Name: generation, amplification, and titration of recombinant respiratory syncytial viruses
Uploaded: 2019-04-04T13:00:00
Description: Watch this Scientific Journal Video about Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses at JoVE.com

作者感谢来自美国马萨诸塞州波士顿 & 波士顿的秦宇博士提供了 AZD4316 药物。作者感谢 Cymages 平台, 以便访问 Scnr 奥林巴斯显微镜, 该显微镜得到了法兰西岛 (DIM one 健康) 地区的资助。作者感谢 INSERM 和凡尔赛圣昆廷大学的支持。

1. 材料制备
<ol><li>购买细胞培养基 (减少血清培养基、最低必需培养基 [MEM]、10倍 MEM 和 Dulbecco 的改性鹰培养基 [DMEM])、转染试剂和微晶纤维素 (见材料表)。</li>
	<li>获取反向遗传学的以下载体: 编码 n 蛋白和聚合酶复合蛋白的基因组载体和表达载体。基因组载体包含来自噬菌体 T7 rna 聚合酶 (tpol) 启动子下游的 RSV-推销 V-GFP (对 Rsv-m2-1gfp) 的全 cDNA 基因组。表达式向量 (指定为 p-N、P-N、P...

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本文提出了一种从五种质粒中抢救重组 Rsv 并对其进行扩增的方法。操纵病毒基因组的能力使病毒学研究彻底改变了实验突变, 并表达了一个额外的基因或标记的病毒蛋白。我们在本文中描述并使用的 RSV 是一种病毒, 表达了记者基因, RSV-GFP (未出版), 并表达了一个 M2-1 蛋白融合到 GFP 标签12。RSV 救援具有挑战性, 需要练习。转染效率至关重要, 这取决于转染试剂的明智选择和转染协...

