This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.

以下のデータの収集に使用されるすべての豚は、オハイオ州立大学施設内動物管理使用委員会の下で保護されていた（動物使用プロトコル番号2009A0134-R1）。 1.ウイルス輸送培地の調製とサンプル収集バイアル<ol><li>精製水900 mlにブレインハートインフュージョンの37グラムを追加し、70℃に加熱し、完全に粉末を溶解する一方で攪拌棒および電磁攪拌機を完全に混合します。 </li><li> 15分間121℃で培養液をオートクレーブ。培養液は、室温になるまで作業台の上に冷却します。 4℃の冷蔵庫で一晩に必要な場合。 </li><li>磁気撹拌棒、ペニシリンGナトリウム塩6.02 gで、室温で精製水50mlに硫酸ストレプトマイシン10gと混合し、溶解します。 100ミリリットルの全量を、追加の精製水を追加します。滅菌ボトルに0.22μmのポリエーテルスルホン膜を通して溶液をろ過。 </li><li>無菌的に冷却されたブレインハートインフュージョンブロス中に濾過ペニシリン及びストレプトマイシン溶液を加え、徹底的に（手で撹拌棒やスワールフラスコ）を混ぜウイルス輸送培地を作るために。 </li><li>無菌的には、ウイルス輸送培地のpHを試験します。必要に応じて、HClまたはNaOHを用いて7.4±0.2にpHを調整します。 </li><li>張とハーモンによって記載されているように、RRT-PCRによりインフルエンザウイルスマトリックスタンパク質をテストすることによって、ウイルス輸送培地上の品質保証テストを実行します。22 </li><li> 8 mlの容量の無菌の高密度ポリエチレンボトルにウイルス輸送培地5mlを分配します。フィールドでの使用のためにバイアルにラベルを付けます。 </li><li>サンプル収集まで-20℃で標準クライオボックスにすぐに使用バイアルに保管してください。 </li></ol>豚の鼻ワイプの2コレクション<ol><li>コレクションをサンプリングする直前にバイアルの適切な数を解凍。 1バイアルは、サンプリングされた豚ごとに必要とされるであろう。部屋で解凍温度は約30〜45分かかります。解凍したバイアルを使用するまで氷上パックに冷静さを保ちます。 </li><li>ドン適切な個人保護具を使用する状況によって決定される（例えばカバーオール、ブートカバー、呼吸器、耳栓）など。 </li><li>動物の面積を入力します。必要に応じて、小さな領域に豚を閉じ込めるませんが、何の拘束ありません。動物休止奮起しないでください。 </li><li>使い捨て検査用手袋のペアに入れてください。豚、人、または無生物に触れて、手袋を汚染しないように注意してください。 </li><li> （中×2。2で）5.08センチメートル×無菌5.08センチメートルを削除するには、手袋をはめた手を使用してそのラッパーから綿ガーゼパッド。滅菌ガーゼパッドは可能な限り使用する必要があります。個別包装のガーゼパッドは、パッケージごとに2つのガーゼパッドで包まれたものを使用するより便利です。 </li><li>サンプリングのための可能な限りパッドのできるだけ多くを露出する一方の手の指先で綿ガーゼパッドを持ちます。 </li><li> 、豚の鼻を横切ってガーゼパッドを拭きとります可能な場合は、外部鼻孔を入力するには細心の注意。同じガーゼパッドに見える鼻汁（約1ml）を収集します。 </li><li>同じ手を使用して、ウイルス輸送培地を含むバイアル中に配置することを容易にするためにそれ自体の上にガーゼパッドを折ります。 </li><li>もう一方の手を使用して、バイアルのキャップを取り外し、バイアルに折り畳まれたガーゼパッドを配置します。バイアルをおさらいし、ガーゼパッドおよびウイルス輸送培地の混合を確実にするために、バイアルを振ります。 </li><li>適切な容器に手袋と場所を削除します。各ブタの間に手袋を変更します。 </li><li>バイアルの識別子を確認してください。レコード豚の識別番号、年齢、性別、臨床徴候、およびその他の注意事項は、調査員が必要と認めました。 </li><li>すぐに収集した後、サンプルを冷やします。複数の凍結融解サイクルを防ぐために凍結する前にサンプルのアリコートすることを検討してください。できるだけ早く収集した後、実験室への輸送のためにドライアイスの上にサンプルを置きます。 -80でバイアルを保存テストが開始されるまでCを°。 </li></ol> A型インフルエンザウイルス核酸の検出3。 <ol><li> 5分間37℃で乾燥したビーズ槽内のサンプルを解凍し、その後20〜30分間室温でベンチ上にサン​​プルを解凍終えます。ビーズには、いくつかのバイアルの同時添加は、浴を加熱する原因となります。バイアルを過熱しないよう注意すること。 </li><li> RNA抽出のためにウイルス輸送培地100μlを削除します。 <ol><li>多数のサンプルからRNAを抽出するためのハイスループット（96ウェルプレート形式）磁気ビーズプラットフォームを使用してください。31 </li></ol></li><li>迅速な検出インフルエンザウイルスのリアルタイム逆転写PCRアッセイを使用してください。31 </li></ol>鼻ワイプからのインフルエンザAウイルスの4.絶縁<ol><li> 3.1節で前述したように、サンプルを解凍</li><li>千（硫酸ゲンタマイシンで解凍したサンプルを扱います/ mlの）、アンホテリシンB（22.5 / mlの）、および硫酸カナマイシン（325 / mlの）。 </li><li>前述のようにマディンDarbyイヌ腎臓（MDCK）上皮細胞に接種する。32 </li></ol>

