この研究の一部は、欧州連合（EU）の研究枠組み7資金プロジェクトは、（契約番号513648）VIROBATHE、VIROCLIME（契約番号は243923で、スペイン政府は&quot;保健省ドEducación Y Ciencia&quot;（プロジェクトAGL2008-05275-C01/ALI）によってサポートされていました）と水のカタロニア庁、AgènciaカタラーナドゥAigua（ACA）、Departamentド制御I Millora DELS Ecosistemes水泳で。開発研究の間マルタルシニョールはカタロニア政府&quot;AGAUR&quot;（FI - DGR）の仲間だった。

1。水試料中に存在するウイルス粒子の濃度<ol><li>水試料の収集とコンディショニング<ol><li> 2の最小値を収集するプロセス制御などの平らな底部及びつの余分なサンプルをプラスチック容器内の試料あたり10 Lの複製。この最後のサンプルは、ウイルス粒子の既知量をスパイクし、コントロールとして使用されます。 注：これは、添加サンプルのための特別な別の材料を（瓶、チューブ、など）を使用することが推奨されます。 </li><li> conductimeterのキャリブレーションを確認し、必要に応じて再キャリブレーション。つの余分なプラスチック製の10リットルの容器で、以前に十分な導電率に調整した水道水を（1.6下記参照）を使用してネガティブコントロールを用意します。 </li><li>添加サンプルの場合：サンプルにコントロールウイルスの標準量（水10Lあたり約10 5ゲノムコピー）を追加します。回避飛散やエアロゾルを攪拌して混ぜる。ポジティブコントロールは、アデノウイルスsの珍しい株で構成されています可能性がHAdV - 35またはMS2のようなバクテリオファージとしてUCH。 </li><li>サンプルは、浮遊物質（砂または他の材料）の高い量を提示した場合、15分間堆積物にしましょう​​。新しい容器に水を移す。 </li><li> 1 N HClの添加によって3.5（± 0.1）に水試料のpHを調整します。このステップでは、ウイルスの濃度が重要であるので、pHを適切に調整されていることを確認してください。 HClを添加しながら激しく撹拌しながらで十分に水を混ぜる。 （注：pH値は3.5追加1 M水酸化ナトリウムより低い場合）。 </li><li>導電性を調整します。サンプルは、1500μS/ cm以上の導電性を持っている場合、この手順は不要です。伝導率が1500μS/ cmの凝集物質の形成（フロック）が保証されていないよりも低い場合は人工的な海の塩（シグマ）を添加することによって1500μS/ cmの導電率を調整する。海の塩を加えながら撹拌することにより、積極的に混ぜる。 </li><li>エアコンの前と後のサンプルのpHを記録するだけでなく、使用される塩酸のsはボリューム。導電率は、pHを調整した後、記録されるべきである。常に新鮮なHCl溶液でpHメーターとconductimeter電極を消毒、10％ナトリウムtiosulphate液で塩素を除去すると、最後に蒸留水ですすいでください。 </li></ol></li><li>あらかじめ凝集1パーセントスキムミルクの準備（PSM） <ol><li> pHメーターとconductimeterのキャリブレーションを確認し、必要に応じて再キャリブレーション。 </li><li> 1リットルの人工海水に10グラム脱脂粉乳（ディフコ）を溶解することによって事前に凝集したスキムミルク溶液（1％PSM、w / v）の準備（脱塩素水道水とオートクレーブ1Lに人工的な海の塩の33.3 gを溶解）し、慎重に1N HClで3.5にpHを調整する。フロックが表示されるはずです。 4で使用または保管される直前に溶液を調製℃で24時間インキュベートする。脱塩素のために水100mlあたり10％のtiosulphate溶液1mlを使用してください。 </li></ol></li><li>水試料中に存在するウイルス粒子の凝集<ol><li> 10 - Lの水サンプルを1％のPSMの100 mlを追加します。 </li><li>フロックに吸着するウイルスを可能にするために80〜10時間のサンプルをかき混ぜる。 80から10時間後に攪拌をオフにするタイマーを使用</li><li>攪拌を停止し、80〜10時間撹拌重力によるフロック沈降をしましょう</li></ol></li><li>フロックを収集し、再溶解する。遠心分離<ol><li>ペリスタルティックポンプとプラスチック製のチューブに接続されているプラ​​スチック製のピペットを用いて上清を取り除く。添加サンプルのために上清は、ボトルと内部手続きに従って消毒に収集する必要があります。すべてのケースではペレットを集めるように注意してください。 </li><li>遠心ボトルにフロックと堆積物（約500 ml）を収集する。 </li><li> PSMのpHを3.5の添加により鉢のバランスをとる。 </li><li> 4時30分、8,000 xgで高速遠心機でポットを遠心℃にとすぐに遠心分離機が停止するように、慎重に遠心から遠心ポットを削除します。 </li><li>非常にグラムently捨て、上清を捨てる。感染性物質のための適切な措置に従ってください。 </li><li>各遠心ボトルにペレットを溶解するリン酸緩衝液7 mlを追加します。 </li><li>フロックが溶解された後、測定し、10 mlの全体積に到達するためのリン酸緩衝液を加える。 </li><li>ボルテックスによるウイルス濃縮物をホモジナイズし、さらなる分析に必要になるまで-80℃で凍結する必要があるきれいなマイクロチューブに10 mlを配布する。 </li></ol></li></ol> 2。