这项研究得到了 nih/NIAID AI112423 和 nih/研究院 GM113885 给本杰明. 陈和 nih/NIAID K08-AI120806 的资助。我们想感谢在西奈山院长的流式细胞术核心的伊坎医学院。

1. 目标细胞线的生成
注意: 此步骤是可选的;如果使用现有的 RG 目标单元格行, 请从步骤2开始。
<ol><li>Cotransfect 70% 汇合 10 cm 板293T 细胞11 [在10毫升 Dulbecco 的被改进的老鹰的媒介 (DMEM) 与10% 胎牛血清 (血清)] 与 pMSCV-loxp-dsRed-loxp-eGFP-普洛 WPRE10和 pCL-10A112 (包装质粒) 在一个1:1 比例 (20 µg 总) 使用钙基转染13。</li>
	<li>收获10毫升的病毒上清 48 h 后转染通过吹打介质从板块成15毫升圆锥管和离心管在 3000 x g 5 摄氏度, 以颗粒任何细胞碎片。</li>
	<li>将0.45 µm 过滤器连接到10毫升注射器上。离心后, 取整个上清液并装入注射器。把样品通过进入一个干净的15毫升管。</li>
	<li>整除过滤的病毒上清到适当的大小 (通常, 0.5–1毫升整除数)。这些可以立即在下一步使用, 或可以冻结在-80 摄氏度和存储。</li>
	<li>使用50–100µL 的病毒上清液感染细胞线的选择, 在一个96井板。文化细胞在37°c 与 5% CO2。RFP 信号应在24小时内出现在荧光显微镜下 (40X 放大, 532 nm 激发, 588 nm 发射), 但可能需要多达 72 h。 注意: 在这些实验中, Jurkat 细胞被使用, 但其他类型也可以使用。</li>
	<li>通过设置一系列10口井并将嘌呤霉素添加到每个嘌呤霉素, 选择转基因细胞, 将至少1的未处理好的控制 (使用从0.5 µg/毫升到5µg/毫升的浓度范围); 这可能会因每个单元格类型而异)。监测细胞的生存能力 (细胞裂解将发生在没有嘌呤霉素抵抗的细胞中) 在数天的过程中与未经治疗的控制相比, 并使用嘌呤霉素集中, 只有 RFP 表达细胞生存。 注: 选择在转染细胞存活时 untransfected 细胞被杀死的浓度。</li>
	<li>使用单细胞流式细胞仪排序或限制稀释 (见步骤 1.8–1.10), 培养从单细胞1发展成克隆细胞线 (这可能需要几个星期的成长)。</li>
	<li>如果使用稀释方法, 用 hemocytometer 计数细胞, 并将样品稀释到大约500细胞/毫升。</li>
	<li>在一个96井板, 吸管50µL 细胞稀释成50µL 媒体 [罗斯威尔公园纪念研究所 (RPMI) 培养基与10% 血清和2% 青霉素-链霉素, 如果使用 Jurkat 细胞], 并执行1:1 串行稀释到11井, 包含50µL 的媒体。为达到最佳效果, 请至少执行10个复制操作。</li>
	<li>通过显微镜 (40X) 在组织培养孵化器 (37 °c 与 5% CO2) 或大约4周, 监测细胞生长。选择从最低的嘌呤霉素浓度与生长 (如显微镜, 40X) 的克隆文化的文化。</li>
	<li>冷冻整除数由离心20毫升的文化 (50万细胞/毫升, 计数与 hemocytometer) 在 800 x g 5 分钟在23°c 和 resuspending 它在500µL 的血清与10% 亚砜。把它放在-80 摄氏度使用一个细胞冻结容器, 然后储存在液氮。</li>
</ol>2. 细胞对细胞病毒的传播
<ol><li>
靶细胞的制备
	<ol>
