我々はVirochipプロトコルの初期の開発と最適化のためにデイヴィッド王、アナUrisman、エイミーキスラー、ケールフィッシャー、パトリック唐、そしてアレキサンダーGreningerに感謝。この作品は、NIH K08助成金とアボットディスカバリー賞（CCまで）とハワードヒューズ医学研究所（JLDまで）によってサポートされています。

Virochipアッセイの手順は、臨床サンプルから（1）核酸の抽出、抽出したRNAの（2）逆転写および第2鎖cDNA合成、ランダムプライミングcDNAの（3）PCR増幅、（3）Cy3の蛍光色素の取り込みを含む、（4）Virochipマイクロアレイの標識物質のハイブリダイゼーション、及び（5）スキャンおよび分析（図1）。以下に示すプロトコルは、インフルエンザ様疾患を持つ子どもから鼻腔スワブサンプルのVirochipアッセイの使いかたを説明します。 プライマー配列プライマー5&#39; - GTTTCCCAGTCACGATA -（N 9）-3&#39; プライマーB 5&#39; - GTTTCCCAGTCACGATA - 3&#39; 1。核酸抽出<ol><li>これらのサンプルは潜在的に感染であるため、臨床検体からの核酸の抽出は、バイオセーフティレベル2（BSL - 2）以上の条件の下で実行する必要があります。 </li><li>鼻腔スワブサンプルは、ウイルス保持媒体で転送されます。 1.5mlのエッペンドルフマイクロ遠心チューブに試料のアリコート200μLの。 </li><li> オプション：0.22μmフィルター（ウイルス粒子のために精製するために）を通過サンプル</li><li> オプション：製造業者の指示に従って、ターボDNアーゼキット（Ambion社）を用いてDNaseで前処理サンプル（ホストのゲノムDNAを分解すると、保護された、capsidに包まれたウイルス核酸のために精製するために） </li><li> Zymo ZRウイルスRNA抽出キットまたは、製造元の指示に従ってキアゲンUltrasensウイルスキットを使用してサンプルを抽出する。 </li></ol> 2。逆転写および第2鎖cDNA合成（&quot;ラウンドA&quot;） <ol><li> RNAを抽出した4ユニットに1 uLの40μL/ pmolのプライマーを追加。 65〜熱が° Cクラー（例：ジーンアンプPCRシステム9700）とその後の5分間室温温度× 5分で冷ます。 </li><li> 2μL5X RTバッファー、1μL12.5mmのdNTPを、1μL水、0.5μL0.1M DTT、および0.5μLSSIII RT（Invitrogen社）で構成されたマスターミックスの5μLを追加。 42℃でインキュベート℃× 60分。 </li><li>プログラム可能なサーマルサイクラーで、94〜熱10℃まで冷却C × 2分（中止に逆転写酵素反応）、° C、パルス遠心分離し、1μL5Xシークエナーゼのバッファで構成される5μLシークエナーゼミックスを追加し、3.8μL水、および0.15μLシーケナーゼ（USB社）。第2鎖合成の場合は、° C〜37 ° Cから8分、8分を保持し、94への熱は、° C × 2分（シークエナーゼを不活性化する）と10 ° C（保持）へのクールな上の10から立ち上がる。 </li><li> 省略可能 ：RD cDNAサンプルは-20℃で、この時点で保存することができます。 </li></ol> 3。ランダムプライミングcDNAのPCR増幅（&quot;ラウンドB&quot;） <ol><li> 5μL10X PCRバッファー、1μL12.5mmのdNTPを 、1μLは100pmol /μLプライマーB、1μLKlenTaq LA（Sigma社）、および37μLの水から成るマスターミックス45μLにRdの5μlのサンプルを追加。 </li><li>次のようにPCRプロトコール実行：94 ° C、2分→（94の25サイクル° C、30秒/ 50℃、45秒/ 72℃、1分）→72℃、5分→10℃（ホールド） </li><li> 1.5％アガロース電気泳動ゲル上のRD Bのサンプルを確認してください。約200から塗抹標本 - 1000 BPは可視でなければなりません（図2A） </li></ol> 4。 Cy3の蛍光色素の取り込み<ol><li> オプション ：別のサンプルは、Cy5の蛍光色素で標識し、同じマイクロアレイにハイブリダイズすることができます。 </li><li> AA - dNTPを組み込むには、5μlの10 × PCRバッファー、1μlの12.5mmのAA - dNTPを （Invitrogen社製）、1μlのは100pmol / ULプライマーB、1から成るマスターミックスの45 ULに路Bのサンプルを5 ULを追加する1μlのKlenTaq LA（Si​​gma社）、および37 uLの水。 