核酸抽出<ol><li>製造元の指示に従って別途準備ウイルスRNAミニキットでの核酸抽出を行います。このキットは、自動化されたプラットフォーム（などQiacubeなど、キアゲン）の使用が可能になります。 </li></ol> 3。ヒトアデノウイルスの定量的PCR（HAdV）、JCのpolyomaviruses（JCPyV）、ブタアデノウイルス（PAdV）とウシpolyomaviruses（BPyV） <ol><li>サンプル中のゲノムコピーの定量化<ol><li>準備のTaqMan環境PCRマスターミックス2倍に（Applied Biosystems社）を使用して、クリーンな別々の領域で定量PCRミックス。反応は、光学接着剤のカバーで覆われた96ウェル光学反応プレートで行われます。マスターミックスの濃度は、プライマーおよびプローブは、表1で説明されています。 </li><li>一度ミックスはよく（3.2参照）、コントロールを含めてそれぞれに、アリコート15μlを準備している。ターゲットの追加後に一つの反応の合計量は、（15μlのミックス+10μlのサンプルまたは標準）25μlのようになります。 </li><li>別のエリア内のサンプル（10μL）からの核酸抽出を追加。直接実行し、重複して各サンプルの精製水で10倍希釈。接着剤カバーの一部のサンプルを含むウェルをカバーする、次のステップのためにカバーの他の部分を保持します。 </li><li>三連で10 0〜10 6GC/10μlからのDNAの標準懸濁液（10μlの）の希釈液を追加し、排他的に標準に使用するマイクロピペットを用いて標準のDNA。プラスミドDNAを破壊し、それぞれの使用後にマイクロピペットをきれいにするためのUV照射を装備した領域での規格を追加することをお勧めします。プレカット接着剤のカバーと基準を含むウェルをカバーしています。 注：ゲノムコピーの定量化に使用される標準的な懸濁液を調製するためには、標的DNA領域はプラスミドと直鎖状にクローン化されるべきである。以下のアドレスでは、定量PCRに使用するプラスミドDNAのテンプレートを使用して標準曲線を作成する方法についての詳細な手順を見つける： <a href="http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf">http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf</a> </li><li>適切なパラメータを（接着剤カバーと各ウェルの総容積の使用、などを考慮して）選択して適切なシステムに定量PCRを行います。 95℃で10分間のAmpliTaq Goldの次の活性化° C、増幅の40サイクル、次のように実行されています：95 ° Cで1​​5秒と1 60 minでHAdV、JCPyVとBPyVのためのC、および95℃、55℃20℃と60℃PAdVのためのCで20歳代で15秒。 </li><li>使用される機器の取扱説明書で説明されているように反応は、ストアのデータと結果を完了したら。 DNA量は、必要に応じて希釈率を修正した後、得られたデータの中央値として定義されます。 </li></ol></li><li>コントロール<ol><li>ポジティブとネガティブコントロールを使用してください。アッセイは、ミックスが蛍光を生成しないことを証明するために複数の非テンプレートコントロール（NTC）を含める必要があります。研究試料中に存在する阻害剤に起因する潜在的な酵素阻害を評価するために、正のプロセス制御を実行することをお勧めします。 </li><li>標準DNAのおよびプロセス制御から2つの異なる希釈液のqPCRアッセイの結果を記録。アッセイの感度および効率に関係する品質管理（QC）プログラムのために管理図を準備するために、結果を使用してください。 </li></ol></li><li>の確認結果は<ol><li>肯定的な結果は、さらに1,5,6,7,9,12を検出した菌株の塩基配列に関する追加データを生成する、アンプリコンのネスト- PCRと塩基配列を用いて確認することができます。 </li></ol></li></ol>説明した手順に従って、人間と動物のウイルスは、入浴の海、海水や河川水2,3で検出し、定量化されています。代表的な例として、硝酸塩の高いレベルを提示する分野から地下水試料は、汚染の発生源を定義するために評価した。十リットルの水のサンプルは、スペイン北東部の農村部における4つの井戸から採取された。五は、プロセス制御として使用されるヒトアデノウイルス2で播種した各ウェルと1つの複製に収集された複製。サンプルを図1に表されたプロトコルに従って処理した。 4人は（値7.7平均PAdVための肯定的な結果を示した研究4つのサイトのいずれかで分析した複製サンプリングサイトを周辺地域における豚スラリーの存在に関連されると地下水の硝酸塩（表2）のソースとして糞便ブタの汚染をサポートする4 × 2 GC / L）。 4。代表的な結果： <img src="/files/ftp_upload/2820/2820fig1.jpg" alt="図1" /> 図1水中ウイルスの検出および定量するための手順。 <img src="/files/ftp_upload/2820/2820table1.jpg" alt="表1" /> qPCRアッセイ用のプライマーとプローブの表は、1。集中。 <img src="/files/ftp_upload/2820/2820table3.jpg" alt="表3" /> 表2。検出と地下水試料中の動物やヒトアデノウイルスとpolyomavirusesの定量化。 のn個の数は、分析複製 正の％の割合では、複製 （ - ）検出された非

