999).</li><li>Whelan, S. P., Ball, L. A., Barr, J. N., Wertz, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+recovery+of+infectious+vesicular+stomatitis+virus+entirely+from+cDNA+clones.">Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (18), 8388-8392 (1995).</li><li>Cureton, D. K., Massol, R. H., Saffarian, S., Kirchhausen, T. L., Whelan, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+enters+cells+through+vesicles+incompletely+coated+with+clathrin+that+depend+upon+actin+for+internalization.">Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization.</a> PLoS Pathogens. 5 (4), e1000394 (2009).</li><li>Duntsch, C. 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=Recombinant+vesicular+stomatitis+virus+vectors+as+oncolytic+agents+in+the+treatment+of+high-grade+gliomas+in+an+organotypic+brain+tissue+slice-glioma+coculture+model.">Recombinant vesicular stomatitis virus vectors as oncolytic agents in the treatment of high-grade gliomas in an organotypic brain tissue slice-glioma coculture model.</a> Journal of Neurosurgery. 100 (6), 1049-1059 (2004).</li><li>Li, Y., Yuan, S., Moyer, 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=The+non-permissive+infection+of+insect+(gypsy+moth)+LD-652+cells+by+Vaccinia+virus.">The non-permissive infection of insect (gypsy moth) LD-652 cells by Vaccinia virus.</a> Virology. 248 (1), 74-82 (1998).</li><li>Seet, B. 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=Poxviruses+and+immune+evasion.">Poxviruses and immune evasion.</a> Annual Review of Immunology. 21, 377-423 (2003).</li><li>Cotter, C. A., Earl, P. L., Wyatt, L. S., 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=Preparation+of+Cell+Cultures+and+Vaccinia+Virus+Stocks.">Preparation of Cell Cultures and Vaccinia Virus Stocks.</a> Current Protocols in Microbiology. 39, 11-18 (2015).</li><li>Andrei, G., 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=Cidofovir+resistance+in+vaccinia+virus+is+linked+to+diminished+virulence+in+mice.">Cidofovir resistance in vaccinia virus is linked to diminished virulence in mice.</a> Journal of Virology. 80 (19), 9391-9401 (2006).</li><li>Dascher, C., VanSlyke, J. K., Thomas, L., Balch, W. E., Thomas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+recombinant+vaccinia+virus+for+expression+of+small+GTPases.">Preparation of recombinant vaccinia virus for expression of small GTPases.</a> Methods in Enzymology. , 174-188 (1995).</li><li>Martin, A., Rex, E. A., Ishidate, T., Lin, R., Gammon, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+Caenorhabditis+elegans+with+Vesicular+Stomatitis+Virus+via+Microinjection.">Infection of Caenorhabditis elegans with Vesicular Stomatitis Virus via Microinjection.</a> Bio-Protocol. 7 (22), (2017).</li><li>Luker, K. E., Hutchens, M., Schultz, T., Pekosz, A., Luker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+vaccinia+virus:+effects+of+interferon+on+viral+replication+and+spread.">Bioluminescence imaging of vaccinia virus: effects of interferon on viral replication and spread.</a> Virology. 341 (2), 284-300 (2005).</li><li>Rozelle, D. K., Filone, C. M., Dower, K., 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=Vaccinia+reporter+viruses+for+quantifying+viral+function+at+all+stages+of+gene+expression.">Vaccinia reporter viruses for quantifying viral function at all stages of gene expression.</a> Journal of Visualizes Experiments. (87), e51522 (2014).</li><li>Cao, 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=Characterization+of+the+transcriptome+of+the+Asian+gypsy+moth+Lymantria+dispar+identifies+numerous+transcripts+associated+with+insecticide+resistance.">Characterization of the transcriptome of the Asian gypsy moth Lymantria dispar identifies numerous transcripts associated with insecticide resistance.</a> Pesticide Biochemistry and Physiology. 119, 54-61 (2015).</li><li>Sparks, M. E., Blackburn, M. B., Kuhar, D., Gundersen-Rindal, 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=Transcriptome+of+the+Lymantria+dispar+(gypsy+moth)+larval+midgut+in+response+to+infection+by+Bacillus+thuringiensis.">Transcriptome of the Lymantria dispar (gypsy moth) larval midgut in response to infection by Bacillus thuringiensis.</a> PloS One. 8 (5), e61190 (2013).</li><li>Sparks, M. E., Gundersen-Rindal, 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=The+Lymantria+dispar+IPLB-Ld652Y+cell+line+transcriptome+comprises+diverse+virus-associated+transcripts.">The Lymantria dispar IPLB-Ld652Y cell line transcriptome comprises diverse virus-associated transcripts.</a> Viruses. 3 (11), 2339-2350 (2011).</li><li>Lin, G., Li, G., Granados, R. R., Blissard, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+cell+lines+expressing+baculovirus+P35:+resistance+to+apoptosis+and+nutrient+stress,+and+increased+glycoprotein+secretion.">Stable cell lines expressing baculovirus P35: resistance to apoptosis and nutrient stress, and increased glycoprotein secretion.</a> In Vitro Cellular &amp; Developmental Biology - Animal. 37 (5), 293-302 (2001).</li><li>Li, W. X., 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=Interferon+antagonist+proteins+of+influenza+and+vaccinia+viruses+are+suppressors+of+RNA+silencing.">Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (5), 1350-1355 (2004).</li><li>Kanost, M. 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=Multifaceted+biological+insights+from+a+draft+genome+sequence+of+the+tobacco+hornworm+moth,+Manduca+sexta.">Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta.</a> Insect Biochemistry and Molecular Biology. 76, 118-147 (2016).</li><li>Paixao, E. S., Teixeira, M. G., Rodrigues, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika,+chikungunya+and+dengue:+the+causes+and+threats+of+new+and+re-emerging+arboviral+diseases.">Zika, chikungunya and dengue: the causes and threats of new and re-emerging arboviral diseases.</a> BMJ Global Health. 3, (2018).</li></ol>

