この記事の出版物は、テネシー大学の研究局からの助成金によって支えられて。著者らは、これらの手順を絞り込むには働いている学生や研究者の前世代に感謝。研究は全米科学財団（NSF - 0851113、NSF - 0825405とNSF - 0550485）からの補助金によって支えられている。

1。 &quot;ウイルスフリー&quot;水（ヴィルヘルム＆Poorvin 2001）を生成するために海水の限外ろ過<ol><li>海水/ lakewaterの約20Lを無菌的に可能な限り収集されます。 </li><li>水はシリアル142 - mm径のポリカーボネートを通じて地域社会の分析のために-20℃で保管することができます0.8μmのフィルタをフィルタ処理されている。より大きな孔径のフィルターは、非常に生産的なシステムのためのこのステップの前に使用することができます。 </li><li> 30kDaのカットオフスパイラルカートリッジはすべてのウイルスを排除するために使用されるアミコンM12システム（ミリポア）、さらに小型のRNAウイルスから限外濾過水を得るために。 </li><li>サンプルは、背圧の〜15から16 kPaで〜25％の速度で処理し、濃縮されています。 </li><li>水の〜500mLの残りのサンプルでは、​​ウイルスのない水の残りの19.5 Lのウイルス産生のアッセイに使用される一方、濃縮ウイルスのコミュニティを（これは他の実験のために保存することができる）が含まれます。 </li><li>使用のそれぞれの一日を過ごした後、アミコンM12システムは、フィルターカートリッジの膜への損傷を防ぐために洗浄する必要があります。 </li><li>もし海水を使用している場合は、30〜45分のための0.1N NaOH溶液で洗浄した後、Milli - Q水の少なくとも6Lで膜をすすいでください。 </li><li>再びMilli - Q水の少なくとも6Lでカートリッジをすすいでください。 </li><li> M12システムの使用を終了するときに、スパイラルのカートリッジは4で0.05MH 3 PO 4水溶液℃で保存してください。 </li></ol> 2。ウイルス産生のためのウイルスの削減手法（ヴィルヘルムら、2002） <ol><li>その上に置かれた0.2μmの公称孔径の低タンパク質結合フィルター（例えば、デュラポア）で取得し、sterifilterユニットに配置され、最大ホストとウイルスの両方で海水/ lakewaterサンプルの500mlに。 </li><li>継続的にフィルターに集中するから、細菌細胞を抑制するために無菌トランスファーピペットを使用してサンプルを再懸濁しながら、サンプルを穏やかに&lt;200 mmHgの時に圧力をかけて真空されています。 </li><li>ゆっくりと限外濾過の三巻が大幅に試料中の遊離ウイルスの数を減らすために細菌懸濁液に添加されています。 </li><li>細菌の割合は、ウイルスフリーの水で500mLに戻って希釈し、3つに分割されている150mLのそれぞれの複製と透明な250ミリリットルポリカーボネートボトルに配置されます。 </li></ol> 3。ウイルス産生のためのタンジェンシャルフローフィルトレーション（TFF）メソッド（Weinbauer ら、2002、ウィンゲットら、2005） TFFは、ウイルス還元法に代わるアプローチを表します。 <ol><li>上記のような天然試料の約500mLを集めています。 </li><li>このサンプルは、0.2μmの公称孔径タンジェンシャルフローろ過システムを用いて濃縮される。 </li><li>細菌の割合が約10〜15 mLに低下すると、限外濾過、ウイルスフリー水を添加し、上記のように配布されています。 </li><li>複製ボトルは、環境槽を用いてその場の条件でインキュベートする。 <ol><li>光レベルは、光強度を減少させるためにネットスクリーニングと青がかったアクリルまたは透明なアクリルを使用して表面状態に変更されます。 </li><li>周囲の表面温度は、しばしば流れる海水デッキインキュベーターを使用することによって得られる。 </li></ol></li><li>細菌とウイルスの資源量推定値のサンプルはcryovialsに追加さ2.0〜2.5パーセント滅菌グルタルアルデヒドの最終濃度で、時間0で撮影されています。これらのサンプルは、直ちに液体窒素で凍結させ、処理されるまで凍結されたフラッシュされています。 <ol><li>液体窒素が使用できない場合は、顕微鏡のスライドは、（次の手順を参照）を調製し、直ちに処理されることがあります。 </li></ol></li><li>サブサンプルは、上記の方法で少なくとも10時間毎に2.5時間を集めています。 <ol><li>この時点で水は定量的PCR解析のために収集されることがあります。最大サンプルを5 mLまで液体窒素中で即座にフラッシュの凍結と無固定剤をクライオバイアルに添加することができる。 </li></ol></li></ol> 4。ウイルスの生産顕微鏡（ノーブル＆ファーマン1998年、温家宝ら 2004） <ol><li> 0.02μmの顕微鏡用にフィルタリングされる凍結試料を氷上で融解してください。 </li><li>滅菌水で原液を1:10に希釈してSYBR Greenのストック溶液を調製。次に、ストック溶液から、滅菌水39 mLに原液の1mlを加えることによって、作業溶液を調製。 50％グリセロール、50％のリン酸塩は生理食塩水をバッファ（PBS、0.05 MのNa 2 HPO 4、0.85％のNaCl、pH7.5）およびp -フェニレンジアミンの新鮮な2.5％の原液は、開始前に準備する必要があります。 4に50％glycerol/50％PBS溶液を保持° CとP - phenylenediam-20伊根の株式△初任まで、暗所で。右の退色防止ソリューションとして使用される最終濃度0.1％を50％glycerol/50％PBSにp -フェニレンジアミンを追加フィルタリングの前に。 </li><li> 0.45μmのMicronStep、セルロース系バッキングフィルターの上に0.02μmのAnodiscフィルター25mmを置きます。完全に乾くまでに20のpKaでAnodiscと真空の上部に固定されたサンプル850μLを加える。場所100滅菌シャーレにSYBRグリーンワーキング溶液μLと、上でも、真空で、慎重にSYBRグリーン上でフィルタ塔と場所からAnodiscを削除します。 20分間室温で暗所でサンプルをインキュベートする。慎重にSYBR Green液からフィルタを削除して、すべての残留色素を除去するキムワイプとフィルターの後ろを放出する。必要に応じて、タワーにフィルターを戻し、余分な汚れを洗い流しますするフィルタを介して0.02μmのフィルタ処理された水または無菌のメディアの800μLまで渡す。 </li><li>顕微鏡のスライドに退色防止ソリューションの小滴を追加し、上にカバースリップを配置。カバーガラスを外し、退色防止ソリューションによる湿式顕微鏡のスライドに乾燥フィルタを追加します。再び、カバーガラスに退色少量の溶液を加えると徐々に形成する可能性のある気泡を取り除くために確認しながら、フィルターの上に置きます。 </li><li>必要になるまで、すぐに（これらはフェージングと低下ウイルスのカウントを防ぐために、数ヶ月以内に使用されるべきである）-20℃でスライドを凍結する</li><li>ウイルスは、広い青いフィルターセット（λ 例 = 450〜490 nmの、λ エム λにおける抑制フィルタ付き= 510 nmの= 510 nm）を（我々の場合ライカDMRXA顕微鏡）蛍光顕微鏡を使用して列挙されます。各フィルタは、フィルタ膜を通過するウイルスを均等に各フィールドのグリッドから合計ウイルスを定量化することを確認し、カウントビューの少なくとも20個のフィールドを持つことになります。 </li><li>三つの独立からのウイルスの再発の平均速度は、計算されると標準偏差は、生産率から決定される複製。 </li></ol> 5。代表的な結果研究者によって収集された生データは、ウイルスの存在量の再発率を生成するための最小限の数学的処理が必要になります。この研究から生じる主要なデータセットには、インキュベーションから二次検体中のウイルスの存在量の再発率である。これらの結果は、試料の各々のウイルスの存在量対時間の独立した回帰を形成する。各サンプルについて、個々のインキュベーションでは、その研究者が同様に変動の推定値（例えば、標準偏差）として速度を計算することができる3つの繰り返し、インキュベーションを行うことにより、一つの治療、（図1を参照。）として機能このプロセスの一つの注意点は、ウイルスの存在量不変の減少は、ウイルスの負担を運んでいるサンプルで宿主細胞の減少につながるということです。この損失を相殺するために、ソース（フィルタ処理されていない海水や湖沼の水）とT = 0インキュベーションのサンプルの両方からの細菌の存在量の列挙が必要です。この情報は、失われた細胞の割合を考慮するために使用することができます。ウイルスの存在量を減少させるプロセスは微生物コミュニティのあらゆるメンバーのためにまたはに対して選択的ではない、この因子がその後、ウイルスのその場の生産率の推定に使用することができると仮定。 <img src="/files/ftp_upload/2196/2196fig1.jpg" alt="図1" /> 図1。落射蛍光顕微鏡を用いた原位置条件下で少なくとも10時間のインキュベーションオーバーウイルス様粒子の生産。サンプルは2008年9月にニュージーランド沖の咲く植物プランクトン中に収集された。 <img src="/files/ftp_upload/2196/2196fig2.jpg" alt="図2" /> 図2。ウイルス産生をアッセイするためのワークフロープロセスの模式図。プロセスは、ウイルスフリー水を生成するために、サンプル水のろ過から始まります。これは、限外ろ過システムを使用して完了します。微生物群集が（感染および非感染細胞の混合物を含有する）保たれる間、パラレル水サンプルに同じサイトから収集され、無料のウイルスがフィルターを通過。このコミュニティは、ウイルスフリーの水に再懸濁し、 その場の状況の下でインキュベートする。ウイルスの再発率は、ウイルス産生の速度を決定するために、次の10時間監視されています。

