我々は、ST-246を提供するためのSIGA Technologies社（オレゴン州、OR）に感謝します。 DKRは、ボストン大学（5T32AI 7309）への免疫学のNIHの訓練助成金によってサポートされていました。この作品は、P41 086180、NIH RO1AI1096159-01、および（JHC）はRO3によって部分的にサポートされていました。

1。プレート細胞<ol><li>プレートからHeLa細胞を解離し、約2.0×10 5細胞/ mlに増殖培地（DMEM、2mMのL-過剰、10％FBS）中に希釈する。黒壁、透明な平底96ウェルプレート（20,000細胞/ウェル）に100μl/ウェルを分注する。 </li><li> +5％のCO 2、37℃のインキュベーターでコンフルエントになるまで24時間細胞をインキュベートします。 </li></ol> 2。細胞に感染<ol><li>ウイルスを希釈し、細胞に感染する。 <ol><li>解凍TRPV（トリプルウイルス;早期金星、中間mCherry、後期TagBFP）とPLV（プロモーターのない金星）は、蛍光ウイルス、5分間超音波処理を使用して脱凝集。あるいは、粗ウイルスストックの感染培地中0.25 mg / mlのトリプシンと1:1で混合し、30分間37℃でインキュベートすることができる。 </li><li> 37℃の感染培地（DMEM、2mMのL-GLUT、2％FBS）中の粗ウイルスストックを希釈する。高MOI感染症（10 PFU /細胞）の場合は、ミリリットル5×10 4と仮定すると、1.0×10 7 PFU /にウイルスストックを希釈細胞/ウェルおよび50μlの接種量。 TRPVおよび各治療に固有のバックグラウンド蛍光を考慮してPLVと同じ治療のための別の3複製ウェルに各処理について3反復ウェルに感染する計画。 </li><li>感染培地に50μl/ウェルに希釈したウイルスを加えることによってウェルに感染。これは、時間= 0時間後に感染（0 HPI）として定義されています。 </li></ol></li><li>感染培地に所望の治療化合物を希釈する。すべての処置のための単一2Xマスターミックスの希釈液を作り、井戸を複製するために適用されるべきである（ 例えば 50μlのそれぞれに6 96ウェル350μlの総量）。 <ol><li>感染培地に実験化合物を希釈する。 </li><li>車両制御溶媒（ 例えば 、PBS、DMSO）は、感染培地に希釈します。注：実験化合物に適用されるように、車両制御用溶媒の同様の最終濃度を使用すべきである（ 例えば、あなたのIBTストックはDMSOで製造し、最終concentratioで使用される場合1μLのNSは/ mlの場合、車両制御などの1μL/ ml）を単独で、DMSOを使用しています。 </li><li>感染培地に制御する化合物を希釈する。推奨制御化合物としては、β-セミカルバゾン（IBT）イサチン3.6μM（1μg/ ml）を1-β-D-アラビノフラノシルシトシン（AraCの）、50μMの（11.7μg/ ml）を、60μM（50μg/ ml）をリファンピシンを、または5μM（1.9μg/ ml）を、ST-246 8,15,16。期待される効果のための代表的な結果を参照してください。注意：これはよく、すでにボリュームに1:1の比率で添加されるので、2倍（2倍）所望の最終濃度この希釈を作る。 </li></ol></li><li>すぐにウイルスを添加した後に、ステップ2.2に希釈したように、所望の治療とコントロールを含む50μlの感染培地を追加します。注意：初期段階の式の前に阻害についてアッセイするために、化合物は、いずれの希釈ウイルス接種（ステップ2.1.2）に直接追加することができますか（ステップ2.1.3の前に）感染する前に宿主細胞。 </li><li> 37＆＃で6月24日時間インキュベートする176;インキュベーター+ C、5％CO 2。 </li></ol> 3。細胞を固定<ol><li>各ウェルに、既に感染培地に100μlの8％パラホルムアルデヒドを添加することにより、細胞を固定。光から保護して室温で15分間インキュベートする。注意：これは、高力価のメディアが固定前に直接廃棄物皿に反転している場合に発生する可能性の感染性ワクのエアロゾル化を防ぐために、感染のメディアに直接2X PFA（8％）を追加することをお勧めします。 </li><li>廃棄物の皿にプレートを反転させて固定液を除去します。 </li><li>室温100μlのPBSを追加。 </li><li>光学的に透明な接着フィルムでプレートを密封する。注：すぐに読み出されていない場合、プレートを4℃で保存することができる。分光光度計の測定値を歪ま結露を防ぐために、読む前に室温にシールしたプレートを返すようにしてください。 </li></ol> 4。ウイルスの増殖を定量化<ol><li> 、金星のために515:53​​0でmCherryため587:610をエンドポイント蛍光を測定し、415:457 FまたはTagBFP（励起：NM発光波長）。 4つの測定は、各チャネルの最適化された利得設定を用いて、ウェル当たり平均化されるべきである。注：適切な発光波長は、プレートリーダーの特定のモデル、フィルタ特性に応じて異なっていてもよい。これは、各チャネルのTRPVおよびPLV間の最大差がある点を決定するために、発光波長スキャンを実行することによって経験的に決定することができる。 </li><li>実験間の比較を容易にするために生データを正規化する。 <ol><li>各チャンネル毎に、複製TRPVおよびPLVウェルの平均を決定する。 </li><li>それぞれの治療のための平均TRPV感染実験測定からの平均PLV感染背景測定値を減算します。 </li><li>よく車専用の値によって各バックグラウンドを差し引いた値で割って、データを正規化します。 </li><li>実験は複数回、一元配置分散分析（一元配置ANOVA）試験および複数cと繰り返された後omparisonポストテストは、治療のいずれかが各チャネルのビヒクルのみの治療統計学的に有意な差を反映しているかどうかを決定するために行うことができる。 </li></ol></li></ol> 5代替プロトコル：キネティックプレートアッセイ<ol><li>処理（ステップ2.3）を添加して、すべての手順を実行します。 </li><li> 37℃に平衡化プレートリーダーチャンバ内の接着フィルムと場所とシールプレート注：特別な注意は、過度の細胞ストレスおよび結露を防ぐために、一貫して37℃でプレートを維持するために注意する必要があります。動的アッセイのために、5％のCO 2を添加せずに適切なpHを維持する緩衝培地（炭酸水素ナトリウムおよびHEPES）を使用することが特に重要である。 </li><li>後の時点での飽和を防ぐために、手動でプレートリーダーゲインを設定する。注：正確な利得の最適化は、同様の条件下で、エンドポイントアッセイを行うことによって得ることができる。 </li><li> 8月24日HPI 1時間ごとの測定値を取得し、シミを使用して正規化エンドポイントアッセイ（ステップ4.2）のように、LARの手順。 </li><li>個々の時点は、前述の終点アッセイと同様の方法を用いて統計的有意性について分析することができる。 </li></ol>

