この作品は一部NIHの助成金R01 A1064826-01、U19 AI062627-01 NO1-HHSN26600500032C（SMに）でサポートされています。私たちは、原稿の重要なレビューのためにティナ·ハイル、議論と技術的な助けを我々の研究室のメンバーに感謝します。我々は、ウイルス培養とPCRのためにデビッド·ビナ、ミルウォーキー衛生局研究所に感謝します。

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+molecular+markers+of+antiviral+resistance+in+influenza+A+(H5N1)+viruses+using+a+pyrosequencing+method.">Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using a pyrosequencing method.</a> Antimicrob. Agents Chemother. 53, 1039-1047 (2009).</li><li>Jonsson, N., Gullberg, M., Lindberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+polymerase+chain+reaction+as+a+rapid+and+efficient+alternative+to+estimation+of+picornavirus+titers+by+tissue+culture+infectious+dose+50%+or+plaque+forming+units.">Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units.</a> Microbiol. 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L., Ferguson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suboptimal+detection+of+influenza+virus+in+adults+by+the+Directigen+Flu+A+B+enzyme+immunoassay+and+correlation+of+results+with+the+number+of+antigen-positive+cells+detected+by+cytospin+immunofluorescence.">Suboptimal detection of influenza virus in adults by the Directigen Flu A+B enzyme immunoassay and correlation of results with the number of antigen-positive cells detected by cytospin immunofluorescence.</a> J. Clin. Microbiol. 41, 3407-3409 (2003).</li><li>Herrmann, B., Larsson, C., Zweygberg, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+and+typing+of+influenza+viruses+A+and+B+by+a+nested+reverse+transcription-PCR:+comparison+to+virus+isolation+and+antigen+detection+by+immunofluorescence+and+optical+immunoassay+(FLU+OIA).">Simultaneous detection and typing of influenza viruses A and B by a nested reverse transcription-PCR: comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay (FLU OIA).</a> J. Clin. Microbiol. 39, 134-138 (2001).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> Am. J. Epidemiol. 27, 493-497 (1938).</li><li>Doerr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins:+into+the+infrared.">Fluorescent proteins: into the infrared.</a> Nat. Meth. 6, 482-483 (2009).</li><li>Shu, B., Wu, K. H., Emery, S., Villanueva, J., Johnson, R., Guthrie, E., Berman, L., Warnes, C., Barnes, N., Klimov, A., Lindstrom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+performance+of+the+CDC+real-time+reverse+transcriptase+PCR+swine+flu+panel+for+detection+of+2009+A+(H1N1)+pandemic+influenza+virus.">Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus.</a> J. Clin. Microbiol. 49, 2614-2619 (2011).</li></ol>

