著者らは、rWSN-PB2-球菌ウイルスの灘Naffakhとマリー·アンRameix-Welti（パスツール研究所）に感謝したいと思います。

<html>プロトコルの概略フローチャートを図に示されています。 1と試薬のテーブルは以下に提示されています。 1。感染症（16から24時間） <ol><li>ダルベッコ改変イーグル培地（DMEM）高さは次の日（約5×10 8細胞）の約80％コンフルエントに達するようなグルコース培地、5 150ミリメートルのプラスチック細胞培養ディッシュにHeLa細胞外シード。細胞がコンフルエントに100％に達していないことが重要です。 </li><li>次の日、にStrep-tag IIタグインフルエンザウイルスストックを希釈3の感染多重度にウシ血清アルブミン0.3％（BSA）を含む生理食塩水（PBS）をリン酸緩衝。 </li><li>細胞から増殖培地を除去し、PBSで1回洗浄し、10 mlのPBSで細胞にウイルスを含む追加します。室温で1時間インキュベートする。 </li><li> DMEM高グルコースの感染媒体を交換し、37℃でインキュベーションを続ける℃に</li></ol>&quot;&gt;インキュベーションの長さは（説明を参照してください）​​勉強する感染症の位相に依存します。 2。細胞内分画（3 H） <ol><li>冷PBSで1回細胞を洗浄し、ゴム製のスクレーパー及び50 mlコニカルチューブに移すと冷PBSで細胞を懸濁する。 </li><li>ペレットは、1,000 rpmで5分間、卓上遠心機で細胞とその後PBSを吸引除去する。 </li><li> 10ミリリットル冷ショ糖バッファー（10 mM HEPES pHは7.9、10mMの塩化カリウム、2mMのMgのアセテート（MgOAc）、3 mMのCaCl 2を 、340 mMスクロース、1 mMジチオスレイトール（DTT）、1％のプロテアーゼ阻害剤ミックス）に再懸濁し、細胞ペレット。サスペンション後、細胞塊を破壊するために10ミリリットルのプラスチック血清ピペットに取り付けられた200μlのピペットチップを介して細胞を渡します。 </li><li>氷上で10分間インキュベートします。穏やかに細胞を再懸濁するために定期的に反転させる。 </li><li> 15秒間高速で0.5％と渦の最終濃度にノニデットP-40（NP-40）を追加します。すぐにテーブルトップ遠心で4℃で3500×gで10分間遠心geがあります。 </li><li> 4℃で上清とストアを削除℃にこれは、細胞質画分である。ペレットは、元の細胞ペレットよりも小さく、白くなければなりません。すべての画分を4℃で少なくとも4時間保存することができます</li><li>ステップ2.5のように5 mlの冷ショ糖バッファー、再遠心機でペレットを洗浄し、上清を捨てる。 </li><li> 1.5ミリリットル冷たいnucleoplasmic抽出緩衝液（50mMのHEPES pHは7.9、150mMのKOAc、1.5mMのMgCl 2、0.1％NP-40、1mMのDTT、1％のプロテアーゼ阻害剤·ミックス）でペレットを（核を含む）を再懸濁します。 </li><li>タイトフィット乳棒を用いて冷却した、すべてのガラス4ミリリットルDounceホモジナイザーに移し、20ストロークで核を均質化する。 均質化時に発泡しないようにしてください。抵抗は約10ストロークの後に増やす必要があります。効率的な均質化は、位相差顕微鏡下で確認することができます。 </li><li> 1.5 mlのマイクロ遠心チューブに均質化した核を移し、4℃で回転するホイール​​の20分​​間インキュベートしC. </li><li> 10分間、4℃で遠心で16000×gで遠心します。 </li><li> 4℃で上清とストアを削除℃にこれはnucleoplasmic画分である。 </li><li> 1.5 mlの消化緩衝液（50mMのHEPES pHは7.9、10mMのNaCl、1.5mMのMgCl 2、1mMのDTT、1％のプロテアーゼ阻害剤ミックス、100 U / mlのベンゾナーゼ（RNA解析のために、20 U / mlのRNaseを持つ代替でペレットを再懸濁しフリーのDNase I））事前に温めた37℃、37℃で10分間インキュベート℃、 </li><li> 5 MのNaCl（150mMのNaClの最終濃度）を42μlを加え、4時20分さらにインキュベート℃、 </li><li> 10分間、4℃で遠心で16000×gで遠心します。 </li><li> 4℃で上清とストアを削除℃にこれはch150画分である。 </li><li>ペレットを再懸濁し、1.5 mlの冷高塩緩衝液（50mMのHEPES pHは7.9、500mMのNaCl、1.5mMのMgCl 2、0.1％NP-40、1mMのDTT、1％のプロテアーゼ阻害剤ミックス）と氷上で20分間インキュベートする。 </li><li> 16000×gで遠心&lt;/ em&gt;は、10分間4℃で遠心インチ</li><li> 4℃で上清とストアを削除℃にこれはch500画分である。 </li></ol> 3。 Strepタグ精製（2時間） <ol><li>細胞質画分（150mMのNaClの最終濃度のために）の5 M NaClを300μlを追加します。 </li><li> 15 mlコニカルチューブに分ごとにパックされたのStrep-Tactinセファロースビーズのアリコートを100μlの。 </li><li>卓上で混合し、1,000×gで5分間遠心するチューブを反転、ビーズに球菌洗浄バッファー（20mMのHEPES pHは7.9、150mMのNaCl（ch500サンプルの500mMのNaCl）、1mMのEDTA）の10冊を追加します。