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二つの特許出願は、PAdVとBPyVの定量化のためのプロトコルの知的財産を保護するために2009年に提起された。

<html>再現性、信頼性の高い、簡単で、費用対効果：説明する手順では、ルーチン、環境および公衆衛生研究所のためのフィッティング方法のための条件を満たすでしょう。プロトコルはシンプルですが、それは注意深く従ってください。凝集のための攪拌時間を大幅に（たとえば5時間未満）減少する場合のように人工海水の塩の要求された濃度を加えることなく、試料中の低導電率が飛躍的...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>高速遠心（8,000 × g）で</td><td>バークマンコールター</td><td>アヴァンティJ - 20XP </td><td></td></tr><tr><td> pHメーター、温度計とconductimeter </td><td> Afora </td><td> LPPC3003 </td><td></td></tr><tr><td>プラスチック製のチューブ100〜200センチメートルの長さ</td><td> Deltalab </td><td> 350059 </td><td></td></tr><tr><td>滅菌は、使い捨てピペットを卒業</td><td> Labclinics </td><td> PN10E1 </td><td></td></tr><tr><td> 1.5 10〜15 mLの滅菌プラスチックチューブ（エッペンドルフ、ファルコンズ、等） </td><td> Afora </td><td> KA298/00 </td><td></td></tr><tr><td>ポットを遠心（500mL中） </td><td>フィッシャーサイエンティフィック</td><td> SE5753512 </td><td></td></tr><tr><td>磁気撹拌器と磁石（ごとにサンプル） </td><td>フィッシャーサイエンティフィック</td><td> 10510 </td><td></td></tr><tr><td>マグネットスターラーの使用を許可するように平らな底を有するガラスまたはプラスチック製の容器</td><td> Deltalab </td><td> 191642 </td><td></td></tr><tr><td>上清を除去するための蠕動ポンプ（またはウォータージェット真空ポンプ） </td><td>ワトソン - マーロウ</td><td> 323E / D </td><td></td></tr><tr><td> 8-10時間後に攪拌をオフにするタイマー</td><td> Deltalab </td><td> 900400 </td><td></td></tr><tr><td>塩酸（1Nと0.1N） </td><td> Panreac </td><td> 141020.1611 </td><td></td></tr><tr><td>水酸化ナトリウム（1N） </td><td> Panreac </td><td> 131687.1211 </td><td></td></tr><tr><td>人工海水の海塩</td><td>シグマ</td><td> S9883 </td><td></td></tr><tr><td>スキムミルク（SM） </td><td>ディフコ</td><td> 232100 </td><td></td></tr><tr><td>リン酸緩衝液のpHを75 </td><td></td><td></td><td> pH7.5ので1:2 v / vの無菌のNa 2 HPO 4 0.2 MのとのNaH 2 PO 4 0,2 M </td></tr><tr><td>チオ硫酸</td><td> Panreac </td><td> 121879.1209 </td><td>水中の10％溶液を作る</td></tr><tr><td>ウイルスRNA Mini Kitを別途準備</td><td>キアゲン</td><td> 52904 </td><td></td></tr><tr><td> 96ウェル光学反応プレート（500台） </td><td>アプライドバイオシステムズ</td><td> 43426659 </td><td></td></tr><tr><td>光学接着剤のカバー（100台） </td><td>アプライドバイオシステムズ</td><td> 4311971 </td><td></td></tr><tr><td>のTaqMan環境PCRマスターミックス2倍</td><td>アプライドバイオシステムズ</td><td> 4396838 </td><td></td></tr></tbody></table>

cost-effective method for microbial source tracking using specific human and animal viruses