<li>解冻一个500µL 瓶 Jurkat RG 记者细胞通过放置到一个37°c 水浴。将细胞从瓶子中注入10毫升的 RPMI 完全介质, 然后将混合物在 800 x g处离心, 在23摄氏度时为5分钟。并用重悬在一个 T-75 瓶中的20毫升 RPMI 完全培养基中的颗粒。隔夜孵化烧瓶 (37 °c 和 5% CO2)。 注: RPMI 完整培养基包含 RPMI 1640, 10% 血清, 2 毫米 l-谷氨酰胺, 100 单位/毫升青霉素, 100 毫克/毫升链霉素。</li>
		<li>第二天, 添加0.5 µg/毫升的嘌呤霉素 (1 µL 2 毫克/毫升的股票每8毫升的媒体)。培养细胞, 保持200,000–800,000 细胞/毫升的密度 (通过hemocytometer 计数)。</li>
		<li>在建立转移化验前的前一天, 将细胞分解为200,000–400,000 细胞/毫升, 在含有0.5 µg/毫升嘌呤霉素的新鲜 RPMI 培养基中。</li>
	</ol>
</li>
	<li>
供体细胞的制备
	<ol>
<li>解冻 untransduced Jurkat 细胞和培养他们在一个 T-75 瓶与20毫升 RPMI 完全培养基 (如步骤2.1 所述), 保持一个密度的200,000–800,000 细胞/毫升。</li>
		<li>离心750万细胞 (800 x g为5分钟) 和并用重悬他们在120µL 的 nucleofection 溶液 V 与补充 (参见材料表)。将细胞转移到电穿孔试管, 加入4.5 µg 地球复兴社区1的 DNA。染细胞 (通过以电穿孔为基础的方法, 看到材料表) 使用适当的程序 (如S-18), 然后立即转移到3毫升的 RPMI 培养基 (10% 血清)。</li>
		<li>允许细胞恢复通过孵化他们隔夜在37°c 与 5% CO2。</li>
		<li>第二天早晨, 离心细胞在 800 x g 5 分钟在23°c 和并用重悬他们在3毫升的 RPMI 完全培养基, 允许他们 2 h 恢复在37°c (5% CO2), 然后继续进行化验。</li>
	</ol>
</li>
	<li>
共同文化
	<ol>
<li>用 hemocytometer 计数细胞, 然后向下旋转5万个细胞 (800 x g 5 分钟, 23 °c), 每井 (包括捐献者和靶细胞) 进行测定。Resuspending 1 x 106细胞/毫升在 RPMI 完成没有嘌呤霉素。</li>
		<li>在一个板块, 增加25µL 的捐献细胞悬浮在每个井;在另一个板块中, 添加25µL 的目标细胞。</li>
		<li>对每个井, 添加25µL 的测试化合物稀释在 RPMI 完全培养基在适当的浓度。用该化合物孵育供体和靶细胞30分钟。 注意: 每一种药物的浓度都会有所不同。如果它有一个已知的 IC50, 使用它作为一个起点, 滴定上面和下面。如果 IC50是未知的, 准备一个200µM 库存的最后浓度为10µM。</li>
		<li>预处理后, 将供体细胞与靶细胞结合, 将一个板块的内容吹打到另一个盘子中, 在37摄氏度的组织培养孵化器中孵化出40小时的细胞, 5% CO2。</li>
	</ol>
</li>
	<li>
流式细胞仪
	<ol>
<li>将多聚甲醛 (粉煤灰) 添加到每个井中, 最终浓度为2%。 注: 对于16% 库存解决方案, 这是14µL 每100µL 井。</li>
		<li>注意: 粉煤灰对暴露的皮肤和眼睛有害。戴上实验室大衣、手套和安全护目镜, 并在安全罩中处理库存解决方案。</li>
		<li>阅读 mCherry 和 FITC 通道的流式细胞仪上的单元格。 注意: 有可能看到在感染细胞中表达 GFP 以上背景的细胞与5–20%。未感染的细胞。</li>
