この作品は、国立医学研究評議会、シンガポールによって資金を供給されています。

1。デング熱ウイルスののAlexa Fluor ®標識<ol><li>ラベリング反応の前に、ショ糖クッションでデング熱ウイルスを精製し、プロトコルに示されるように必要な試薬や機器を準備する。 </li><li>ラベルとフィルタは0.2μmのシリンジフィルターで殺菌する直前に、新鮮な0.2M炭酸水素ナトリウム緩衝液、pH 8.5（標識バッファー）、および1.5Mヒドロキシルアミン緩衝液、pH 8.3（試薬を停止する）準備。 </li><li> 2ミリリットルチューブ内のバッファを標識する1mlのデング熱ウイルスを精製したのは約3 × 10 8プラーク形成単位（pfu）を希釈する。これは、ウイルスのバッチの標識のために比例してスケールアップすることができます。 </li><li>再構成する凍結乾燥のAlexa Fluor ® 594（AF594）標識反応直前にバッファのラベル付けさ1mmのスクシンイミジルエステル。のAlexa Fluor ®色素シリーズからの他の蛍光色素は、自分のニーズに応じて使用することができます。このステップ以降から光への曝露を最小限に抑えることができます。 </li><li>ピペットの先端で軽く攪拌しながら希釈したウイルスに1mMのAF594染料液100μlを追加。 </li><li>暗所で1時間、室温で標識反応ミックスをインキュベートする。穏やかな逆転であらゆる15分を混ぜる。 </li><li>卓上遠心機でチューブを軽くスピンし、ピペットの先端で軽く攪拌しながら反応混合物に停止試薬100μLを加える。 </li><li>暗闇の中でさらに1時間室温でインキュベートする。穏やかな逆転であらゆる15分を混ぜる。 </li></ol> 2。浄化のAlexa Fluor ®は、デング熱ウイルスを標識し<ol><li>その間に、選択肢のバッファーで精製カラムを平衡化します。この実験では、PD - 10カラムは、使用前に、pH7.4のHNEのバッファの25ミリリットル（5mMのヘペス、150mmのNaClを、0.1mmのEDTA）で平衡化する。 </li><li>列の先頭にラベルの付いたウイルスを適用し、ラベルの付いたウイルスが行列に入ると、フロースルーを収集を開始。すべてのラベルの付いたウイルスが行列を入力した後、HNEのバッファーでカラムを埋める。フロースルーの最初の2.5ミリリットルを破棄し、標識ウイルスの割合の次の2ミリリットルを収集する。 </li><li> -80でAF594というラベルのデング熱ウイルスを精製分注し、店舗° C、離れて光源から。 </li></ol><ol><li> oneアリコートを解凍し、標識ウイルスのバッチを使用する前に、プラークアッセイで標識されたウイルスの力価を決定する。 </li><li>シード5 × 10 4ウェルあたりの感染の前によく日の底面上にカバースリップの4ウェルプレートでM - 199の増殖培地で増殖させたVero細胞、、。 </li><li>で37ウェル中に培養上清を除去し、10分間100μlのボリューム（必要に応じてM - 199維持培地で希釈）で標識したウイルスの1の感染多重度で細胞に感染℃に</li><li> inoculumsを削除し、1xPBSで二度カバーグラスを洗う。 </li><li> 30分3％paraformalydehydeで細胞を固定する。 </li><li> 1xPBSでカバーグラス3回洗浄する。 </li><li> 30分のための1xPBSで0.1％サポニンと5％のBSAを含む透過液と細胞を透過処理。 </li><li>原液光から保護するために、湿度の高い部屋で1時間のために3H5モノクローナル抗体のハイブリドーマの培養上清を遠心分離、清澄化した細胞をインキュベートする。 </li><li> （1xPBSは、1mMの塩化カルシウム、1mMの塩化マグネシウムと0.1％のサポニンを含む）洗浄バッファーでカバースリップ3回洗浄する。 </li><li>遮光して湿気の多い部屋で45分、のためのAF488抗マウスIgG抗体、透過液中に希釈1:100、で細胞をインキュベートします。 </li><li>カバーガラスの洗浄バッファーで3回洗浄し、脱イオン水で一度すすいでください。 </li><li> DAB8μlMowiol 4〜88を含む2.5％DABCOで余分な水分を排出し、スライドガラスにマウントするためにペーパータオルに対するカバースリップの端。 </li><li>ツァイス共焦点顕微鏡を使用して表示する前に一晩4に設定してマウントソリューション° Cを許可する。シーケンシャルの買収は、付随して検出器の間の時間とスイッチングでエキサイティングな蛍光団を実行する必要があります。 </li><li>画像は、ラベリングの度合いを推定するためにツァイスLSM禅ソフトウェアを用いて標識ウイルスのE蛋白質の抗体染色の共局在のために分析されます。 </li></ol> 3。代表的な結果 AF594色素で標識されたデング熱ウイルスの収量の例を図2に示されています。通常は、初期の力価からわずか10倍ドロップが成功したラベリング後に観察されるべきである。しかし、それはすべてのバッファがラベル成功させるために、それらが水溶液8の非反応性の遊離アミノ酸に加水分解としてのAlexa Fluorスクシンイミジルエステルが再構成後すぐに使用する必要があるために新鮮に準備する必要があることに留意すべきである。 次に、標識されたウイルスは、実験で使用する前に、十分な蛍光をチェックする必要があります。シンプルな免疫蛍光アッセイは、Vero細胞で行われ、ラベリングの度合いは、抗E蛋白質の抗体染色で標識したウイルスの共局在から推定することができる。断ち切るらの細胞を調べたところ、典型的な共焦点画像を図3に示されています。 LSM禅ソフトウェアを使用して画像の共局在の解析は、ビリオンの約65〜80％が色素で標識されたことを示唆し、0.65から0.8までの係数を重ねる示した。 <img src="/files/ftp_upload/3168/3168fig1.jpg" alt="図1" /> 図1.Overallスキームは、デング熱ウイルスの手続きののAlexa Fluor ®色素のラベルを描いた。まず、関連するバッファーと精製されたデング熱ウイルスが用意されている。のAlexa Fluor ®色素は、再構成し、バッファをラベルに希釈したデングウイルスに追加されます。反応は、その後、停止試薬を加えると1時間後に停止されます。その後、標識されたウイルスは、無料の色素を除去するためにサイズ排除カラムを通して精製される。最後に、標識されたウイルスは、プラークアッセイにより再滴定し、蛍光のためにテストされています。 <img src="/files/ftp_upload/3168/3168fig2.jpg" alt="図2" /> 図2。 AF594ラベリングの前と後のプラークアッセイで決定される実行可能なビリオン（PFU / ml）の数を意味する。AF594というラベルのデング熱ウイルスのアリコートを解凍し、プラークアッセイにより再滴定し、それが通常はより小さいよりも10倍ドロップを示しています開始タイター。エラーバーは、重複の標準偏差を示している。 <img src="/files/ftp_upload/3168/3168fig3.jpg" alt="図3" /> 図3。前日カバーグラス上で成長させたVero細胞におけるデングウイルスEタンパク質とAF594ラベルの共局在。細胞を37℃で10分間1のMOIでのAF594というラベルのデング熱に感染していた℃、細胞は、その後修正及び抗E抗体で標識し、Eのタンパク質の共局在（緑）とAF594ラベル（赤）を調べた。蛍光シグナルはツァイスLSM 710共焦点顕微鏡を用いて63X倍率で可視化した。スケールバーは、10μmである。挿入図に示すように、黄色、共局在の領域を示します。

