这项工作全部或部分得到了美国国立卫生研究院的支持（R01 AI121315，R01 AI146017，R01 AI148382，R01 AI120860，R37 AI076119和U54 AI150472）。我们感谢Raymond F. Schinazi博士和Sadie Amichai提供感染甲型流感病毒的细胞。

1.盖玻片或腔室载玻片上的种子细胞（悬浮液 与 贴壁细胞）（提供的方案使用盖玻片）。
<ol><li>悬浮细胞的接种<ol><li>通过首先将盖玻片在乙醇（EtOH）中孵育5分钟（灭菌盖玻片并去除任何残留物）来制备聚-D-赖氨酸（PDL）包被的盖玻片（促进悬浮细胞粘附到盖玻片上）。然后在磷酸盐缓冲盐水（PBS）中洗涤2x，然后在室温（RT）下将盖玻片在PDL（20μg/ mL）中孵育30分钟（分钟）。</li>
		<li>去除 PDL 并在 PBS 中洗涤 2 次。沉淀细胞（先前根据所需的成像条件感染/处理）并将 106 个细胞重悬于 50 μL PBS 中。在玻璃上点缀 50 μL 细胞（PDL 包衣），并在室温下孵育 30 分钟。</li>
	</ol></li>
	<li>贴壁细胞的接种<ol><li>将细胞放在无菌盖玻片上培养，置于6孔培养皿中，用病毒颗粒感染细胞或用感兴趣的化合物处理。在成像实验的样品制备之前，让细胞达到50-70%的汇合度。</li>
	</ol></li>
</ol>2. 细胞固定:保存细胞形态以进行荧光成像研究
注意:在细胞固定前，将 4% PFA 和 PBS 保持在室温至少 30 分钟。
<ol><li>吸出细胞培养基，在PBS中在盖玻片上洗涤细胞3次，并将细胞固定在4%多聚甲醛（PFA）中30分钟。吸出PFA并在PBS 2x中洗涤细胞。 注意:如果实验者希望在另一个日期恢复实验，细胞现在可以脱水和储存。固定细胞在储存前可以在EtOH中脱水。<ol><li>细胞固定后洗涤后去除PBS，并用500μL的50%EtOH（水中v / v）替换。在室温下孵育5分钟。</li>
		<li>去除 50% 的 EtOH，并用 500 μL 的 70% EtOH 代替。在室温下孵育5分钟。</li>
		<li>去除 70% 乙醇，并用 500 μL 100% 乙基醇代替。在室温下孵育5分钟。</li>
		<li>去除100%环氧乙烷，并用新鲜的100%乙醇代替。 注意:脱水细胞可以在-20°C下储存6个月。储存前用胶带或封口膜密封板，以防止乙二醇蒸发。</li>
		<li>对细胞进行再水化以转移到细胞/病毒的多重荧光标记上。</li>
		<li>去除 100% 乙醇，并用 500 μL 70% 乙基醇代替。在室温下孵育2分钟。 注意:任何时候都不要让细胞变干。始终使用足够的溶液来淹没所有细胞。</li>
		<li>去除 70% 的 EtOH，并用 500 μL 的 50% EtOH 代替。在室温下孵育2分钟。</li>
		<li>去除 50% 的 EtOH，并用 1x PBS 代替。 注意:在蛋白酶处理（步骤4） 和目标探针应用（步骤5）之前准备蛋白酶稀释液，杂交探针和洗涤缓冲液。有关试剂制备的具体说明在每个相应步骤的开头提供。</li>
	</ol></li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>试剂</td>
			<td>其他注意事项</td>
		</tr><tr><td>蛋白酶溶液</td>
			<td>在 1X PBS 中制备</td>
		</tr><tr><td>目标寡核探针</td>
			<td>在杂交缓冲液中稀释</td>
		</tr><tr><td>洗涤缓冲液</td>
			<td>按照靶标杂交步骤洗涤</td>
		</tr><tr><td>杂交缓冲液</td>
			<td>配方如表3所示</td>
		</tr></tbody></table>表 1:MICDDRP 方案中的关键试剂。
3. 细胞透化:增加大分子（抗体、核酸杂交探针）进入细胞和细胞器的通道
<ol><li>细胞固定或补液后取出 1x PBS，并在 1x PBS 中用 500 μL 的 0.1% （v/v） 吐温-20 替换。在室温下孵育10分钟。逐个替换。用 500 μL 1x PBS 洗涤一次，然后加入新鲜的 1x PBS。</li>
</ol>4. 玻片盖 玻片 固定和 蛋白酶处理 ，去除固定病毒/细胞核酸中的核酸结合蛋白，提高杂交效率
注意:在细胞透化过程中制备稀释的蛋白酶III（参见 材料表中的试剂细节）溶液。让蛋白酶在透化前达到室温10分钟。
<ol><li>将一小滴指甲油放在无菌载玻片上。干燥背部（没有细胞层的一面），并将盖玻片的边缘放在指甲油滴上，粘附细胞朝上的一侧朝上。在固定的盖玻片上加入几滴PBS以防止干燥。<ol><li>使用疏水屏障笔（使试剂定位在细胞上的憎水笔）在现在粘附在载玻片上的盖玻片周围画一个圆圈（距离盖玻片约 3 毫米）。</li>
	</ol></li>
	<li>在 1x PBS（100 μL/盖玻片）中稀释蛋白酶 III。 注意:蛋白酶浓度可能需要根据细胞类型、探针或靶核酸的差异通过经验优化进行调整（参见 讨论）。为了在不同病毒系统中实现最有效的RNA / DNA标记，我们成功地进行了下表2所示的以下稀释，稀释范围为（1至 2 ）-（1至15）（蛋白酶至1x PBS）。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>探测目标</td>
			<td>蛋白酶稀释在 1X PBS 中（蛋白酶 III 至 1X PBS）</td>
		</tr><tr><td>HIV-1 DNA/RNA</td>
			<td>1 到 5</td>
		</tr><tr><td>HTLV-1 脱氧核糖核酸/核糖核酸</td>
			<td>1 到 5</td>
		</tr><tr><td>乙肝病毒核糖核酸和总乙肝病毒核糖核酸</td>
			<td>1 到 15</td>
		</tr><tr><td>亚病毒核酸核糖核酸</td>
			<td>1 到 15</td>
		</tr><tr><td>齐克病毒核糖核酸</td>
			<td>1 至 2</td>
		</tr></tbody></table>表2:用于病毒核酸杂交的PBS中的蛋白酶III稀释液。
<ol start="3"><li>固定后在盖玻片上倒出1x PBS，并应用稀释的蛋白酶III。</li>
	<li>在40°C的加湿烤箱中孵育15分钟。</li>
	<li>倒出蛋白酶溶液并将载玻片浸没在 1x PBS 中。在室温下用摇摆盘搅拌2分钟，用新的1x PBS重复洗涤。<ol><li>对于仅DNA检测，用无核酸酶水洗涤样品三次，每次2分钟，然后用在PBS中稀释的5mg / mL RNase A在37°C下孵育30分钟。</li>
		<li>倒出RNase A溶液，用超纯水洗涤3次2分钟。继续对目标探针进行 ISH。 注意:杂交缓冲液可在不影响所提供结果的vRNA染色效率的情况下改进vDNA检测（代表性结果）。用杂交探针 1:1 稀释 DNA 通道 1 （C1） 探针。在杂交缓冲液中稀释RNA C2和C3探针。我们的成像研究中使用的C2和C3探针采用50X溶液（1:50;靶探针 到 杂交缓冲液）。</li>
	</ol></li>
	<li>按照以下分步程序在无核酸酶水中制备杂交缓冲液:<ol><li>在 15 mL 管中，加入 700 μL 无核酸酶水、300 μL 50%（重量/体积 （w/v））葡聚糖硫酸盐、300 μL 5 M NaCl、125 μL 200 mM 柠檬酸钠 （pH 6.2） 和 375 mg（粉末）碳酸乙烯酯。</li>
		<li>使用涡旋充分混合以溶解碳酸乙烯酯（确保所有粉末溶解并澄清溶液）。</li>
		<li>加入 25 μL 10%（体积/体积 （v/v））吐温-20 和足够的无核酸酶水以完成 2.5 mL（2x 溶液）。 注意:所示杂交缓冲液的配方可在下面的 表3 中找到。溶液稳定一周。确保吐温-20洗涤剂充分混合，同时注意防止溶液起泡。</li>