我々は、開示することは何もありません。

東南アジアにおける鳥インフルエンザと新型インフルエンザのパンデミックの世界的な恐怖の最近の流行の流行は、公衆衛生と安全に多大な懸念を課す。重要な焦点は、インフルエンザウイルスの既存および新しい株のワクチンを生成するのに専念してきました。正確な検出と正確なウイルス力価計算のための迅速なハイスループット技術の可用性は飛躍的に治療を向上させます。ウイルス力価を計算するために最もよく使用される技術は、プラーク形成、インフルエンザ赤血球凝集アッセイが含まれています。プラーク形成アッセイは、最初のバクテリオファージの力価を推定するために開発され、動物のウイルス力価4を計算するためにレナート·ダルベッコで採択された。このアッセイを実行するために、6ウェルプレートでのインフルエンザの在庫や、BAL液などの動物標本の10倍希釈液を調製し、0.1ミリリットルのアリコートを、MDCK細胞の単層に接種されています。ウイルスは、広告に続いてMDCK細胞に吸着する許可されている栄養培地及びアガロースのdition。インキュベーション中に、元の感染した細胞にのみ、隣接するセルに広がるウイルスの子孫を解放します。インフルエンザウイルス感染の細胞変性効果はプラークと呼ばれる感染細胞の円形のゾーンを生成します。各プラークは、おそらくウイルスの複製に続いて​​、単一のウイルス粒子を持つ単一の細胞の感染後に形成されています。生きている細胞とプラークの間にコントラストがそのようなクリスタルバイオレットなどの色素によって強化することができます。このアッセイの主な欠点は、再現性と実験中で巨大なバリエーションの欠如である。インフルエンザウイルスの力価を決定するために使用される別のアッセイは、赤血球凝集アッセイである。インフルエンザウイルスの表面の赤血球凝集素タンパク質が存在すると、効率的な哺乳類と鳥類の赤血球1のN-アセチルノイラミン酸含有タンパク質に結合する。インフルエンザウイルスと赤血球との間のこの相互作用は、格子状に凝集につながります。リットル凝集のevelは、試験片におけるインフルエンザウイルスの力価を推定するために使用されています。赤血球凝集アッセイは、既知のウイルスの滴定を決定するためにのみ使用され、株の同定を目的として使用することはできません。このアッセイは、ライブや死んだウイルスを区別しませんので、ウイルス力価の正確な計算を得ることが困難である。 インフルエンザ感染症の現在の臨床診断は血清学的検査、ポリメラーゼ連鎖反応、直接試料の免疫と細胞培養1,6に基づいています。ここで説明した新しいメソッドでは、臨床および実験室診断可能性を秘めています。インフルエンザ検出キットディレクインフルエンザ（BD Biosciences社、カリフォルニア州サンノゼ）とBinaxNow（Binax社、スカーバラ、ME）の使用イムノクロマトアッセイのような核タンパク質7のようなウイルス抗原を検出する。これらのキットを使用して、ウイルスの検出は、15分以内に達成することができますが、すべての負のサンプルは、さらにsとする必要があります細胞培養法によりcreened。様々な研究はまた、感染が軽度または8低く、特にこれらのキットの感度が低い、報告されています。提案手法では、我々は一貫してウイルスの100 PFUのように低い検出します。これは感染症が軽度の場合でも、私たちの技術は、臨床サンプル中のH1N1ウイルスを検出するのに有用であることを示唆している。ウイルス抗原を検出する蛍光ベースの方法のいずれか鼻咽頭吸引検体を直接分析したか、ウイルスの増殖とその後の分析の9の感受性細胞の単層に接種したところ、報告されています。しかし、これらの方法は、診断目的のために限られており、ウイルス力価の計算には適していませんされています。 Q-PCRは、ウイルスRNAの存在を識別するのに役立ち、ライブウイルスの感染性を推定することはありません。これらのアッセイに比べて、IR染料ベースのアッセイシステムは、臨床検査試料中に検出し、ウイルス力価の列挙に役立ちます。インフルエンザの流行の場合には、それは、短い期間で数千のサンプルを診断することが重要。 384ウェルプレートおよびIRスキャナに基づいて、提案手法では、したがって、他の直接蛍光法に比べて大きな利点を提供し、時間（&gt; 2000サンプル）で6プレートをスキャンすることができます。臨床サンプルの数千人の迅速な処理の実現可能性は、アッセイ時間とコストの長さを試験のばらつきを減らすことができます。 M2-特異的抗体の使用は、HとN蛋白質の系統差に依存しないという利点を提供しています。 M2蛋白質は、比較的人間に感染するほとんどのインフルエンザ株に保存されている。循環汚れに特異的な抗体は、我々のシステムと組み合わされた場合は、1つは、ウイルスの両方の力価を測定し、ひずみを識別することができます。 M2はアマンタジンのような薬物のターゲットであるため、しかし、変異体は、ここで使用されるモノクローナル抗体の結合部位を変更するかもしれない発生する可能性があります。これは、これらの薬剤によって治療されている個人で、このシステムの潜在的な制限を表しています。 複数アッセイは、試験サンプル中のインフルエンザ抗体価を列挙するために使用されています。ウイルス力価、50％組織培養感染量（TCID 50）と卵の尿膜腔液ベースアッセイが広く使用されている1,10を計算することができます。ただし、複数の日の要件は、これらの方法は信頼性が低くなります。さらに、これらの方法でプラーク数の50％の変化に基づいて計算が正確に理論的ではなくウイルスの滴定を提供しています。アッセイをプラーク形成は、96時間以内に行うことができますが、再現性の欠如は、ウイルスの力価計算の主要な関心事である。ここで説明した本手法は、いくつかの明確な利点を持っています。まず、一貫性のある結果と再現性の高い手法である。第二に、ウイルス力価の計算は、定義された標準曲線と比較される蛍光強度のシンプルな定量化に基づいています。第三に、二つ以上の一次抗体は、与えられたサンプル中の複数の血清型を診断するために使用することができます。また、IR色素標識抗体は、ミニムを持っているら蛍光とは、細胞イメージング11に使用されています。高い量子収率内因性の蛍光物質の存在に起因する高い細胞自家蛍光のUVや視覚的な放射線（300から600 nM）の結果。これらは芳香族アミノ酸、リポ顔料、ピリジンヌクレオチド（NADPH）、エラスチン、コラーゲン、フラビン補酵素が含まれています。細胞のこの本質的なプロパティは、プローブが特定のタンパク質の発現を定量化するとして、それは困難な放射のこれらのソースを利用できるようになります。対照的に、近赤外色素は、励起と670から1000 nmの間放出されます。多くの宿主細胞分子や多環芳香族炭化水素は、この波長で励起すると低光散乱のために、極めて低いバックグラウンド蛍光を放出しない。さらに、背景と特異的検出との間のウィンドウは非常に近赤外蛍光色素の高い消光係数による改善されています。光安、優れた信号対雑音比は、ウイルス力価の定量化に非常に関連があります。 microflの使用uidic室は、ロボットコントロールを介してスクリーニングプロセスを自動化し、私たちは容易に非常に伝染性の臨床サンプルをテストする可能性があります。したがって、これらのメソッドの近赤外色素の利用は、組織サンプル中のウイルス力価を列挙するのに理想的で信頼性の高いです。