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	<li>Olenych, S. G., Claxton, N. S., Ottenberg, G. K. &. a. m. p. ;. a. m. p., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fluorescent+Protein+Color+Palette.">The Fluorescent Protein Color Palette.</a> Current Protocols in Cell Biology. 21.5, 1-34 (2007).</li><li>Panchuk-Voloshina, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alexa+dyes,+a+series+of+new+fluorescent+dyes+that+yield+exceptionally+bright,+photostable+conjugates.">Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.</a> J Histochem Cytochem. 47, 1179-1188 (1999).</li><li>Shaner, N. C., Patterson, G. H., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+fluorescent+protein+technology.">Advances in fluorescent protein technology.</a> J Cell Sci. 120, 4247-4260 (2007).</li><li>Lippincott-Schwartz, J., Patterson, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+use+of+fluorescent+protein+markers+in+living+cells.">Development and use of fluorescent protein markers in living cells.</a> Science. 300, 87-91 (2003).</li><li>Kuhn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+dengue+virus:+implications+for+flavivirus+organization,+maturation,+and+fusion.">Structure of dengue virus: implications for flavivirus organization, maturation, and fusion.</a> Cell. 108, 717-725 (2002).</li><li>Aguilar, J. S., Roy, D., Ghazal, P., Wagner, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethyl+sulfoxide+blocks+herpes+simplex+virus-1+productive+infection+in+vitro+acting+at+different+stages+with+positive+cooperativity.+Application+of+micro-array+analysis.">Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis.</a> BMC Infect Dis. 2, 9-9 (2002).</li><li>Zhang, S. L., Tan, H. C., Hanson, B. J., Ooi, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+Alexa+Fluor+dye+labelling+of+dengue+virus.">A simple method for Alexa Fluor dye labelling of dengue virus.</a> J Virol Methods. 167, 172-177 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Alexa Fluor Succinimidyl Esters (Molecular Probes. , (2009).</li><li>Huang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+proteins+stained+by+Alexa+dyes.">Analysis of proteins stained by Alexa dyes.</a> Electrophoresis. 25, 779-784 (2004).</li><li>Freistadt, M. S., Eberle, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+poliovirus+for+flow+cytometric+cell+surface+binding+studies.">Fluorescent poliovirus for flow cytometric cell surface binding studies.</a> J Virol Methods. 134, 1-7 (2006).</li></ol>