		<li>通过荧光显微镜 (40X) 在 gfp 特定通道上可视化 gfp 信号 (带有 400 nm 的励磁过滤器和 508 nm 的发射过滤器)。</li>
	</ol>
</li>
</ol>3. 无细胞病毒感染的替代议定书
<ol><li>
靶细胞的制备
	<ol>
<li>通过放入37°c 水浴, 解冻一个500µL 瓶 Jurkat RG 记者细胞。将细胞从瓶子中注入10毫升的 RPMI 完全培养基, 然后离心 RPMI 完全培养基在 800 x g 5 分钟在23摄氏度。并用重悬在一个 T-75 瓶中的20毫升 RPMI 完全培养基中的颗粒。隔夜孵化烧瓶 (37 °c 和 5% CO2)。</li>
		<li>第二天, 添加0.5 µg/毫升的嘌呤霉素 (1 µL 2 毫克/毫升的股票每8毫升的媒体)。培养细胞, 保持200,000–800,000 细胞/毫升的密度 (通过hemocytometer 计数)。</li>
		<li>在建立转移化验前的前一天, 将细胞分解为200,000–400,000 细胞/毫升, 在含有0.5 µg/毫升嘌呤霉素的新鲜 RPMI 培养基中。</li>
	</ol>
</li>
	<li>
无细胞病毒的生产
	<ol>
<li>染70% 汇合 10 cm 板293T 细胞11 (在10毫升 DMEM 与10% 血清) 与5µg 地球复兴社区1质粒使用钙基转染13。 注: 这一比例将产生10毫升的病毒, 这是足够的约四96井板, 根据效率的转染。</li>
		<li>通过将介质从盘子中吹打到15毫升圆锥管中, 并离心在 3000 x g处为5分钟, 将所有细胞碎片全部切片, 以48小时收割整个病毒上清液。</li>
		<li>将0.45 µm 过滤器连接到10毫升注射器上。离心后, 取整个上清液并装入注射器。把样品通过进入一个干净的15毫升管。</li>
		<li>使用病毒立即感染目标细胞 (步骤 3.3) 或冻结并将病毒存储在-80 摄氏度 (在使用前解冻样品)。</li>
	</ol>
</li>
	<li>
感染
	<ol>
<li>使用 hemocytometer 计算 RG Jurkat 目标单元格 (从步骤 3.1.3), 并在 800 x g处向下旋转5万个目标单元格5分钟, resuspending 1 x 106细胞/mL 中的细胞, RPMI 完全培养基中没有嘌呤霉素。</li>
		<li>在一个板块, 添加25µL 的 RG Jurkat 目标细胞的每一个井;在一个单独的板块, 添加25µL (2 ng) 病毒每一个井。</li>
		<li>对每一个井, 添加25µL 的测试化合物, 稀释在介质中适当的浓度。用化合物孵育细胞和病毒30分钟。 注: 测试浓度会因药物而异。如果它有一个已知的 IC50, 使用它作为一个起点, 滴定上面和下面。如果未知, 准备一个200µM 的股票培养基, 最终浓度为10µM。</li>
		<li>预处理后, 将病毒/复合物的全部内容 (50 µL) 从水井添加到目标细胞, 并在37摄氏度与 5% CO2的组织培养孵化器中孵化一切40小时。</li>
	</ol>
</li>
	<li>
流式细胞仪
	<ol>
<li>将粉煤灰添加到每个井中, 最终浓度为2%。 注: 对于16% 库存解决方案, 这是14µL 每100µL 井。</li>
		<li>阅读 mCherry 和 FITC 通道的流式细胞仪上的单元格。gfp 信号也可以通过荧光显微镜在 gfp 通道上进行可视化。 注: 一旦可以期望看到5–20% 的细胞表达 GFP 以上背景的感染细胞vs。未感染的细胞。</li>
	</ol>
</li>
</ol>

