这项工作得到了科学和高等教育部对SARS-CoV-2研究的支持，并得到了国家科学中心的赠款（向K.P.和UMO-2018/30/E/NZ1/00874赠款给A.K.-P.）。

1. 缓冲区准备
<ol><li>对于 500 mL 的 2 倍 PHEM 缓冲器，将 18.14 克管道拉辛 -N，N+-二（2-乙烷硫酸）（PIPES），6.5 克 4-（2-羟基苯甲酸） 1-管道丙烷硫酸（HEPES）、3.8克乙二醇四乙酸（EGTA）和0.99克硫酸镁（MgSO4）。用蒸馏水（dH2O）使体积达到~400毫升，搅拌，然后使用10M氢氧化钾（KOH）或氢氧化钠（NAOH）将pH调整到7.0。使最终音量达到 500 mL，然后拆分为 50 mL 的别名。储存在-20°C，直到需要。 注:缓冲区在 pH 达到 7.0 之前不会清晰。</li>
	<li>准备 37% 的库存溶液，其中不含甲醛 （PFA）。对于 50 毫升，将 18.5 克 PFA 和 35 毫升 dH2O 混合在玻璃瓶中。将瓶子放在带加热的磁性搅拌器上。加入 900 μL 的 1 M KOH 或 NaOH，搅拌，直到解决方案变得清晰。允许冷却并转移到50mL圆锥离心机管:充值至50毫升与dH2O。解决方案可存储在 -20 °C，直到需要为止。 注意:甲醛应在戴防护手套和实验室外套时用烟熏罩处理。</li>
	<li>准备固定缓冲区（3.7%PFA缓冲与PHEM）。对于 50 mL，在 50 mL 锥形离心机管中组合 5 mL 的 37% PFA 解决方案、25 mL 的 2 倍 PHEM 缓冲器和 20 毫升 dH2O。储存在-20°C，直到需要。 注意:解冻后，缓冲区可存储在 4 °C 以至 3 个月之久。</li>
	<li>准备PBST（0.1%补间-20在1x磷酸盐缓冲盐水[PBS]）。对于 50 mL，添加 50 μL 的 100% 补间-20 至 50 mL 的 1x PBS，并很好地混合。 注:解决方案可在室温下存储 （RT）。</li>
	<li>通过将 MeOH/PBST 组合在 3:1、1:1 和 1:3 的比例（每份最终体积为 100 mL;参见 表 1）来准备补液缓冲器。 注意:MEOH是非常有毒和易燃的。因为它可以损害视神经，它应该在烟雾罩处理，避免任何明火。由于混合MeOH和PBST会产生外热反应，因此将冰上的溶液组合在一起。</li>
	<li>对于 1000 毫升的 20x SSC，将 175.3 克氯化钠 （NaCl） 和 88.2 克柠檬酸钠混合在烧瓶中，并加满 800 毫升的 dH2O. 搅拌至溶解，并使用 NaOH 将 pH 调整为 7.2。将 dH2O 添加到总体积为 1000 mL 中，然后通过 0.22 μm 滤镜将高压灭菌器或滤芯过滤到自封瓶中。</li>
	<li>对于 50 毫升的 5 倍 SSCT，将 20 倍 SSC 缓冲区的 12.5 mL 与 37.5 mL 的 dH2O 组合在一起，并添加 50 μL 的 100% Tween-20。混合好。</li>
	<li>对于 50 mL 的 50% 5x SSCT/50% PBST，将 25 mL 的 5 倍 SSCT 与 25 mL 的 PBST 组合在一起。</li>
	<li>对于 2x SSC 的 50 毫升，将 5 毫升的 10 倍 SSC 缓冲区与 45 毫升的 dH2O. 混合好。 注意:所有SSC缓冲区应存储在黑暗中的RT。</li>
</ol>2. 目标定义、探头和放大器
<ol><li>使用制造商网站上可用的工具来设计放大器和探头。确保探针与SARS-CoV-2 N基因的反向DNA链（补充图1）相辅相成。</li>
	<li>确定最佳探头集大小（一组 20 个探头足以可视化）。</li>
	<li>设置用于目标RNA检测的放大器。<ol>
<li>使用标有亚历克萨氟647的HCR放大器B1。 注:HCR 放大器包括可转移的 HCR 发夹 h1 和 h2。使用此协议可以设计多路复用实验。如果计划这样做，请选择不同的HCR放大器（B1，B2，...），以便在同一样本中对每个目标RNA进行成像（例如，目标1的放大器B1，目标2的放大器B2，...）。</li>
	</ol>
</li>
</ol>3. 细胞培养和感染SARS-CoV-2
<ol><li>培养维罗（猴子肾上皮细胞）细胞在杜尔贝科的修改鹰介质含有5%的胎儿牛血清。<ol>
