This work was funded by both the&#160;Canadian Institutes of Health Research&#160;and&#160;Natural Sciences and the Engineering Research Council of Canada. M. Bisaillon is a Chercheur Boursier Senior from the Fonds de Recherche en Sant&#233; du Qu&#233;bec and also a member of the Centre de Recherche Clinique du Centre Hospitalier Universitaire de Sherbrooke. We thank Dr. Ralf Bartenschlager for the generous gift of the HCV replicon system. We also thank Dr. Charles Rice and Dr. Daniel Lamarre for kindly providing the hepatic cell line. We also want to thank Guillaume Tremblay for technical assistance.

1.细胞制备与处理<ol><li>在一个24孔板，把一个玻璃盖在孔的底部。用磷酸盐缓冲盐水（PBS）洗涤各孔。 </li><li>种子Huh-7细胞在每孔配套的HCV亚基因组复制到5×10 4个细胞的密度/孔。如果没有治疗做，细胞可以在更高的密度接种。培养基是Dulbecco氏改良的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），2mM L-谷氨酰胺，1mM丙酮酸钠和250毫克/毫升的G-418（以维持复制子）。 </li><li>在37℃和5％的CO 2。第二 ​​天，处理细胞以3微米的hLF的从人乳纯化。 </li></ol> 2.多聚甲醛/蔗糖制备（12％的PAF和12％蔗糖） <ol><li>把500个毫升的PBS在一个烧杯中，并加热它在20℃至30℃。溶解60克蔗糖的PBS中。溶解60克的多聚醛（PAF）在PBS /蔗糖溶液中。慢慢加入氢氧化钠2N（3至7毫升）中获得澄清溶液为止。 </li><li>调节pH至7.4，用HCl。完整体积至500ml，用PBS。通过重力的滤纸过滤器过滤。在使用前，稀释用PBS溶液至4％的PAF和4％蔗糖（寄存4℃，1周最大）的终浓度。 </li></ol> 3.免疫<ol><li>在所需的时间（0小时，2小时或24的治疗小时），洗涤细胞两次（1分钟），用PBS并用PBS / 4％PAF / 4％蔗糖固定20分钟，在室温。所述细胞通常是在30-40％汇合和1.5×10 5个细胞的使用。 </li><li>透化在PBS中的10％正常山羊血清（NGS）将细胞用0.15％的Triton X-100的PBS进行5分钟，在RT和块20分钟。 </li><li>稀释的感兴趣的蛋白质的一次抗体，在10％NGS（例如:主小鼠抗体hLF抗体稀释1:1000）。应用第一抗体的细胞。 </li><li> 4小时后在室温下或O /北，4℃，洗细胞三次，持续5分钟，用10％NGS。细胞染色与标记荧光染料稀释在10％NGS的二级抗体（例如:荧光染料具有488纳米连接于抗体抗小鼠1的激发波长:1,000）在RT在黑暗1小时。 </li><li>一次5分钟，用PBS洗涤细胞，并将细胞染色用4&#39;，6-二脒基-2-苯基吲哚（DAPI）（1微克/ ml）在室温15分钟在黑暗中。 </li><li> 5分钟两次，用PBS洗涤细胞。盖山在SlowFade安装介质眼镜通过显微镜可视化之前。这些细胞是现在可以通过荧光或共聚焦显微镜分析。 </li><li>使用不同的控制，以确保检测的特异性。例如，所有抗体可为了检测自体荧光被省略。第一抗体可以被省略，以确定由所述第二抗体的非特异性染色。最后，如​​果可能，控制不这样做的细胞或组织表达也可用于感兴趣的分子。 </li><li>可视化使用适当的荧光显微镜（epifluorescence的或共焦）和过滤器组适合于所使用的荧光标记的样品。幻灯片也可以存储在&lt;-20℃供以后检查一个滑动箱。 </li></ol> 4.荧光显微镜<ol><li>保持样品在黑暗中以防止光漂白。打开所选择的显微镜（萤光或共焦）的光源。 </li><li>打开显微镜。打开相机的显微镜上。把滑动显微镜上进行可视化。添加浸油在滑动以增加物镜的数值孔径。 </li><li>选择适当的过滤器。调整的重点和合适的目标。调节百叶窗以便检测适当的荧光染料，但防止光漂白。请顶灯关在看样品，以尽量减少背景光。 </li><li>变更日ë滤波器可视化的各种荧光染料（例如DAPI和为488纳米的激发波长下）。拍照与摄像头。 </li></ol>