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</ol>

The authors have nothing to disclose.

ポリエステルが先端に付いた鼻腔​​スワブを用いて、ブタからサンプルを収集すると、IAVの監視を行う際に有用であることが判明しました。しかし、鼻腔スワブ手順を使用すると、拘束用スネアの必要な使用による監視の努力を妨げています。鼻ワイプは、サンプル収集中の人や豚のストレスを最小限にするために、現在の豚のサンプリング技術の洗練を表します。この方法は、のために?...

<html>インフルエンザAウイルス（IAV）は家禽、ブタ、およびヒトを含む多くの種において、呼吸器疾患を引き起こします。迅速なウイルスの進化が発生する可能性があり、新たなIAV株が頻繁に出現するセグメント化されたIAVゲノムの再集合のために。豚は、複数のホスト種からIAVsの再集合のための混合容器としての役割を果たすことができる種である。1は 、一般的にヒトから北米の豚（H1N1、H1N2、H3N2）が、複数のIAVの紹介の中で循環するIAVの三大サブタイプはまだありきこれらのサブタイプ内の大規模なIAVの多様性につながった。豚に感染IAVsの2急速な進化は、人間、鳥や豚のウイルスからの遺伝子セグメントを含むトリプル再集合IAV 3は、米国で豚の間で広まった1998年以来、明らかになっている。4つの内部遺伝子そのトリプル再集合体のIAVのセグメントは現在、豚に感染IAVsの間で非常に普及している残る。5 &quot;&gt;世界的に、IAVは、典型的な臨床徴候は、発熱、食欲不振、倦怠感、咳、呼吸苦しい、くしゃみ、鼻汁や不十分な体重増加が含まれている豚の呼吸器疾患の重要な原因である。IAVはどこ繁殖農場をまくために特にコストがかかる可能性が原因IAV誘発される発熱と弱い生まれの子豚への障害が報告されている。6,7を米国内では、IAVは、一般的に商業豚の群れとIAV感染豚の間で広範な抗原およびゲノム多様性と進化を続ける中で検出されたが、この制御を妨げていますウイルス。8-11 トリプル再集合体北米の豚の系統とユーラシアの鳥のような豚系統からの遺伝子セグメントを含む豚系統のIAVは、世界中の大流行を引き起こしたときに、2009年に実現された豚に再集合に起因するパンデミックIAV株の出現についての公衆衛生上の懸念ヒトでは12パンデミックウイルス（A（H1N1）pdm09が）ので、持っています流行豚で再集合IAVは13,14の株およびこれらの新たに再集合株のいくつかは、人間に戻って送信されてきた。15再集合イベントやパンデミック可能性を秘めた新しいIAV株の出現豚不可欠でIAVウイルス循環の積極的な監視を行う頻度を、特に豚ヒト界面で。 豚ヒューマンインタフェースは、IAVの双方向の種間伝播のために重要です。ヒト - 豚商業豚生産で発生する伝送は、豚の人口に現在存在IAV多様性の大規模な量を担当しています。農業フェアは、米国の人々と豚のcominglingの最大設定であり、IAVの人獣共通伝送のための部位を知られている。2012年には15〜21、バリアントH3N2 IAVの発生時に、例93％の出席を報告しました前疾患の発症日の農業フェア。15ゲノム解析人間分離株と比較して展示豚からのウイルス分離株のは、人獣共通感染伝送を確認した。IAVに感染した21展覧会の豚は、多くの場合、直接診断テストの必要性を示す病気、21〜23の臨床徴候を示さありません。 目に見えて病気豚のサンプリングだけでは成功した豚でIAVの有病率を特定せず、豚の中で新興IAVの新しい株を同定するために依拠することはできません。アクティブサーベイランスは、ブタにおけるIAVの新興株の検出のために絶対に必要であると豚と公衆衛生の両方のための彼らの脅威を評価します。ほとんどのIAVの監視活動は自発的であり、したがって、最小限の破壊方法が必要とされています。 IAV感染豚のための3つの主要な生前のサンプル収集の手順は以下のとおりです。鼻腔スワブ、口腔液、および鼻ワイプ。 IAVリストに合成繊維の挿入を検出するために、個々の豚をサンプリングするための現在の推奨事項は、好ましい方法として鼻孔に綿棒をひっくり返し鼻汁および上皮細胞を収集する。24,25豚は、この手順を回避しようとする可能性があるため、訓練を受けた人材のチームが手動または動物の大きさに応じて、スネアと豚を抑える必要があります。26の拘束プロセスがために手間がかかります人事、および豚のためにストレスのたまります。競技動物に追加されたストレスの認識が監視努力の所有者が耐性にすることができるように加えて、展覧会の豚は、多くの場合、公正に複数の競技に関与しています。 IAVで80〜100％の範囲のIAV検出の確率が群れに感染すると、口腔液は、豚の集団におけるIAVの分子検出のための綿棒を鼻にする一般的な代替となっている。27,28さらに、口腔液は、より広い窓を提供することができます初期感染後の鼻腔スワブよりI​​AV検出。しかし、口腔液からIAV分離は、IAで得られたウイルス分離の試みの50％のみで問題がありましたV回復。29 豚におけるIAV監視中に鼻腔スワブの代わりに鼻ワイプを使用すると、上記の制限を克服します。鼻ワイプは拘束スネアの使用を必要としないと手続きの動物や証人を強調することなく実行することができます。最小限の技術研修を監視サンプルを収集するために、豚の所有者を含む非獣医専門家を可能にする、鼻ワイプを収集するために必要とされています。鼻ワイプは、以前にインフルエンザの検出および単離ウイルス30鼻腔スワブと比較した、サンプリングのこの非侵襲的方法の詳細なプロトコルは、以下に記載されています。