<li>种子 50，000 细胞到盖片 （No. 1， 15 × 15 毫米） 在 12 井板.</li>
	</ol>
</li>
	<li>在透气跨井插入支架上用 膜（直径 = 6.5 mm） 和支气管上皮生长介质培养培养物，以备不时之用。</li>
	<li>病毒感染<ol>
<li>接种SARS-CoV-2的细胞，每mL组织培养中位数感染剂量（TCID50）</li>
		<li>在37°C下孵化细胞2小时。</li>
		<li>用 PBS 清洗两次细胞以去除未绑定病毒。</li>
		<li>培养细胞为48小时。</li>
	</ol>
</li>
</ol>4. 在盖片培养的维罗细胞中的 SARS-CoV-2 RNA-FISH
第 1 天
<ol><li>修复和渗透细胞<ol>
<li>在 RT 使用 3.7% 的 w/v PFA 解决方案修复受感染的细胞，使用 1 小时。</li>
		<li>吸气 3.7% PFA 溶液，使用 1 倍 PBS 洗涤细胞两次。</li>
		<li>在 RT 中用 PBST 溶液使细胞渗透 10 分钟，并激动地进行。</li>
		<li>吸气PBST，用1倍PBS洗两次细胞。</li>
	</ol></li>
	<li>检波<ol>
<li>吸气1倍PBS溶液，并在RT用2x SSC洗两次细胞。</li>
		<li>吸气 2x SSC 溶液，并通过添加至少 300 μL 的探针杂交缓冲来预混合样品。盖住含有细胞的井，在37°C孵育30分钟。</li>
		<li>通过在探头混合缓冲区中加入 1.2 pmol 的探针混合物来准备探针解决方案。<ol>
<li>使用 1 μM 探头库存的 1.2 μL 来准备 300 μL 的工作库存。</li>
		</ol>
</li>
		<li>取出预贿赂溶液，并将盖片转移到加湿室。<ol>
<li>将探针溶液的 30-50μL 吹到胶片上，形成单个液滴。</li>
		</ol>
</li>
		<li>在 37 °C 处过夜（12-18 h） 孵化样品。 第2天</li>
		<li>将盖片重新转移到 12 井板中，并在 37 °C 下用 400 μL 的探头清洗缓冲器清洗 4 x 5 分钟，去除多余的探头溶液。 注意:作为将 30-50μl 别名放在底片上和覆盖膜下进行孵化的替代方案，请直接添加 300 μL 的探针溶液，以覆盖 12 井板中的盖片。此过程更简单，但需要大量的试剂。使用前将探头洗涤缓冲器加热至 37 °C。计算所需的缓冲区量，并将其转移到 15 mL 锥形离心机管中。</li>
		<li>在RT使用5x SSCT清洗样品2 x 5分钟。</li>
		<li>将 5 倍 SSCT 解决方案替换为 1 倍 PBS，并将样品存储在 4 °C，直到放大。</li>
	</ol></li>
	<li>放大<ol>
<li>从井中取出 1 倍 PBS 溶液，并在每个井中加入至少 300 μL 的放大缓冲。在 RT 孵化样品 30 分钟。</li>
		<li>通过在单独的管中快速冷却所需的体积来准备每个HCR发夹（h1和h2）。<ol>
<li>要准备 300 μL 的放大溶液，请使用每个发夹的 18 pmol（例如，对于 300 μL，使用 3 μM 库存发夹溶液的 6 μL）。</li>
			<li>将发夹溶液转移到管中。</li>
			<li>在95°C孵育90年代。</li>
			<li>在黑暗中冷却到RT30分钟。</li>
		</ol>
</li>
		<li>通过在放大缓冲区中添加快速冷却的"h1"和"h2"发夹来准备发夹混合物。</li>
		<li>将30-50μL的发夹混合物滴入胶片上。</li>
		<li>在 RT 的黑暗中在夜间 （12-18 h） 孵化样品。 第3天</li>
		<li>将盖片移回 12 个井板，并通过在 RT 中搅拌 5 x 5 分钟，用 5 倍 SSCT 去除多余的发夹。</li>
		<li>吸气 5 倍 SSCT 缓冲区，代之以 1 倍 PBS 。 注意:如有必要，请在标准免疫荧光检测中使用RNA-FISH样本，然后对核进行染色（见第5节）。</li>
	</ol></li>
	<li>核染色和滑动安装<ol>
<li>吸气 1x PBS 解决方案，并在 1x PBS 解决方案中将其替换为 4+，6-二恶英-2-苯林多（DAPI，0.2μg/mL）。</li>
		<li>在黑暗中在RT孵化样品10分钟。</li>