AF594染料がこのレポートで使用されていますが、のAlexa Fluorスクシンイミジルエステルシリーズの蛍光体の広い範囲で同様のラベリングの化学で使用可能です。これは、イメージングを超えてラベリングアプリケーションを拡張することができます。フローサイトメトリーは、FACSの機械によって励起して検出できる蛍光物質のラベリングの度合いを推定するための共焦点顕微鏡の代替として?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>重炭酸ナトリウム</td><td>シグマアルドリッチ</td><td> S6297 </td><td></td></tr><tr><td>ヒドロキシルアミン</td><td>シグマアルドリッチ</td><td> 159417 </td><td></td></tr><tr><td>水酸化ナトリウム</td><td>メルク</td><td> 106498 </td><td></td></tr><tr><td> AF594スクシンイミジルエステル</td><td>分子プローブ、インビトロジェン</td><td> A20004 </td><td></td></tr><tr><td> PD - 10カラム</td><td> GEヘルスケア</td><td> 17-0851-01 </td><td></td></tr><tr><td> HEPES </td><td>シグマアルドリッチ</td><td> H6147 </td><td></td></tr><tr><td> NaClの</td><td>シグマアルドリッチ</td><td> S3014 </td><td></td></tr><tr><td> EDTA </td><td>シグマアルドリッチ</td><td> E9884 </td><td></td></tr><tr><td> M - 199 </td><td>インビトロジェン</td><td> 11150 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070.03 </td><td></td></tr><tr><td> 4ウェルプレート</td><td>ヌンク</td><td> 176740 </td><td></td></tr><tr><td>カバースリップ</td><td> Einst </td><td> 0111520 </td><td></td></tr><tr><td>顕微鏡のスライド</td><td>ブランドセイル</td><td> 7105 </td><td></td></tr><tr><td> 3H5ハイブリドーマ</td><td> ATCC </td><td> HB46 </td><td></td></tr><tr><td> 10倍PBS </td><td>第一基地</td><td> BUF - 2040 - 10X1L </td><td></td></tr><tr><td>サポニン</td><td>シグマアルドリッチ</td><td> S4521 </td><td></td></tr><tr><td> BSA </td><td>シグマアルドリッチ</td><td> A7906 </td><td></td></tr><tr><td>マグネシウムの塩化</td><td>シグマアルドリッチ</td><td> M2670 </td><td></td></tr><tr><td>カルシウム塩化</td><td>シグマアルドリッチ</td><td> C3306 </td><td></td></tr><tr><td> Paraformaldehye </td><td>シグマアルドリッチ</td><td> 15,812-7 </td><td></td></tr><tr><td> Mowiol 4から88 </td><td>カルビオケム</td><td> 475904 </td><td></td></tr><tr><td> DABCO </td><td>シグマアルドリッチ</td><td> D27802 </td><td></td></tr><tr><td>卓上遠心機</td><td>エッペンドルフ</td><td> 5424 </td><td></td></tr><tr><td>共焦点顕微鏡</td><td>ツァイス</td><td> LSM 710 </td><td></td></tr></tbody></table> M - 199の増殖培地を準備するには、FBS、M - 199の500ミリリットルの非必須アミノ酸のピルビン酸ナトリウムと5ミリリットルの5ミリリットル、滅菌フィルター50mlのを追加します。 M - 199維持培地を準備するには、FBS、M - 199の500ミリリットルの非必須アミノ酸のピルビン酸ナトリウムと5ミリリットルの5ミリリットル、滅菌フィルターの15ミリリットル加える。