作者没有什么可透露的。

在这里, 我们描述了简单的荧光和发光的检测, 以筛选条件, 拯救 VSV 复制在限制性的鳞翅目细胞培养。VSV 在鳞翅目细胞中的流产感染在 VSV 基因表达的检测中产生了良好的信噪比。例如, 裂解物从单 VSV 感染中检测到的 LU 信号比模拟感染的裂解物高1000倍, 然而这些信号在72小时的时间内只改变了大约两个。相比之下, VSV 的复合感染与 VACV 增强的 LU 信号相比, 300 倍于单 VSV-卢克感染 72 hpi。因此, VSV 救...

病毒在特定宿主中进行高效复制的能力, 部分取决于支持病毒输入和复制1的宿主细胞因子的可用性。病毒宿主范围也可以由病毒抵御细胞抗病毒防御的能力决定, 否则会阻碍病毒复制2,3。这是这些复杂的病毒宿主交互的结果, 最终决定病毒是否能够在特定主机上完成其生命周期。鉴于病毒复制的出现可能会对宿主造成致病性后果, 制定实验策略以进一步了解可能导致流产和生产效率平衡的关键病毒宿主相互作用至关重要。感染。阐明病毒宿主相互作用的分子特征将有助于制定新的和替代的抗病毒治疗策略。
随着 RNA 干扰 (核糖核酸)4、5和基因组编辑工具 (CRISPR-Cas9、锌指核酸、TALENs)6、7的出现, 改变细胞因子在全基因组范围内的表达, 并探讨这些改变对病毒复制的影响。事实上, 在无脊椎动物和脊椎动物宿主细胞类型中进行了大量的基于 rna 和基因组编辑的屏幕, 揭示了病毒宿主相互作用的新方面8、9、10,11,12. 这些屏幕通常使用病毒编码的记者, 如萤火虫荧光素酶 (卢克) 或荧光蛋白 (e., GFP, DsRed), 提供了方便的手段定量评估病毒基因表达作为一个读数病毒复制9,12。这一策略允许研究人员确定宿主因素, 它们要么促进或拮抗病毒复制, 在病毒报告信号9,12中分别增加或减少。然而, 在绝大多数情况下, 这些屏幕使用的病毒是很好地适应了他们正在研究的宿主细胞类型。虽然这一策略对于理解病毒病原体及其自然宿主之间的进化关系是很重要的, 但对于它们在揭示宿主抗病毒因子方面的应用却构成了根本的担忧。在这些情况下, 正在寻找病毒报告器上的信号增强, 或者是一种通常会阻碍病毒复制的细胞因子的失活。首先, 如果病毒已经能够在控制条件下检查的宿主细胞中进行强力复制, 则屏幕的动态范围 (即区分背景和增强的病毒报告信号的能力) 可能会受到限制。第二, 这个问题进一步复杂化的情况下, 病毒是良好的适应宿主细胞, 并有效地对抗主机防御路径, 是在屏幕上的目标。
由于上述对传统病毒宿主交互筛选方法的关注, 我们开发了一种新的模式来研究病毒宿主相互作用, 利用鳞翅目昆虫细胞中自然流产的虫媒病毒感染。这一策略来源于一项观察, 即研究良好的人类虫媒病毒, VSV, 在来自吉普赛蛾 (L. 蛾)13的细胞中经历了一次流产感染。VSV 是自然传播的 dipteran 昆虫 (即, 沙蝇) 的哺乳动物宿主, 并已证明实验性感染广泛的无脊椎动物和脊椎动物宿主在细胞培养和体内14。VSV 的 11 kb 负感单链 RNA 基因组编码五 subgenomic 基因, 它们分别转化为构成包络病毒的蛋白质。然而, VSV 反向遗传系统已经允许创建复制能力的菌株编码卢克或荧光蛋白, 除了五自然 VSV 基因产品15,16,17。由于这些记者的蛋白质没有纳入 VSV 病毒, 他们提供了一个方便的读数 VSV 基因表达, 发生后进入。我们以前曾表明, 使用 VSV 菌株编码 GFP 或卢克, VSV 基因表达严重限制在 LD652 细胞的进入, VSV 的滴度不会增加72小时后感染 (hpi)。相比之下, LD652 细胞与 VSV 和哺乳动物痘, 痘苗病毒 (VACV) 的复合感染, 导致在 VSV 基因表达和效价的这一时间点的对数增加。VACV 在 LD652 细胞感染中经历早期基因表达、DNA 复制和晚期基因表达, 但 VACV 复制周期最终由于不完全病毒形态发生18而流产。VACV 编码 > 200 蛋白的大 ~ 192 kb DNA 基因组, 其中许多显示免疫调节特性, 通过抑制宿主免疫应答19来促进病毒复制。因此, 我们假设 VACV 复合感染在 LD652 细胞中 VSV 复制的 "营救" 可能是由 VACV 免疫调节剂引起的, 抑制L. 蛾响应通常限制 VSV 复制。为支持这一点, LD652 细胞与宿主 RNA 聚合酶 II 抑制剂放线菌 D 的治疗也拯救 VSV 在 LD652 细胞复制, 表明转录依赖主机响应阻止 VSV 复制后进入13。
上述观察表明, LD652 细胞对 VSV 感染的自然限制性, 在筛选增强 VSV 编码的报告信号的条件时可能提供相对较低的背景 (即那些抑制宿主抗病毒防御)。在这里, 我们提供的方法, 以荧光或卢克为基础的化验, 以筛选条件, 以缓解 VSV 限制鳞翅目细胞。首先, 我们展示了如何使用这些检测方法来识别维拉利编码的免疫调节因子, 在复合感染实验中或通过候选病毒因子的异位表达打破 VSV 限制。举例来说, 我们说明了我们如何使用这些筛选技术来识别痘编码的 A51R 蛋白作为一种新的免疫调节因子家族, 在没有其他痘因素13的情况下拯救 VSV 复制。其次, 我们说明了如何在限制性 VSV-LD652 细胞感染中进行 rna 干扰筛选, 以直接识别虫媒病毒限制13中所涉及的真核宿主因素。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>CELLTREAT</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>CELLTREAT</td>
			<td>229124</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm tissue culture dishes</td>
			<td>Corning</td>
			<td>C430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#8217;s Insect Medium</td>
			<td>Sigma</td>
			<td>G8142</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 420</td>
			<td>Sigma</td>
			<td>14420C</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Optima</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth medium</td>
			<td></td>
			<td></td>