<ol>
	<li>Yen, J., Golan, R., Rubins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+infection+&amp;+temporal+analysis+of+virus+gene+expression:+part+1.">Vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1.</a> J Vis Exp. 26 (26), (2009).</li><li>Hansen, S. G., Cope, T. A., Hruby, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BiZyme:+a+novel+fusion+protein-mediating+selection+of+vaccinia+virus+recombinants+by+fluorescence+and+antibiotic+resistance.">BiZyme: a novel fusion protein-mediating selection of vaccinia virus recombinants by fluorescence and antibiotic resistance.</a> BioTechniques. 32 (5), 1182-1187 (2002).</li><li>Popov, S., Mirshahidi, S., Essono, S., Song, R., Wang, X., Ruprecht, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+recombinant+vaccinia+viruses+via+green+fluorescent+protein+selection.">Generation of recombinant vaccinia viruses via green fluorescent protein selection.</a> DNA and Cell Biology. 28 (3), 103-108 (2009).</li><li>Bartee, E., Mohamed, M. R., Lopez, M. C., Baker, H. V., McFadden, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+addition+of+tumor+necrosis+factor+plus+beta+interferon+induces+a+novel+synergistic+antiviral+state+against+poxviruses+in+primary+human+fibroblasts.">The addition of tumor necrosis factor plus beta interferon induces a novel synergistic antiviral state against poxviruses in primary human fibroblasts.</a> Journal of Virology. 83 (2), 498-511 (2009).</li><li>Ward, B. M., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+intracellular+movement+of+vaccinia+virus+virions+containing+a+green+fluorescent+protein-B5R+membrane+protein+chimera.">Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera.</a> Journal of Virology. 75 (10), 4802-4813 (2001).</li><li>Johnson, M. C., Damon, I. K., Karem, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+high-throughput+vaccinia+virus+neutralization+assay+for+testing+smallpox+vaccine+efficacy+based+on+detection+of+green+fluorescent+protein.">A rapid, high-throughput vaccinia virus neutralization assay for testing smallpox vaccine efficacy based on detection of green fluorescent protein.</a> Journal of Virological Methods. 150 (1-2), 14-20 (2008).</li><li>Dower, K., Rubins, K. H., Hensley, L. E., Connor, J. 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+of+Vaccinia+reporter+viruses+for+rapid,+high+content+analysis+of+viral+function+at+all+stages+of+gene+expression.">Development of Vaccinia reporter viruses for rapid, high content analysis of viral function at all stages of gene expression.</a> Antiviral Research. 91 (1), 72-80 (2011).</li><li>Dower, K., et al. <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+pyridopyrimidinone+inhibitor+of+orthopoxviruses+from+a+diversity-oriented+synthesis+library.">Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library.</a> Journal of Virology. 86 (5), 2632-2640 (2012).</li><li>De Clercq, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Potential+of+the+Acyclic+Nucleoside+Phosphonates+Cidofovir,+Adefovir,+and+Tenofovir+in+Treatment+of+DNA+Virus+and+Retrovirus+Infections.">Clinical Potential of the Acyclic Nucleoside Phosphonates Cidofovir, Adefovir, and Tenofovir in Treatment of DNA Virus and Retrovirus Infections.</a> Clinical Microbiology Reviews. 16 (4), 569-596 (2003).</li><li>Prichard, M. N., Kern, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthopoxvirus+Targets+for+the+Development+of+New+Antiviral+Agents.">Orthopoxvirus Targets for the Development of New Antiviral Agents.</a> Antiviral Research. 10, (2012).</li><li>Fenner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+successful+eradication+campaign.