この作品は、オーストリア科学基金（FWF、BZにP20619とP22988）によってサポートされていました。

マイクロ波アシスト試料調製<ol><li>試料調製1の開始は、電子顕微鏡のための自動マイクロ波組織プロセッサ（ライカEM AMWは、ライカマイクロシステムズ、ウィーン、オーストリア）をプログラムする必要があります前に、TEM用マイクロ波アシスト試料調製には、次のプロトコル（表1）： <img src="/files/ftp_upload/2950/2950table1.jpg" alt="表1" /> 表1マイクロ波照射を用いた試料調製のためのプロトコル。別の列（左から右へ）表示： <ol><li>バイアル番号（ バイアル番号nr。）バイアルは、プロセッサのカルーセルにロードされる順序を表します。 </li><li>実際のプロセスのステップ。 </li><li>バイアル中の試薬。 </li><li>実際の工程の時間。 </li><li>マイクロ波照射が切れる前にバイアルに到達する最高温度。 </li><li>マイクロ波照射の設定：連続=急激な温度刻みASE、設定温度を保持する。スロープ=穏やかな温度上昇は、最後に到達した最終的な温度は、温度が5を落としてまで、パルス=急速な温度上昇、電源がオフになって℃、電源、温度に到達するために再開した。 </li><li>マイクロ波照射の最大電力。 </li></ol></li><li>たての試料調製プロトコル（寒天100エポキシ樹脂のため1.7を参照）で説明されているさまざまな手順のためのソリューションを用意し、プログラムされたプロトコル（表1）によると、指定バイアルにそれらを埋める、カルーセルにバイアルをロードしてから、INSERTマイクロ波組織プロセッサにカルーセル、最終的にモノモード室に最初のバイアルをロードします。 </li><li> 60MMソレンセンの3％グルタルアルデヒド（寒天サイエンティフィック（株）、スタンステッド、イギリス）のドロップでモデリングワックスプレート上にかみそりの刃でタバコモザイクウイルス（TMV）に感染したタバコタバコの葉の小さなセクション（1ミリメートル2）を切り取る室温でリン酸塩緩衝液（pH7.2）erature。 </li><li>約200μmのメッシュ幅は指定バスケットにすぐに細かいピンセットでセクションを転送します。互いの上にバスケットを積み重ねるとモノラルモードチャンバーに挿入してください。ケアは、サンプルは常に彼らが出て乾燥させないようにロードして、バスケットのスタッキングで固定液で覆われているように注意しなければならない。 </li><li>固定、脱水、浸透のために予めプログラムされたマイクロ波アシスト試料調製プロトコルを起動します。 </li><li>試料調製は、マイクロ波組織プロセッサによって自動的に実行されますが、第3条（ネガティブ染色）で説明したように、残りの植物材料とネガティブ染色を続行します。 </li><li>たての説明に従って、以下の成分を混合することにより寒天100エポキシ樹脂を準備します。フィル24グラム寒天100、16グラムドデセニルコハク酸無水物、及びプラスチックカップで10グラムメチルナジック酸無水物（すべてのコンポーネントの寒天科学（株）、スタンステッド、イギリスを参照）、熱をそれへの40℃とよく混ぜる。ベンジルジメチルアミン1.2グラムを加え、よく混ぜる。試料調製プロトコルは、エンド（表1のステップ22中など）に来る直前に指定された重合形に寒天100エポキシ樹脂を充填します。 </li><li>プロトコルが終了した後（表1のステップ22の後に）モノモード室からカルーセルの最後のバイアルの中に浸透したサンプルを含む積み重ねバスケットを離します。マイクロ波装置からカローセルを取り外し、バスケットのスタックを解除し、指定された重合形に細かいピンセットを使用してそれらをロードします。ケアは、サンプルは常に彼らが出て乾燥させないように、アンスタックとロード中に寒天100エポキシ樹脂で覆われていることを注意しなければならない。 </li><li>互いの上に重合フォームを積み重ねる。ケアは、サンプルは常に彼らが出て乾燥させないように積み重ね、ロード中に寒天100エポキシ樹脂で覆われていることを注意しなければならない。 </li><li>マイクロ波TISSのカルーセルから以前使用してバイアルを取り外しUEプロセッサは、積み重ねられたバスケットでそれをロードし、マイクロ波組織プロセッサにカルーセルを挿入します。 </li><li>あらかじめプログラムされた重合プロトコル（表1）を起動します。 </li><li>重合は、マイクロ波組織プロセッサによって自動的に行われているが、透過型電子顕微鏡でネガティブ染色グリッドを調べる[例えばフィリップスCM10、TEM、FEI社（旧フィリップス）、アイントホーフェン、オランダ]、セクション3と4（で説明したように画像解析を行うネガティブ染色と画像解析）。 </li><li>プロトコルスタック解除後モノモード室から重合形態、重合フォームを削除して、サンプルを含む重合ブロックを削除完了です。彼らは今、ミクロトームで切片化する準備が整いました。 </li></ol> 2。トリミングとセクショニング<ol><li>極薄のがホル​​ダーの外に約1cm上部に付着試料とセクショニングの個別のサンプルホルダーに1つ以上のブロックを挿入します。 </li><li>TEM（例えばライカライヘルトUltratrim、ライカマイクロシステムズ）の試料トリマーを使用してブロックを整えるように最大のブロック面。長さと（顔の大きさをブロックダイヤモンドナイフの大きさに調整する必要があります）を実現しており、できるだけ多くの葉材料を含んで幅200μmの中で1ミリメートル。 </li><li> 45°（例えばDiatomeウルトラ45、Gröpl、Tulln、オーストリア）のナイフの角度でダイヤモンドナイフを使用することにより、ウルトラミクロトーム（ライカマイクロシステムズなどライヘルトライカUltracut S）と節ブロックを。切片厚は約70〜90nmのに調整されるべきであり、切削速度は1mm / s程度である必要があります。 formvar（寒天サイエンティフィック（株））でコーティングされた銅やニッケル200平方メッシュグリッドを使用して複数のセクションを拾う。 </li><li>部分的に2フリーCOを作成するためのNaOHでいっぱいシャーレ内で5分間、クエン酸鉛（42ミリリットル再蒸留水と8ミリリットルを1N NaOHに溶解した1.1グラムクエン酸鉛寒天科学製）を用いてグリッド上のセクションを後染色環境と15マイルのための室温で蒸留水に溶解した1％酢酸ウラニル（寒天サイエンティフィック株式会社）とnutes。各ポスト染色工程の間に1分間蒸留水でグリッドを洗ってください。空気がグリッドボックスでグリッドを乾燥させてください。 </li><li>透過型電子顕微鏡（例えばフィリップスCM10、TEM、FEI社、アイントホーフェン、オランダ）を持つセクションを調べます。 </li></ol> 3。逆染色法<ol><li> TMV感染葉材料として100mg約収穫と60mmSøfrensenリン酸塩緩衝液（pH7.2）100μlの中で顕微鏡スライド上にかみそりの刃で2分のための材料を均質化することにより粗汁液を準備します。 </li><li> 4ウェル以上（あるいはそれはパラフィルムの一部にホモジネートを転送することも可能である）を持つテフロン顕微鏡スライドの最初のウェルに得られたホモジネート20μlのを転送します。 </li><li>ドロップおよびインキュベーションに向いformvar（寒天サイエンティフィック株式会社）コーティングされた側とのホモジネートの上formvarコーティングされたグリッドを配置5分間、それを脱灰する。 </li><li> 200μlの60MMソレンセンリン酸緩衝液（pH7.2）を2滴の上にグリッドを配置することによって、2分ごとにグリッド2回洗浄する。 </li><li> 60MMソレンセンリン酸塩緩衝液（pH6.5）中で新しく調製した2％リンタングステン酸（寒天サイエンティフィック株式会社）溶液で1分間グリッドをインキュベートする。 </li><li>グリッドを削除し、それがグリッドボックスで空気乾燥することができます。 </li><li>透過型電子顕微鏡（; FEI社、アイントホーフェン、オランダ例えばフィリップスCM10 TEM）でグリッドを調べます。 21000X以降の主な倍率での透過型電子顕微鏡を用いてランダムに選ばれたネガティブ染色したウイルス粒子の少なくとも10個のイメージが（少なくとも10以上ビリオンは各画像を表示する必要がある）を取る。ケアは、すべてのイメージが同じ倍率を持っていることを注意しなければなりません。 </li></ol> 4。画像解析<ol><li>から採取した顕微鏡写真で少なくとも100ランダムに選択された単一のウイルス粒子の長さと幅を測定ネガティブ染色試料を任意の画像解析用コンピュータソフトウェアを使用して[例えばセルD（オリンパス、生命およびマテリアルサイエンスヨーロッパGmbH、Hamburg、ドイツ）粒子分析ツールを使用してまたはOptimas 6.5.1-（Media Cyber​​netics社株式会社メリーランド州ベセスダ米国）]。 </li><li>ネガティブ染色法とTEMにより可視化したウイルス粒子の平均の長さと幅を達成するために、平均値と標準偏差を計算します。 </li><li>明らかにウイルス病を識別するために、文献で知られているウイルス疾患によって誘発されるウイルスおよび超微細構造変化の大きさと長さと微細構造の特徴を（2.5）で得られた比較。 </li></ol> 5。代表の結果： などのマイクロ波アシスト試料調製典型的なTMV誘起微細構造の変化の後に並列状に配列ビリオンを含む大きな領域は、感染した喫煙者のサイトゾルに透過型電子顕微鏡で観察することができたタバコ細胞（図1A）。さらに、TMVに感染葉の粗汁液でTMV粒子は負染色（図1B）の後屈曲、棒状構造として観察することができた。 100ウイルス粒子画像解析の幅が長さ17nmと280nmのである（図2）、TMVの平均サイズを明らかにした。 TMV感染細胞における超微細構造と本研究で観察されたビリオンのサイズはタバコと以前に文献9月15日に報告されたTMV粒子の大きさの範囲内で、TMV誘起微細構造の特性と一致していることがわかった。 <img src="/files/ftp_upload/2950/2950fig1.jpg" alt="図1" /> TMVに感染した葉の細胞およびビリオンの図1透過型電子顕微鏡写真。 a）画像は、マイクロ波アシスト植物試料調製後のタバコタバコのTMVに感染した葉肉葉細胞の超微細構造を示しています。細胞質に蓄積された平行に整列ビリオンの大面積に注意してください。でんぷんを使用したC =葉緑体（ST）、M =ミトコンドリア、核、N = V =液胞、バー=2μmの。 B）のイメージは、感染した葉の樹液でネガティブ染色により検出したビリオンを示しています。 <img src="/files/ftp_upload/2950/2950fig2.jpg" alt="図2" /> 図2ビリオンの相対的な大きさ。彼らは電子顕微鏡で登場しましたように、負は、感染した葉の樹液を染色した後、TMV粒子の長さと幅の相対的な分布。 （長さ/幅の平均値±標準偏差）平均値は、100ビリオンから算出した。