<ol>
	<li>Odell, I. D., Cook, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+techniques.">Immunofluorescence techniques.</a> J Invest Derm. 133 (1), e4 (2013).</li><li>Portugal, J., Waring, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assignment+of+DNA+binding+sites+for+4&#8217;,6-diamidine-2-phenylindole+and+bisbenzimide+(Hoechst+33258).+A+comparative+footprinting+study.">Assignment of DNA binding sites for 4&#8217;,6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study.</a> Biochim Biophys Acta. 949 (2), 158-168 (1988).</li><li>Lichtman, J. W., Conchello, J. -. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Nat Methods. 2 (12), 910-919 (2005).</li><li>St Croix, C. M., Shand, S. H., Watkins, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy:+comparisons,+applications,+and+problems.">Confocal microscopy: comparisons, applications, and problems.</a> BioTechniques. 39, S2-S5 (2005).</li><li>Yi, M., Kaneko, S., Yu, D. Y., Murakami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+envelope+proteins+bind+lactoferrin.">Hepatitis C virus envelope proteins bind lactoferrin.</a> Journal of virology. 71, 5997-6002 (1997).</li><li>Wakabayashi, H., Oda, H., Yamauchi, K., Abe, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactoferrin+for+prevention+of+common+viral+infections.">Lactoferrin for prevention of common viral infections.</a> J Inf Chem. 20 (11), 666-671 (2014).</li><li>Picard-Jean, F., Bouchard, S., Lariv&#233;e, G., Bisaillon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+inhibition+of+HCV+replication+represents+a+novel+mechanism+of+action+by+the+innate+immune+Lactoferrin+protein.">The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein.</a> Antiviral research. 111, 13-22 (2014).</li><li>Johnson, G. D., Davidson, R. S., McNamee, K. C., Russell, G., Goodwin, D., Holborow, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fading+of+immunofluorescence+during+microscopy:+a+study+of+the+phenomenon+and+its+remedy.">Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy.</a> J Immunol Methods. 55 (2), 231-242 (1982).</li><li>Green Remington, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein:+a+perspective.">fluorescent protein: a perspective.</a> Protein Sci. 20 (9), 1509-1519 (2011).</li></ol>

The authors declare that they have no competing financial interests.

丙型肝炎病毒的流行仍然是一个全球性的威胁，有80％的新感染者发展为慢性感染的，这使他们肝硬化，肝衰竭或肝癌的危险。直接作用的靶向HCV复制和成熟抗病毒剂代表素抗HCV药物，如最近由两个NS3 N-末端蛋白酶抑制剂（用boceprevir和替拉瑞韦）的监管机构批准证明。所述的hLF抗HCV活性的目前认为表现为病毒进入抑制剂，结合循环病毒粒子和阻止它们进入到他们的肝细胞靶。我们对铁蛋白的研究提出了一个行动新颖的细胞内抗HCV机制（从细胞外病毒中和不同）7。 免疫可以有效地用于可视化靶分子的分布和/或定位在生物样品中。由于发现在许多其他的荧光技术，主要职业之一与免疫相关的blems被漂白（光化学荧光染料的破坏）8。这种活性损失造成的漂白可以通过使用更健壮的荧光染料是不易于漂白或降低强度或时间范围的光曝光来规避。此外，由于抗体不能穿过细胞膜，免疫荧光一般只限于固定的细胞。替代方法来监测活细胞中蛋白质的分布/本地化因此一般依靠含有荧光蛋白结构域的蛋白的表达，如绿色荧光蛋白（GFP）9。然而，加入这类的GFP的表位的有时与在蛋白的定位和活性的修改相关联。免疫荧光也可以在许多不同的样本，包括组织切片，培养的细胞系，或单个细胞用它也经常用于监控蛋白质，多糖和小分子的本地化/分布。该技术的主要优点驻留在它的简单的事实，它可以被组合使用，荧光染色（例如，DNA染色）的其它的，非抗体的方法。此外，商业化生产的二级抗体是相对便宜和可以在一个颜色繁多。 在目前的研究中，肝细胞支持的HCV亚基因组复制的免疫荧光揭示处理后定性的降低与HCV染色强度用的hLF。这是一个有趣的观察，因为这亚基因组复制系统缺少的HCV E1和E2包膜糖蛋白，这是体hLF的普遍接受的药理学靶点。不像细胞外病毒颗粒中和活性，的hLF将需要由丙型肝炎病毒感染的肝细胞，以削弱病毒复制内在化。使用fuorescence显微镜允许我们evalu吃了这种可能性。在对HCV感染的肝细胞预孵育，外源性的hLF成功内在化，并显示出削弱的细胞内的病毒复制。这种新颖的铁蛋白的抗病毒活性的理解是向前迈出了一步，抑制这种先天免疫蛋白的全球（可能多效）机制的理解。

