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.細胞の調製および治療<ol><li> 24ウェルプレートにおいて、ウェルの底部にカバーガラスを置きます。リン酸緩衝生理食塩水（PBS）を各ウェルを洗浄します。 </li><li> /ウェル5×10 4細胞の密度に各ウェル中のHCVサブゲノムレプリコンを支援するシードのHuh-7細胞。行うには治療法がない場合、細胞は、より高い密度で播種することができます。培地は、ダルベッコ改変イーグル培地、10％ウシ胎児血清（FBS）、2mMのL-グルタミン、1mMピルビン酸ナトリウムおよび250 mg / mlのG-418（レプリコンを維持するため）を補足した（DMEM）です。 </li><li> 37℃で培養し、5％CO 2翌日、ヒトの乳から精製されたHLFの3μMで細胞を処理します。 </li></ol> 2.パラホルムアルデヒド/ショ糖の製造（12％PAFおよび12％ショ糖） <ol><li>ビーカーに500mlのPBSに入れ、20°Cと30°Cの間に加熱します。 PBS中のショ糖の60グラムを溶解します。 paraformalの60グラムを溶かしPBS /スクロース溶液中のアルデヒド（PAF）。透明な溶液が得られるまでゆっくりと2NのNaOH（3~7 ml）を加えます。 </li><li> HClでpHを7.4に調整します。 PBSで500ミリリットルに、完全なボリューム。ワットマンのフィルター上の重力によってフィルタします。使用前に、4％PAFおよび4％スクロース（ストレージ1週間に最大4°C）の最終濃度にPBSで溶液を希釈します。 </li></ol> 3.免疫蛍光<ol><li>所望の時間（0時間、2時間または治療の24時間）で、PBSで2回（1分）細胞を洗浄し、室温で20分間、PBS / 4％PAF / 4％スクロースで固定します。細胞が30-40％コンフルエントであり、典型的には1.5×10 5個の細胞が使用されます。 </li><li> 20分間、PBS中10％正常ヤギ血清（NGS）中で室温で5分間、PBS中の0.15％のトリトンX-100で細胞を透過性とブロック。 </li><li> 10％NGS中で目的のタンパク質の一次抗体を希釈する（例：1,000：一次マウス抗HLF抗体の1希釈）。細胞に一次抗体を適用します。 </li><li> RTまたはOで4時間後/4℃でのNは、10％NGSで5分間、細胞を3回洗浄します。 10％NGS中で希釈した蛍光色素で標識した二次抗体を用いた細胞染色（例：蛍光色素を抗体抗マウス1に連結された488nmでの励起波長で1,000）暗所でRTで1時間。 </li><li> PBSで5分間、細胞を1回洗浄し、暗所で室温で15分間、4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）（1μg/ ml）で細胞を染色します。 </li><li> PBSで5分間、細胞を2回洗浄します。マウントは、顕微鏡を通して視覚化する前にメディアをマウントSlowFadeのメガネをカバーしています。細胞は今落射蛍光または共焦点顕微鏡による分析のために準備ができています。 </li><li>検出の特異性を確保するために、別のコントロールを使用します。例えば、すべての抗体は、自己蛍光を検出するために省略することができます。一次抗体を、二次抗体による非特異的染色を決定するために省略することができます。ない最後に、可能な場合は、コントロール細胞または組織目的の分子を用いることも可能で発現します。 </li><li>使用される蛍光標識に適した適切な蛍光顕微鏡（落射蛍光または共焦点）とフィルターセットを使用してサンプルを可視化します。スライドはまた、&lt;-20°Cの場合、後の検査でスライドボックスに保存することができます。 </li></ol> 4.蛍光顕微鏡<ol><li>光退色を防止するために、暗闇の中でサンプルを保管してください。選択した顕微鏡（落射蛍光または共焦点）の光源をオンにします。 </li><li>顕微鏡の電源をオンにします。顕微鏡用カメラの電源をオンにします。可視化のための顕微鏡のスライドを入れてください。対物レンズの開口数を増加させるために、スライド上の液浸油を加えます。 </li><li>適切なフィルタを選択します。フォーカスし、適切な目標を調整します。適切な蛍光色素を検出するが光漂白を防ぐためにシャッターを調整します。サンプルを見ているときに、背景光を最小化するためにオーバーヘッドライトをオフにしてください。 </li><li>目を変更電子フィルタは、（例えばDAPIおよび488nmでの励起波長のための）様々な蛍光色素を可視化します。カメラで写真を撮ります。 </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.

