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?...

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 border="0" cellpadding="0" cellspacing="0">
	<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.&#160;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Wisent</td>
			<td>311-425-CL</td>
			<td>Without Ca2+ &#38; Mg2+</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&#160;mutagenic&#160;and&#160;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?...

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

Research

JoVE Journal

Immunology and Infection

13.2K 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

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

羧基荧光素二醋酸琥珀酰亚胺酯（CFSE）用于监视淋巴细胞增殖

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

科研

免疫与感染

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

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

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

为检验新型抗病毒药物的鉴定对蓝舌病病毒

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

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 ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.