		<li>吸气 DAPI 溶液，用 1 倍 PBS 洗两次细胞。</li>
		<li>放置两滴10μL的安装介质:确保将滴子充分分离，以便将两个盖片放置在单个幻灯片上。</li>
		<li>通过敲击干净毛巾上的盖子，去除多余的液体，然后将它们置于反法德安装介质中，细胞朝下。</li>
		<li>将安装的样品放在干燥平坦的黑暗表面，让它们进行固化。</li>
		<li>固化后，用 VALAP 密封剂或指甲油密封盖片的边缘，以防止样品干燥。</li>
	</ol></li>
</ol>5. HAE 文化中的 SARS-CoV-2 RNA-鱼
第 1 天
<ol start="1"><li>修复和渗透 HAE 文化<ol>
<li>吸气介质，并在RT使用3.7%PFA溶液修复受感染的细胞1小时。</li>
		<li>吸气 3.7% PFA 溶液，用 1 倍 PBS 洗两次细胞。<ol>
<li>在跨井插入下，用 1 倍 PBS 替换 3.7% 的 PFA 解决方案。</li>
		</ol>
</li>
		<li>丢弃 PBS，然后使用 2 x 5 分钟洗涤器使样品脱水，100% MeOH 预制为 -20 °C。</li>
		<li>第二次洗涤后，用新鲜的冷MeOH代替缓冲区，在Transwell插入下渗透。在-20°C过夜。</li>
	</ol>
</li>
</ol>第2天
<ol start="2"><li>再水化 通过冰上分级的 MeOH/PBST 解决方案系列（每组 5 分钟）补充样品水分:75% MeOH/25% PBST、50% MeOH/50% PBST、25% MEOH/75% PBTS 和 100% PBST（两次）。<ol>
<li>在冰上用 50% 5 倍 SSCT/50% PBST 清洗细胞 5 分钟。</li>
		<li>用5倍SSCT在冰上清洗细胞5分钟。</li>
		<li>用 1 倍 PBS 替换 5 倍 SSCT 缓冲区。</li>
	</ol>
</li>
	<li>检波<ol>
<li>在冰上用100μL的探针杂交缓冲器孵化细胞（在Transwell插入器内）5分钟。接下来，在37°C（预灌木化）下将盘子转移到孵化器30分钟。 注意:探头混合缓冲区在使用前必须预热至 37 °C。计算所需的音量:单个跨井插入需要 100 μL。</li>
		<li>准备探头解决方案。由于 1 mL 的探头解决方案需要 4 pmol 的探针，因此在 1 mL 的探针杂交缓冲区中加入 1 μL 的 1 μM 探头库存解决方案，并很好地混合。 注意:对于RNA检测，请使用每个跨井插入100μL的探针溶液。将探针溶液留在冰上，直到预贿赂步骤结束。</li>
		<li>删除预贿赂解决方案，并添加探头解决方案。</li>
		<li>在 37 °C 处隔夜（12-18 h） 孵化细胞。 第3天</li>
		<li>通过在 37 °C 下使用 100 μL 的探头清洗缓冲器清洗 4 x 15 分钟来去除多余的探头。</li>
		<li>在RT使用5x SSCT清洗样品2 x 5分钟。</li>
		<li>将 5 倍 SSCT 替换为 1 倍 PBS，并将样品存储在 4 °C，直到放大。</li>
	</ol>
</li>
	<li>放大<ol>
<li>通过在 RT 上用放大缓冲器孵化样品 30 分钟来预加样品。</li>
		<li>通过在单独的管中快速冷却所需的体积来准备每个发夹。<ol>
<li>要准备 500 μL 的放大溶液，请使用每个发夹的 30 pmol（例如，对于 500 μL，使用 10 μL 的 3 μM 库存发夹溶液）。</li>
			<li>将发夹溶液转移到管子上。</li>
			<li>在95°C孵育90年代。</li>
			<li>在黑暗中冷却到RT30分钟。</li>
		</ol>
</li>
		<li>通过在 RT 将所有扣冷发夹添加到 500 μL 的放大缓冲器中，准备发夹解决方案。</li>
		<li>删除预加放大解决方案，并添加完整的发夹解决方案。</li>
		<li>在黑暗中在 RT 过夜 （12-18 h） 孵化样品。</li>
		<li>通过在 RT 中用 5 倍 SSCT 清洗去除多余的发夹，具体如下: 2 x 5 分钟、2 x 15 分钟和 1 x 5 分钟。</li>