人 RSV 是全世界婴儿急性呼吸道感染住院的主要原因1。此外, RSV 与成人的疾病负担相当, 与流感相当, 老年人的住院和死亡负担大多为2。目前还没有针对 rsv 的疫苗或特定的抗病毒药物, 但有希望的新药正在开发中 3,4。RSV 乘法定量技术的复杂性和繁重程度阻碍了寻找抗病毒药物或疫苗的工作, 尽管目前作出了相当大的努力。RSV 体外增殖的定量一般是基于费力、耗时、昂贵的方法, 主要是通过显微镜、免疫染色、斑块还原量、定量等方法对细胞病理效应进行分析。逆转录酶 (qRT)-聚合酶链反应 (PCR) 和酶联免疫吸附试验。具有修饰基因组和表示记者基因的病毒, 如 GFP 编码的病毒, 更适合于此类筛选。结合自动化平板阅读器的使用, 记者基因携带重组病毒可以使这些检测更适合标准化和高通量的目的。
RSV 是一种包覆的、非分段的负感 RNA 病毒, 属于肺炎家族的正交肺炎病毒属, 顺序mononegavirales5。RSV 基因组是一种单链、负感 RNA, 约为 15 kb, 它包含一个位于 3 ' 和 5 ' 末端的非编码区域, 称为 "引线" 和 "预告片", 以及10个编码11蛋白的转录单元。基因顺序如下: 3 '-NS1、NS2、N、p、M、SH、g、f、M2 (m2-1 和 M2-2 蛋白编码) 和 L-5 '。基因组 RNA 由核蛋白 N 紧密包装, 以包塞化基因组 RNA 为模板, 病毒 rna 相关 RNA 聚合酶 (RdRp) 将确保病毒 RNA 的转录和复制。病毒 Rrp 由携带核苷酸聚合酶活性的大蛋白 L、其强制性辅因子磷蛋白 P 和作为病毒转录因子6的 m2-1 蛋白组成。在受感染的细胞中, RSV 诱导细胞质体内含物的形成, 称为包涵体 (IBs)。在几个mononegavirales7,8, 9,10中观察到了类似的形态类似的细胞质内含物。最近关于狂犬病毒、泡状口炎病毒 (vsv)、埃博拉病毒和 rsv 的研究表明, 病毒 rna 合成发生在 ibs 中, 因此可以被视为病毒工厂8,9,11,12. 病毒工厂集中了病毒 rna 合成所需的 rna 和病毒蛋白, 还含有细胞蛋白 13、14、15、16、17. ibs 表现出一个称为 IB-associated 颗粒 (iba) 的功能亚隔间, 该亚隔间集中了新合成的新生病毒 mrna 以及 mRNA 蛋白。在 Ibag 中没有检测到基因组 Rna 和 L、P 和 N。Ibag 是 IBs 内部的小动态球状结构, 表现为液体细胞器12的性质。尽管 IBs 在病毒增殖中发挥着核心作用, 但对这些病毒工厂的性质、内部结构、形成和运营了解甚少。
来自 cDNA 的脊髓灰质炎病毒基因组的表达使 1981年18年首次感染病毒克隆得以产生.对于单链阴性 RNA 病毒, 直到1994年才产生第一种狂犬病病毒, 然后将质粒转染成细胞19 。1995年20年公布了第一个基于血浆的 rsv 反向遗传系统.反向遗传学在病毒学领域取得了重大进展。在病毒基因组中引入特定修饰的可能性为 RNA 病毒的复制和发病机制提供了重要的见解。这项技术还通过有针对性的一系列修改, 使疫苗的开发能够产生具体的衰减。基因组修饰允许快速定量的病毒增殖大大改善了抗病毒筛选和研究其作用模式。
虽然前面描述过, 但获得转基因 Rsv 仍然很微妙。在这里, 我们详细介绍了一种创建两种类型的重组 HRSV 的协议, 分别表示 RSV-GFP 或 RSV-M1-GFP。在该协议中, 我们描述了抢救新的重组病毒所需的转染条件, 以及它们的扩增, 以获得高滴度的病毒库存, 适合可重复的实验。这里没有描述反向遗传学载体本身的构造。我们确实描述了最佳收集和冷冻病毒种群的方法。量化病毒感染颗粒最准确的方法仍然是斑块检测。细胞感染了连续稀释的分析悬浮液和孵育覆盖, 禁止扩散自由病毒颗粒在上清液。在这种情况下, 病毒只会感染连续的细胞, 形成每个初始传染性颗粒的 "斑块"。在传统的 RSV 滴定法中, 斑块通过免疫染色结果, 并在显微观察下计数。这种方法既昂贵又耗时。在这里, 我们描述了一个非常简单的协议 RSV 斑块检测使用微晶纤维素覆盖, 使斑块的形成可见肉眼。我们展示了如何使用 RSV-GFP 来测量 RSV 复制, 从而量化抗病毒药物的影响。结合反向遗传学和活体成像技术, 我们展示了 RSV-M1-GFP 如何允许科学家在活细胞中想象 M2-1, 并跟踪细胞内病毒结构的动态, 如 IBs。