	</ol></li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>试剂</td>
			<td>库存集中度</td>
			<td>附加说明</td>
		</tr><tr><td>无核酸酶水</td>
			<td>那</td>
			<td></td>
		</tr><tr><td>葡聚糖硫酸盐</td>
			<td>50% （w/v）</td>
			<td>非常粘稠</td>
		</tr><tr><td>氯化钠</td>
			<td>5 米</td>
			<td></td>
		</tr><tr><td>柠檬酸钠（pH 6.2）</td>
			<td>200 毫米</td>
			<td>储存在4°C</td>
		</tr><tr><td>碳酸乙烯酯</td>
			<td>那</td>
			<td>粉</td>
		</tr><tr><td>吐温-20</td>
			<td>10% （V/V）</td>
			<td></td>
		</tr></tbody></table>表3:试剂杂交缓冲液列表。
5. 与 DNA/RNA 靶标杂交探针孵育:靶寡核苷酸探针与目标区域结合，形成用于前置放大器、放大器和荧光探针结合的分子支架。
注意:如果没有包括RNase处理，则在40°C下加热DNA / RNA探针10分钟（在细胞透化期间），并冷却至RT至少10分钟。如果在靶标探针杂交之前需要RNase处理或进一步的样品处理，请相应地加热探针。升温后旋转C2和C3探针，并在杂交缓冲液中稀释。稀释后，可以短暂加热C2和C3探头。在40°C下温热50x洗涤缓冲液（详见 材料表 ）10-20分钟，并在分子生物学级水中稀释至1x。
<ol><li>在加入杂交探针之前，将 200 μL 杂交缓冲液在 67 °C 下孵育 10 分钟。在杂交缓冲液中稀释探针（ 配方如表2所列）。加入 50 μL/盖玻片。</li>
	<li>在40°C的加湿炉中孵育2小时（h）。倾析探针并将载玻片浸没在 1x 洗涤缓冲液中。在室温下摇动培养皿搅拌2分钟，用新的1x洗涤缓冲液重复洗涤。</li>
</ol>6. 扩音器（放大器）1-FL 杂交:添加与靶 DNA/RNA 探针尾序列互补的前置扩音器（步骤 5）
注意:放大器在使用前应处于室温状态。使用前 30 分钟将每个单独的放大器从冰箱中取出，然后放在室温工作台上。
<ol><li>从 1x 洗涤缓冲液中取出载玻片并轻拍/吸收以去除多余的液体。</li>
	<li>在盖玻片上加入 1 滴 Amp 1-FL。在40°C的加湿炉中孵育30分钟。</li>
	<li>倒出放大器 1-FL 并浸没在 1x 洗涤缓冲液中。在室温下摇动培养皿2分钟搅拌，用新的1x洗涤缓冲液重复洗涤。</li>
</ol>7. Amp 2-FL 杂交:与具有同源识别序列的信号放大器一起孵育到前置放大器 （Amp 1-FL）
<ol><li>从 1x 洗涤缓冲液中取出载玻片并轻拍/吸收以去除多余的液体。</li>
	<li>在盖玻片上加入 1 滴 Amp 2-FL。在40°C的加湿炉中孵育15分钟。</li>
	<li>倒出安培 2-FL 并浸没在 1x 洗涤缓冲液中。在室温下摇动培养皿2分钟搅拌，用新的1x洗涤缓冲液重复洗涤。</li>
</ol>8. Amp 3-FL 杂交:与第二个信号放大器一起孵育
<ol><li>从 1x 洗涤缓冲液中取出载玻片并轻拍/吸收以去除多余的液体。</li>
	<li>在盖玻片上加入 1 滴 Amp 3-FL。在40°C的加湿炉中孵育30分钟。</li>
	<li>倾析放大器 3-FL 并浸没在 1x 洗涤缓冲液中。在室温下摇动培养皿2分钟搅拌，用新的1x洗涤缓冲液重复洗涤。</li>
</ol>9. Amp 4-FL杂交:荧光标记和最终杂交步骤
注意:首先，请参阅表 4，根据目标探头的通道选择合适的 Amp 4（A、B 或 C）- FL。评估标记目标 DNA/RNA 所需的 Amp 4-FL。下表提供了一个示例:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td>
			<td>一个</td>
			<td>B</td>
			<td>C</td>
		</tr><tr><td>DNA 通道 1 （C1）</td>
			<td>488</td>
			<td>550</td>
			<td>550</td>
		</tr><tr><td>DNA/RNA 通道 2 （C2）</td>
			<td>550</td>
			<td>488</td>
			<td>647</td>
		</tr><tr><td>核糖核酸通道 3 （C3）</td>
			<td>647</td>
			<td>647</td>
			<td>488</td>
		</tr></tbody></table>表 4:用于多重 ISH 的 Amp 4（A、B 或 C）-FL 荧光探针的选择。
注意:对于多路复用 FISH（多个靶标），选择正确的 Amp 4（A、B 或 C）- FL 对于正确标记感兴趣的靶标至关重要。目标探针（步骤5）具有不同的颜色通道（C1、C2或C3），这些通道根据所选的Amp 4-FL决定其各自的荧光标记（Alexa 488，Atto 550或Alexa 647）。选择荧光团组合进行多重成像的示例如图 2 和 图3所示。作为另一个示例，选择 Amp 4B-FL 将选择性地使用 Atto 550 标记 DNA C1 探针，使用 Alexa 647 标记 RNA C3 探针。
<ol><li>从 1x 洗涤缓冲液中取出载玻片并轻拍/吸收以去除多余的液体。</li>
	<li>在盖玻片上加入 1 滴 Amp 4-FL。在40°C的加湿炉中孵育15分钟。 注意:按照步骤9.2，盖住样品，避光。</li>
	<li>倾析放大器 4-FL 并浸没在 1x 洗涤缓冲液中。在室温下摇动培养皿搅拌2分钟，用新的1x洗涤缓冲液重复洗涤。用 1x PBS（2 分钟）清洗并存放在 PBS 中。</li>
</ol>10. 蛋白质免疫染色:标记感兴趣的蛋白质
<ol><li>倒出 PBS 并向盖玻片中加入 200 μL 封闭缓冲液（1% w/v BSA，10% v/v FBS 在 PBS 中，0.1% v/v 吐温-20 （PBST））。在室温下孵育1小时。</li>
	<li>倒出封闭缓冲液并应用在PBST + 1% w/v BSA中稀释的200 μL一抗。在室温下孵育1小时。</li>
	<li>用PBST在室温下摇动洗涤载玻片两次10分钟。</li>
	<li>在室温下以PBST + 1% w / v BSA应用所选的二抗1小时。</li>
	<li>用PBST在室温下摇动洗涤载玻片10分钟。</li>
</ol>11. 核染色:免疫染色后的复染细胞核
<ol><li>倒出PBST并在室温下应用DAPI或所选的核染色剂1分钟。</li>
	<li>用PBS在室温下摇动洗涤载玻片两次10分钟。</li>
</ol>12. 安装
<ol><li>将 1 滴抗淬灭溶液（例如 Prolong Gold）放在新的无菌载玻片上（首先，用 EtOH 清洁载玻片并晾干以确保玻璃上没有残留物）。使用相同的尖端，铺开抗淬灭溶液滴剂以覆盖大约盖玻片大小的区域。 注意:防淬灭溶液非常粘稠，可能难以移液。在移液前切断 200 μL 吸头可能会缓解这些问题。</li>
	<li>从载玻片上取下带有细胞样品的盖玻片，然后浸入PBS中，以使用镊子和PBS去除背部残留的指甲油。使用Kimwipe擦干镊子和盖玻片的背面。</li>
	<li>将盖玻片轻轻嵌入抗淬灭溶液液滴中，将盖玻片的样品侧（带有细胞层的一侧）放在液滴上）。</li>
	<li>让样品干燥过夜。</li>
</ol>13. 成像
<ol><li>用落射荧光显微镜成像。</li>
</ol>