<ol>
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作者没有什么可透露的。

地球复兴社区的实验证明对筛选可能抑制病毒复制融合步骤的药物候选者非常有用。在执行此检测时, 获得良好信号的最重要步骤类似于大多数病毒感染化验。第一个关键步骤是产生高质量的病毒的高滴度。这一步要求293T 细胞频繁传代 (至少每48小时 1x), 这样它们就不会变成 overconfluent 并聚集在一起。此外, 它可能值得尝试不同的转染方法, 因为一些研究人员发现, 磷酸钙转染提供了更好的结果, 而...

新的抗逆转录病毒疗法的需要, 促使开发高通量的屏幕抑制剂的 HIV-1 进入。地球复兴社区报告的研究是为了识别细胞-细胞感染系统融合步骤中病毒进入的抑制剂, 具体测量与宿主细胞膜1的病毒膜融合。研究了一种新的抑制剂的筛选, 具体表现在 HIV-1 感染的早期阶段, 直至病毒膜融合点。测量细胞感染的一个挑战是最初的接种含有受感染的供体细胞和 ininfected 靶细胞, 因此测量新的感染很难从输入细胞中辨别出来。理想的系统将涉及一个外源基因标记, 可诱导的目标细胞感染的启动, 并没有出现在捐献细胞。一种流行的病毒含量混合检测基于病毒蛋白-酶融合, 砰-Vpr, 可以有效, 但有限制高通量, 细胞筛选2。这种检测包括一个β-酶 (砰) 报告基因, 融合到 HIV-1 Vpr, 包装成病毒, 并交付到靶细胞后, 病毒膜融合。基质 CCF2-AM 被装入靶细胞的细胞质中, 在卵裂时进行荧光转移。CCF2-AM 基板成本高昂, 可用于高吞吐量屏幕。为了区分供体和靶细胞, 有必要对目标种群进行染色标记。最后, 该协议需要几个洗涤和孵化步骤, 这可能是繁重和昂贵的测试大量的化合物。
以地球复兴社区为解决这些问题的方法, 开发了高通量的细胞间融合抑制剂筛选。此系统不需要将基板加载到单元格中。该检测也可用于无细胞研究, 并适用于其他病毒融合研究使用伪类型 HIV-1 地球复兴社区病毒粒子。艾滋病病毒的细胞融合检测可以测量病毒的 HIV-1 蛋白介导的细胞融合, 然而这些不使用实际的病毒粒子作为地球复兴社区的实验3,4,5。这种检测可以用流式细胞术或荧光显微镜阅读。它已成功地用于筛选 FDA 图书馆以及一个小图书馆的嘌呤抑制剂1,6。其他调查人员还通过一种经过改进和优化的高通量融合检测发现了 HIV-1 融合中的嘌呤抑制剂, 报告将病毒封装的β-酶转移到细胞质7、8.
地球复兴社区病毒的设计与 iGFP 病毒类似的方法, 插入 recombinase 之间的基质和衣壳, 两侧由 HIV-1 蛋白酶网站9。当 HIV-1 蛋白酶在新生的病毒微粒中激活时, 该酶是作为插入在 polyprotein 中的前驱体而制成的。因此,通过病毒膜融合过程将蛋白酶活化的 HIV-1 粒子的含量传递到目标细胞中, 该方法的交付是依赖的。这里提供了两个版本的协议。首先使用 HIV-1 感染的人群感染靶细胞, 研究 HIV 的直接细胞间传播。第二个版本使用无细胞病毒来研究无细胞病毒感染。细胞对细胞的透射检测需要7天的时间完成从细胞解冻的当天, 或5天, 如果细胞已经解冻和传代。如果细胞需要解冻, 如果细胞解冻和传代, 可以在5天内进行无细胞感染检测。还可以使用 Clevers 实验室10创建的质粒, 在所需的单元格类型中生成一个 rg (红到绿) 目标单元格线 (如果未使用现有的 rg 目标单元格线)。建议在本试验中采用病毒和病毒表达细胞进行适当的生物安全防护。我们在 BSL2+ 组织培养设施中进行这种检测的传染性部分。细胞固定后, 可在标准流式细胞仪和显微术中进行分析。
在这里, 我们描述了这种检测的应用, 以筛选新的化合物, 抑制 HIV-1 病毒膜融合 (图 1)。嘌呤受体是亲炎症介质。我们的实验室已经证明, 非选择性的嘌呤受体抑制剂作为 HIV-1 病毒膜融合的抑制剂6。我们报告说, 这种检测在高通量的利用可以识别新的 HIV-1 病毒膜融合抑制剂。我们证明, 嘌呤类受体抑制剂是一种新型的 HIV-1 病毒膜融合抑制剂。