Immunofluorescence is a technique that uses a fluorescence microscope to visualize the distribution and/or localization of a target molecule in a biological sample. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell1. It is typically used on tissue sections and cultured cell lines in order to analyze the distribution/localization of various biological molecules such as proteins, nucleic acids and glycans. It should be noted that immunofluorescence is often used in combination with other non-antibody methods of fluorescent staining such as the 4',6-diamidino-2-phenylindole (DAPI) stains which are typically used to label DNA2. Moreover, this technique involves fixation of the cells which allows the analysis of cells at a specific time.
Different types of microscopes can be used to analyze immunofluorescence samples. The simplest is the epifluorescence microscope (Figure 1) for which excitation of the fluorochrome and detection of the fluorescence are done through the same light path3. Because most of the excitation light is transmitted through the sample, only reflected excitatory light can reach the objective together with the emitted light. This approach unfortunately leadsto a frequent high signal to noise ratio.In contrast, confocal microscopy (Figure 2) offers a distinct advantage for increasing optical resolution and contrast by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light4. This approach allows the reconstruction of three-dimensional structures from the obtained images. However, since an important fraction of the light from the sample is blocked at the pinhole, long exposures are often required.
There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect). Direct immunofluorescence involves a primary antibody linked with a fluorochrome (Figure 3). This method is less frequently used because it provides lower signal, involves higher cost and less flexibility1. Moreover, such antibodies are generally harder to find commercially. On the other hand, the direct attachment of the fluorochrome to the antibody significantly reduces the number of steps in the procedure, saving time and frequently reducing non-specific background signal. This also limits the possibility of antibody cross-reactivty.
Indirect immunofluorescence involves a primary unlabelled antibody which is specific for the epitope of interest1. A secondary antibody which carries the fluorochrome then recognizes the primary antibody and binds to it (Figure 3). Although indirect immunofluorescence is more complex and time consuming than direct immunofluorescence, it is frequently used because of its high sensitivity and it also provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In addition, a vast array of commercial secondary antibodies is available at affordable prices.
Hepatitis C virus (HCV) is a major public-health problem with 130-170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion5. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. This manuscript presents a study in which immunofluorescence assays were performed to monitor the internalization of hLF, an important component of the immune system6, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus (HCV).