			<td>1:1 mixture of Grace&#39;s Insect Medium and EX-Cell 420 Serum-Free Medium also containing 1 % antibiotic-antimycotic solution and 10 % Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution (100&#215;)</td>
			<td>Sigma</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td>Sigma</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum Free Media (SFM)</td>
			<td>Thermo Fisher</td>
			<td>10902096</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine arabinoside</td>
			<td>Sigma</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagent</td>
			<td>Thermo Fisher</td>
			<td>10362100</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning cellgro DMSO (Dimethyl Sulfoxide)</td>
			<td>Corning</td>
			<td>25950CQC</td>
			<td></td>
		</tr>
		<tr>
			<td>Reporter lysis buffer 5X</td>
			<td>Promega</td>
			<td>E3971</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase Assay Reagent</td>
			<td>Promega</td>
			<td>E1483</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Microplates</td>
			<td>Corning</td>
			<td>3915</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-FLAG antibody</td>
			<td>Wako</td>
			<td>014-22383</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-firefly luciferase antibody</td>
			<td>Abcam</td>
			<td>ab21176</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-actin antibody</td>
			<td>Sigma</td>
			<td>A2066</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-VSV M</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Dr. John Connor (Boston University)</td>
		</tr>
		<tr>
			<td>Mouse anti-VACV I3L</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Dr. David Evans (University of Alberta)</td>
		</tr>
		<tr>
			<td>8-well Chambered dish</td>
			<td>Lab-Tek II</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell viability dye</td>
			<td>Thermo Fisher</td>
			<td>C12881</td>
			<td></td>
		</tr>
		<tr>
			<td>FLUOstar microplate reader</td>
			<td>BMG Labtech</td>
			<td>FLUOstar</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i-LIV</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Olympus</td>
			<td>v1.18</td>
			<td>cellSens software</td>
		</tr>
		<tr>
			<td>Eppendorf 5702 ventilated centrifuge</td>
			<td>Eppendorf</td>
			<td>22628102</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Fc Infrared Imaging System</td>
			<td>Li-COR Biosciences</td>
			<td>Odyssey Fc</td>
			<td></td>
		</tr>
		<tr>
			<td>LD652 cells</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Dr. Basil Arif (Natural Resources Canada)</td>
		</tr>
		<tr>
			<td>BSC-40 cells</td>
			<td>ATCC</td>
			<td>CRL-2761</td>
			<td></td>
		</tr>
		<tr>
			<td>BHK cells</td>
			<td>ATCC</td>
			<td>CCL-10</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>BSC-1 cells</td>
			<td>ATCC</td>
			<td>CCL-26</td>
			<td></td>
		</tr>
		<tr>
			<td>in vitro transcription and purification kit</td>
			<td>Thermo Fisher</td>
			<td>AM1626</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
	</tbody>
</table>