遠心します。 </li><li> 、上清を捨て、ステップ3.3回繰り返し、その後、連鎖球菌の洗浄緩衝液を等量にビーズペレットを再懸濁します。 </li><li>各分画するために洗浄のStrep-Tactinビーズスラリー（nuceloplasmicのために1.5 mlのマイクロ遠心チューブ、ch150、ch500と分画と細胞質画分の15 mlコニカルチューブ）200μlを添加します。 </li><li>回転させるチューブを4℃1H°回転ホイール上のC。 </li><li>チューブ4で1,000×gで1分間℃を遠心分離上清を捨てる。 </li><li>各チューブに1 mlの冷球菌の洗浄緩衝液を追加します。 1.5 mLのチューブに15 mLの試験管（細胞質画分）からビーズを転送します。 4で全てのチューブ1分°C、ステップ3.7のようにした後、遠心分離を洗う。二回の洗浄ステップを繰り返します。 </li><li>最後の洗浄ステップの後、平らなピペットチップを使用して、洗浄緩衝液の痕跡をすべて削除します。 100μlの溶出バッファー（2.5mMのdesthiobiotinを含む連鎖球菌洗浄バッファー）を加え、穏やかに混ぜる。 </li><li>氷上で15分間インキュベートします。優しくチューブの底をタップすることで時折ビーズを混ぜます。 </li><li>遠心チューブ4で1000×gで1分°Cマイクロインチを含む上清を溶出したStrep-tag IIタグvRNPsを収集します。 </li></ol> 4。代表的な結果分別の効率は最高のa​​ntibを使用して分画し、溶解物のウエスタンブロット分析により判断される細胞内マーカー（ 図2）特定のodies。具体的には、成功した核内分別がnucleoplasmic、または可溶性核画分4のほとんど、あるいはまったく細胞のRNAポリメラーゼII（Pol II）を示すべきであり、ほとんどは150mMのNaCl 5で抽出する必要があります。はPol IIの劣化も再現文献6と一致して非感染細胞に比べて、インフルエンザウイルス感染細胞で観察されています。 図細胞質溶出液のために示されるようにvRNPsの精製は、最高の、銀またはクマシー染色により判断される。 3。我々の経験では、ほとんどのStrep-PB2が完全に形成されたvRNPの一環として、すなわち小さな水溶性ポリメラーゼがキャプチャされたで精製されています。銀またはクマシー染色により観察PA/PB1/PB2比：これは、高NPに反映されます。いくつかのユビキタスRNアーゼが（例えば、RNアーゼAなど）dissociのような方法で切断し、ウ​​イルスRNAによく知られているような低い比率は、RNaseのでバッファの混入を示唆しているNP多量7からポリメラーゼを食べました。興味深いことに、単独でベンゾナーゼによる治療は、ウイルスRNAを消化するのに十分ながら、vRNPをタンパク質の化学量論比に影響を及ぼさないように見えます。我々はこの現象を説明することはできませんが、我々はベンゾナーゼは、特定の構造的に重要なRNA領域はそのまま残しておく可能性が示唆された、他のRNase（データは示さず）で消化後vRNPsの破壊を観察した。細胞質と核質（ヌクレアーゼ消化せずに実行）からvRNPsの精製した後、高分子量で8かすかなしかし、離散的なバンドは、8インフルエンザウイルスゲノムセグメントの予測分子量に相当する銀染色法、（リファレンスを参照してください。3で観察することができます。） 9時間の感染後に各画分から精製vRNPsの相対量は、 図に示されています。 4。 vRNPsの分布は、初期の時点でch150分で最強の蓄積と、感染の経過とともに変化し、後半の時点で核質と細胞質内蓄積を増加させた。 <img src="/files/ftp_upload/4028/4028fig1.jpg" alt="図1"/> 細胞内分画の図1のフローチャート。 refから適応した。 3。 <img src="/files/ftp_upload/4028/4028fig2.jpg" alt="図2"/> 図2インフルエンザウイルス感染細胞からの細胞内画分のウェスタンブロット分析。 10 9 HeLa細胞は、細胞内分画の前に9時間のインフルエンザウイルス株のWSNを感染させた。総タンパク質の等しい量は、各レーンにロードされ、示された抗体を用いて分析した。 RNAポリメラーゼII（Pol II）は、C末端ドメイン（hypophosphorylatedバンドが最も顕著である）のすべてのフォームを認識するクローン8WG16を使用して、検出された。 refから適応した。 3。 <img src="/files/ftp_upload/4028/4028fig3.jpg" alt="図3"/> 図3。精製vRNPsの銀染色分析。 HeLa細胞は、細胞質画分から連鎖球菌精製に続いて​​、9時間rWSN（ネガティブコントロール）またはrWSN-PB2-連鎖球菌に感染させた。アスタリスクはRNase消化後表示されていないバンドを示します。 refから適応した。 3。 <img src="/files/ftp_upload/4028/4028fig4.jpg" alt="図4"/> 図4：異なる細胞分画から精製されたvRNPsの銀染色分析。 10 9 HeLa細胞はHが細胞内分画およびStrep精製に続いて9にrWSN-PB2-連鎖球菌またはrWSNを感染させた。各画分から溶出液の等量がロードされた。 refから適応した。 3。