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ウイルスは、海洋微生物群集にどのように影響するかを理解する上で重要な部品とは、ウイルス粒子が生成されるレートを決定することです。 （ヴィルヘルムら、1998）水生システムに迅速に存在量はほとんどのシステムでは（多かれ少なかれ）静的であることを考えると（ヴィルヘルム＆サトル1999、Weinbauer 2004）、そしてそのウイルスは、非感染性の除去またはレンダリングされ、その後、生産率は、でなければならない相対的な急速な失われた粒子を交換する。 死亡率のウイルスは微生物群集の原因と推定することは、ウイルスが細胞を（&quot;バーストサイズ&quot;）溶解するたびに、生成される多くのウイルスの知識が必要です。天然試料中のウイルスのバーストサイズは​​大きく異なる可能性があります。バーストサイズは​​、透過型電子顕微鏡（例えば、Weinbauer＆Peduzzi 1994）によって直接決定されるが、これは必ずしも現実的ではない与えられた実験室の能力を超えて多くの場合、またはすることができます。それらは経験的に決定することができない状況では、溶解イベントあたり24ウイルスの文献値は、海洋システムや淡水系（パラダほか 2006）のための34のために使用することができます。ウイルス産生の割合がこの数値によって分割されている場合、結果は毎日、ウイルスによって破壊された体積あたりの細胞の豊富さです。値を溶解微生物は、問題のシステムのためのウイルスによる死亡率をもたらす細菌の存在量の立っている株式によって分けることができます。数パーセントからほぼ全人口に既存の推計範囲及び当該のシステムではしばしば他の要因に依存しています（ヴィルヘルム＆マッテソン2008）。総死亡率の割合を決定するためにこの数は、多くの場合、両者（細胞の50％が再現するに行くことを前提で仕事をし、細胞の50％が失われ、Weinbauer 2004）によって乗算されます。 となっている栄養素や微量元素の生物学的利用能（例えば、N、P、Fe）の一次生産の速度を制限し、そのような炭素フラックスとして、水生システムを通じて、そしてこの過程でウイルス駆動型微生物の死亡率の役割を理解できることを考える海洋地球化学への関心の。いくつかの推定値は、現在（2009年、2008年、ヒギンズらロウら ）ウイルスが日常的に水の列に戻って養分の有意な濃度を放出示唆それが存在し、これらの要素が急速に微生物群集によって同化されること（Poorvin らら2004年、ミら、2005）。環境への養分フラックスの割合は、栄養素ごとのセル（&quot;クォータ&quot;と表記）の量によって破壊された細胞の数を乗じて求めることができます。この情報は、微生物食物網は、水系全体でどのように機能するかについての我々の理解に不可欠なコンポーネントを提供することができます。 継続的な開発：現在の努力の研究グループの一連では、コミュニティ内の特定のウイルスを列挙すると、次のような、ウイルスの活動によって影響を受けているどのように特定の微生物を決定するために上記の戦略を適応させる伴う。これを行うには、研究者は全体のウイルスのコミュニティの推定値に並行して特定のウイルスのグループや家族の存在量を推定する定量的ポリメラーゼ連鎖反応（定量PCR）を使用してください。結果は、直接、特定のプランクトンのグループのためのウイルスの死亡率、栄養売上高などの推定値を提供するために適用されます。この強力な新しいアプローチは、今後数年間で研究者が実験室のシステムの制約を超えて特定のウイルス - 宿主の社会の相互作用を定量化するためにウイルスの生態学に関連するプロセスや、初めて、にはるかに深く掘ることができます。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2.5% p-phenylenediamine</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>130575000</td>
<td>Stock for Antifade</td>
</tr>
<tr>
<td>Amicon Proflux M12 system</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>N/A</td>
<td>Any ultrafiltration device may be used for this step</td>
</tr>
<tr>
<td>SYBR Green I nucleic acid gel stain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S-7563</td>
<td>&nbsp;</td>
<tr>
<td>Helicon S10 30kDa Filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>CDUF010LT</td>
<td>&nbsp;</td>
<tr>
<td>Pelicon XL filters 0.22 &mu;m</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>PXGVPPC50</td>
<td>&nbsp;</td>
<tr>
<td>GE 0.2 &mu;m PCTE membrane filters (47mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-732-35</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Labscale Tangential Flow Filtration System</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>XX42LSS11</td>
<td>Other TFF systems may be used for this step</td>
</tr>
<tr>
<td>0.45 &mu;m Micronstep, Cellulosic, white plain filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>E04WP02500</td>
<td>&nbsp;</td>
<tr>
<td>GE Whatman 0.02 &mu;m Anodisc filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>68-09-6002</td>
<td>&nbsp;</td>
<tr>
<td>Leica DMRXA microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Any epifluorescence microscope with a blue filter set may be used</td>
</tr>
<tr>
<td>20L polycarbonate carboys</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP229-4</td>
<td>&nbsp;</td>
<tr>
<td>PBS (0.05 M Na2HPO4, 0.85% NaCl, pH 7.5)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP329-500, S640-500</td>
<td>&nbsp;</td>
<tr>
<td>50% Glutaraldehyde </td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G151-1</td>
<td>&nbsp;</td>
<tr>
<td>Corning 2 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-71</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td>Corning 5 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-74</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td> 85% H3PO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A242-212</td>
<td>Spiral Membrane storage</td>
</tr>
<tr>
<td>NaOH pellets</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>S318-3</td>
<td>M12 cleaning</td>
</tr>
<tr>
<td>Graduated Cylinders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopore 0.8-&mu;m pore-size membrane filter (142mm)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ATTP14250</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Stainless Steel Pressure Filter Holder (142mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>YY30 090 00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