PCRプロトコールを以下のように実行する：94 ° C、2分→（94の15サイクル° C、30秒/ 50℃、45秒/ 72℃、1分）→72℃、5分→10℃（ホールド） </li><li>メーカーの指示ごとにクリーンとコンセントレータ- 5キット（Zymo研究）DNAと路Cのサンプルを清掃してください。 10μLで溶出します。 </li><li>路Cのサンプルに1M炭酸水素1 UL（Sigma社）および1μlのCy5と（GE Healthcare）を追加します。 </li><li>暗所でインキュベート× 1時間。 </li><li>メーカーの指示に従ってDNAクリーンとコンセントレータ- 5キット（Zymo研究）とCy3標識サンプルを清掃してください。 12μLで溶出します。 </li></ol> 5。 Virochipマイクロアレイへのハイブリダイゼーション<ol><li> オプション：（サンプルの正規化のための）光度計分光光度計に組み込まれたcDNA濃度と色素の量をチェックするために1.5μLを使用してください</li><li> PCRストリップチューブに25μLの全体積（アジレント）に1μL25Xフラグメンテーションバッファ、5μL5Xブロッキング緩衝液、Cy3標識サンプルの9.5μL（または正規化する量）、及び水を加える</li><li> 2X GEXのハイブリダイゼーションバッファー（アジレント）の25μLを追加。気泡を除去するために一時的にスピンダウン。 </li><li>ガスケットスライド上にサンプル40μLをロードする。 </li><li>場所アジレント印刷さVirochipの配列（Californi大学、サンフランシスコ/アジレント）（ダウン&quot;アジレント&quot;マーク側）ガスケットのスライドの上とは、しっかりとネジを締めます。 </li><li> 65で一晩ハイブリダイズ℃のオーブン、10 rpmで速度設定。 </li><li> 37℃に配列、予熱バッファ2（アジレント）を洗浄するバッファ1（アジレント）のガスケットのスライド/配列サンドイッチ水中を離れてください。バッファ1 × 1分を洗ってください。予熱されたバッファ2 × 1分で洗浄する。徐々に（直接配列のアクティブな側面に触れないように注意しながら余分な水分を逃がすためにキムワイプを使用）気泡に注意しながら、バッファ2からスライドを削除する</li><li>スライドホルダーにス​​ライドを配置し、アレイスキャナーに挿入。 </li></ol> 6。 Virochipスキャンと分析<ol><li>スキャンCy3標識測定器の標準のスキャンプロトコル（図2B）によるとは5μmまたは2μm以下で配列。 </li><li> E -予測11、目視検査、クラスター分析、または自動化されたVirochip解析プログラムによってVirochipマイクロアレイを解析する。これらの3つの方法のそれぞれによって、インフルエンザウイルスは、容易に鼻腔スワブサンプル（インフルエンザ様疾患を持つ子から）（図2C）で検出される</li></ol> 7。代表的な結果： プロトコルが正しく行われている場合、スミアは、RD Bのステップ（図2A）の後にアガロースゲル電気泳動で見られるべきである。 Agilentのプラットフォーム上でVirochipマイクロアレイは、（図2B）視覚的に分析することは困難になりますが、ウイルスが存在しているなら、それは容易に目視検査、クラスター分析、および/またはE - Predictは（図1C）で識別する必要があります。アッセイの陽性コントロールとして、サンプルは、既知のウイルス（MS2バクテリオファージなどのタバコモザイクウイルス）からの核酸の測定された量で添加することができます。 <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="図1" /> 図1。 Virochipマイクロアレイの処理と分析の概略図。臨床サンプルから核酸を抽出は無作為に、増幅蛍光色素で標識し、Virochipマイクロアレイにハイブリダイズさせる。マイクロアレイは、2μmの解像度でスキャンし、E -予測を含め、計算ツールのさまざまな方法を使って分析されます。 <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="図2" /> 図2。 Virochipアッセイの手順は、ランダムPCR、200の塗抹標本による増幅後- 。1000bpのゲル電気泳動によって可視化することができます（）（B）8アレイ/スライドガラスの三Virochipマイクロアレイアウトは、小さな地域で、示されている。右下の隅に挿入で吹き、最大1マイクロアレイの。臨床検体中のインフルエンザウイルスの存在を明らかにE -予測。使用して（C）自動化されたマイクロアレイウイルス分析高い類似度スコア（S = 0.55）ため、この非常に重要な予測を作る、ゼロに近いp値に対応しています。