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Sigma Aldrich</td>
			<td>D5546</td>
			<td>Media for 293 Cells</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma Aldrich</td>
			<td>R0883</td>
			<td>Media for Jurkats</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Albumin</td>
			<td>Gibco</td>
			<td>16140-071</td>
			<td>Serum for 293 Cells</td>
		</tr>
		<tr>
			<td>Cosmic Calf Serum</td>
			<td>Hyclone</td>
			<td>SH30087.03</td>
			<td>Serum for Jurkat Cells</td>
		</tr>
		<tr>
			<td>Hyclone Pennecillin Streptomycin solution</td>
			<td>GE Healthcare Life sciences</td>
			<td>SV30010</td>
			<td>Penn/Strep used in both media</td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>Corning</td>
			<td>3073</td>
			<td>Used for Culture of Jurkat Cells</td>
		</tr>
		<tr>
			<td>10cm Tissue Culture Plates</td>
			<td>Corning</td>
			<td>430167</td>
			<td>Used for Culture of 293 Cells</td>
		</tr>
		<tr>
			<td>96 Well Plates (tissue culture Treated)</td>
			<td>Corning</td>
			<td>3595</td>
			<td>Used for fusion assay</td>
		</tr>
		<tr>
			<td>Polyjet Transfection Reagent</td>
			<td>Signagen</td>
			<td>SL100688</td>
			<td>Used to transfect 293 Cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537-100ML</td>
			<td>Used in wash steps</td>
		</tr>
		<tr>
			<td>Hyclone Trypsin Protease</td>
			<td>GE Healthcare Life sciences</td>
			<td>SH30042.01</td>
			<td>For Trypsinization of 293 cells</td>
		</tr>
		<tr>
			<td>Amaxa Cell Line Nucleofector Kit V</td>
			<td>Lonza</td>
			<td>VACA-1003</td>
			<td>For Nucleofection of Jurkats</td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>GE Healthcare Life sciences</td>
			<td>17144002</td>
			<td>For Nucleofection of Jurkats</td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Fisher Brand</td>
			<td>13-678-11E</td>
			<td>For all tissue culture</td>
		</tr>
		<tr>
			<td>Pipettor Tips</td>
			<td>Denville Scientific</td>
			<td>P3020-CPS</td>
			<td>For all tissue culture and liquid handling steps</td>
		</tr>
		<tr>
			<td>Millex Syringe Filter (0.45 micron)</td>
			<td>Millipore</td>
			<td>SLHA033</td>
			<td>For filtration of virus</td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile syringes</td>
			<td>BD Diagnostics</td>
			<td>309656</td>
			<td>For filtration of virus</td>
		</tr>
		<tr>
			<td>Amaxa Nucleofector</td>
			<td>Lonza</td>
			<td>2b</td>
			<td>for Nucleofection of Jurkats (various models available)</td>
		</tr>
		<tr>
			<td>BD Fortessa Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>for flow cytometry analyss of samples</td>
		</tr>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>Various models</td>
			<td>Fortessa 2</td>
			<td></td>
		</tr>
		<tr>
			<td>pMSCV-loxp-dsRed-loxp-eGFP-Puro-W PRE</td>
			<td>Addgene</td>
			<td>32702</td>
			<td>Koo BK et al. Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods 9:81-83 (2011).</td>
		</tr>
		<tr>
			<td>pCL10a1</td>
			<td>Novus Bio</td>
			<td>NBP2-29542</td>
			<td>Naviaux, RK, Costanzi, E, Haas, M and Verma, I. The pCL vector system: Rapid production of helper-free, high titer, recombinant retroviruses. Journal of Virology 70: 5701-5705 (1996)</td>
		</tr>
		<tr>
			<td>Gag-iCre</td>
			<td>Benjamin Chen Lab</td>
			<td></td>
			<td>Esposito AM et al. A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry. Virology 490:6-16 (2016).</td>
		</tr>
	</tbody>
</table>

a high-throughput cre-lox activated viral membrane fusion assay to identify inhibitors of hiv-1 viral membrane fusion

本试验旨在通过流式细胞术或荧光显微镜检测绿色荧光蛋白 (GFP) 的表达, 具体报告 HIV-1 融合。一个 HIV-1 报告病毒 (HIV-1 地球复兴社区) 是通过插入 recombinase 到 HIV-1 基因组之间的母体和外壳蛋白的 polyprotein。这导致了 recombinase 的包装成病毒微粒, 然后被释放入目标细胞线稳定地表达 recombinase 激活的红色荧光蛋白 (RFP) 到 GFP 交换盒。在基本状态下, 此卡带只表示 RFP。在 recombinase 进入目标细胞后, RFP 由 loxP 站点两侧切除, 导致 GFP 表达。该方法可用于检测无细胞和细胞对细胞感染系统中的任何病毒进入抑制剂 (特别是融合步骤), 并被用来鉴定一类嘌呤受体拮抗剂作为新的 HIV-1 病毒膜融合抑制剂。

未感染的 RG Jurkat 细胞展示了一个低级别的背景 GFP 信号 (0.3%) 与一个非常强的 RFP 信号 (图 2A, 未感染的专栏)。地球复兴社区的感染导致 GFP 信号 (24.9%) 的增加, 而 HIV-1 融合抑制剂 AMD3100 (20 µM) 的存在抑制了这一信号的发展, 使其下降到未感染的背景水平 (0.34%)。当使用反向转录抑制剂 AZT (2 µM) 的后融合事件抑制剂时, 信号不受显著影响 (23.5%), 表明该?...

Watch this Scientific Journal Video about A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion at JoVE.com

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

通过流式细胞术或荧光显微镜检测绿色荧光蛋白的表达, 我们描述了以细胞为基础的 HIV-1 融合报告。它可以用来测试病毒进入的抑制剂 (特别是在融合步骤) 在无细胞和细胞对细胞感染系统。

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

a-high-throughput-cre-lox-activated-viral-membrane-fusion-assay-to

Research

JoVE Journal

Immunology and Infection

6.3K 次观看.  Icahn School of Medicine at Mount Sinai, Immunology Institute. 通过流式细胞术或荧光显微镜检测绿色荧光蛋白的表达, 我们描述了以细胞为基础的 HIV-1 融合报告。它可以用来测试病毒进入的抑制剂 (特别是在融合步骤) 在无细胞和细胞对细胞感染系统。

视频: A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

科研

免疫与感染

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 ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学:生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

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. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

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 ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.