<table><tbody><tr><td>BBL Brain Heart Infusion</td><td>Becton, Dickinson and Company</td><td>211059</td><td></td></tr><tr><td>Penicillin G Sodium Salt</td><td>MP Biomedicals, LLC</td><td>021194537</td><td>1500 u/mg</td></tr><tr><td>Streptomycin Sulfate</td><td>AMRESCO LLC</td><td>0382</td><td></td></tr><tr><td>Gentamicin Solution</td><td>Mediatech, Inc.</td><td>30-005-CR</td><td>50 mg/ml</td></tr><tr><td>Amphotericin B Solution</td><td>Fisher</td><td>S 24 25</td><td>250 ug/ml</td></tr><tr><td>Kanamycin Sulfate</td><td>Teknova</td><td>K2105</td><td>5000 ug/ml</td></tr><tr><td>TPP Rapid Filtermax System</td><td>TPP Techno Plastic Products AG&amp;nbsp;</td><td>99150</td><td></td></tr><tr><td>Nalgen Diagnostic Bottles</td><td>Thermo Scientific</td><td>342002-9025</td><td>HDPE with white PP closure</td></tr><tr><td>Dermacea Gauze Sponge, 8 ply</td><td>Covidien</td><td>441211</td><td>5.08 cm &amp;times; 5.08 cm (2 in. &amp;times; 2 in)&amp;nbsp;</td></tr><tr><td>Nitrile Exam Gloves</td><td>Saftey Choice</td><td>19-170-010 (A-D)</td><td></td></tr></tbody></table>