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利害の衝突が宣言されません。

多くの研究が最近、個々のタンパク質やインフルエンザウイルス感染8に関与する細胞のネットワークを識別しているが、これらの相互作用の大部分の機能的意義は不明である。インフルエンザウイルスRNA合成と核9の複雑な生物物理学的および生化学的性質のためにクロマチンベースの関数の絶対的な依存性を考えると、新技術は、これらの機能を解明する必要があります。 ...

<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> DMEM高グルコース</td><td>ギブコ</td><td> 11965-092 </td><td></td></tr><tr><td> BSA </td><td>シグマ</td><td> A9418 </td><td></td></tr><tr><td>プロテアーゼ阻害剤ミックスG </td><td> Serva </td><td> 39101 </td><td></td></tr><tr><td>ベンゾナーゼヌクレアーゼ</td><td> Novagen社</td><td> 71206 </td><td> 25 U /μlの</td></tr><tr><td>のDNase I、RNaseフリー</td><td> ThermoScientific </td><td> EN0523 </td><td> 50 U /μlの</td></tr><tr><td>ダウンスホモジナイザー</td><td>ホイートン</td><td> 432-1271 </td><td> &quot;B&quot;乳棒を入力して使用</td></tr><tr><td>のStrep-Tactinセファロース</td><td> IBA GmbH社</td><td> 2-1201-025 </td><td> 50％懸濁液カラムフォーマットを使用することもできます</td></tr><tr><td> Desthiobiotin </td><td> IBA社</td><td> 2-1000-002 </td><td></td></tr></tbody></table>

affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells

すべてのマイナス鎖RNAウイルスと同様に、インフルエンザウイルスのゲノムは一本鎖ゲノムは核タンパク質（NP）で包まれ、構成される三量体ポリメラーゼ複合体と関連付けされているウイルスのリボ核タンパク質複合体（vRNPを）の形でパッケージ化されていますPA、PB1、PB2サブユニットとの。しかし、ほとんどのRNAウイルスとは対照的に、インフルエンザウイルスは、感染細胞の核内にウイルスのRNA合成を実行します。興味深いことに、ウイルスmRNAの合成は、プライマーとして、携帯プレmRNAを使用しており、それは、このプロセスは、クロマチン1日に行われることが提案されている。ウイルスポリメラーゼ及び宿主RNAポリメラーゼIIと同様に、NPとホストヌクレオソーム間の相互作用はまた、1,2特徴づけられている。 最近では、One-Strepタグをコードする組換えインフルエンザウイルスの発生は遺伝的であるウイルスポリメラーゼのPB2サブユニットのC末端（rWSN-PB2-球菌3）にした融合enは説明します。これらの組換えウイルスは、感染細胞からのvRNPsなど、PB2を含む複合体の精製を可能にします。精製されたvRNPsを取得するために、培養細胞が感染されており、vRNPsは、親和性、これらの細胞から派生したライセートから精製されています。ただし、日付に使用される溶解の手順は、一般的なヌクレアーゼの存在にもかかわらず、しばしば唯一の非効率的にクロマチンに結合した物質を抽出し、ワンステップの洗剤溶解に基づいてされています。 私たちの予備的な作業は、核vRNPsの大部分は伝統的な細胞溶解中に抽出されなかったことを示唆し、したがって、アフィニティー精製することができませんでした。この抽出効率を高めるために、非クロマチン結合型核vRNPsからクロマチンに結合分離するために、我々は、インフルエンザウイルス感染細胞へ段階的に細胞内抽出プロトコルを適応した。簡単に言うと、このプロシージャは、最初のセルから核を分離し、（ここでは &quot;nucleoplasmic&quot;分数と呼ばれる）水溶性核タンパク質を抽出します。残りの不溶性の核物質は、その後ベンゾナーゼ、二つの塩の抽出手順に続いて非特異的なDNA / RNAヌクレアーゼで消化されます：最初の150mM NaClを（ &quot;ch150&quot;と呼ばれる）を使用し、500mMのNaCl（ &quot;ch500&quot;）（ 図1 。）これらの塩抽出の手順はまだ親和性マトリックスへのタグ付けvRNPsの結合を許可500mMのNaCl、まだ核vRNPsの85％以上を可溶化するのに十分であったという我々の観察に基づいて選ばれました。 感染細胞の細胞内分画後、それは、それぞれ個々の画分からPB2-タグvRNPsの親和性を精製し、それらの蛋白質およびウエスタンブロットおよびプライマー伸長を用いてRNA成分を分析することが可能である。最近、我々は3 500mMのNaCl（ch500）で抽出したクロマチン画分で感染した後に遅れてのポイントの間にそのvRNPをエクスポートする複合体の形を発見するためにこのメソッドを使用しました。

Watch this Scientific Journal Video about Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells at JoVE.com

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

インフルエンザウイルスは、宿主細胞のクロマチンに関連して、そのRNAゲノムを複製します。ここでは、感染細胞のクロマチンから無傷のウイルスのリボ核タンパク質複合体を精製する手法を提案する。ウイルスの複合体を精製し、それぞれウェスタンブロット、タンパク質およびRNA含量のプライマー伸長の両方で解析することができます。

感染細胞のクロマチンからのインフルエンザウイルスのリボ核タンパク質複合体のアフィニティ精製

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

affinity-purification-influenza-virus-ribonucleoprotein-complexes

Research

JoVE Journal

Immunology and Infection

13.0K ビュー.  Universitätsklinikum Freiburg. インフルエンザウイルスは、宿主細胞のクロマチンに関連して、そのRNAゲノムを複製します。ここでは、感染細胞のクロマチンから無傷のウイルスのリボ核タンパク質複合体を精製する手法を提案する。ウイルスの複合体を精製し、それぞれウェスタンブロット、タンパク質およびRNA含量のプライマー伸長の両方で解析することができます。

ビデオ: Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

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

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, ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

病原体胞の精製から<em&gt;レジオネラ菌</em&gt;感染した食細胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

バキュロウイルス発現系を用いた新規H7N9インフルエンザウイルス由来の機能組換えヘマグルチニンとノイラミニダーゼタンパク質の発現

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

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 ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

インフルエンザAウイルス核を持つマウスMx1のタンパク質の共免疫沈降

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

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 ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA精製は、細胞内から成長します<em&gt;リステリア菌</em&gt;マクロファージ細胞で

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表現型および慢性リンパ球性脈絡髄膜炎ウイルスに感染したマウスから単離された活性化制御性T細胞の機能解析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

細菌や真菌によってウイルス感染細胞上のバイオフィルムの開始の決意

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

感染細胞のクロマチンからのインフルエンザウイルスのリボ核タンパク質複合体のアフィニティ精製

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