<ol>
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作者没有什么可透露的。

同时可视化 RNA、DNA 和蛋白质通常需要广泛的优化。两种常用的方法是5-乙炔基-2-脱氧尿苷（EdU）标记和DNA FISH。EdU标记已被应用于同时可视化病毒DNA和蛋白质，因为EdU被掺入新生DNA中，随后通过点击化学 用 含叠氮化物的荧光染料标记。因此，EdU标记可用于监测DNA病毒的天然病毒复制动力学或具有DNA模板的病毒进行复制12。然而，EdU标记的一个缺点是，在分裂细胞中，复制基因组将结合EdU，产生高背景和混杂图像分析。DNA FISH可以通过将核酸探针直接杂交到相应的靶标来规避这些问题，而不管细胞周期如何。然而，传统的FISH通常依靠高温来实现高效的探针杂交，阻碍了免疫染色甚至同时进行RNA染色13。MICDDRP可以潜在地规避这些问题，在各种细胞系统中对DNA，RNA和蛋白质进行强大的同时荧光标记。
虽然我们已经证明可以使用MICDDRP方案同时标记蛋白质和核酸，但需要跨不同系统进行优化。我们必须优化的第一个主要参数是蛋白酶处理。我们在不同的条件下改变了蛋白酶III浓度。蛋白酶处理的优化是经验性的，因为我们使用不同的稀释度来评估产生最大杂交效率的方法，而不会影响免疫染色效率。并排进行适当的对照，以评估探针特异性和蛋白酶处理引起的蛋白质染色效率的变化。接下来需要优化的主要参数是探针设计和探针杂交。
捕获和扩增探针的正确设计对于实现bDNA技术的灵敏度和特异性至关重要。预测非特异性杂交事件概率的软件包可用于改进探针设计7，11。bDNA探针以及随附的前置放大器、放大器和荧光标记探针现在可以商业购买，以确保与bDNA成像试剂盒的兼容性。用户可以以fasta文件的形式为制造商提供目标区域的序列信息（~300-1000个碱基对），用于表示核苷酸序列的基于文本的格式。生成的靶探针与提供的序列>90%的序列同源性。
对于DNA标记，我们发现在协议步骤5中描述的杂交缓冲液中稀释探针可改善DNA杂交。当标记DNA和RNA时，RNA探针可以在杂交缓冲液中稀释。在没有RNase的情况下进行DNA标记不能排除观察到的核酸包含靶向链RNA的可能性。可能还必须调整温度以提高杂交效率。温度升高可能会影响蛋白质染色效率，因为温度升高可能会促进蛋白质变性，从而消融一抗的表位识别。在我们提供的代表性数据中，我们在40°C下进行了ISH。
与传统的DNA鱼相比，MICDDRP提供了一种改进的程序，可以同时标记DNA，RNA和蛋白质，以通过荧光显微镜 进行 可视化。一个潜在的限制是探针的选择可能会影响杂交效率和定量比较探针之间数据的能力。该协议在我们手中的不同细胞和病毒系统中有效，在不同条件下只需要进行微小的优化。最近的知名出版物利用我们的方法来研究HIV整合位点选择14 和HIV逆转录动力学15。MICDDRP的未来应用可能包括病毒核酸的可视化以及特定细胞基因和细胞蛋白的核酸序列。

虽然数以千计的商业抗体可用于通过常规免疫染色方法特异性标记蛋白质，并且虽然融合蛋白可以使用光优化荧光标签进行工程设计以跟踪样品中的多种蛋白质1，但蛋白质的显微可视化通常与传统的DNA荧光原位杂交（FISH）不兼容2.使用基于荧光的方法同时可视化DNA、RNA和蛋白质的技术限制阻碍了对病毒复制的深入了解。在感染过程中跟踪病毒核酸和蛋白质使病毒学家能够可视化病毒复制和组装的基本过程3，4，5，6。
我们开发了一种成像方法，即基于d na，rNA和Protein的成像方法m ultiplex immunofluorescent cell（MICDDRP）3，该方法利用支链DNA（bDNA）原位技术来提高核酸检测的灵敏度7，8，9.此外，该方法利用成对探针来增强特异性。bDNA序列特异性探针使用分支前置放大器和放大器DNA产生强烈的局部信号，改进了以前的杂交方法，该方法依赖于靶向DNA9中的重复区域。临床环境中的受感染细胞通常不含丰富的病毒遗传物质，为诊断环境中荧光核酸检测的灵敏方法提供了商品。通过RNAscope7和ViewRNA10等方法实现bDNA技术的商业化填补了这一空白。bDNA荧光成像的灵敏度在细胞生物学中也具有重要的用途，可以检测细胞培养模型中稀缺的核酸种类。灵敏度的巨大提高使基于bDNA的成像方法适用于研究病毒。然而，一个潜在的缺点是这些方法专注于可视化RNA或RNA和蛋白质。所有复制细胞和许多病毒在其复制周期中都具有DNA基因组或形成DNA，因此非常需要能够对RNA和DNA以及蛋白质进行成像的方法。
在MICDDRP协议中，我们使用RNAscope方法进行bDNA FISH以检测病毒核酸，并修饰7。该方案的主要修改之一是化学固定后蛋白酶处理的优化。蛋白酶处理有助于去除与核酸结合的蛋白质，从而提高探针杂交效率。蛋白酶处理后用支链寡核苷酸探针孵育。应用bDNA探针后，洗涤样品，随后与信号前置放大器和放大器DNA一起孵育。多重原位杂交（ISH），标记多个基因靶标，需要具有不同颜色通道的靶标探针进行光谱分化7。用DNA扩音器孵育后进行免疫荧光（IF）。
bDNA ISH通过放大靶标特异性信号来改善信噪比，并减少非特异性杂交事件的背景噪声7，11。靶探针是使用公开可用的软件程序设计的，这些软件程序预测非特异性杂交事件的概率，并计算探针-目标杂交7，11的熔解温度（Tm）。靶探针包含一个与靶DNA/RNA序列互补的18至25碱基区域、一个间隔序列和一个14个碱基的尾部序列。一对靶探针，每个探针具有不同的尾序列，杂交到目标区域（跨越~50个碱基）。两个尾序列形成前置放大器探针的杂交位点，其中包含20个放大器探针的结合位点，此外还包含20个标记探针的结合位点。例如，核酸分子上的一千碱基（kb）区域被20个探针对靶向，从而创建一个分子支架，用于与前置放大器、放大器和标记探针进行顺序杂交。因此，这可以使每个核酸分子的理论产量达到8000个荧光标记，从而能够检测单个分子，并且比传统的FISH方法7有了巨大的改进（bDNA信号扩增示意图见图1A）。要为多路复用 ISH 设置探针，每个目标探针必须位于不同的颜色通道（C1、C2 或 C3）中。这些具有不同颜色通道的目标探针具有不同的14碱基尾序列。这些尾部序列会将不同的信号放大器与不同的荧光探针结合，从而实现跨多个目标的光谱分化。在所提出的方案中，步骤9中的表4提供了有关荧光标记靶探针的更多信息。此外，图2和图3提供了我们如何选择合适的放大器4-FL（A，B或C）（荧光探针和最终杂交步骤）的示例，以实现HIV-1和HTLV-1感染后多个病毒核酸靶标的特异性荧光标记。
我们已经展示了同时对RNA，DNA和蛋白质进行荧光可视化的几种应用，以高时空分辨率3，4，5观察病毒复制的关键阶段。例如，病毒RNA，细胞质和核DNA以及蛋白质的同时单细胞可视化使我们能够可视化HIV-1感染期间的关键事件，包括在核进入和前病毒DNA3整合之前跟踪细胞质中含有核心的RNA。此外，我们还应用MICDDRP来表征宿主因子和药物治疗对病毒感染和复制的影响4，5。在Ukah等人中，我们跟踪了用不同潜伏逆转剂处理的潜伏期细胞模型中HIV-1转录的再激活，以可视化HIV转录和潜伏期逆转4。此外，MICDDRP可以让我们可视化与小分子治疗或宿主因子限制引起的抗病毒抑制相关的表型变化。作为我们方法稳健性和广泛适用性的概念证明，我们已经证明，我们可以使用我们的方案的修改来有效地标记病毒核酸，以跟踪感染，不仅在人类免疫缺陷病毒（HIV）-1，而且在人类嗜T淋巴细胞病毒（HTLV）-1，乙型肝炎病毒（HBV），丙型肝炎病毒（HCV）， 寨卡病毒（ZIKV）和甲型流感病毒（IAV）。由于HIV-1生命周期由病毒DNA和RNA物种组成，因此我们已经根据HIV-1复制动力学对MICDDRP进行了大部分优化。然而，此外，我们已经证明，我们可以跟踪ZIKV，IAV，HBV和HCV等病毒中或两种感觉（+）和反义（-）链的不同病毒RNA转录本的合成，以监测病毒转录和复制3，4，5，6。这些研究旨在提高对几种病毒过程的机制理解，并作为将这种荧光成像技术应用于各种细胞模型的指南。