+Global+eradication+of+smallpox.">A successful eradication campaign. Global eradication of smallpox.</a> Reviews of Infectious Diseases. 4 (5), 916-930 (1982).</li><li>Breman, J. G., Henderson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+dilemmas--monkeypox,+smallpox,+and+biologic+terrorism.+The+New+England.">Poxvirus dilemmas--monkeypox, smallpox, and biologic terrorism. The New England.</a> Journal of Medicine. 339 (8), 556-559 (1998).</li><li>Baker, R. O., Bray, M., Huggins, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+antiviral+therapeutics+for+smallpox,+monkeypox+and+other+orthopoxvirus+infections.">Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections.</a> Antiviral Research. 57, 1-2 (2003).</li><li>Rimoin, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+increase+in+human+monkeypox+incidence+30+years+after+smallpox+vaccination+campaigns+cease+in+the+Democratic+Republic+of+Congo.">Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo.</a> Proceedings of the National Academy of Sciences of the U S A. 107 (37), 16262-167 (2010).</li><li>Sodeik, B., Griffiths, G., Ericsson, M., Moss, B., Doms, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+vaccinia+virus:+effects+of+rifampin+on+the+intracellular+distribution+of+viral+protein+p65.">Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.</a> Journal of Virology. 68 (2), 1103-1114 (1994).</li><li>Grosenbach, D. W., Jordan, R., Hruby, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+small-molecule+antiviral+ST-246+as+a+smallpox+therapeutic.">Development of the small-molecule antiviral ST-246 as a smallpox therapeutic.</a> Future Virology. 6 (5), 653-671 (2011).</li><li>Broyles, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+transcription.">Vaccinia virus transcription.</a> Journal of General Virology. 84 (9), 2293-2303 (2003).</li><li>Condit, R. C., Niles, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+viral+transcription+elongation+and+termination+during+vaccinia+virus+infection.">Regulation of viral transcription elongation and termination during vaccinia virus infection.</a> Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1577 (2), 325-336 (2002).</li><li>D&#237;az-Guerra, M., Rivas, C., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+expression+of+the+2-5A+synthetase/RNase+L+system+results+in+inhibition+of+vaccinia+virus+replication.">Inducible expression of the 2-5A synthetase/RNase L system results in inhibition of vaccinia virus replication.</a> Virology. 227 (1), 220-228 (1997).</li><li>Cohrs, R. J., Condit, R. C., Pacha, R. F., Thompson, C. L., Sharma, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+ppp(A2&#8217;p)nA-dependent+RNase+by+a+temperature-sensitive+mutant+of+vaccinia+virus.">Modulation of ppp(A2&#8217;p)nA-dependent RNase by a temperature-sensitive mutant of vaccinia virus.</a> Journal of Virology. 63 (2), 948-951 (1989).</li><li>S&#225;nchez-Puig, J. M., S&#225;nchez, L., Roy, G., Blasco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+different+leukocyte+cell+types+to+Vaccinia+virus+infection.">Susceptibility of different leukocyte cell types to Vaccinia virus infection.</a> Virology Journal. 1 (1), (2004).</li><li>Bengali, Z., Satheshkumar, P. S., Yang, Z., Weisberg, A. S., Paran, N., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+S2+cells+are+non-permissive+for+vaccinia+virus+DNA+replication+following+entry+via+low+pH-dependent+endocytosis+and+early+transcription.">Drosophila S2 cells are non-permissive for vaccinia virus DNA replication following entry via low pH-dependent endocytosis and early transcription.</a> PLoS One. 6 (2), (2011).</li></ol>