<table>
	<tbody>
		<tr>
			<td>35mm &#181; dish for live cell imaging</td>
			<td>Ibidi</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>ATCC CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Avicel RC-591</td>
			<td>FMC BioPolymer</td>
			<td>Avicel RC-591</td>
			<td>Technical and other information on Avicels is available at http://www.fmcbiopolymer.com. Store at room temperature. Protocol in step 4 is optimized for this reagent.</td>
		</tr>
		<tr>
			<td>BSRT7/5</td>
			<td></td>
			<td>not commercially available</td>
			<td>See ref 22. Buchholz et al, 1999</td>
		</tr>
		<tr>
			<td>Crystal violet solution</td>
			<td>Sigma</td>
			<td>HT90132</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope for observations</td>
			<td>Olympus</td>
			<td>IX73 Olympus microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope for videomicroscopy</td>
			<td>Olympus</td>
			<td>ScanR Olympus microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>HEp-2</td>
			<td>ATCC</td>
			<td>ATCC CCL-23</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES &#8805;99.5%</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>LIPOFECTAMINE 2000 REAGENT</td>
			<td>ThermoFisher Scientific</td>
			<td>11668019</td>
			<td>Protocol in step 2.3. is optimized for this reagent.</td>
		</tr>
		<tr>
			<td>MEM (10X), no glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>11430030</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM, GlutaMAX Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>41090-028</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 ReagentPlus, &#8805;99.5%</td>
			<td>Sigma</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>51985-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde Aqueous Solution, 32%, EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td>not commercially available</td>
			<td>see ref 21. Rameix-Welti et al, 2014</td>
		</tr>
		<tr>
			<td>See Saw Rocker</td>
			<td>VWR</td>
			<td>444-0341</td>
			<td></td>
		</tr>
		<tr>
			<td>Si RNA GAPDH</td>
			<td>Dharmacon</td>
			<td>ON-TARGETplus siRNA 
			D-001810-10-05</td>
			<td>SMARTpool and 3 of 4 individual siRNAs designed by Dharmacon.</td>
		</tr>
		<tr>
			<td>Si RNA IMPDH2</td>
			<td>Dharmacon</td>
			<td>ON-TARGETplus siRNA IMPDH2 Pool- Human 
			L-004330-00-0005</td>
			<td>SMARTpool of 4 individual siRNAs designed by Dharmacon. 
			Individual references and sequences 
			J-004330-06: GGAAAGUUGCCCAUUGUAA; 
			J-004330-07: GCACGGCGCUUUGGUGUUC; 
			J-004330-08: AAGGGUCAAUCCACAAAUU; 
			J-004330-09: GGUAUGGGUUCUCUCGAUG;</td>
		</tr>
		<tr>
			<td>Si RNA RSV N</td>
			<td>Dharmacon</td>
			<td>ON-TARGETplus custom siRNA</td>
			<td>UUCAGAAGAACUAGAGGCUAU and UUUCAUAAAUUCACUGGGUUA</td>
		</tr>
		<tr>
			<td>SiRNA NT</td>
			<td>Dharmacon</td>
			<td>ON-TARGETplus Non-targeting Pool</td>
			<td></td>
		</tr>
		<tr>
			<td>SiRNA transfection reagent</td>
			<td>Dharmacon</td>
			<td>DharmaFECT 1 Ref: T-2001-03</td>
			<td>Protocol in steps 5.1.and 5.1.2 are optimized for this reagent.</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate 7.5% solution</td>
			<td>ThermoFisher Scientific</td>
			<td>25080094</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrofluorometer</td>
			<td>Tecan</td>
			<td>Tecan infinite M200PRO</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation, amplification, and titration of recombinant respiratory syncytial viruses

重组病毒的使用已成为基础或应用病毒学的关键。反向遗传学已被证明是一项极其强大的技术, 既可以破译病毒复制机制, 也可以研究抗病毒药物, 也可以为疫苗提供开发平台。然而, 为呼吸合胞病毒 (RSV) 等负链 RNA 病毒构建和操作反向遗传系统仍然很微妙, 需要特殊的专门知识。RSV 基因组是一种单链、负感 RNA, 约为 15 kb, 可作为病毒 RNA 复制和转录的模板。我们的反向遗传学系统使用人类 RSV 长株基因组 (HRSV) 的 cDNA 拷贝。这种 cDNA, 以及编码聚合酶复合物 (L、P、N 和 M2-1) 病毒蛋白的 Cdna, 被放置在 T7 聚合酶控制序列下的单个表达载体中。这些元素在 BSR-T展望5细胞中的转染, 稳定地表达 T7 聚合酶, 使重组 RSV 的细胞质复制和转录, 从而产生转基因病毒。一种新的 RSV, 存在于细胞表面和 bsrt75 的培养上清液中, 聚集在感染人 HEp-2 细胞进行病毒扩增。需要两轮或三轮扩增, 以获得含有 1 x10 6 至 1 x 10 7 斑块形成单元的病毒储存量。本文详细介绍了病毒库存的最佳收获、冷冻和滴定方法。我们通过创建两种分别表示游离绿色荧光蛋白 (RSV-GFP) 或病毒 M2-1 融合到 GFP (RSV-M1-GFP) 的重组病毒来说明本文所介绍的协议。我们展示了如何使用 RSV-GFP 量化 RSV 复制和 RSV-M1-GFP 可视化病毒结构, 以及病毒蛋白动态的活细胞, 通过视频显微镜技术。

