この作品は、パリ市から医療研究助成金、フランス国立助成金ANR - 09 - JCJC - 0118 - 1、およびno ERCの開始グラントRNAvirusPopDivNVaxプロジェクトからの資金によって支えられている。 242719。

<ol>
	<li>Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+RNA-dependent+RNA+polymerase+(in)fidelity+revealed+by+kinetic+analysis+of+the+polymerase+encoded+by+a+foot-and-mouth+disease+virus+mutant+with+reduced+sensitivity+to+ribavirin.">Determinants of RNA-dependent RNA polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a foot-and-mouth disease virus mutant with reduced sensitivity to ribavirin.</a> J Virol. 82, 12346-12355 (2008).</li><li>Arnold, J. J., Vignuzzi, M., Stone, J. K., Andino, R., Cameron, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+site+control+of+an+active+site+fidelity+checkpoint+in+a+viral+RNA-dependent+RNA+polymerase.">Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase.</a> J Biol Chem. 280, 25706-25716 (2005).</li><li>Korneeva, V. S., Cameron, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+of+the+viral+RNA-dependent+RNA+polymerase:+fidelity,+replication+speed,+and+initiation+mechanism+determined+by+a+residue+in+the+ribose-binding+pocket.">Structure-function relationships of the viral RNA-dependent RNA polymerase: fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket.</a> J Biol Chem. 282, 16135-16145 (2007).</li><li>Pfeiffer, J. K., Kirkegaard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+mutation+in+poliovirus+RNA-dependent+RNA+polymerase+confers+resistance+to+mutagenic+nucleotide+analogs+via+increased+fidelity.">A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity.</a> Proc Natl Acad Sci U S A. 100, 7289-7294 (2003).</li><li>Pfeiffer, J. K., Kirkegaard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+fidelity+reduces+poliovirus+fitness+and+virulence+under+selective+pressure+in+mice.">Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice.</a> PLoS Pathog. 1, e11-e11 (2005).</li><li>Vignuzzi, M., Stone, J. K., Arnold, J. J., Cameron, C. E., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quasispecies+diversity+determines+pathogenesis+through+cooperative+interactions+in+a+viral+population.">Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population.</a> Nature. 439, 344-348 (2006).</li><li>Vignuzzi, M., Wendt, E., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+attenuated+virus+vaccines+by+controlling+replication+fidelity.">Engineering attenuated virus vaccines by controlling replication fidelity.</a> Nat Med. 14, 154-161 (2008).</li><li>Crotty, S., Cameron, C., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribavirin's+antiviral+mechanism+of+action:+lethal+mutagenesis.">Ribavirin's antiviral mechanism of action: lethal mutagenesis.</a> J Mol Med. 80, 86-95 (2002).</li><li>Coffey, L. L., Vignuzzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+alternation+of+chikungunya+virus+increases+fitness+while+restricting+population+diversity+and+adaptability+to+novel+selective+pressures.">Host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures.</a> J Virol. 85, 1025-1035 (2011).</li><li>Ciota, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+mutant+spectrum+in+adaptation+and+replication+of+West+Nile+virus.">Role of the mutant spectrum in adaptation and replication of West Nile virus.</a> J Gen Virol. 88, 865-874 (2007).</li><li>Ibarra, K. D., Pfeiffer, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+ribavirin+antiviral+efficacy+via+nucleoside+transporter-mediated+drug+resistance.">Reduced ribavirin antiviral efficacy via nucleoside transporter-mediated drug resistance.</a> J Virol. 83, 4538-4547 (2009).</li><li>Levi, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fidelity+variants+of+RNA+dependent+RNA+polymerases+uncover+an+indirect,+mutagenic+activity+of+amiloride+compounds.">Fidelity variants of RNA dependent RNA polymerases uncover an indirect, mutagenic activity of amiloride compounds.</a> PLoS Pathog. 6, e1001163-e1001163 (2010).</li><li>Sierra, S., D&#225;vila, M., Lowenstein, P. R., Domingo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+foot-and-mouth+disease+virus+to+increased+mutagenesis:+influence+of+viral+load+and+fitness+in+loss+of+infectivity.">Response of foot-and-mouth disease virus to increased mutagenesis: influence of viral load and fitness in loss of infectivity.</a> J Virol. 74, 8316-8323 (2000).</li><li>Ruiz-Jarabo, C. M., Ly, C., Domingo, E., de la Torre, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+mutagenesis+of+the+prototypic+arenavirus+lymphocytic+choriomeningitis+virus+(LCMV).">Lethal mutagenesis of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV).</a> Virology. 308, 37-47 (2003).</li><li>Sierra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foot-and-mouth+disease+virus+mutant+with+decreased+sensitivity+to+ribavirin:+implications+for+error+catastrophe.">Foot-and-mouth disease virus mutant with decreased sensitivity to ribavirin: implications for error catastrophe.</a> J Virol. 81, 2012-2024 (2007).</li></ol>