<table><tbody><tr><td>DMEM</td><td>Wisent</td><td>319-005-CL</td><td></td></tr><tr><td>PAF</td><td>BioShop</td><td>PAR070.1</td><td>Flammable solid, skin irritant, lungs and eyes&amp;nbsp;</td></tr><tr><td>PBS</td><td>Wisent</td><td>311-425-CL</td><td>Without Ca&lt;sup&gt;2+ &lt;/sup&gt;&amp;amp; Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>NGS</td><td>Wisent</td><td>053-150</td><td></td></tr><tr><td>AlexaFluor 488-labeled anti-mouse</td><td>Invitrogen</td><td>A11017</td><td></td></tr><tr><td>AlexaFluor 568-labeled anti-rabbit</td><td>Invitrogeb</td><td>A21069</td><td></td></tr><tr><td>Wheat germ agglutinin Alexa Fluor 488 conjugate (WGA)</td><td>Invitrogen</td><td>W11261</td><td>Potentially mutagenic</td></tr><tr><td>Anti-NS5A rabbit</td><td>Abcam</td><td>ab2594</td><td></td></tr><tr><td>Anti-hLF mouse</td><td>Abcam</td><td>ab10110</td><td></td></tr><tr><td>SlowFade</td><td>Invitrogen</td><td>S36937</td><td></td></tr><tr><td>Hoechst stain</td><td>Life Techn.</td><td>H1399</td><td>Potentially&amp;nbsp;mutagenic&amp;nbsp;
and carcinogenic</td></tr><tr><td>hLF</td><td>Sigma</td><td>L0520</td><td></td></tr><tr><td>Nikon Eclipse visible/epifluorescence Microscope</td><td>Nikon</td><td>TE2000-E</td><td></td></tr><tr><td>epifluorescence/confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr></tbody></table>

immunofluorescence to monitor the cellular uptake of human lactoferrin and its associated antiviral activity against the hepatitis c virus

免疫是通常用于研究生物学的许多方面实验室技术。它通常用于可视化目标分子的细胞和组织中的分布和/或定位。免疫荧光依赖于对小区内其相应的抗原的荧光标记的抗体的特异性。直接和间接的免疫荧光方法可以用于其中依靠使用带有荧光联抗体。直接免疫是不经常使用的，因为它提供较低的信号，涉及到较高的成本和更少的灵活性。与此相反，间接免疫荧光更常用，因为其高灵敏度和提供放大的信号，因为一个以上的次级抗体可以附加到每个主抗体。在这份手稿，无论是荧光显微镜和共聚焦显微镜被用来监测人乳铁蛋白的内化，免疫的重要组成部分系统，进入肝细胞。此外，我们监测了使用免疫荧光的丙型肝炎病毒的细胞内复制的hLF的抑制潜能。都与这些方法相关的优点和缺点进行了讨论。