HCVの流行は、肝硬変、肝不全や肝細胞癌の危険にそれらを置くこと、新たに感染した患者の80％が慢性感染症を発症して、世界的な脅威のまま。最近2 NS3 N末端プロテアーゼ阻害剤（ボセプレビルおよびテラプレビル）の規制当局の承認によって実証されるようにHCV複製および成熟をターゲットに直接作用型抗ウイルス剤は、プライム抗HCV剤を表します。 HLF抗HCV活性は、現在、ビリオンを循?...

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

免疫蛍光は、一般的に生物学の多くの側面を研究するために使用される実験技術です。それは、典型的には、細胞及び組織中の標的分子の分布および/または局在化を可視化するために使用されます。免疫蛍光は、細胞内でのそれらの対応する抗原に対する蛍光標識抗体の特異性に依存しています。直接的および間接的の両方の免疫アプローチは、蛍光色素に結合された抗体の使用に依存しているものを用いることができます。それは、より低い信号を提供し、より高いコスト、より少ない柔軟性を必要とするので、直接免疫蛍光法は、あまり頻繁に使用されます。対照的に、間接免疫蛍光法は、より一般的に、その高い感度を使用し、複数の二次抗体は、それぞれ一次抗体に付着することができるので、増幅された信号を供給する。本稿では、落射蛍光顕微鏡及び共焦点顕微鏡法の双方は、ヒトラクトフェリンの内在化、免疫の重要な構成要素を監視するために使用しました。肝細胞へのシステム。また、我々は、免疫蛍光法を用いて、C型肝炎ウイルスの細胞内複製にHLFの阻害可能性を監視しました。これらのアプローチに関連する利点と欠点の両方が議論されています。

外因的に投与さHLFを内在する肝臓のHuh-7細胞株の能力を、共焦点miscroscopeに免疫蛍光を用いてモニターしました。 HLFは、培養培地に添加し、インターナリゼーションは、その後、細胞外HLFをPBSで洗浄し、結合したHLF残留膜を5分間トリプシン処理（1 ml）で分解し、24時間おきました。細胞は、再播種し、免疫蛍光染色の前に18時間再接着させました。細胞質の限界を概説するために、細胞膜を48...

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.

C型肝炎ウイルスに対するヒトラクトフェリンの細胞取り込みおよびそれに関連する抗ウイルス活性を監視するために、免疫蛍光

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 ビュー.  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.

ビデオ: 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.

リンパ球増殖を監視するためのカルボキシフルオレセインジアセテートスクシンイミジルエステル（CFSE）の使用

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

免疫・感染学

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.

TCRとそれに関連するシグナル伝達タンパク質のリアルタイムの同時イメージングのための全反射顕微鏡法

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.

タングウイルスに対する新規抗ウイルス剤の同定のためのアッセイ

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.

C型肝炎ウイルスの複製を分析するためのプロトコル

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.

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

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.

複製サイクルの初期段階を監視するためにB型肝炎ウイルスのレポーターシステムの開発

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.

抗ウイルス薬をスクリーニングし、宿主の免疫細胞応答を特徴づけるために、ヒトインフルエンザAウイルス感染のゼブラフィッシュモデルを使用して、

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.

エスケープ中和抗体でインフルエンザ ウイルスの亜種の世代

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.

ヒトの過剰発現と精製<em&gt;シス</em&gt; - プレニルトランスフェラーゼ<em&gt;大腸菌</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.