<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>ウシ血清アルブミン</td><td>アトランタバイオ</td><td> S11750 </td><td></td></tr><tr><td> PR8ウイルス</td><td></td><td></td><td>博士トーマス·M·モランから</td></tr><tr><td>ヤギ抗マウスIRDye @ 800 </td><td> LI-COR Biosciences社</td><td> 926から32210 </td><td> 1:200希釈</td></tr><tr><td> LI-CORオデッセイIRスキャナ</td><td> LI-COR-バイオサイエンス</td><td> 9201から01 </td><td></td></tr><tr><td> 384ウェル平底プレート</td><td> Nalge Nunc社国際</td><td> 164730 </td><td></td></tr><tr><td> 96ウェル平底プレート</td><td> Nalge Nunc社国際</td><td> 165305 </td><td></td></tr><tr><td> DMEM培地</td><td> Invitrogen </td><td> 11965126 </td><td></td></tr><tr><td> RPMI培地</td><td>インビトロジェン</td><td> 11875135 </td><td></td></tr><tr><td>重炭酸ナトリウム</td><td>インビトロジェン</td><td> 25080 </td><td></td></tr><tr><td> L-グルタミン100倍</td><td>インビトロジェン</td><td> 25030 </td><td></td></tr><tr><td> Trypson-EDTA </td><td>インビトロジェン</td><td> R001100 </td><td></td></tr><tr><td>ピルビン酸ナトリウム</td><td>インビトロジェン</td><td> 11360070 </td><td></td></tr><tr><td>抗M-2抗体</td><td></td><td></td><td>博士トーマス·M·モランから</td></tr><tr><td>抗NPモノクローナル抗体</td><td> CDC </td><td> V2S2208 </td><td> MTAを用いて得られた</td></tr></tbody></table>