estimating virus production rates in aquatic systems

ウイルスは、海洋および淡水システムの広範な成分であり、微生物の死亡率の重要な薬剤であることが知られている。我々はその後、微生物群集の構造と機能の優れたモデルを開発するだけでなく、ウイルスが水生生物地球化学サイクルを変更する仕組みについての我々の理解を進めることができるように、このプロセスの定量的な推定を開発することが重要です。ウイルスの低減技術は、研究者は、ウイルス粒子が風土病微生物群集から放出される速度を見積もることができます。微生物群集が付近の周囲の濃度に維持しながら簡単に言えば、フリー（細胞外）ウイルスの存在量は、サンプルに低減されます。微生物群集は、その後、フリーのウイルスや落射蛍光顕微鏡または、特定のウイルスの場合で、定量的で定量化することができるウイルスサンプルの再発（コミュニティのすでに感染しているメンバーの溶解を通じて）レートの存在下でインキュベートするPCR。これらのレートは、その後、ウイル​​ス媒介細胞溶解に起因する微生物の死亡率を推定するために使用することができます。

Watch this Scientific Journal Video about Estimating Virus Production Rates in Aquatic Systems at JoVE.com

Estimating Virus Production Rates in Aquatic Systems

海洋および淡水系におけるウイルスの離職率は低下し、再発の手法によって推定することができる。データは、研究者は、水系におけるウイルス媒介微生物の死亡率を推測することができます。

水系でのウイルス産生率を予測

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

estimating-virus-production-rates-in-aquatic-systems

Research

JoVE Journal

Immunology and Infection

12.5K ビュー.  University of Tennessee. 海洋および淡水系におけるウイルスの離職率は低下し、再発の手法によって推定することができる。データは、研究者は、水系におけるウイルス媒介微生物の死亡率を推測することができます。

ビデオ: Estimating Virus Production Rates in Aquatic Systems

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

のAlexa Fluor ®標識を通じてデング熱ウイルスの可視化

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

免疫・感染学

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

アデノウイルス由来のシステムの助けを借りて効率的な組換えパルボウイルスの生産

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

神経侵襲性脳炎ウイルスのin vivoイメージングシステム（IVIS）検出で

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

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 ...

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 ...

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

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

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

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

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

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

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

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

迅速・安全・簡易マニュアル ベッドサイド核酸抽出の全血試料中のウイルス検出

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

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

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

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

全血豚で表されるコーディングおよび非コーディングの RNA クラスの識別

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

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

機能向上レポーター・システムによる特異的マイコバクテリア・誤訳率の測定

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

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

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

狂犬病ワクチンの効力を評価するインビトロELISA試験

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