著者らは、開示することは何もないし、何の特許は、ウイルスやスクリーニング法のために保留されていません。

ここでは、ウイルス複製の再現可能な、リアルタイムのフィードバック対策を提供し、多段ワクシニアウイルスレポーター（TRPV）の実際の使用を記載している。明確に定義されたポックスウイルス阻害剤を使用することにより、我々はTRPVは、各阻害剤の作用機序を理解と一致した様式で応答することを示すことができた。 TRPVウイルスはウイルスステージの進行に関する最も包括的な情報を...

伝統的に、ウイルス発現は、分子生物学技術（ 例えばノーザンブロッティング、ウェスタンブロッティング、マイクロアレイハイブリダイゼーション、 等 ）1を用いて研究されている。これらの方法は個々のmRNAまたはタンパク質の発現の変化を分類に関する詳細な情報を提供することができるが、これらは典型的には、リアルタイムのハイスループットプロセスに適していない。ポックスウイルスを扱うとき蛍光ベースのレポーターを使用して別のアプローチは、以前適用されている。しかし、その開発と使用法は様々な目的が動機とされています。いくつかのそのような方法は、組換えウイルスの2,3の選択のために設計した。これらの技術は、適切な取り込みおよび組換えワクシニアクローンからの外因性DNAの発現の際に、EGFPを発現する。同様に、いくつかのワク株は、安定的に、可溶性EGFPを発現しているか、ネイティブのワクシニアプロモーター下で発現GFPタグタンパク質は、広く使用されてきた。これらは代表値であるICALLYは、ウイルス侵入および複製抗体ベースの中和アッセイ中に、化学阻害、または抗ウイルス効果4-6との比較を定量化するために使用される。これらのウイルスは有用であることが証明されてきたが、これらは曖昧なため、単一の初期/後期ウイルスプロモーターのそれらの使用に阻害の点についての詳細な情報を提供する能力が制限されている。以前の方法はまた、次善の信号対雑音特性を有するEGFPタンパク質の使用を行った。 によるポックスウイルスの複製およびウイルス遺伝子発現のリアルタイムの変化をアッセイするために利用できる既存のツールの欠如が複雑であるため、我々は、単一および複数段レポーターウイルス7,8一式を開発した。以前の刊行物に記載されるように、これらのウイルスは、感染の初期、中間、または後期段階の間に一つ、二つ、または天然のワクシニアプロモーターから三スペクトル的に異なる蛍光タンパク質を発現する。これらのウイルスは、私たちすることができます蛍光顕微鏡を用いてウイルス複製の進行の指標としてオピニオン、それらは、ハイスループットプレート·アンド·リーダーベースのアッセイのために等しく良好に適している。これらのウイルスは、同等の力価が7に到達するために同様の速度論で成長し、野生型ウイルスの代わりに使いやすい。これらのウイルスの計画と作成中に、多くの注意が発現の変化（金星、mCherryとTagBFP）への迅速なフィードバックと信頼性の定量化を容易にするために、優れたフォールディング効率の高い信号対バックグラウンド特性を持つ蛍光プローブを選択する際に撮影された。さらに、ウイルスプロモーターの組み合わせがあいまいとは対照的に、高忠実度、明確な段階特異的発現、早期（C11Rプロモーター）のすべての範囲についての情報を提供して、中間体（G8Rプロモーター）と後期（F17Rプロモーター）の遺伝子発現を生成するものを選択し、初期/後期プロモーター。 これらのウイルスはINVEためのツールと​​して使用することができる原型ポックスウイルス、ワクシニアウイルスのライフサイクルをstigating。多くはまだ研究の長い歴史にもかかわらず、宿主 - ウイルス相互作用に関する知られていない。