<table><tbody><tr><td>4% PFA</td><td></td><td></td><td></td></tr><tr><td>50% dextran sulfate</td><td>Amreso</td><td>198</td><td>For DNA hybridization buffer</td></tr><tr><td>50X wash buffer</td><td>ACD Bio</td><td>320058</td><td></td></tr><tr><td>6-well plates</td><td></td><td></td><td></td></tr><tr><td>Amplifier 1-FL</td><td>ACD Bio</td><td></td><td></td></tr><tr><td>Amplifier 2-FL</td><td>ACD Bio</td><td></td><td></td></tr><tr><td>Amplifier 3-FL</td><td>ACD Bio</td><td></td><td></td></tr><tr><td>Amplifier 4-FL</td><td>ACD Bio</td><td></td><td>Consult Amp-4 table in protocol</td></tr><tr><td>Anti-HCV NS5a antibody</td><td>Abcam</td><td>ab13833</td><td>Mouse monoclonal; works with HCV genotypes 1a, 1b, 3, and 4</td></tr><tr><td>Anti-HIV-1 p24 monoclonal antibody</td><td>NIH AIDS Reagent Program</td><td>3537</td><td></td></tr><tr><td>Anti-Mov10 antibody</td><td>Abcam</td><td>ab80613</td><td>Rabbit polyclonal</td></tr><tr><td>Anti-PB1 antibody</td><td>GeneTex</td><td>GTX125923</td><td>Antibody against flu protein</td></tr><tr><td>Bovine serum albumin</td><td></td><td></td><td>Blocking reagent for immunostaining</td></tr><tr><td>Cell media with supplements</td><td></td><td></td><td>Media appropriate for cell model</td></tr><tr><td>Coverslips</td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>ACD Bio</td><td></td><td>Nuclear stain (RNAscope kit from ACD Bio)</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (1X PBS)</td><td>Gibco</td><td>14190250</td><td>No calcium and magnesium</td></tr><tr><td>Ethylene carbonate</td><td>Sigma</td><td>E26258</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td></td><td></td><td>Use specific FBS based on what serum secondary antibody was raised in (e.g goat FBS)</td></tr><tr><td>Fisherbrand colorfrost plus microscope slides</td><td>Fisher Scientific</td><td>12-550-17/18/19</td><td>Precleaned</td></tr><tr><td>HCV-GT2a-sense-C2 probe</td><td>ACD Bio</td><td>441371</td><td>HCV(+) sense RNA probe</td></tr><tr><td>HIV-gagpol-C1</td><td>ACD Bio</td><td>317701</td><td>HIV-1 cDNA probe</td></tr><tr><td>HIV-nongagpol-C3</td><td>ACD Bio</td><td>317711-C</td><td>HIV-1 RNA probe</td></tr><tr><td>HybEZ hybridization oven</td><td>ACD Bio</td><td>321710/321720</td><td></td></tr><tr><td>ImmEdge hydrophobic barrier pen</td><td>Vector Laboratories</td><td>H-4000</td><td></td></tr><tr><td>Nail polish</td><td></td><td></td><td>For immobolizing coverslip to slide prior to protease treatment</td></tr><tr><td>Nuclease free water</td><td>Ambion</td><td>AM9937</td><td></td></tr><tr><td>Poly-d-lysine (PDL)</td><td></td><td></td><td>Coat coverslips in 20 &amp;micro;g/mL of PDL for 30 minutes</td></tr><tr><td>Probe diluent</td><td>ACD Bio</td><td>300041</td><td>For diluting RNA C2 or C3 probes</td></tr><tr><td>Prolong gold antifade</td><td>Invitrogen</td><td>P36930</td><td></td></tr><tr><td>Protease III</td><td>ACD Bio</td><td>322337</td><td></td></tr><tr><td>RNAscope&amp;reg;&amp;nbsp;Probe- V-Influenza-H1N1-H5N1-NP</td><td>ACD Bio</td><td>436221</td><td></td></tr><tr><td>RNase A</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Secondary antibodies</td><td></td><td></td><td></td></tr><tr><td>Slides</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td></td><td></td><td>For DNA hybridization buffer</td></tr><tr><td>Sodium citrate, pH 6.2</td><td></td><td></td><td>For DNA hybridization buffer</td></tr><tr><td>Tween-20</td><td></td><td></td><td>For DNA hybridization buffer and PBS-T</td></tr><tr><td>V-HBV-GTD</td><td>ACD Bio</td><td>441351</td><td>Total HBV RNA</td></tr><tr><td>V-HBV-GTD-01-C2</td><td>ACD Bio</td><td>465531-C2</td><td>HBV pgRNA probe</td></tr><tr><td>V-HCV-GT2a probe</td><td>ACD Bio</td><td>441361</td><td>HCV(-) sense RNA probe</td></tr><tr><td>V-HTLV-HBZ-sense-C3</td><td>ACD Bio</td><td>495071-C3</td><td>HTLV-1 (-) sense RNA probe targetting HBZ</td></tr><tr><td>V-HTLV1-GAG-C2</td><td>ACD Bio</td><td>495051-C2</td><td>HTLV-1 DNA probe</td></tr><tr><td>V-HTLV1-GAG-POL-sense</td><td>ACD Bio</td><td>495061</td><td>HTLV-1 (+) sense RNA probe</td></tr><tr><td>V-Influenza-H1N1-H5N1-NP</td><td>ACD Bio</td><td>436221</td><td>IAV RNA probe</td></tr><tr><td>V-ZIKA-pp-O2</td><td>ACD Bio</td><td>464531</td><td>Zika(+) sense RNA probe</td></tr><tr><td>V-ZIKA-pp-O2-sense-C2</td><td>ACD Bio</td><td>478731-C2</td><td>Zika(-) sense RNA probe</td></tr></tbody></table>