high-throughput detection method for influenza virus

インフルエンザウイルスは、世界の複数の部分では毎年罹患率と死亡率の高い学位を引き起こす呼吸器病原体である。したがって、臨床サンプルの膨大な数の感染株と迅速なハイスループットスクリーニングの正確な診断は、パンデミックの感染の広がりを制御することが重要です。インフルエンザ感染症の現在の臨床診断は血清学的検査、ポリメラーゼ連鎖反応、直接試料の免疫および細胞培養1,2に基づいています。 ここでは、ライブのインフルエンザウイルスを検出するために用いられる新規な診断技術の開発について報告する。我々は、MDCK細胞4を使用して、この手法の有効性をテストするためにマウスに適合した人間A/PR/8/34（PR8、H1N1）ウイルス3を使用していました 。 MDCK細胞（ウェルあたり10 4または5×10 3）96中で培養した-またはPR8とウイルスタンパク質に感染した384ウェルプレートでは、IR色素標識秒、続いて抗M2を使用して検出された二次抗体。 M2 5ヘマグルチニン1は 、さまざまな診断アッセイに使用される2つの主要なマーカー蛋白質である。採用したIR-色素コンジュゲート二次抗体は、他の蛍光色素に関連付けられた蛍光を最小限に抑える。抗M2抗体の使用は、私たちは、ウイルス量の直接的な指標として抗原特異的な蛍光強度を使用することができました。蛍光強度を列挙するには、我々は、LI-CORオデッセイベースの赤外線スキャナを使用していました。このシステムは、蛍光物質を識別し、バックグラウンドノイズと区別するために2つのチャネルのレーザーベースのIRの検出を使用しています。最初のチャンネルは680 nmで励起し、バックグラウンドを定量化するために700 nmで発光する。番目のチャネルは780 nmで蛍光体を励起を検出し、800 nmで発光する。 IRスキャナでPR8感染MDCK細胞のスキャンは、ウイルスの力価に依存明るい蛍光を示した。 10 2 -10 5 PFUから始めてウイルス力価の蛍光強度の正の相関がCONかもしれませんsistently観察した。一貫して10 2 -10 3 PFU PR8ウイルス力価で見られた最小限のしかし、検出可能な陽性は近赤外色素の高感度を示した。信号対雑音比はモック感染またはアイソタイプ抗体処理MDCK細胞を比較することによって決定されました。 96からの蛍光強度を使用して - または384ウェルプレートフォーマットを、我々は標準的な滴定曲​​線を構築した。番目の変数は、蛍光強度でありながら、これらの計算では、最初の変数は、ウイルス力価である。したがって、我々は、ウイルス力価および蛍光強度の間に多項式の関係を決定するためにカーブフィットを生成するために指数分布を使用していました。総称して、我々はIR染料ベースのタンパク質検出システムは、ウイルス株を感染の診断と正確に感染する病原体の力価を列挙助けることができると結論する。

Watch this Scientific Journal Video about High-throughput Detection Method for Influenza Virus at JoVE.com

High-throughput Detection Method for Influenza Virus

この方法は、高感度で感染マウスの気管支肺胞洗浄（BAL）液中のH1N1を検出するための赤外線染料ベースのイメージングシステムの使用方法について説明します。試験材料の&lt;10μlの体積を必要とし、複数の病原体の同時スクリーニングのための可能性を秘めている、または384ウェルプレート - この方法論は、96で行うことができ

インフルエンザウイルスのためのハイスループット検出方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

high-throughput-detection-method-for-influenza-virus

Research

JoVE Journal

Immunology and Infection

26.0K ビュー.  Blood Research Institute. この方法は、高感度で感染マウスの気管支肺胞洗浄（BAL）液中のH1N1を検出するための赤外線染料ベースのイメージングシステムの使用方法について説明します。試験材料の

ビデオ: High-throughput Detection Method for Influenza Virus

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

プラスミドDNAの組換えインフルエンザウイルスの生成

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

免疫・感染学

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

の間に相乗効果を調査するために生物発光イメージングを使用して肺炎球菌幼児マウスで>とインフルエンザウイルス

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

インフルエンザのためのDNAマイクロアレイ上ampliPHOX比色検出

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

鶏卵卵におけるインフルエンザウイルスの伝播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

豚からのインフルエンザAウイルスの検出および単離のための鼻ワイプ

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

勾配遠心によるインフルエンザAウイルスキャプシドのインビトロ分解で

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

ナノスケールPCRによる動物標本における呼吸器病原体のハイスループット検出

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

感染症のマウスモデルでの A 型インフルエンザ ウイルス研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

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

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

エスケープ中和抗体でインフルエンザ ウイルスの亜種の世代

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

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 ...