		<li>将 5 倍 SSCT 解决方案替换为 1 倍 PBS，并在 4 °C 中存储不超过 2-3 天，或直接进行核染色。 注意:如有必要，请在标准免疫荧光检测中使用RNA-FISH样本，然后进行核染色（见第6节）。</li>
	</ol>
</li>
	<li>核染色和滑动安装<ol>
<li>吸气 1x PBS 解决方案，并在 1x PBS 中将其替换为 DAPI 解决方案 （0.2μg/mL）。</li>
		<li>在黑暗中在RT孵化样品10分钟。</li>
		<li>吸气 DAPI 溶液，用 1 倍 PBS 洗两次细胞。</li>
		<li>将 Transwell 插入的切口膜放置在 10 μL 的抗法德安装介质上，将细胞朝上，并在膜上添加额外的安装介质 （5 μL）。</li>
		<li>用盖子盖住膜。</li>
		<li>在黑暗中将安装的样品涂在干燥平坦的表面上。</li>
		<li>固化后，用 VALAP 密封剂或指甲油密封盖片的边缘，以防止样品干燥。</li>
	</ol>
</li>
</ol>6. 维罗细胞和HAE培养物的免疫荧光分析
注意:在第3天对细胞系进行免疫荧光检测，或对HAE培养物进行第4天免疫荧光检测。对每个模型使用不同的方法。所有差异都清楚表明。
<ol><li>从井中吸气 1 倍 PBS 解决方案。</li>
	<li>在 PBST 溶液中用 1% w/v 牛血清白蛋白在 37 °C 下孵化 30 分钟来阻止样品。</li>
	<li>通过在阻断溶液中准备适当的稀释并孵育，准备主要抗体。<ol>
<li>对于盖片上的维罗细胞:<ol>
<li>将30-50μL的抗体溶液滴入湿度室的胶片上。</li>
			<li>将盖子盖在抗体滴上，细胞朝下。</li>
		</ol>
</li>
		<li>对于 HAE，用抗体溶液 （100 μL） 替换插入物中的阻塞剂，并将样品孵化在加湿室中。 注意:用原抗体调整每次孵化的时间和温度。典型的参数是2小时在RT或过夜在4°C。</li>
	</ol>
</li>
	<li>使用PBST清洗样品3 x 5分钟。<ol>
<li>对于盖片上的细胞，转移到12井板，并添加PBST解决方案。</li>
		<li>对于 HAE 文化，用 PBST 解决方案替换抗体解决方案。</li>
	</ol>
</li>
	<li>检查可用于对焦显微镜的光源和过滤器。</li>
	<li>根据宿主物种选择次生抗体。根据可用光源选择荧光参数（激发和发射波长、光谱宽度和激发效率）。 注:光谱参数可以使用在线工具进行建模（参见 材料表）。</li>
	<li>通过用阻塞溶液稀释适当的二次抗体解决方案，准备适当的二次抗体解决方案。</li>
	<li>用二次抗体孵育（如在6.3），但在37°C孵育1小时。</li>
	<li>将样品按步骤 6.4 清洗。</li>
	<li>核染色和滑动安装<ol>
<li>用于盖片上的单元格<ol>
<li>吸气PBST，并在1x PBS解决方案中将其替换为DAPI（0.2微克/mL）。</li>
			<li>在黑暗中在RT孵化样品10分钟。</li>
			<li>吸气 DAPI 溶液，用 1 倍 PBS 洗两次细胞。</li>
			<li>将盖片放在 10 μL 的安装介质滴上，细胞朝下。</li>
			<li>用指甲油密封盖子。</li>
		</ol>
</li>
		<li>对于HAE文化<ol>
<li>吸气1倍PBST，并在1x PBS溶液中替换为DAPI（0.2μg/mL）。</li>
			<li>在黑暗中在RT孵化样品10分钟。</li>
			<li>吸气 DAPI 溶液，用 1 倍 PBS 洗两次细胞。</li>
			<li>将切出的膜放在 10 μL 的安装介质滴上，将细胞朝上，并在膜上添加额外的 （5 μL） 安装介质。</li>
			<li>用盖子盖住膜。</li>
			<li>用指甲油密封盖子。</li>
		</ol>
</li>
	</ol>
</li>
</ol>7. 聚焦显微镜
<ol><li>通过指定使用的氟磷来定义轨道。</li>
	<li>选择扫描模式和速度。</li>
	<li>通过将每个氟磷与各自的负控制进行比较来调整每个荧光素的激光功率、增益和偏移值:用于病毒、模拟感染细胞:对于细胞蛋白，样品沾染了来自适当宿主的异型控制抗体。</li>
	<li>要获取 3D 图像，请激活 z 堆栈模式，并设置顶部和底部限制。设置步数/数字。 注:有关冠状病毒成像的更多详细信息，请参阅19。</li>
</ol>