single-cell multiplexed fluorescence imaging to visualize viral nucleic acids and proteins and monitor hiv, htlv, hbv, hcv, zika virus, and influenza infection

由于缺乏能够灵敏和同时标记病毒核酸和蛋白质的强大原位杂交（ISH）技术，捕获病毒的动态复制和组装过程受到了阻碍。传统的DNA荧光原位杂交（FISH）方法通常与免疫染色不兼容。因此，我们开发了一种成像方法 MICDDRP （multiplex immunofluorescent c ell-basedd etection of D NA， RNA和 Protein），该方法能够同时进行DNA，RNA和蛋白质的单细胞可视化。与传统的DNA鱼相比，MIDDRP采用支链DNA（bDNA）ISH技术，可显著提高寡核苷酸探针的灵敏度和检测。MICDDRP的微小修饰能够同时对病毒蛋白进行成像，同时具有不同链的核酸（RNA或DNA）。我们已经应用这些协议来研究多种病毒病原体的生命周期，包括人类免疫缺陷病毒（HIV）-1，人类嗜T淋巴细胞病毒（HTLV）-1，乙型肝炎病毒（HBV），丙型肝炎病毒（HCV），寨卡病毒（ZKV）和甲型流感病毒（IAV）。我们证明了我们可以有效地标记各种病毒中的病毒核酸和蛋白质。这些研究可以为我们提供对多种病毒系统的机理理解，此外，还可以作为在广泛的细胞系统中应用DNA，RNA和蛋白质的多重荧光成像的模板。