在这项工作中, 我们描述了一个详细的协议, 以产生表达荧光蛋白的重组 RSV 病毒 (图 2)。在全球同步转法中, GFP 基因是在 P 基因和 M 基因之间引入的, 这在先前发表的研究21中对樱桃基因进行了描述。在 Prsv-m1-gfp 中, M2 基因没有被触及, 并在 SH 和 G 基因12之间插入了 m1-gfp 的附加基因编码。第一步与 bsrt毒5个细胞中病...

Watch this Scientific Journal Video about Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses at JoVE.com

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

我们描述了一种生成和扩增转基因呼吸道合胞病毒 (Rsv) 的方法和 Rsv 的优化斑块检测方法。我们通过创建两种重组病毒来说明这一协议, 它们分别允许对 RSV 复制进行定量, 并对 RSV 包涵体和包涵体相关颗粒动力学进行实时分析。

重组呼吸道合胞病毒的生成、扩增和滴定

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

使用重组病毒是一项强大的技术，既可以破译病毒复制机制，又为疫苗提供开发平台。同样，从最基本的研究到应用研究，生成高质量的病毒存量对于每一个病毒研究都至关重要。拯救重组病毒、最佳收获、冷冻和 RSV 库存滴定是所有病毒学研究的关键方法。它们可能被视为传统，但仍然很微妙，需要特定的专门知识。转染前一天，以每毫升浓度0.5百万细胞，将BSR-T7/5细胞悬浮在完整的介质中。在六井板中分配每井两毫升的细胞悬浮液。在37摄氏度的二氧化碳中孵育，直到第二天达到80-90%的汇合。为了拯救每种病毒，首先将解冻的反向遗传学载体混合在管子中，然后按照转染试剂制造商的协议进行转染。接下来，在混合载体中加入250微升的减少血清介质。在另一根管子中，在减少血清介质的250微升中稀释10微升这种转染试剂。轻轻旋转两个管子，等待五分钟。混合两个管的内容，并在室温下等待20分钟。同时，用一毫升的减少血清介质冲洗BSR-T7/5细胞，并免费分配1.5毫升的最低必需介质抗生素，每井补充10%的胎儿小牛血清。如有必要，在5%的二氧化碳环境中在37摄氏度下孵育。经过20分钟的孵育，在每一个井中加入500微升的转染混合物。在5%的二氧化碳环境中在37摄氏度下孵育细胞三天。为了监测救援效率，在倒置荧光显微镜下以每天20倍的放大倍率观察GGFP荧光。在转染的第三天，在转染BSR-T7/5六井板的每个井中刮伤细胞。将每井的细胞和上流液转移到无菌的两毫升微离心管中。要从细胞膜中释放被救出的病毒，每个管子的涡流至少30秒。对于第一次扩增被救出的病毒，首先从前一天播种的HEp-2板中去除培养培养剂，然后快速添加500微升每井的新鲜通道-零病毒悬浮液。将 HEp-2 板在 37 摄氏度的跷跷板摇杆上放置两个小时的软搅拌。要产生被拯救病毒的第一个通道，丢弃500微升的中分，并加入两毫升的 MEM 与 2%FCS。在37摄氏度的二氧化碳中孵育37摄氏度，为期三天。要以20倍的放大率在倒荧光显微镜下监测感染情况，观察感染通道零悬浮液的HEp-2细胞的GGFP荧光。在亮场显微镜下，观察小同步和细胞分离的外观，这反映了RSV细胞病变效应。为了放大被救的病毒，首先稀释无FCS MEM的病毒悬浮液，以每毫升5万PFU获得三毫升悬浮液，然后从HEp-2烧瓶中去除介质。快速添加三毫升病毒悬浮液，并在 37 摄氏度的跷跷板摇杆上孵化烧瓶，进行软搅拌两个小时。接下来，取出并丢弃 inoculum，并添加 15 毫升 MEM 与 2%FCS。在37摄氏度的二氧化碳中孵育2至4天。要估计收获病毒的准确时间，请在 20 倍放大的倒荧光显微镜下检查细胞形态和 GFP 荧光。请注意，这通常是当 50% 到 80% 的 HEp-2 细胞层由于感染后 48 到 72 小时之间发生的 RSV 细胞病变效应而分离时。接下来，使用单元格刮刀刮取所有单元格。将细胞和上一液收集在一起，并转移到50毫升的离心管。添加 10X RSV 保护解决方案的 1/10 体积。用力旋转管5秒钟，在200倍g下离心5分钟，以澄清悬浮液。将上一液转移到50毫升的管子上。