環境の微生物汚染は重大な健康上のリスクを表しています。古典的な細菌の糞便指標が重要な限界があることが示されている、ウイルスが汚染源に通知していない多くの不活性化プロセスと標準の糞便の指標に対してより耐性があります。費用対効果の高い水からのウイルスの濃度のための方法と分子アッセイの開発は、糞便汚染の指標として、微生物源の追跡（MST）ツールとしてウイルスの適用を容易にします。アデノウイルスとpolyomavirusesは、ヒトを含む特定の脊椎動物に感染するDNAウイルスであり、永続的に糞及び/または調査したすべての地理的領域における尿中に排泄される。以前の研究では、我々は人間の糞便汚染の指標として定量PCR（qPCR）により、ヒトアデノウイルスの定量化（HAdV）とJCのpolyomaviruses（JCPyV）を提案した。最近、我々は、PORの特定の定量化のためのqPCRアッセイを開発しました。試験管当たり1〜10ゲノムコピーの感度と汚染の動物糞便のマーカーとしてシネアデノウイルス（PAdV）とウシpolyomaviruses（BPyV）。本研究では、我々はこれらのツールを使用して水試料中の汚染源を特定するために従うべき手順を示します。代表的な成果の例として、硝酸塩の高いレベルを提示する地下水のウイルスの分析が示されています。 低または中程度に汚染された水域でのウイルスの検出は、はるかに少ない量に水の少なくとも数リットルからのウイルスの濃度、通常は直列の2つの濃度のステップが含まれている手順が必要です。この少々面倒な手順やウイルスの回収率で観察された変動は大幅に水の多数のサンプルの同時処理を妨げる。 我々は以前のstudieで開発したワンステップのプロトコルを適用した二段階の手続きによって生じるボトルネックを解消するためにsと水マトリックスの多様性に適用される。十リットルの水試料の酸性化、スキムミルクで凝集、凝集物質の重力沈降、沈殿と遠心分離のコレクション、10 mlのリン酸緩衝液中の沈殿物の再懸濁を：手順が含まれています。ウイルス濃縮液は、ウイルス核酸の抽出に使用され、関心のある特定のアデノウイルスとpolyomavirusesは、定量PCRにより定量化されています。サンプル数が多いが、同時にこの低コストの濃縮法を用いて解析することができます。 手順は、入浴の海、海水や河川水の分析に適用し、本研究では、我々は、地下水のサンプルを分析結果を提示されています。このハイスループット定量法は、信頼性の高い簡単な、そして費用対効果の高いです。

Watch this Scientific Journal Video about Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses at JoVE.com

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

研究では、MSTのツールと​​して提案したヒト/ブタ/ウシDNAウイルス、アデノウイルスとpolyomaviruses、特定の定量化のために定量PCRを用いて水中の硝酸塩による尿/糞便汚染または汚染源の同定のための費用対効果の高い方法を説明します。

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 ...

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12.1K ビュー.  University of Barcelona. 研究では、MSTのツールと​​して提案したヒト/ブタ/ウシDNAウイルス、アデノウイルスとpolyomaviruses、特定の定量化のために定量PCRを用いて水中の硝酸塩による尿/糞便汚染または汚染源の同定のための費用対効果の高い方法を説明します。

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

粘着テープベースのサンプリングと蛍光の組み合わせその場で</emの迅速検出のための>ハイブリダイゼーションサルモネラ生鮮食品について

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

免疫・感染学

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

HIV / AIDSの包括的＆コスト効率に優れた研究室の監視：アフリカのロールモデル

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

鉄源としてヒトヘモグロビンを用いた黄色ブドウ球菌の成長

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

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 ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

ヒト免疫不全ウイルスの複製フィットネスを決定するためのペアごとの成長競合アッセイ

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

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 ...

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 ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

試金を使用して SIAT1 MDCK 細胞のインフルエンザ a (h3n2) ウイルス中和によるひと血清中の抗体を中和することを測定

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

水サンプル中のウイルス検出のための利用ポイント方法VirWaTest

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

カルボキシフルオレスチンスクシニミジルエステルを用いたヒト抗原特異的CD4+T細胞の増殖の定量化

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...