MICDDRP的示意图如图 1所示。标记DNA和RNA之后进行免疫染色。支化放大器的使用增加了信号，允许检测单个核酸分子。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61843/61843fig1v5.jpg"> 图 1:MICDDRP 和分步工作流程示意图。 （A）bDNA信号通过分支前置放大器和放大器DNA放大，以增强对病毒DNA（1）和RNA物种（2）的检测。寡核苷酸探针成对（原理图中的ZZ）杂交到感兴趣的靶标，为前置放大器（Amp 1-FL，协议中的步骤6），放大器（Amp 2-和3-FL）和荧光探针（Amp 4，协议中的步骤9）创建支架。请参阅表 4，为多路复用 ISH 选择合适的放大器 4（A、B 或 C）-FL。标记核酸后对感兴趣的蛋白质进行免疫染色（3）。（B）MICDDRP协议中的13个主要步骤，并估计每个步骤的持续时间。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61843/61843fig1v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
应用MICDDRP研究HIV-1感染过程一直是跟踪原代细胞中病毒复制动力学的有用工具。作为该程序的概念证明，HIV-1 DNA，RNA和蛋白质在单细胞水平上同时进行标记和显微镜可视化（图2）。两个HIV-1 DNA基因组在单个细胞中可视化，因为它们正在积极转录病毒RNA（vRNA）。vRNA已通过核孔复合物输出，病毒蛋白在细胞质中合成。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61843/61843fig02.jpg"> 图2:感染HIV-1的原代血单核细胞（PMBC）的MICDDRP。 PMBC感染HIV-1（NL4.3）的多重感染（MOI）为2。细胞在感染后48小时固定（hpi）。（A）HIV-1 bDNA FISH探针（用ATTO 550标记;图中为红色）。该探针与模板（3'<—5'）vDNA链杂交，以防止与意义（+）vRNA串扰。（B）未剪接的HIV-1 RNA（用Alexa 647标记;图中绿色）。HIV-1 vRNA探针与以5'—>3'方向转录的病毒转录物杂交。（C）HIV-1衣壳（p24）蛋白（与Alexa 488偶联的二抗;图中白色）的免疫染色。（D） 合并图像。比例尺代表 5 μm。细胞核用DAPI（蓝色）染色。为了分别用ATTO 550标记HIV-1 DNA（DNA C1探针）和用Alexa 647标记HIV-1 RNA（RNA C3探针），将样品与Amp 4-FL 'B'一起孵育（参见协议中的表4，为杂交探针选择合适的通道颜色）。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61843/61843fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
此外，我们还进行了双病毒DNA（vDNA）和vRNA染色，以跟踪HTLV-1感染。为了优化核酸标记，我们严格遵守为HIV感染的多重荧光成像开发的vDNA / VRNA染色程序。我们已经证明，我们可以同时特异性地标记HTLV-1 DNA和RNA。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61843/61843fig3v2.jpg"> 图 3:MT-2 细胞中 HTLV-1 vDNA 和 vRNA 的同时标记。 （A）HTLV-1 vDNA（用ATTO 550标记;图中为红色）（B）未剪接HTLV-1感觉（+）RNA（RNA C2探针并用Alexa 488标记;图中为绿色））。（C）HTLV-1 HBZ反义（-）vRNA（RNA C3探针和（用Alexa 647标记;图中白色））。（D） 合并图像。比例尺代表 20 μm。细胞核用DAPI（蓝色）染色。使用Amp 4-FL 'B'（参见协议中的表4）来实现HTLV-1（"+"和"-"）RNA的多重标记。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61843/61843fig3v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61843/61843fig04.jpg"> 图 4:用于评估靶探针特异性和背景的对照实验。 在淋巴细胞系（未感染的Jurkat T细胞，HTLV-1感染细胞（MT2）和HIV-1感染细胞（H9111B））感染后评估HIV-1和HTLV-1靶探针的特异性。比例尺代表10μm。 （A）用HTLV-1 RNA探针处理细胞。（B）用HIV-1（+）RNA探针（C-D）处理细胞。进一步证明HTLV-1探针的特异性，与HIV-1无交叉反应性。 图4C 中的白色箭头表示HTLV-1 DNA（红色）。（E-F）。vRNA染色（绿色）+/-核糖核酸酶处理。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61843/61843fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
为了验证杂交探针的特异性并评估ISH标记方法如何影响蛋白质染色效率和整体背景，我们进行了关键控制，以确保实验的最高水平严谨性和可重复性。例如，在图4A-D中，我们验证了HIV-1和HTLV-1 vDNA和vRNA探针的特异性，因为我们显示两种病毒的探针组之间几乎没有交叉反应性。尽管两种逆转录病毒具有探针交叉反应性的潜力，但HIV-1探针仅对HIV-1遗传物质具有特异性，而不是HTLV-1。HTLV-1探头也有同样的趋势。此外，在图4F中，我们表明，如果我们在样品制备过程中对细胞进行RNA酶处理，我们可以消除vRNA染色。为了评估由于ISH（蛋白酶处理（协议中的步骤4和图1B）和杂交条件而导致蛋白质染色效率衰减的可能性，这可能导致表位识别消融或背景信号增加，我们证明了核斑点标记物sc-35的蛋白质染色效率在常规IF方法和MICDDRP协议期间的免疫染色中具有可比性。在传统的IF方案中，细胞用0.1%Triton X-100透化15分钟，而不是用0.1%吐温-20透化10分钟，后者用于MICDDRP协议（步骤3在协议和工作流程中，如图1B）。