使用免疫荧光共聚焦miscroscope肝的Huh-7细胞系的内化外生有管理的体hLF的能力进行监测。的hLF加入到培养液中并内化被允许进行24小时，在这之后，细胞外的hLF用PBS洗涤和结合体hLF残留膜降解，5分钟胰蛋白酶处理（1ml）中。将细胞再接种，并使其重新附着的免疫荧光染色前1​​8小时。为了概括的胞质限制，将细胞的膜进行染色的488纳米缀合物的激发波长的麦胚凝集素（WGA）的荧光染料。所述的hLF染色使用抗体hLF第一抗体并用633 nm波长连接到二抗荧光染料来实现的。为了确定体hLF的精确定位，将细胞进行成像以下顺序0.5微米的水平横截面通过共聚焦显微镜。 图4表示一个选择对应于中间单元厚度的iCal横截面。在加入外源性的hLF，被肝细胞的细胞质内检测出的显著量的hLF。没有铁蛋白是使用相同的工具设置时，未经处理的细胞中观察到。总之，这些数据证实，肝细胞必须从它们的胞外环境取得的hLF的能力。 使用荧光显微镜铁蛋白抑制丙型肝炎病毒在细胞内的复制是明年的能力监控。 Huh-7细胞的HCV支承亚基因组复制（表达HCV NS3，NS4A，NS4B，NS5A NS5B蛋白）的复制具有3微米的hLF共治疗0,2或24小时。双免疫荧光染色铁蛋白和HCV NS5A的蛋白质是由荧光显微镜进行分析。这些结果表明，HCV亚基因组复制染色（如由丙型肝炎病毒非结构蛋白5A的免疫荧光测定）减少与铁蛋白处理的细胞中。 图5 </s<html>仲&gt;揭示的HCV染色强度质减少大约50％的hLF治疗24小时后。这是一个有趣的观察，因为这亚基因组复制系统缺少的HCV E1和E2包膜糖蛋白，这是体hLF的普遍接受的药理学靶点。伴随的HCV染色的褪色，铁蛋白积累开始2小时后进行检测，并强的hLF积累24小时后观察到。 <img alt="图1" src="/files/ftp_upload/53053/53053fig1.jpg" /> 图1原理落射荧光显微镜。在这种类型的显微镜，激发滤光片选择特定波长激发样品（皮膜组织或细胞携带荧光标记的抗体）。此外，发射滤波器被选择为匹配用于标记样品中的荧光团的光谱发射特性。加载/ 53053 / 53053fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53053/53053fig2.jpg" /> 图2.原理共聚焦显微镜的。这种类型的显微镜使用的发射针孔来消除失焦光信号。由于只有通过荧光非常接近焦平面产生的光可以被检测，图像的光学分辨率比落射荧光显微镜的要好得多。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53053/53053fig3.jpg" /> 图3.原理的直接和间接免疫荧光。直接免疫荧光涉及只有一种类型的抗体的标记荧光染料是特定于感兴趣（一级抗体）的分子。与此相反，间接免疫荧光涉及标记的荧光染料是特定的未标记第一抗体的物种的二抗。不止一个二级抗体可以连接到每个主抗体，增加了荧光信号。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53053/53053fig4.jpg" /> 外源性的hLF。Huh-7细胞的图4.肝癌摄取与0微米或3μM的hLF共治疗24小时，洗涤两次，提交到5分钟胰蛋 ​​白酶处理，允许重新附着18小时，并进行免疫荧光。细胞核染色瓦特第i个DAPI（蓝色通道），血浆膜标记的WGA（绿色通道）和体hLF染色用抗体hLF抗体（红色通道）。在60倍的放大倍率原被收购共聚焦显微镜图像。的水平光学截面表示中间厚度的细胞。比例尺，10微米。从（11）修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="Figure5" src="/files/ftp_upload/53053/53053fig5.jpg" /> 图5.体hLF治疗降低HCV染色。Huh-7细胞的HCV支承亚基因组复制的复制用3微米的hLF共治疗0,2，或24小时，并进行免疫荧光。细胞核用DAPI染色（蓝色通道），丙型肝炎病毒揭示通过抗NS5A抗体（红色信道）和体hLF染色用抗体hLF一个ntibody（绿色通道）。在60倍的放大倍率原被收购通过荧光显微镜图像。比例尺，10微米。从（11）修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus at JoVE.com

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

免疫监视人乳铁蛋白的细胞摄取及其相关抗病毒活性抗丙型肝炎病毒

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

immunofluorescence-to-monitor-cellular-uptake-human-lactoferrin-its

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.3K 次观看.  Université de Sherbrooke. The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

视频: Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

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, ...

科研

免疫与感染

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

两种异核体的形成方法，发现丙型肝炎病毒的制约因素

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

一个协议，用于分析丙型肝炎病毒复制

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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 ...

生物学

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

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

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

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 ...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Source: Bailey, M. J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56256">A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.</a> J. Vis. Exp. (2018).
This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.

测量 HA 抗体对 Fc 介导的效应子功能的激活

1. Expression of Influenza Virus Hemagglutinin via Transfection

	Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

This video demonstrates an immunofluorescence assay designed to measure the neutralization of Human Cytomegalovirus (HCMV). Virus-specific antibodies are mixed with the virus and incubated with human fibroblast cells. The antibodies neutralize the virus, rendering it unable to infect the host cells, while non-neutralized viruses infect the cells. After incubation, the extent of neutralization is determined by quantifying the virus-infected cells using immunofluorescence.

一种测量人巨细胞病毒中和作用的免疫荧光方法

1. Preparation of target cells for infection

	Seed target cells the day prior to performing the experiment in a 96-well plate (the &#39;target&#39; ...

Immunodiagnostics

Imaging and Visualization Techniques

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

用于筛选抗病毒化合物的病毒附着测定

NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...