<ol>
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特別な利害関係は宣言されません。

TEM用マイクロ波アシスト植物試料の調製は、数時間以内に3,4,16、高速かつ信頼性の高い超微細構造のデータを提供することが実証されています。本研究で使用した方法で達成小器官と膜の微細構造保持には、3日以上〜約2時間サンプルの固定と包埋時間の大幅な削減という利点を持つ5,15従来とcryofixedサンプルに類似していた。これは文学で現在利用可能なTEM用最速試料調製プロトコルを表します。本研究で述べた方法は、TMV誘起微細構造の変化およびウイルス剤自体の明確かつ迅速な同定を可能にしたネガティブ染色法とTEM用マイクロ波アシスト植物試料調製を組み合わせました。 TMVは超微細構造変化が電気伝送での固定の開始後約4時間以内に、トリミングセクショニングと後染色した後に調査される可能性が誘導されるトロン顕微鏡。完全自動試料作成モードを使用すると、ウイルス剤のサイズと幅を決定するために、暫定的にネガティブ染色を実施する研究者を解放した。したがって、私たちは、この方法は植物植物病理学における農業と科学的な実験では、将来の使用のために非常に重要である約半日での植物ウイルス病の明確かつ迅速な診断を可能と結論付けることができます。この方法はまた、動物とヒトの疾患の迅速な診断のために使用することができるとして、それは医療や獣医病理学の将来のアプリケーションのための大きな可能性を秘めています。

<table border="1"><tbody><tr><td> 試薬/機器の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>グルタルアルデヒド</td><td>寒天サイエンティフィック株式会社</td><td> R1312 </td><td></td></tr><tr><td>四酸化オスミウム</td><td>寒天サイエンティフィック株式会社</td><td> R1022 </td><td></td></tr><tr><td>寒天100樹脂</td><td>寒天サイエンティフィック株式会社</td><td> R1043 </td><td></td></tr><tr><td>ドデセニルコハク酸無水物</td><td>寒天サイエンティフィック株式会社</td><td> R1051 </td><td></td></tr><tr><td>メチルナジック酸無水物</td><td>寒天サイエンティフィック株式会社</td><td> R1081 </td><td></td></tr><tr><td>ベンジルジメチルアミン</td><td>寒天サイエンティフィック株式会社</td><td> R1060 </td><td></td></tr><tr><td>クエン酸鉛</td><td>寒天サイエンティフィック株式会社</td><td> R1210 </td><td></td></tr><tr><td>酢酸ウラニル</td><td>寒天サイエンティフィック株式会社</td><td> R1260A </td><td></td></tr><tr><td>リンタングステン酸</td><td>寒天サイエンティフィック株式会社</td><td> R1213 </td><td></td></tr><tr><td> Formvar </td><td>寒天サイエンティフィック株式会社</td><td> R1202 </td><td></td></tr><tr><td colspan="4"></td></tr><tr><td>ライカEM AMW </td><td>ライカマイクロシステムズ</td><td></td><td></td></tr><tr><td>ライカ（ライヘルト）Ultratrim </td><td>ライカマイクロシステムズ</td><td></td><td>新しいモデルが利用可能です</td></tr><tr><td>ライカ（ライヘルト）Ultracut S </td><td>ライカマイクロシステムズ</td><td></td><td>新しいモデルが利用可能です</td></tr><tr><td> Diatomeウルトラ45 </td><td> Gröpl </td><td></td><td></td></tr><tr><td>フィリップスCM10、TEM </td><td> FEI社（旧フィリップス） </td><td> &lt;/ TD&gt; <td>新しいモデルが利用可能です</td></tr><tr><td>セルD </td><td>オリンパス株式会社</td><td></td><td></td></tr><tr><td> Optimas 6.5.1 </td><td>メディアサイバネティックス株式会社</td><td></td><td>新しいバージョンが利用可能です</td></tr></tbody></table>

microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy

ウイルスによって誘発される超微細構造変化の調査は、しばしば明確植物におけるウイルス性疾患を識別する必要があります。透過型電子顕微鏡（TEM）のための従来の試料調製とそのような調査は数日間1,2を取ることができ、したがって、植物ウイルス病の迅速診断には適していません。マイクロ波固定が大幅に従来の試料調製3月5日後に観察されたと同様の超微細構造のTEM観察結果と調査のためのサンプル調製時間を削減するために使用することができます。さまざまなカスタムメイドのマイクロ波デバイスは、成功した固定およびTEM調査5-8生物試料の埋め込 ​​みに使用することができる現在利用可能です。本研究において、我々は、それがTのためのマイクロ波アシスト試料調製を使用して約半日で葉の微細構造の変化を診断することが可能であることをタバコモザイクウイルス（TMV）感染タバコタバコ植物に実証EM。それは多くのステップがまだ手動で6月8日に実行されなければならないため、より多くの時間と労力を消費している他の利用可能なデバイスとは対照的に、ほぼ完全に自動的に5試料調製を行いますので、我々は市販のマイクロ波デバイスと、この研究を行うことを選択しました。サンプルの準備が完全に自動的に行われるように、残りのTMV感染葉や超微細構造およびサイズ​​は、次の検査の樹液でウイルス粒子の負染色は固定と包埋時に行うことができます。

Watch this Scientific Journal Video about Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy at JoVE.com

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

本研究では、透過型電子顕微鏡と負染色法のためのマイクロ波アシスト植物試料調製の組み合わせを使用して約半日での植物ウイルス病の迅速かつ明確な診断を可能にする方法を説明します。

マイクロ波は、透過型電子顕微鏡による植物ウイルス病の迅速診断をアシスト

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

microwave-assisted-rapid-diagnosis-plant-virus-diseases-transmission

Research

JoVE Journal

Immunology and Infection

13.4K ビュー.  University of Graz. 本研究では、透過型電子顕微鏡と負染色法のためのマイクロ波アシスト植物試料調製の組み合わせを使用して約半日での植物ウイルス病の迅速かつ明確な診断を可能にする方法を説明します。

ビデオ: Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

野鳥の鳥インフルエンザウイルスの迅速な診断：フィールドのポータブルRRT - PCR及び凍結乾燥試薬の使用

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

免疫・感染学

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Vero細胞培養アッセイによるフィールドコレクト蚊から感染性ウイルスの検出

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

クライオ電子顕微鏡によるHIV - 1キャプシドアセンブリの構造とヘリカル実空間の反復再構成

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

上のシステイン残基の溶媒アクセシビリティの分析<em&gt;トウモロコシrayadoフィノウイルス</emで生産&gt;ウイルス様粒子<em&gt; Nicotiana benthamianaの</emVLPは、ペプチドの&gt;植物と架橋

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

の高分解能電子顕微鏡<em&gt;ヘリコバクター·ピロリ</em鉄の可用性の変化する条件で生産&gt; CAG IV型分泌系線毛

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Tetracistronicミニゲノムを含むウイルス様粒子とバイオセーフティレベル2の条件の下ではエボラウイルスのライフサイクルのモデル化

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

研究のための方法論<em&gt; B。枯草菌</em小分子バイオフィルム阻害剤を特徴付けるためのモデルとしての&gt;バイオフィルム

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

コットンボールワームでデンソウイルスのホストフィットネス上のホスト組織分布、伝送モード、および効果を調査するためのプロトコル

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

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 ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

インクジェットプリンター支援によってテスト自動チェッカー ボード配列および手動で時間を殺す方法抗菌の相乗効果

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...