两种条件下的蛋白质染色（IF与MICDDRP）产生的信号比对照（无一抗的MICDDRP）大几个数量级，进一步证明了该协议产生的低背景和蛋白质表位的保存以实现有效的免疫染色。如前所述3进行每个细胞sc-35信号的平均综合荧光强度的图像定量（图5E）。对于显示的所有成像结果，我们确保了探针和抗体的特异性，并评估了由于我们的ISH方法而导致的任何可能的扰动或高于正常背景噪声。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61843/61843fig05v2.jpg"> 图 5:常规 IF 与 MICDDRP 后的蛋白质染色效率比较。细胞蛋白sc-35是核斑点的生物标志物，在Jurkat T细胞中进行了免疫染色。所有比例尺代表10μm。 （A）未感染的Jurkat T细胞接受MICDDRP方案，并用上面图 2 和 图4 中使用的HIV-1 DNA / RNA杂交探针处理。在免疫染色过程中，未添加一抗。未感染的Jurkat T细胞接受常规IF，标记sc-35（白色）。用0.1%Triton X-100透化细胞15分钟。（C）. 对HIV感染的Jurkat T细胞进行MICDDRP。vRNA被标记为绿色，vDNA被标记为红色，sc-35被标记为白色。（D）. HIV感染细胞中vRNA，vDNA和蛋白质标记（sc-35）的特写。（E）在不同免疫染色条件下每个sc-35细胞的平均积分荧光信号的定量。y 轴采用对数刻度。虚线表示未感染细胞的背景信号，这些细胞经过MICDDRP方案，其中未添加一抗。MICDDRP与传统 IF相比 ，MICDDRP后的信号没有显着差异。对500多个细胞进行采样以进行定量，以达到相应的条件。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61843/61843fig05v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
该方法还可用于研究可能包含也可能不包含用于病毒复制的DNA模板的RNA病毒。例如，可以设计链特异性bDNA探针来监测病毒（如HBV，HCV，IAV和ZIKV）中义（+）或反义（-）链RNA和不同vRNA物种的表达。在感染过程中不同RNA物种的可视化可以深入了解各种病毒系统的复制动力学。
根据HBV感染的时间过程，我们可以看到HBV基因组前RNA（pgRNA）和总HBV RNA的数量随时间增加。此外，我们同时对细胞宿主因子MOV10进行了免疫染色（图6）。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61843/61843fig06.jpg"> 图6:乙肝病毒。3E8细胞HBV感染的时间进程。每个细胞感染300个HBV基因组。病毒复制显示在三个时间点（24、48 和 72 hpi）。pgRNA（用ATTO 550标记;图中为红色），总HBV RNA（用Alexa 647标记;图中为绿色）和MOV10（与Alexa 488偶联的二抗;图中为白色）。细胞核用蓝色的DAPI染色。合并图像上的比例尺代表感染后数小时的 10 μm. hpi。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61843/61843fig06large.jpg" target="_blank">请点击此处查看此图的大图。</a>
使用60倍油浸物镜通过共聚焦显微镜进行成像。用于检测DAPI、Alexa 488、ATTO 550和Alexa 647的激发/发射带通波长分别设置为405/420-480、488/505-550、550/560-610和647/655-705 nm（图7、图8和图9）。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61843/61843fig7v2.jpg"> 图7:Huh-7.5.1细胞HCV感染的时间过程。 Huh-7.5.1细胞感染丙型肝炎病毒（HCV）Jc1 / Gluc2A，MOI为0.5。在指示的时间间隔内，将细胞固定并依次探测感觉（+）vRNA，反义（-）vRNA和NS5A（HCV蛋白）。细胞核用DAPI染色。来自每个时间点的代表性合并图像，以绿色显示（+）RNA（用Alexa 647标记）（-）红色RNA（用Atto 555标记），NS5A为白色（与Alexa 488偶联的二级山羊抗小鼠），以蓝色显示细胞核。比例尺代表 10 μm。下面的图像是从相应的时间点放大的剪切。HPI，感染后数小时。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61843/61843fig7v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61843/61843fig08.jpg"> 图 8:感染甲型流感病毒的 A549 细胞的链特异性 bDNA FISH 和免疫染色。 固定感染PR8 Flu A病毒的A549细胞并探测（A）IAV核蛋白（NP）RNA（用Alexa 488标记;图中绿色）和（B）IAV聚合酶蛋白（PB1）（二级山羊抗小鼠Atto 550;图中红色）和细胞核用DAPI（蓝色）染色。（C） 合并图像。比例尺代表 10 μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61843/61843fig08large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61843/61843fig9v2.jpg"> 图9:寨卡病毒（ZIKV）感染细胞中的链特异性bDNA FISH。 Vero细胞以0.1的MOI感染ZIKV。细胞固定在48 hpi。（A）同时对细胞进行感觉（+）vRNA（用Alexa 488标记;图中为绿色）和反义（-）vRNA（用Atto 550标记;图中为红色）染色。细胞核用DAPI染色。在（A）中，白框表示同时具有（+）和（-）vRNA的区域。插图呈现（+）vRNA（绿色）和稀缺（-）vRNA物种（红色）的特写。（B） 感觉 （+） vRNA （绿色）。（C）反义（-）vRNA（红色）。（A） 中的比例尺代表 10 μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61843/61843fig9v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infectio