<ol>
	<li>Choi, H. M. 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=Third-generation+in+situ+hybridization+chain+reaction:+multiplexed,+quantitative,+sensitive,+versatile,+robust.">Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.</a> Development. 145, (2018).</li><li>Milewska, A., 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=Replication+of+severe+acute+respiratory+syndrome+coronavirus+2+in+human+respiratory+epithelium.">Replication of severe acute respiratory syndrome coronavirus 2 in human respiratory epithelium.</a> Journal of Virology. 94, (2020).</li><li>Banach, B. S., 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=Human+airway+epithelial+cell+culture+to+identify+new+respiratory+viruses:+coronavirus+NL63+as+a+model.">Human airway epithelial cell culture to identify new respiratory viruses: coronavirus NL63 as a model.</a> Journal of Virological Methods. 156 (1-2), 19-26 (2009).</li><li>Milewska, A., 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=Entry+of+human+coronavirus+NL63+into+the+cell.">Entry of human coronavirus NL63 into the cell.</a> Journal of Virology. 92, (2018).</li><li>El Baba, R., Herbein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+epigenomic+networks+entailed+in+coronavirus+infections+and+COVID-19.">Management of epigenomic networks entailed in coronavirus infections and COVID-19.</a> Clinical Epigenetics. 12, 118 (2020).</li><li>Pinto, B. G. 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=ACE2+expression+is+increased+in+the+lungs+of+patients+with+comorbidities+associated+with+severe+COVID-19.">ACE2 expression is increased in the lungs of patients with comorbidities associated with severe COVID-19.</a> Journal of Infectious Diseases. 222 (4), 556-563 (2020).</li><li>Verdecchia, P., Cavallini, C., Spanevello, A., Angeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pivotal+link+between+ACE2+deficiency+and+SARS-CoV-2+infection.">The pivotal link between ACE2 deficiency and SARS-CoV-2 infection.</a> European Journal of Internal Medicine. 76, 14-20 (2020).</li><li>Gordon, D. E., 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+SARS-CoV-2+protein+interaction+map+reveals+targets+for+drug+repurposing.">A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.</a> Nature. 583, 459-468 (2020).</li><li>Ferron, F., Decroly, E., Selisko, B., Canard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viral+RNA+capping+machinery+as+a+target+for+antiviral+drugs.">The viral RNA capping machinery as a target for antiviral drugs.</a> Antiviral Research. 96 (1), 21-31 (2012).</li><li>Mehta, S., Jeffrey, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+receptors+and+signaling:+epigenetic+factors+in+the+regulation+of+innate+immunity.">Beyond receptors and signaling: epigenetic factors in the regulation of innate immunity.</a> Immunology and Cell Biology. 93 (3), 233-244 (2015).</li><li>Poppe, 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=The+NF-kappaB-dependent+and+-independent+transcriptome+and+chromatin+landscapes+of+human+coronavirus+229E-infected+cells.">The NF-kappaB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells.</a> PLoS Pathogens. 13, 1006286 (2017).</li><li>Schafer, A., Baric, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+landscape+during+coronavirus+infection.">Epigenetic landscape during coronavirus infection.</a> Pathogens. 6 (1), 8 (2017).</li><li>Carletti, 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=Viral+priming+of+cell+intrinsic+innate+antiviral+signaling+by+the+unfolded+protein+response.">Viral priming of cell intrinsic innate antiviral signaling by the unfolded protein response.</a> Nature Communications. 10, 3889 (2019).</li><li>Heil, F., 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=Species-specific+recognition+of+single-stranded+RNA+via+toll-like+receptor+7+and+8.">Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.</a> Science. 303 (5663), 1526-1529 (2004).</li><li>Sze, A., 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=Host+restriction+factor+SAMHD1+limits+human+T+cell+leukemia+virus+type+1+infection+of+monocytes+via+STING-mediated+apoptosis.">Host restriction factor SAMHD1 limits human T cell leukemia virus type 1 infection of monocytes via STING-mediated apoptosis.</a> Cell Host and Microbe. 14 (4), 422-434 (2013).</li><li>Kikkert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+evasion+by+human+respiratory+RNA+viruses.">Innate immune evasion by human respiratory RNA viruses.</a> Journal of Innate Immunity. 12, 4-20 (2020).</li><li>Kennedy, E. 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=Posttranscriptional+m(6)A+editing+of+HIV-1+mRNAs+enhances+viral+gene+expression.">Posttranscriptional m(6)A editing of HIV-1 mRNAs enhances viral gene expression.</a> Cell Host and Microbe. 22 (6), 830 (2017).</li><li>Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R., Randell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-differentiated+human+airway+epithelial+cell+cultures.">Well-differentiated human airway epithelial cell cultures.</a> Human Cell Culture Protocols in Methods in Molecular Medicine. 107, 183-206 (2005).</li><li>Milewska, A., Owczarek, K., Szczepanski, A., Pyrc, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+coronavirus+entry+into+cells.">Visualizing coronavirus entry into cells.</a> Methods in Molecular Biology. 2203, 241-261 (2020).</li></ol>