在标有耐酒精标签的低温管中，涡流短暂地和等同。将管子浸入预冷、零下80摄氏度的酒精中至少一小时，并将其存放在零下80摄氏度。要进行斑块滴定测定，首先通过用无菌水稀释商用10X MEM并加入L-谷氨酰胺、每毫升青霉素1000个单位和每毫升链霉素1毫克，使至少2倍的必需介质。大力摇动稀释液并将其储存在4摄氏度，然后将900微升的无FCS MEM添加到6个管中。解冻病毒等分，并大力旋转五秒钟。为了进行连续稀释，首先在第一管的900微升介质中加入100微升病毒。将盖子放在管子上，通过涡旋混合几秒钟，然后在第一个稀释的100微升中加入第二管中的900微升介质。把盖子放在管子上，然后旋涡。重复此过程，直到第六管。在HEp-2 12孔板上写上病毒名称和全部稀释。添加一个标记来匹配板及其盖，因为它们可能在染色过程中分离。从板材上拆下介质，每孔分配 400 微升稀释剂。在37摄氏度下孵育板，用于病毒吸附两小时。在病毒吸附过程中，首先将2X MEM的pH值调整为7.2左右，在颜色指示器之后用7.5%的无菌碳酸氢钠溶液准备微晶纤维素覆盖。为了获得100毫升的覆盖物，加入10毫升2X MEM，10毫升2.4%单晶纤维素悬浮液，80毫升的M......辅以2%的FCS，并大力混合。在两个小时的孵育结束时，在12孔板的每个孔中加入两到三毫升的覆盖物，而不去除孵化。在37摄氏度的二氧化碳中孵育6天。要用水晶紫罗兰溶液染色细胞，首先轻轻摇动板，以脱下微晶纤维素覆盖物。取出超自然剂，用1X PBS清洗细胞两次，然后加入1至2毫升的水晶紫罗兰溶液，等待10至15分钟。拆下溶液，该溶液可重复使用，用于后续的板染色。最后计算病毒滴度作为干板井中可见斑块数的一小部分，以及烧液量和稀释。对负对照进行斑块测定，仅转染N、P、L和M2-1的表达质粒，在最低稀释时未发现斑块。如果救援是有效的，从转染细胞获得的滴答声预计高于每毫升100PFU。在通道上增加的滴答声在通道上达到每毫升100万至1000万PFU。由于这些荧光病毒，可以容易地观察和测量针对蛋白质N和细胞蛋白IMPDH的siRNA对RSV表达的抑制。相比之下，观察和测量了与非靶向siRNA或与siRNA对细胞蛋白GAPDH进行转染的控制细胞上的强GP信号。同样，这些病毒能够通过抗病毒药物化合物轻松观察RSV乘法的抑制作用。跟踪HEp-2感染细胞中的荧光蛋白M2-1表明IB和IB相关颗粒是非常动态的结构。被观察到的MB是移动和球形结构，能够融合并形成更大的球形内含物。IB相关颗粒经历了连续的组装-分解周期，形成与IB相关的小颗粒，这些颗粒生长，融合成大型IB相关颗粒，然后消失。使用微晶纤维素覆盖的RSV斑块滴定协议可以很容易地适应其他细胞和/或其他病毒。这将需要调整微晶纤维素的浓度。

Research

JoVE Journal

Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Protein Misfolding Cyclic Amplification of Prions

朊病毒蛋白质错误折叠循环扩增

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

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

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

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 ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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

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 ...

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

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

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