arbovirus infections as screening tools for the identification of viral immunomodulators and host antiviral factors

RNA 干扰-和基因组编辑为基础的筛选平台已广泛用于识别宿主细胞因子, 限制病毒复制。然而, 这些屏幕通常进行的细胞, 自然纵容的病毒病原体正在研究中。因此, 在控制条件下的病毒的健壮复制可能会限制这些屏幕的动态范围。此外, 如果病毒适应宿主并能够抵抗抗病毒防御, 这些屏幕可能无法轻易识别出限制病毒复制的细胞防御通路。在本文中, 我们描述了一个新的范式, 以探索病毒宿主相互作用, 通过使用的屏幕, 中心的自然流产感染的虫媒病毒, 如水泡性口炎病毒 (VSV)。尽管 VSV 能够在广泛的 dipteran 昆虫和哺乳动物寄主中进行复制, VSV 还是经历了进入后, 在鳞翅目昆虫 (毒蛾) 的各种细胞系中流产后的感染。然而, 当宿主细胞抗病毒防御被破坏时, 这些流产的 VSV 感染可以被 "拯救"。我们描述如何 VSV 菌株编码方便的报告基因和限制性的L. 蛾细胞系可以配对设置屏幕, 以确定宿主因素参与虫媒病毒限制。此外, 我们还显示了这些筛选工具的效用, 以确定维拉利编码因素, 拯救 VSV 复制期间复合感染或通过异位表达, 包括那些编码的哺乳动物病毒。VSV 复制在L. 蛾细胞中的自然限制在筛选促进 VSV 救援的条件时提供了高信噪比, 从而使使用简单的荧光和荧光检测方法能够监测VSV 复制的更改。这些方法对于了解宿主抗病毒反应与病毒免疫规避因素之间的相互作用是有价值的。

作为活体细胞成像应用监测 VSV 救援 VACV 复合感染的例子, LD652 细胞被镀在一个8井腔内的盘子里, 然后在 VACV--GFP (语言 = 25) 的存在或不存在的情况下, 在 VSV-DsRed (语言 = 1) 中被模拟感染或感染。因为 VSV-DsRed 表达 DsRed 作为一个自由的蛋白质, 并没有融合到结构 VSV 蛋白 (图 1A), 它只是检测后, VSV 进入和基因表达启动。所有细胞都被标记为细胞活性染料, ?...

Watch this Scientific Journal Video about Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors at JoVE.com

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

在这里, 我们提出了识别 1) 病毒编码的免疫调节剂的协议, 以促进虫媒病毒复制和 2) 限制虫媒病毒复制的真核宿主因素。这些荧光和发光的方法, 使研究人员能够迅速获得定量读数的虫媒病毒复制在简单的检测, 低信噪比。

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

arbovirus-infections-as-screening-tools-for-identification-viral

Research

JoVE Journal

Immunology and Infection

6.8K 次观看.  University of Texas Southwestern Medical Center. 在这里, 我们提出了识别 1) 病毒编码的免疫调节剂的协议, 以促进虫媒病毒复制和 2) 限制虫媒病毒复制的真核宿主因素。这些荧光和发光的方法, 使研究人员能够迅速获得定量读数的虫媒病毒复制在简单的检测, 低信噪比。

视频: Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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 ...

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. ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

摘要

探索更多视频

此视频中的章节

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

在细菌粘附的研究剪应力

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

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定

生成和多表型高内涵筛选

早期的病毒进入测定的鉴定和抗病毒化合物的评价

冻干环介导等温扩增试剂的检测中的发展

人胎盘及蜕膜器官培养研究在母胎界面感染

在参与宿主因素的高通量筛选基于显微镜的检测方法