作者没有利益冲突可以申报。

免疫-RNA-FISH是RNA和细胞蛋白双重染色的可靠方法。在这里，描述了一个经过修改的免疫-RNA-FISH协议，允许检测SARS-CoV-2RNA和细胞蛋白的细胞系和HAE培养物。此协议可适用于不同的细胞模型，包括细胞单层或特定组织。该方法依赖于由适当的探头本地化启动的HCR的概念。接下来，使用分发启动器探头开始放大荧光标记放大器的信号，当使用共聚焦显微镜观察时，可产生极小到无背景荧光。放大器可以...

SARS-CoV-2是一种新型的人类β可罗纳病毒，于2019年底出现，几个月后引发了前所未有的大流行。由于该病毒是科学的新生病毒，因此其大部分生物学及其对宿主细胞的影响仍不得而知。因此，如果要了解病毒细胞和组织在感染过程中的三胞头，其基本的生物学特征及其对宿主的影响就很重要。使用多种技术来检查病毒宿主之间的相互作用，包括生化、生物和物理检测。原位杂交是一种常用方法，它采用标记的互补DNA、RNA或改性核酸探针，该探针定位到细胞或组织中的特定DNA或RNA序列。
一种新的RNA荧光原位杂交（RNA-FISH）方法已经开发出来，它结合了修改，通过HCR1放大信号与噪声比来增加灵敏度。HCR 允许在单细胞级别上研究 RNA 本地化。由于其特异性、灵敏度和分辨率高，这种方法不仅对基础科学研究有用，而且对应用项目（如诊断）也很有用。最近，该方法的可行性被证明用于检测SARS-CoV-2RNA本地化为完全分化的3D人类气道上皮（HAE）培养物2中的附属细胞。HAE文化是用于研究"自然感染"微环境3、4背景下病毒感染的最先进工具之一。
包括SARS-CoV-2在内的几份关于人类冠状病毒（HCoV）的报告强调了表观遗传学对HCoV感染和病理生理学（在5中回顾）的重要性，例如编码血管紧张素转换酶2（ACE-2）受体6、7的基因的甲基化模式。有趣的是，质谱筛查发现了几个与SARS-CoV-2蛋白体8相互作用的表观遗传因素。更具体地说，非结构蛋白 5 （NSP5） 与表观遗传调节器、高石脱乙酰酶 2 结合，催化不活跃 NSP5 （C145A） 与 tRNA 甲基转移酶 1 （24） 相互作用。此外，NSP16甲基转移酶活性被甲基转移酶抑制剂西内芬金9阻断。然而，这些表观遗传因素在COVID-19中的确切作用尚不清楚。HCoV的复制发生在受感染细胞的细胞质中，并触发由表观遗传修饰调节的炎症反应10。
例如，HCoV-229E微调核因子-卡帕B信号，并通过在某些区域增加H3K36和H4K5的乙酰化，对宿主细胞色度图进行深刻重新编程。中东呼吸系统综合症相关冠状病毒感染增加了H3K27me3的水平，并耗尽了H3K4me3在特定干扰素敏感基因12子集的促进区域。此外，病毒RNA触发细胞免疫反应，如病毒13，逆转录病毒14，15和冠状病毒16证明。病毒RNA上的表观遗传标记可能起到细胞传感器识别的作用，如人类免疫缺陷病毒-1 RNA17的m7A甲基化所示。然而，问题依然存在:SARS-CoV-2RNA对免疫反应有何影响，是否涉及表观遗传标记？
在这里，介绍了一种优化的RNA-FISH方法，以及细胞系和3D组织（完全分化HAE）的免疫荧光分析。虽然细胞学方法，如FISH和免疫荧光，被广泛使用，这种基于HCR的新一代原位杂交方法从未用于病毒检测（除了在最近的出版物）2。一般来说，免疫染色和鱼氧化物需要以下步骤:渗透，使探针或抗体穿透:固定其中细胞材料固定和保存;检测其中应用抗体或核酸探针;最后，安装样品进行可视化。
虽然现有协议具有这些通用功能，但它们在所涉及的参数方面存在显著差异。在这里，一个优化的，简单的，免疫-RNA-FISH协议已经描述检测SARS-CoV-2RNA在HAE培养和维罗细胞。该技术包括以下步骤:（1） 用甲醛固定细胞:（2） 用洗涤剂或甲醇（MeOH） 渗透:（3） 通过一系列分级的 MeOH 解决方案（仅限 HAE 文化）进行补液:（4） 检测:（五）利用HCR技术检测SARS-CoV-2RNA的放大:（六）免疫学:和（7）在共焦显微镜下成像。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Equipment</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Confocal Microscope LSM 880</td>
			<td >ZEISS </td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Grant Bio, Mini Rocker- Shaker</td>
			<td >Fisher Scientific</td>
			<td>12965501</td>
			<td></td>
		</tr>
		<tr >
			<td >Incubator Galaxy170R</td>
			<td >New Brunswick</td>
			<td>CO170R-230-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermomixer Comfort</td>
			<td >Eppendorf</td>
			<td>5355 000 011</td>
			<td></td>
		</tr>
		<tr >
			<td>Materials</td>
			<td></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >15 mm x 15 mm NO. 1 coverslips</td>
			<td >LabSolute</td>
			<td >7695022</td>
			<td ></td>
		</tr>
		<tr >
			<td >1.5 mL tubes</td>
			<td >FL-MEDICAL</td>
			<td >5.350.023.053</td>
			<td ></td>
		</tr>
		<tr >
			<td >12-well plate</td>
			<td >TTP</td>
			<td >92412</td>
			<td ></td>
		</tr>
		<tr >
			<td >Conical centrifuge tube</td>
			<td >Sarstedt</td>
			<td >5.332.547.254</td>
			<td ></td>
		</tr>
		<tr >
			<td >parafilm</td>
			<td >Sigma</td>
			<td >P7793-1EA</td>
			<td ></td>
		</tr>
		<tr >
			<td >serological pipets</td>
			<td >VWR Collection</td>
			<td >612-5523P, 612-5827P</td>
			<td ></td>
		</tr>
		<tr >
			<td >slide glass</td>
			<td >PTH CHEMLAND</td>
			<td >04-296.202.03</td>
			<td ></td>
		</tr>
		<tr >