nasal wipes for influenza a virus detection and isolation from swine

インフルエンザウイルスは急速に豚集団に進化し、新しい株が絶えず出現しているので、豚におけるA型インフルエンザウイルスのためのサーベイランスは、ヒトおよび動物の健康にとって非常に重要です。豚インフルエンザの多様な系統によってそれら新型インフルエンザウイルス株の出現と維持に重要ホスト作るウイルスを感染させることができます。このような商業豚農場、農業フェア、および生きた動物市場などの多様な設定で豚をサンプリングすると、現在IAV株を循環させる包括的なビューを提供することが重要です。現在のゴールドスタンダード生前サンプリング技術（鼻腔スワブのすなわちコレクション）は、豚の物理的な拘束を必要とするため、労働集約的です。鼻ワイプは、動物の無拘束に最小限で豚の鼻を横切っての生地片をこすり伴います。鼻の手順は実行が簡単で、プロの獣医や動物の取り扱い訓練を受けた人材を必要としないワイプ。 W鼻腔スワブよりわずかに小さい敏感hileは、ウイルス検出と分離率は、鼻が低応力サンプリングの方法が必要な場合は、個々の豚をサンプリングするための実行可能な代替を払拭作るために十分です。進行プロトコルは、個々の豚からワイプ実行可能な鼻を収集するために必要な手順を説明します。

この方法の使用の成功は、RRT-PCRは、任意の環境破片がサンプリング中に拾ったからRNA抽出およびRRT-PCRの間の内部コントロールの使用に伴う、ショーのサンプルはPCR阻害物質が含まれていなかった、ということの結果が得られます。サンプル接種後、ウイル​​ス分離ウェルは、サンプルから見える環境の破片があってはなりません。 PCRはウイルス核酸、必ずしも生存ウイルスを検出したた?...

Watch this Scientific Journal Video about Nasal Wipes for Influenza A Virus Detection and Isolation from Swine at JoVE.com

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

nasal-wipes-for-influenza-a-virus-detection-and-isolation-from-swine

Research

JoVE Journal

Immunology and Infection

9.4K ビュー.  The Ohio State University. The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. 

ビデオ: Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

すべてのインフルエンザA型の定量的な分析シンプルなスロットブロットアッセイでユニバーサル抗体を用いたウイルスHemagglutininsとノイラミニダーゼ

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

免疫・感染学

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

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, ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

陰イオン交換樹脂を用いるバイオエアロゾルからのウイルスの検出

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

環境学

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

インフルエンザウイルスAおよびBの急速な分子の検出と分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

蛍光ベースのノイラミニダーゼ阻害アッセイ抗ウイルスのノイラミニダーゼ阻害剤クラスにインフルエンザウイルスの感受性を評価します

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

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 ...

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 ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

イメージング 2 光子生体顕微鏡を用いたインフルエンザ ウイルス感染症の中に気管粘膜の細胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

インフルエンザ特異抗体価を定量化する最適化された赤血球凝集抑制 (HI) 試験

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

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

要約

さらに動画を探す

すべてのインフルエンザA型の定量的な分析シンプルなスロットブロットアッセイでユニバーサル抗体を用いたウイルスHemagglutininsとノイラミニダーゼ

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

陰イオン交換樹脂を用いるバイオエアロゾルからのウイルスの検出

インフルエンザウイルスAおよびBの急速な分子の検出と分化

蛍光ベースのノイラミニダーゼ阻害アッセイ抗ウイルスのノイラミニダーゼ阻害剤クラスにインフルエンザウイルスの感受性を評価します

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

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

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

イメージング 2 光子生体顕微鏡を用いたインフルエンザウイルス感染症の中に気管粘膜の細胞相互作用

インフルエンザ特異抗体価を定量化する最適化された赤血球凝集抑制 (HI) 試験

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

要約

さらに動画を探す

すべてのインフルエンザA型の定量的な分析シンプルなスロットブロットアッセイでユニバーサル抗体を用いたウイルスHemagglutininsとノイラミニダーゼ

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

陰イオン交換樹脂を用いるバイオエアロゾルからのウイルスの検出

インフルエンザウイルスAおよびBの急速な分子の検出と分化

蛍光ベースのノイラミニダーゼ阻害アッセイ抗ウイルスのノイラミニダーゼ阻害剤クラスにインフルエンザウイルスの感受性を評価します

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

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

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

イメージング 2 光子生体顕微鏡を用いたインフルエンザ ウイルス感染症の中に気管粘膜の細胞相互作用

インフルエンザ特異抗体価を定量化する最適化された赤血球凝集抑制 (HI) 試験

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

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

イメージング 2 光子生体顕微鏡を用いたインフルエンザウイルス感染症の中に気管粘膜の細胞相互作用