<ol>
	<li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+and+genotyping+of+viral+pathogens.">Microarray-based detection and genotyping of viral pathogens.</a> Proc Natl Acad Sci U S A. 99, 15687-15692 (2002).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+a+critical+respiratory+illness+caused+by+human+metapneumovirus+by+use+of+a+pan-virus+microarray.">Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.</a> J Clin Microbiol. 45, 2340-2343 (2007).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+detection+of+human+parainfluenzavirus+4+infection+associated+with+respiratory+failure+in+an+immunocompetent+adult.">Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.</a> Clin Infect Dis. 43, e71-e76 (2006).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+DNA+microarrays+for+detection+of+viruses+in+acute+respiratory+tract+infections+in+children.">Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.</a> J Pediatr. 153, 76-83 (2008).</li><li>Kistler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-viral+screening+of+respiratory+tract+infections+in+adults+with+and+without+asthma+reveals+unexpected+human+coronavirus+and+human+rhinovirus+diversity.">Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity.</a> J Infect Dis. 196, 817-825 (2007).</li><li>Rota, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+coronavirus+associated+with+severe+acute+respiratory+syndrome.">Characterization of a novel coronavirus associated with severe acute respiratory syndrome.</a> Science. 300, 1394-1399 (2003).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+discovery+and+sequence+recovery+using+DNA+microarrays.">Viral discovery and sequence recovery using DNA microarrays.</a> PLoS Biol. 1, e2-e2 (2003).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+Gammaretrovirus+in+prostate+tumors+of+patients+homozygous+for+R462Q+RNASEL+variant.">Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.</a> PLoS Pathog. 2, 25-25 (2006).</li><li>Kistler, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+divergent+avian+bornaviruses+from+cases+of+proventricular+dilatation+disease:+identification+of+a+candidate+etiologic+agent.">Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.</a> Virol J. 5, e25-e25 (2008).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardioviruses+related+to+Theiler's+murine+encephalomyelitis+virus+in+human+infections.">Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections.</a> Proc Natl Acad Sci U S A. 105, 14124-14129 (2008).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Predict:+a+computational+strategy+for+species+identification+based+on+observed+DNA+microarray+hybridization+patterns.">E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.</a> Genome Biol. 6, R78-R78 (2005).</li></ol>

利害の衝突は宣言されません。

ここで説明するようにVirochipプロトコルは複雑であり、細心と熟練した研究の技術者が必要です。 PCR、ラベリング、ハイブリダイゼーションのための適切な試薬の濃度と条件が非常に​​重要です。 Virochipプロトコルは、簡単にそのような核酸の抽出のためのトリゾール（Invitrogen社）などの組織抽出法を用いて患部組織の分析に対応するように変更することができます。プローブは、適用範囲の所望のスペクトルと幅広さを得るために、必要に応じて追加または削除することができます。例えば、細菌および真菌などの非ウイルス標的の検出のためのプローブは、&quot;オンザフライ&quot;に設計して追加することができます。現在開発中のVirochipアッセイのいくつかのアプリケーションでは、臨床検査、アウトブレイク調査、薬やワクチンの純度のスクリーニング、および新たなウイルスの病原体の発見の広いスペクトルのウイルス診断が含まれています。

<table border="1"><tbody><tr><th>試薬/材料の名称</th><th>試薬/材料の名称</th></tr><tr><td>プリメーラ</td><td> 5&#39; - GTTTCCCAGTCACGATA -（N9）-3&#39; </td></tr><tr><td>プライマーB </td><td> 5&#39; - GTTTCCCAGTCACGATA - 3&#39; </td></tr><tr><td rowspan="5"> RTマスターミックス（5μL） </td><td> 2μL5X RTバッファー</td></tr><tr><td> 1μL12.5mmのdNTPを（Invitrogen社） </td></tr><tr><td> 1μLの水</td></tr><tr><td> 0.5μL0.1M DTT </td></tr><tr><td> 0.5μLSSIII RT（Invitrogen社） </td></tr><tr><td rowspan="3">シークエナーゼミックス（5μL） </td><td> 1μL5Xシークエナーゼバッファ</td></tr><tr><td> 3.8μLの水</td></tr><tr><td> 0.15μLシークエナーゼ（USB株式会社） </td></tr><tr><td rowspan="5"> RD B PCRマスターミックス（45μL） </td><td> 5μL10X PCRバッファー</td></tr><tr><td> 1μL12.5mmのdNTPを（Invitrogen社） </td></tr><tr><td> 1μLを100μL/ pmolのプライマーB </td></tr><tr><td> 1μLKlenTaq LA（シグマ） </td></tr><tr><td> 37μL水</td></tr><tr><td rowspan="5"> RD C PCRマスターミックス（45μL） </td><td> 5μlの10 × PCRバッファー</td></tr><tr><td> 1μL12.5mmのdNTPを（Invitrogen社） </td></tr><tr><td> 1μLを100μL/ pmolのプライマーB </td></tr><tr><td> 1μLKlenTaq LA（シグマ） </td></tr><tr><td> 37μL水</td></tr><tr><td rowspan="4">ハイブリダイゼーション緩衝液（25μL） </td><td> 1μL25Xフラグメンテーションバッファ（アジレント） </td></tr><tr><td> 5μL5Xブロッキング緩衝液（アジレント） </td></tr><tr><td> Cy3標識サンプルの9.5μL（または正常化する量） </td></tr><tr><td> 25μLの全体積に水を追加します。 </td></tr><tr><td>アジレントVirochipマイクロアレイ</td><td>ライセンス申請中の、チウ研究所、UCSFから入手可能</td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