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

这里介绍的是荧光成像方法的 协议，multiplex immunofluorescent cELL 基于d etection of DNA， RNA和 Protein（MICDDRP），该方法能够同时对不同类型和链的病毒蛋白和核酸进行荧光单细胞可视化。这种方法可以应用于各种系统。

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

single-cell-multiplexed-fluorescence-imaging-to-visualize-viral

Universidad San Sebastian

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JoVE Journal

Biology

2.7K 次观看.  Emory University School of Medicine. 这里介绍的是荧光成像方法的 协议，multiplex immunofluorescent cELL 基于d etection of DNA， RNA和 Protein（MICDDRP），该方法能够同时对不同类型和链的病毒蛋白和核酸进行荧光单细胞可视化。这种方法可以应用于各种系统。

视频: Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

科研

免疫与感染

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

生物化学

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

生物学

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

猕猴免疫缺陷病毒感染 CD4+ T 细胞单细胞定量的 mRNA 和表面蛋白的表达

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

使用多参数光伏平台对钙流入和HIV-1感染进行单细胞特征描述

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

<meta charset="utf8"></head><body>使用双报告基因和多重技术分析 SARS-CoV-2

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

<meta charset="utf8"></head><body>作者聚焦:通过新型免疫捕获和质谱技术推进抗病毒策略

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

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此视频中的章节

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

流感病毒的高通量检测方法

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位

猕猴免疫缺陷病毒感染 CD4⁺ T 细胞单细胞定量的 mRNA 和表面蛋白的表达

荧光检测病毒RNA

使用多参数光伏平台对钙流入和HIV-1感染进行单细胞特征描述

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位