Name: arbovirus infections as screening tools for the identification of viral immunomodulators and host antiviral factors
Uploaded: 2018-09-13T14:30:00.000+00:00
Duration: 6 min 2 s
Description: Watch this Scientific Journal Video about Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors at JoVE.com

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>

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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><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&amp;rsquo;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&#039;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&amp;times;)</td><td>Sigma</td><td>A5955</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;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>&lt;em&gt;in vitro&lt;/em&gt; 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

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 ...

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

全基因组的 rna 干扰筛查以确定调节溶瘤病毒治疗的宿主因素

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

癌症研究

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

为检验新型抗病毒药物的鉴定对蓝舌病病毒

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

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 ...

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

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

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. ...

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 ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

从小鼠多个器官分离和定量寨卡病毒

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Use of Viral Entry Assays and Molecular Docking Analysis for the Identification of Antiviral Candidates against Coxsackievirus A16

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for the identification of candidate viral entry inhibitors. Here, we present the experimental approaches for the identification of small molecules capable of blocking infection by the non-enveloped coxsackievirus A16 (CVA16) through targeting the virus particles or specific steps in early viral entry. Assays include the time-of-drug-addition analysis, flow cytometry-based viral binding assay, and viral inactivation assay. We also present a molecular docking protocol utilizing virus capsid proteins to predict potential residues targeted by the antiviral compounds. These assays should help in the identification of candidate antiviral agents that act on viral entry. Future directions can explore these possible inhibitors for further drug development.

The goal of the protocol is to illustrate the different assays relating to viral entry that can be used to identify candidate viral entry inhibitors.

利用病毒进入检测和分子对接分析鉴定抗病毒候选药物,对抗Coxsackie病毒A16

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for ...

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of the disease.&#160;To study such processes, researchers can use high-throughput sequencing techniques (RNA-seq) that measure the dynamic changes of the host transcriptome at different stages of infection, clinical outcomes, or disease severity.This investigation can lead to a better understanding of the diseases, as well as uncovering potential drug targets and treatments. The protocol presented here describes a complete pipeline to analyze RNA-sequencing data from raw reads to functional analysis. The pipeline is divided into five steps: (1) quality control of the data; (2) mapping and annotation of genes; (3) statistical analysis to identify differentially expressed genes and co-expressed genes; (4) determination of the molecular degree of the perturbation of samples; and (5) functional analysis. Step 1 removes technical artifacts that may impact the quality of downstream analyses. In step 2, genes are mapped and annotated according to standard library protocols.&#160;The statistical analysis in step 3 identifies genes that are differentially expressed or co-expressed in infected samples, in comparison with non-infected ones. Sample variability and the presence of potential biological outliers are verified using the molecular degree of perturbation approach in step 4. Finally, the functional analysis in step 5 reveals the pathways associated with the disease phenotype. The presented pipeline aims to support researchers through the RNA-seq data analysis from host-pathogen interaction studies and drive future&#160;in vitro or in vivo experiments, that are essential to understand the molecular mechanism of infections.

The protocol presented here describes a complete pipeline to analyze RNA-sequencing transcriptome data from raw reads to functional analysis, including quality control and preprocessing steps to advanced statistical analytical approaches.

用于研究宿主-病原体相互作用的高通量转录组分析

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of ...