多くの感染症のウイルスの原因の診断は、ウイルスの本来の配列の多様性だけでなく、従来の方法で検出できないSARSコロナウイルスと2009年のパンデミックH1N1インフルエンザウイルス、などの新しいウイルス性病原体、の継続的な進展に伴い、困難です。これらの課題に対処するために、我々が以前に開発され、汎ウイルスのマイクロアレイプラットフォームを検証して保存された配列の相同性1のベースですべての既知のウイルスだけでなく、小説のバリエーションを検出する能力とVirochipと呼ばれる。 Virochipを使用して、我々は、同等または従来の臨床試験2-5に優れた感度を持つ入院患者、原因不明の重症疾患の場合を含む呼吸器感染症、関連付けられているウイルスの完全なスペクトルを同定した。 Virochipは、SARSコロナウイルス6,7、小説ライノウイルスのクレード5、XMRV（前立腺癌にリンクされているレトロウイルス）8、鳥bornavirus（オウムの消耗性疾患の原因）9、を含む、新規のウイルスを識別するために使用されています呼吸器と下痢10の子供のと小説cardiovirus。 Virochipの現在のバージョンは、アジレントのマイクロアレイプラットフォームに移植され、2009年12月のようにGenBankの以上〜1,500ウイルス由来〜36000のプローブで構成されている。ここでは、サンプル核酸の抽出、ランダムプライマーを用いたPCR増幅、蛍光色素の取り込み、及びマイクロアレイのハイブリダイゼーション、スキャニング、及び分析を含む、（〜24時間のターンアラウンドタイム）を最初から最後までVirochipアッセイの処理に必要なステップを示しています。

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Virochipは同時に保存された配列の相同性に基づいてすべての既知のウイルスだけでなく、新たなウイルスを検出するように設計されたパンウ​​イルスマイクロアレイです。ここでは、既知および未知のウイルスの存在のために臨床サンプルを解析するVirochipアッセイを実行する方法を示しています。

ウイルス性病原体のために臨床サンプルをスクリーニングするためにパンウイルスマイクロアレイアッセイ（Virochip）を使用して、

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

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JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

18.7K ビュー.  University of California, San Francisco. Virochipは同時に保存された配列の相同性に基づいてすべての既知のウイルスだけでなく、新たなウイルスを検出するように設計されたパンウ​​イルスマイクロアレイです。ここでは、既知および未知のウイルスの存在のために臨床サンプルを解析するVirochipアッセイを実行する方法を示しています。

ビデオ: Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

呼吸器病原体に対する宿主細胞応答を研究するために偏ヒト気道上皮Calu-3細胞の液体に覆われた文化の確立<em&gt;インビトロ</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

免疫・感染学

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

宿主細胞のRNA合成でウイルス感染の効果を測定するためにクリックケミストリーを用いた

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

アフリカトリパノソーマにグライコダイナミクスを監視する蛍光タンパク質を使用した

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

インフルエンザA型ウイルスのヘマグルチニンの結合力および特異性を評価するための小型糖鎖マイクロアレイアッセイ

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

酵素結合レクチンアッセイによってインフルエンザノイラミニダーゼ阻害抗体価を測定します

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Biocontainmentにおけるウイルスサンプルの陰性染色のためのカプセルの利用

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

病原性細菌に対する免疫学的応答を評価するための Opsonophagocytic 殺害アッセイ

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

ルシファーゼベース成長アッセイを用いた細胞内トキソプラズマゴンディ成長に対する化学阻害剤の効率の測定

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

フローサイトメトリーベースアッセイによる熱帯 熱マラリア原 虫に対する自然獲得食細胞症誘導抗体の測定

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

等温増幅法を用いたヒト呼吸器細菌感染症診断

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

著者スポットライト:マイクロ流体チップを使用した呼吸器病原体の迅速な検出の進歩 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

マイクロ流体チップベースのループ媒介等温増幅による下気道感染症を引き起こす細菌性病原体の迅速な検出

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...