ワクは免疫調節され、拮抗ホストその多くは200以上のユニークなタンパク質を産生する、複雑です。感染時に、ワクシニアウイルスは、直ちに初期のmRNA転写を開始する。これはビリオン中にパッケージングウイルスゲノム上にロードされ、その後の感染れるまで一時停止状態に保持されたRNAポリメラーゼおよび転写因子によって促進される。この初期の発現は、主に宿主の免疫系（のmRNA脱キャップは、dsRNAの隔離、およびデコイ受容体タンパク質だけでなく、アポトーシスの阻害剤、ストレス応答、およびToll、イリノイ、およびNF-κBシグナル伝達）とゲノム複製の抑制に必要なタンパク質を生成します。初期発現はまた、中間体発現に必要な転写因子を生成する。中間表現は、​​後期転写因子の発現を含む。この発現カスケードは、成熟したワクシニアウイルス粒子の完全なアセンブリのために必要な感染症の後期段階の間の構造的および酵素的ウイルスタンパク質の産生をもたらす。 蛍光レポーターウイルスの私達のセットはポックスウイルス生物学の理解でなされるべき急速な進歩を可能にします。ウイルス学の分野で最も一般的で時間のかかる方法の一つは、増殖アッセイである。これは、典型的には、細胞に感染する感染細胞を溶解することによって治療、収穫ウイルスの系列を制定し、プラークアッセイにより得られたウイルス力価を定量することを含む。ここに記載のレポーターウイルスを使用して、容易にアッセイと並行して行う多数の処置の間で比較することができるウイルス増殖のリアルタイム測定を可能にする。我々は、試薬のセットは、薬物治療、RNAiをkに応じて、ウイルス遺伝子発現の変化を同定するための様々なプロトコルで使用される予見nockdown、又は宿主範囲制限。 また、この方法は、関心のある、具体的に定義されたターゲットのステージのためのハイスループット抗ウイルス薬物スクリーニングを可能にこれまで利用可能なより大きなスケールでの高内容分析を可能にします。ポックスウイルス感染症と戦うために、多数の潜在的な治療は同定されているが、唯一のFDAは、ポックスウイルス感染を治療するための有効な療法を承認した非環状ヌクレオシドホスホネート、シドフォビル、およびワク免疫グロブリン9,10での処理である。 1977 11における天然痘の撲滅にもかかわらず、ポックスウイルスは、人間の健康12に重大な脅威のまま。天然痘ウイルスに対する広範なワクチン接種の中止は、他のポックスウイルス13に対する感受性の増加につながっている。たとえば、以前にワクチン接種により保護されて中央アフリカの領域はサル痘ウイルス感染14の急増を経験している。重大な懸念もされている天然痘ウイルスの意図的なリリースに潜在感受性に関して提起。により現在利用可能な限られた治療に対する、新規な治療法の開発が急務とされている。これらのレポーターウイルスは、ウイルス複製の特定の段階を阻害するための迅速かつハイスループットな小分子阻害剤のスクリーニングを可能にする。阻害のない現在標的ウイルス発現段階を標的阻害剤の同定は、効力の増加との併用療法の開発を容易にする。 多くの各段の遺伝子発現の変化を観察することによってワクシニア細胞生物学について収集することができる。化学的または感染宿主細胞の遺伝子操作による減衰は、典型的には減少し、ウイルス力価で表現される。しかし、ウイルス発現カスケードの各段階の変化を比較することによって、人は、ウイルス適応度がどのようにインパクトのより完全な理解を得ることができる特定の治療によって編これらのデータは、従来のウイルス力価の出力とよく相関するが、より詳細な機構的情報だけでなく、高いスループット能力7を提供することが示されている。