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細胞株の選択は。RNA変異原性物質として塩基類似体の効果は、異なる細胞型11によるそれらの相対的な取り込みと相関する。通常、ウイルスの継代に使用される細胞株は、取り込みや敏感すぎ（高い細胞毒性を）変異原性物質に不応性であることが判明した場合、それはこれらの要件を満たしていると、まだウイルス複製に許容である別の細胞株を使用する必要があります。変異原性の抵抗のバリアントが分離されると、特性の残りの部分はオリジナルの、好ましい細胞ラインで実行することができます。我々の経験では、HeLa細胞は容易に変異を取る、BHK細胞は変異の取り込みに抵抗性の10倍高い濃度およびVero細胞にまで必要とする。 変異原性の複数のタイプが使用されている場合は変異原性物質の選択は。変異処理により忠実度の亜種を分離しようとするには、成功の可能性を高めることができます。誤って複製中にゲノムに組み込まれている別の構造の基本アナログ変異原性物質は、主にその後の複製サイクルにおける突然変異の特定のサブセットの結果を誘発する：リバビリンの治療はGtoAとCtoU遷移変異12を支持し、5 -アザシチジンはと、同じようなバイアスを持っていますCtoGとGtoCトランスバージョン13の加算は、5 -フルオロウラシル優先エイトグと宇徳遷移14を誘導する。また、Mg 2 +のまたはMn 2の高濃度は、+ 12、上記のバイアスなしでRNAウイルスの全体的な突然変異の頻度を増加させる培地に補足することができます。ウイルスのコドンの配列、および忠実度のバリアントを生成するために必要なコドンの変化に応じて、これらの条件のいくつかは他のものに比べてこの変種の出現を優先します。忠実度の高いポリオウイルスG64SおよびコクサッキーウイルスA372V、コドンのサイトで必要なエイトグの遷移が主にこのリバビリンによって生成される突然変異に相当するので、最も容易に亜種のために選択されたリバビリンの治療のために。 MOI対人口サイズ。ウイルス感染症領域では、組織培養感染のプロトコルはのために、欠陥干渉粒子（低MOI）の蓄積を避けるために、またはウイルス（高MOI）との間で組換えを促進するため、感染の多重度（MOI）に特に注意を払う例。継代シリアルオーバー出現のイベントのために選択するには、ウイルスの人口規模を考慮することも重要です。耐性変異株は、当初は低頻度で存在するので、それは各通路にこれらの新興の変種を失うのを避けるために（10 5 -10 6ウイルス、など）次の1つの通路から、できるだけ多くの人口のサイズとして転送することが最善です。よくまたはフラスコの大きさ（感染した細胞の数）をスケールアップすること、これが問題になる場合MOIの増加を最小限に抑えるために役立つことがあります。一方、変異原性物質に対するウイルスの感受性がテストされている実験のために、低MOI感染は実験で発生する複製のサイクル数を増やすこととによってより高い適応度のゲノムで変異ゲノムの救助を避けるために行われ共同感染細胞における相補。複製の最初のラウンドの間に子孫のゲノム上で生成された変異がすぐに検出されないので、これは重要です。これらの変異RNAのほとんどは、依然としてビリオンにパッケージ化されます。それはこれらのゲノムに存在する致死突然変異が中止された複製サイクルにつながる、とウイルス力価の低下することを感染症の次のラウンドになります。それは致死突然変異誘発の有意な効果が観察される前に、変異の蓄積のいくつかのラウンドを可能にするために必要となる場合があります。最後に、変異原性物質の存在下で継代のシリーズを介して、ウイルスの力価が絶滅するまで低下し続ける場合は、研究者は徐々に変異原性物質（非常に低濃度から開始する）の量を増やすことで、ウイルスを継代してみてください。 RNA変異-抵抗性個体群からRNA変異原性物質耐性クローンの単離と世代。RNA変異原性物質は、それぞれのゲノムに複数のランダムな突然変異を導入するが、抵抗の選択は、唯一の（とコンセンサス配列に修正）耐性変異を豊かにする。この変異を識別するには、我々の配列変異の抵抗性の人口（人口のコンセンサス）ではなく、個々のウイルス。したがって、変異原によって作成された単一の、ランダムな突然変異が連続して検出されていない、唯一の変異は選択した後のコンセンサスの変化にその結果が、発見されています。我々の経験では、1つまたは2つだけそのようなコンセンサス配列の変更を識別します。変異原物質抵抗性個体群が得られ、耐性変異が同定されれば、それはこの亜種の多くの純粋な株式を生成する必要があります。上記の、我々は、プラーク精製の手順を説明した。また、興味のあるウイルスが容易に識別できるプラークを生成しない場合は、希望するバリアントはpurif可能性があります限界希釈法によってIED。このアプローチは、本質的にウイルスストックは、井戸の50％未満が感染しているように希釈して96ウェルフォーマット、のTCID 50です。この希釈を用い、上記と同様のアプローチは、10個の変種まで分離し、そのシーケンスを確認するには、取得されます。前述したように、最善のケースでは、ウイルス株の感染性cDNAクローンは入手可能です。変異体の分離は、このように必要ではないでしょう。我々の経験では、忠実度の亜種は、単一のアミノ酸置換の結果であり、したがってそのようなQuikchange（アジレント）のような単純な、市販の変異導入キットを使用して生成することができます。 secondaryオプションは、密接に関連菌株のcDNAクローンを使用することです。しかし、関連株が使用されている場合、我々は強く我々は2つ​​の密接に関連ウイルスに突然変異を変える同じ忠実度は、必ずしも同じ効果を持っていないことを発見したので、このアプローチとウイルスの分離（例えば、プラーク精製）の両方を使用することをお勧めします。 フィデリティと複製。RNA変異原性物質耐性変異株の選択は、野生型の対応4,12,15に似ている成長の特性を持つ上位と下位忠実バリアントの両方の分離をもたらしました。現在、ポリメラーゼ活性率と忠実度の間のリンクは完全には分かっていない。低忠実度の亜種は、より高速な処理1-3,12を持っている傾向がある一方、in vitroで精製したRNAポリメラーゼを用いて生化学的研究は、より高い忠実度の亜種が遅い処理速度を有することが示されている。組織培養では、これらの違いは、律速段階である、そのリソースの可用性ではなく、内因性ポリメラーゼ活性の動態を示唆し、通常は明らかではありません。忠実度のバリアントは、野生型と有意差はない速度で複製する場合は、それらの変異の頻度の比較を直接行うことができます。レプリケーションの速度で非常に重要な変更が存在する場合、データは複製を同じサイクル数を受けたウイルスを比較することによって、例えば運動の違い、を考慮して正規化する必要があります。ワンステップの成長速度に有意差が野生型と高忠実度の亜種間で観察されなかったものの、我々の経験では、、（内々には、より高い忠実度の亜種が一貫して野生型に比べて（1ログ内の）より高い力価が観察しかし、彼らはわずかにRNAを作る同程度の大きさ）は、さらにそれらが生み出すゲノムが少ない突然変異を含む多くの感染、従ってであることを示唆している。 サンプル調製とシーケンシングは。これらのプロトコルのすべての手順については、それは彼らが生物学的に関連する変異と区別できないので、高忠実度、プルーフリーディング酵素は、さらなる変異を導入制限するためにPCRおよびRT - PCRに使用されることが不可欠です。それは比較されるウイルスの集団は十分な出発材料ができたことを確認することも重要であると同じ条件（流路の歴史、組織培養培地、温度、RNA抽出法、RT - PCRのプロトコル、等）で準備されていることが重要です強力なバンドがRT - PCRによって生成されるように、RNAの抽出から得られる。 RNAサンプルの1 / 100希釈は、試料が（何度も同じゲノムを増幅する）表現のバイアスを避けるために、RNA分子の十分な数字が含まれていることを示す、検出可能なRT - PCRのバンドを与える必要があります。変異頻度がディストリビューションなので、一つは前述のバイアス発生していないを提供し、同様の値に関係なく、人口規模の得られることを期待。図6に示すように、ウイルスストックの10 5倍希釈では、親の株と大幅に異なるがない突然変異の頻度を与えます。 TopoTAクローニングのための最適な条件が見つかるまで、シーケンスの前にコロニーPCRによって、青/白スクリーニング後の挿入の有無を確認する。である（ウイルスのゲノムに対応するのin vitro転写されたRNAの変異ノイズ（RT - PCRおよびシークエンシングによって導入された変異）、プラスミドベアリングから同じウイルス配列をPCR産物をクローンおよび/またはクローンとシーケンスRT - PCR産物のためのコントロールとしてin vitro転写酵素で異なるが）別のエラー率を持っていると、プロシージャの実際のバックグラウンドでエラーに有用な情報を得られない可能性があることに注意。いくつかのウイルスの配列は、バクテリアに有毒かもしれないので、突然変異の頻度のために配列決定されるウイルスゲノムの領域を決定する前にこれを確認することが重要です。 TopoTAによって得られた配列を分析する際に、各クローンは1つだけ挿入/配列を含む必要があることに注意して。ダブルピークが混合集団を示唆し、観察される場合、それは2つの隣接する細菌のコロニーが選択されている可能性があります。非常に低い突然変異は細菌培養におけるプラスミドの増幅の間に導入されたこと、細菌の複製に低い突然変異の周波数を与えられたが、それは、も可能です。プラークpのurified集団、ダブルピークが重なってプラーク、またはプラークの開発中に新たな変異を獲得したり突然変異を戻しているウイルスを表してもよい。一貫性と数えるかどうか、これらの変異をカウントするかどうかを決定します。 最後に、ここで使用される突然変異の頻度は相対値であることに留意してください。彼らは、同じ条件下で栽培したウイルス集団の比較でのみ有効であり、同じ領域に塩基配列を決定！これらは、突然変異率、または全体としてのゲノムの変異頻度の絶対値として解釈されるべきではない。しかし、条件が制御されている場合、それらは、突然変異の分布と周波数の差の再現性、定量的な比較を許可しません。