			<td >Transwell ThinCerts</td>
			<td >Grainer bio-one</td>
			<td >665641</td>
			<td ></td>
		</tr>
		<tr >
			<td >Reagents</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Alexa fluorophore 488-conjugated secondary antibodies </td>
			<td>Invitrogen</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >&#946;5-tubulin </td>
			<td>Santa Cruz Biotechnology</td>
			<td > sc-134234</td>
			<td ></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Thermo Scientific</td>
			<td >D1306</td>
			<td ></td>
		</tr>
		<tr >
			<td >Disodium phosphate</td>
			<td >Sigma</td>
			<td >S51136-500G</td>
			<td ></td>
		</tr>
		<tr >
			<td >EGTA</td>
			<td >BioShop</td>
			<td >EGT101.25</td>
			<td ></td>
		</tr>
		<tr >
			<td >HCR Amplification Buffer</td>
			<td >Molecular Instruments, Inc.</td>
			<td >BAM01522</td>
			<td >Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr >
			<td >HCR amplifier B1-h1 Alexa Fluor 647</td>
			<td >Molecular Instruments, Inc.</td>
			<td >S013922</td>
			<td ></td>
		</tr>
		<tr >
			<td >HCR amplifier B1-h2 Alexa Fluor 647</td>
			<td >Molecular Instruments, Inc.</td>
			<td >S012522</td>
			<td ></td>
		</tr>
		<tr >
			<td >HCR Probe Hybridization Buffer</td>
			<td >Molecular Instruments, Inc.</td>
			<td >BPH03821</td>
			<td >Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr >
			<td >HCR probe set for SARS-CoV-2 Ncapsid</td>
			<td >Molecular Instruments, Inc.</td>
			<td >PRE134</td>
			<td></td>
		</tr>
		<tr >
			<td >HCR Probe Wash Buffer</td>
			<td >Molecular Instruments, Inc.</td>
			<td >BPW01522</td>
			<td >Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr >
			<td >HEPES</td>
			<td >BioShop</td>
			<td >HEP001.100</td>
			<td ></td>
		</tr>
		<tr >
			<td >Magnesium sulfate heptahydrate</td>
			<td >Sigma</td>
			<td >63138-250G</td>
			<td ></td>
		</tr>
		<tr >
			<td >Methanol</td>
			<td >Sigma</td>
			<td >32213-1L-M</td>
			<td ></td>
		</tr>
		<tr >
			<td >Monopotassium phosphate</td>
			<td >Sigma</td>
			<td >P5655-100G</td>
			<td ></td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Sigma</td>
			<td >P6148-1KG</td>
			<td ></td>
		</tr>
		<tr >
			<td >PIPES</td>
			<td >BioShop</td>
			<td >PIP666.100</td>
			<td ></td>
		</tr>
		<tr >
			<td >Potassium Chloride</td>
			<td >Sigma</td>
			<td >P5405-250G</td>
			<td ></td>
		</tr>
		<tr >
			<td >Prolong Diamond Antifade Mounting Medium</td>
			<td >Invitrogen</td>
			<td >P36970</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sodium Chloride</td>
			<td >BioShop</td>
			<td >SOD001.5</td>
			<td ></td>
		</tr>
		<tr >
			<td >Trisodium Citrate 2-hydrate</td>
			<td >POCH</td>
			<td > 6132-04-3</td>
			<td ></td>
		</tr>
		<tr >
			<td >Tween-20</td>
			<td >BioShop</td>
			<td >TWN580.500</td>
			<td ></td>
		</tr>
		<tr >
			<td ></td>
			<td >Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescence Spectraviewer</td>
			<td ></td>
			<td></td>
			<td>Modeling spectral parameters</td>
		</tr>
		<tr >
			<td >ImageJ Fiji</td>
			<td ></td>
			<td ></td>
			<td >Acquiring and processing z-stack images</td>
		</tr>
	</tbody>
</table>