<table border="0" cellpadding="0" cellspacing="0" width="1249">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:747px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:104px;">11995-065</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fetal Bovine Serum - Optima, Heat Inactivated (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">200 mM L-Glutamine, (L-glut)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3712</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Trypsin, 2X, Sterile, Irradiated</td>
			<td>Worthington Biochemical Corp.</td>
			<td>TRLVMF</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl Sulfoxide, Cell Culture (DMSO)</td>
			<td>American Bioanalytical</td>
			<td>AB03091</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1-&#946;-D-Arabinofuranosylcytosine&#160; (AraC), MW= 280 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>C6645</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">isatin &#946; -thiosemicarbazone (IBT), MW= 234 g/mol</td>
			<td>Fisher Scientific</td>
			<td>NC9075202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Rifampicin, MW= 823 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">ST-246, MW= 376 g/mol</td>
			<td>SIGA Labs, Corvallis, OR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">8% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TempPlate RT optically clear film</td>
			<td>USA Scientific</td>
			<td>2978-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

vaccinia reporter viruses for quantifying viral function at all stages of gene expression

ポックスウイルスは、サル痘、軟属腫contagiousum、およびContagaloウイルスのような積極的なヒト病原体が含まれる二本鎖DNAウイルスのファミリーである。家族はまた、天然痘ウイルス、痘瘡が含まれています。原因ポックスウイルス複製の複雑さのために、多くの疑問がまだ彼らの遺伝子発現戦略に関する残る。この記事では、ハイスループットフォーマットでウイルス遺伝子発現の単一および複数のステージのリアルタイム測定を可能にする組換えワクシニアウイルスの概念および使用を記載している。これは、ウイルス複製の3段階のそれぞれのためのレポーターとしてスペクトルが異なる蛍光タンパク質を使用することで有効になっています。段階特異的発現パターンを維持しながら、これらのウイルスは、プレートベースのアッセイおよびウイルス増殖および複製の顕微鏡観察を可能にする、高い信号対雑音比を提供する。これらのツールは、ウイルス検出、ウイルス - 宿主相互作用の研究、および進化の生物のための用途がありますでれっと。

これらのウイルスの典型的な使用法の一例として、三重蛍光レポーターウイルスは、いくつかの明確に定義されたポックスウイルス阻害剤の阻害の点を比較した。 HeLa細胞を、黒色壁透明平底96ウェルプレートを処理し、一晩培養し、組織培養にプレーティングした。プレートマップ（ 図1）に詳述されるように、またはプロモーターのないビーナス（PLV）、...

Watch this Scientific Journal Video about Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression at JoVE.com

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

我々は、スペクトル的に別個のレポーターフルオロフォアの段階特異的発現を介して、ウイルス感染および遺伝子発現のリアルタイム測定を可能にする蛍光レポーターワクシニアウイルスの使用を記載している。我々の詳細を正確にウイルス複製を阻害する小分子に応答して影響される段階を識別するためのプレートベースの方法。

遺伝子発現のすべての段階でウイルス機能を定量化するためのワクレポーターウイルス

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

vaccinia-reporter-viruses-for-quantifying-viral-function-at-all

Research

JoVE Journal

Immunology and Infection

11.2K ビュー.  Boston University School of Medicine. 我々は、スペクトル的に別個のレポーターフルオロフォアの段階特異的発現を介して、ウイルス感染および遺伝子発現のリアルタイム測定を可能にする蛍光レポーターワクシニアウイルスの使用を記載している。我々の詳細を正確にウイルス複製を阻害する小分子に応答して影響される段階を識別するためのプレートベースの方法。 

ビデオ: Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

同種造血幹細胞移植後のウイルス感染を予防/治療するMultivirus -特異的T細胞の生成

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

免疫・感染学

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

カテゴリの検出細菌性病原体のための&quot;生物発光&quot;レポーターファージ

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

すべてのインフルエンザA型の定量的な分析シンプルなスロットブロットアッセイでユニバーサル抗体を用いたウイルスHemagglutininsとノイラミニダーゼ

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

特定のヒトと動物のウイルスを用いた微生物ソースのトラッキングのための費用対効果の高い方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

ウイルスの変異頻度のRNAウイルスと特性の忠実性の変異の分離

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

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 ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

マウス胎児肝臓培養システムは、ターミナル赤血球の初期および後期段階での標的遺伝子の機能を分析する

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

キメラマウスモデルにおける組換えインフルエンザワクチンの鼻腔内投与は、粘膜免疫を研究するために、

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

突然変異体ワクシニアウイルスベクターを構築するためのシンプルで効率的なアプローチ

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

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), ...