visualizing dengue virus through alexa fluor labeling

ウイルスと細胞の間の相互作用の初期段階でのイベントは、感染の結果に大きな影響を持つことができます。この相互作用に影響を与える要因を決定することは疾患の病因の理解の向上につながるため、ワクチンや治療薬の設計に影響を与える可能性がある。したがって、この相互作用を調べるための方法の開発は有用であろう。蛍光体の開発1-3とイメージング技術4の最近の進歩は、デング熱発症機序に関する現在の知識を向上させるため、毎年発生するデング熱感染数百万人を削減する道を開くために悪用される可能性があります。 包まれたデング熱ウイルスは、単一のプラス鎖RNAゲノム5を含むヌクレオカプシドシェルを、保護する90エンベロープ糖タンパク質（E）二量体で構成される外部足場を持っています。ウイルスの表面上に同一のタンパク質サブユニットは、このようにアミン反応性色素で標識し、免疫蛍光顕微鏡法によって可視化することができます。ここで、我々はAlexa Fluorスクシンイミジルエステル色素がラベルの後に非常に実行可能なウイルスをもたらした重炭酸ナトリウム緩衝液中で直接溶解でデング熱ウイルスの標識の簡単な方法を提示する。生きているウイルスやタンパク質標識は、通常、ジメチルスルホキシド中の染料の再構成を必要とするために、既存の製造業者のプロトコルのラベリングのための標準化された手順はありません。ジメチルスルホキシドの存在は、さらに微量で、ウイルスの増殖感染を遮断し、また細胞毒性6を誘発することができます。このプロトコルのジメチルスルホキシドの使用の排除は、このように、この可能性を減少させた。のAlexa Fluor ®色素は、優れた光安定性を持ち、細胞の取り込みとウイルスのエンドソーム輸送の研究に最適です、あまりpH感受性などフルオレセインやローダミン2のような一般的な染料、よりです。のAlexa Fluor ®色素のコンジュゲーションは、ウイルス特異的抗体と7宿主細胞内でのその推定受容体による標識デング熱ウイルスの認識に影響を与えなかった。このメソッドは、ウイルス学的研究に有用なアプリケーションを持つことができます。

Watch this Scientific Journal Video about Visualizing Dengue Virus through Alexa Fluor Labeling at JoVE.com

Visualizing Dengue Virus through Alexa Fluor Labeling

蛍光体の開発とイメージング技術、ウイルスはウイルスと細胞の間で早期に相互作用を視覚化するために考案されたデング熱ののAlexa Fluor ®標識の簡単な方法の進歩を活用して。

のAlexa Fluor ®標識を通じてデング熱ウイルスの可視化

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

visualizing-dengue-virus-through-alexa-fluor-labeling

Research

JoVE Journal

Immunology and Infection

13.7K ビュー.  DSO National Laboratories. 蛍光体の開発とイメージング技術、ウイルスはウイルスと細胞の間で早期に相互作用を視覚化するために考案されたデング熱ののAlexa Fluor ®標識の簡単な方法の進歩を活用して。

ビデオ: Visualizing Dengue Virus through Alexa Fluor Labeling

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

免疫・感染学

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

遺伝子または薬物療法で使用するためのHIV-1産生を標的RNAの有効性および毒性の評価

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

新世界Zikaウイルス感染クローンからの組換えウイルスの救出とキャラクタリゼーション

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

共焦点顕微鏡を用いた可視化マクロファージ細胞外トラップ

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

単純なフローサイトメトリーによるジカ回復期血清を用いたデング ウイルスのIn Vitro抗体依存強化測定法

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

迅速・安全・簡易マニュアル ベッドサイド核酸抽出の全血試料中のウイルス検出

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

量的なリアルタイムのポリメラーゼの連鎖反応による感染細胞の培養上清中のデング ウイルス RNA を測定

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

全血豚で表されるコーディングおよび非コーディングの RNA クラスの識別

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

デングウイルス誘発急性ウイルス脳炎様疾患のマウスモデル

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

狂犬病ワクチンの効力を評価するインビトロELISA試験

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...