visualization of sars-cov-2 using immuno rna-fluorescence in situ hybridization

本手稿提供了原位杂交链反应 （HCR） 与免疫荧光的协议，可直观地将严重急性呼吸系统综合征冠状病毒 2 （SARS-CoV-2） RNA 在细胞系中和人类气道上皮的三维 （3D） 培养物中。该方法允许高度具体和敏感的病毒RNA可视化，依靠HCR启动的探针本地化。拆分启动器探头通过荧光标记的放大器帮助放大信号，导致共焦显微镜中可忽略不计的背景荧光。用不同的荧光染料标记放大器有助于同时识别各种目标。这反过来又使组织中的感染图谱能够更好地了解单细胞水平的病毒发病机能和复制。将这种方法与免疫荧光相结合，可以促进更好地了解宿主-病毒相互作用，包括宿主表观基因组和免疫反应通路的交替。由于敏感和特定的HCR技术，此协议也可以用作诊断工具。同样重要的是要记住，该技术可能很容易修改，以便能够检测任何RNA，包括将来可能出现的非编码RNA和RNA病毒。

本手稿中描述的免疫-RNA-FISH协议使用两个蜂窝系统执行:维罗细胞系和3D HAE培养物。两个蜂窝模型的主要步骤都显示在 表 2 中。RNA-FISH 在 HAE 培养中可视化 SARS-CoV-2 的协议包括组织样本的典型步骤，即通过一系列 MeOH-PBS 和 0.1% Tween 解决方案，以 100% MeOH 渗透和补液。免疫荧光是在RNA-FISH完成之后进行的。获取和处理了 Zstack 图像。
<p class="jove_content" fo:keep-toget...

Watch this Scientific Journal Video about Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization at JoVE.com

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

在这里，我们描述了一个简单的方法，结合RNA荧光原位杂交（RNA-FISH）与免疫荧光可视化严重急性呼吸系统综合症冠状病毒2（SARS-CoV-2）RNA。该协议可以增加对SARS-CoV-2 RNA-主机在单细胞水平上的分子特征的理解。

使用免疫RNA-荧光原位杂交对SARS-CoV-2进行可视化

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

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Research

JoVE Journal

Immunology and Infection

5.9K 次观看.  Jagiellonian University. 在这里，我们描述了一个简单的方法，结合RNA荧光原位杂交（RNA-FISH）与免疫荧光可视化严重急性呼吸系统综合症冠状病毒2（SARS-CoV-2）RNA。该协议可以增加对SARS-CoV-2 RNA-主机在单细胞水平上的分子特征的理解。

视频: Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

科研

免疫与感染

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

使用假病毒感染的高通量荧光成像检测SARS-CoV-2中和抗体

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

基于多路复用珠子的流量分析系统的快速、多路双记者 IgG 和 Igm SARS-CoV-2 中和检测

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

使用基于HiBiT的生物报告器检测SARS-CoV-2受体结合域抗体

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

通过荧光 原位 杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

使用报告基因表达重组SARS-CoV-2对K18 hACE2转基因小鼠的病毒感染进行实时成像和定量

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

利用开源移液机器人的高效SARS-CoV-2定量逆转录酶PCR唾液诊断策略

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

定量 SARS-CoV-2 变体中疫苗诱导的中和抗体

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

作者聚焦：用于评估 SARS-CoV-2 研究中 Omicron 亚变体和中和抗体的假型病毒系统

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

Ha-CoV-2假病毒在SARS-CoV-2变异株和中和抗体快速定量中的应用

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...