<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</th></tr><tr><td>リバビリン</td><td>シグマ</td><td> R9644 - 10mgの</td><td></td></tr><tr><td> 5 - フルオロウラシル</td><td>シグマ</td><td> F6627 - 1G </td><td></td></tr><tr><td> 5 - アザシチジン</td><td>シグマ</td><td> A2385 - 100mgの</td><td></td></tr><tr><td> MgCl 2の </td><td>シグマ</td><td> M1028 - 100ML </td><td></td></tr><tr><td> MnCl 2を </td><td>シグマ</td><td> M1787 </td><td></td></tr><tr><td>トリパンブルー</td><td>シグマ</td><td> T8154 - 20mlの</td><td></td></tr><tr><td> TopoTAクローニングキット</td><td>インビトロジェン</td><td> 10351021 </td><td></td></tr><tr><td> Quikchange変異導入キット</td><td>アジレント</td><td> 200516 </td><td> cDNAの感染性クローンは入手可能である場合</td></tr><tr><td> 96ウェルミニプレップキット</td><td>マシュレ-ナーゲル</td><td> 740625 </td><td></td></tr><tr><td> Lasergene、Sequencher </td><td> DNASTAR、ジーンコード株式会社</td><td> <a href="www.dnastar.com">www.dnastar.com</a> <a href="www.genecodes.com">www.genecodes.com</a> </td><td>または他のアラインメントソフトウェア</td></tr></tbody></table>

isolation of fidelity variants of rna viruses and characterization of virus mutation frequency

RNAウイルスは、自分のゲノムを複製するRNA依存性RNAポリメラーゼを使用してください。これらの酵素の本質的に高いエラー率は、ウイルスの適応と進化を容易に極端な人口の多様性の世代に大きな貢献しています。証拠を増やすと、RNAウイルスの固有誤差率、および結果として得られる変異の頻度は、ウイルスのポリメラーゼに微妙なアミノ酸の変化によって変調することができることを示しています。生化学的アッセイは、定款の忠実度の定量的測定を可能にするいくつかのウイルスRNAポリメラーゼのために存在しますが、ここで我々は忠実変える変異を同定する生化学的なアプローチな限り正確であることが証明されてRNAウイルスの変異の頻度を測定する簡単な方法を説明します。アプローチは、ほとんどの生物学の実験室で行うことができる、従来のウイルス学的およびシーケンシング技術を使用しています。異なるウイルスの数の経験に基づいて、我々は、忠実度の亜種を分離して統計的有意性のデータを生成する可能性を高めるために最適化する必要がある重要な手順を特定している。忠実変える変異の単離および特性は、ポリメラーゼの構造と機能1月3日に新たな洞察を提供することができます。さらに、これらの忠実度の亜種は、ウイルスの適応と進化4-7のメカニズムを特徴付けるのに有用なツールになります。

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

現在の記事は、RNAウイルスとどのように組織培養の忠実度の変化を確認するために、変異頻度のデータを使用するためのRNAポリメラーゼの忠実度の亜種を分離し、特徴付けるために必要な手順を説明します。

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 ...

isolation-fidelity-variants-rna-viruses-characterization-virus

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

29.5K ビュー.  Institut Pasteur. 現在の記事は、RNAウイルスとどのように組織培養の忠実度の変化を確認するために、変異頻度のデータを使用するためのRNAポリメラーゼの忠実度の亜種を分離し、特徴付けるために必要な手順を説明します。

ビデオ: Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

のRNAの単離緑膿菌マウス消化管に定着

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

免疫・感染学

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNAウイルスの広域スペクトルの化学抑制剤のためのハイスループットスクリーニング

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

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 ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

抗腫瘍特性を有する好中球の単離とキャラクタリゼーション

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

細菌人工染色体：プラス鎖RNAウイルスの研究のための機能ゲノミクスツール

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

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 ...

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 ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

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

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

エスケープ中和抗体でインフルエンザ ウイルスの亜種の世代

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

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

分離と機能研究の好中球由来の微粒子のキャラクタリゼーション

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

分離と大人住血吸虫から細胞外小胞のキャラクタリゼーション

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...