The present study was supported by the NIH grants HL120947 (P.C.), HL103868 (P.C.), and the American Heart Association Grant-in-Aid (P.C.)

注：総論：無菌条件下で卵の操作に関連するすべての手順を実行し、滅菌技術がそれに応じて使用する必要があります。使用前に70％エタノールを用いたプレクリーンなすべての機器。一般的には、インフルエンザウイルス接種および収穫はBSL-2の実験室で実施されるべきである。しかし、このプロトコルは、多くの病原性インフルエンザウイルス（ 例えば 、パンデミックおよびプレパンデミック株は、家禽および家畜への危険をもたらす菌株、高病原性鳥インフルエンザ株、1918 H1N1株）、高い生物学的安全性レベルを伝播するために使用された場合にいくつかのケースでは、バイオセキュリティの注意事項および権限が必要です。地元の機関バイオセーフティ委員会は、インフルエンザウイルスが関与するすべての研究を承認する必要があります。 接種のための卵の調製<ol><li>適切なベンダーからの血清病原体フリー（SPF）たての受精卵鳥類の卵を入手します。一般的に、ニワトリの卵が使用されます。卵は私たちでなければなりません到着後すぐにエド。 </li><li>定期的に卵を回転させる自動卵ターナー加湿卵のインキュベーターで到着地の卵時に。自動卵ターナーが使用できない場合は、手動で（手袋を着用し、できるだけ無菌のすべてを維持することを確認してください）​​は、少なくとも1日2〜3回卵を回転させる。 </li><li> 10〜11日間、37℃で55〜60％の湿度で卵をインキュベートする。 </li></ol> 2.検卵<ol><li>インキュベーションの約7〜8日後にキャンドリングによって不妊のための卵を確認するために、暗い部屋で明るい光に対して卵を検査するために卵キャンドラーを使用してください。 <ol><li> 70％エタノールで卵キャンドラーを拭きます。多くの場合、手袋を変更して病原体による汚染の危険性を最小限にするために70％エタノールで消毒する。 </li><li>インキュベーターから卵を取り出し、空の卵のカートンに配置します。 </li><li>部屋の照明をオフにします。 </li><li>キャンドラーに対して一度に各卵1の大端を持ってください。 </li><li>決定する際に、卵を守ってEそれは肥沃または不妊の場合は。 注：豆の形胚につながる細い血管がはっきりと見えるはずです。未受精卵は、目に見える卵黄の小さな血液スポットとして表示されます。 </li></ol></li><li>未受精卵である卵を破棄し、インキュベーターに実行可能な卵を返す。 30分以上のためのインキュベーターの外に卵を放置しないでください。卵の状態が確認できない場合は、それをマークし、後でそれを観察する。 </li></ol>ウイルス接種の調製<ol><li> 10 mMのHEPES（pHは7.4）を含有する滅菌PBS中で10 4 pfu / mlで-約10 3インフルエンザウイルスストックを希釈する。重要なステップ：使用直前にウイルスストックを希釈し、氷の上のすべての時間を希釈したウイルスを保つ。 </li></ol>尿膜経路を介し4.インフルエンザウイルス接種<ol><li>ウイルス接種（10日または11）の日には、再び卵をキャンドル、デッド胚を排除する。ライブ胚を区別する卵内部の動きを探して。 </li><li>インキュベーターから3卵を取り出し、気嚢を上にして別々のホルダーに卵を配置する。 </li><li>永久的なマーカーで卵のエアスペースをマークします。 </li><li> 70％エタノールで空気層の上に卵の殻を洗ってください。 </li><li>卵のカートンとバイオセーフティーフード（BSL-2）への空気のスペースを上にして卵を入れてください。 </li><li>各卵の気嚢上の殻に小さな穴をパンチするために無菌の18G針を使用してください。胚または卵黄を刺し避けるためにあまりにも深く針を挿入しないように注意してください。 </li><li>無菌の1mlシリンジに希釈したインフルエンザウイルスを策定し、22Gの針を取り付けます。 </li><li>尿膜腔に45°の角度で針で注射器を慎重に進める。 </li><li>希釈されたインフルエンザウイルスを0.2ml注入する。 </li><li>他の二つの卵を注射を繰り返し、その後、注射器と針を捨てる。 </li><li>グルーガンから接着剤の滴（または溶融パラフィンワックス）で穴をシール。 B電子接着剤が卵の中に漏れないように注意してください。 </li><li>エアスペースを上に指摘して卵のインキュベーターに卵をバックに配置します。 </li><li>すべての卵が接種されてきたまでは4.12 - を繰り返して、4.2を繰り返します。 </li></ol> 5.培養時間<ol><li> 37℃、〜60％の湿度で切らずに感染した卵をインキュベートする。 </li><li>インフルエンザウイルスの種類に応じて最適なインキュベーション時間を選択する。 48時間インフルエンザAをインキュベートする。 72時間インフルエンザBとCをインキュベートする。 </li></ol> 6.インフルエンザウイルスの収穫<ol><li>インキュベーション期間の後、胚を死滅させ、血液に感染した尿膜液を汚染する危険性を低減するために、血管を収縮するために4時間（またはO / N）の最小4℃で卵を冷やす。収量が減少したウイルスを、吸収するから赤血球を防ぐためにこれを行います。 -20℃で30分間、あるいは、冷却卵。しかし、これは血液汚染のリスクを増大させる。 </li><li>卵が十分に冷えたされた後に、バイオセーフティフードに卵を移す。気嚢が上を向くようにしてしっかりしたホルダーに卵を置きます。 70％エタノールで卵の表面を清掃してください。 </li><li>気嚢の上に卵の殻を開くために、無菌のハサミを使用してください。漿尿膜を破壊しないように注意しながら気嚢の周りにシェルを削除します。 </li><li>無菌の鈍鉗子で絨毛尿膜を開きます。そっと小さなへらやスプーンを持つ胚と卵黄嚢を脇に移動します。卵黄を破裂しないように注意してください。 </li><li> 1ミリリットルエッペンドルフピペットを用いて、慎重に尿膜液を集める。必要に応じて、できるだけ多くの液体を収集するためにビット側に卵を傾ける。 </li><li> 50ミリリットルプラスチック製コニカルチューブにすべての卵から尿膜液を組み合わせる。ウイルス収集中、常に氷上にチューブを保管してください。わずかに黄色がかった流体10mlの - One卵利回り5。 </li><li> 4で10分間千×gでウイルス含有尿膜液をスピン6; Cは破片をペレット化する。新しい50mlチューブに透明な流体を移送氷上に保持。 BSL-2廃棄物容器に卵を処分してください。 </li></ol> 7.ストレージ<ol><li>すぐに明らかになっ尿膜液（ 例えば、50、100または500 ulのアリコート）をアリコートし、スナップ凍結し、液体窒素中。凍結されたウイルスストックの長期保存のために-80℃の冷凍庫に転送します。 </li></ol>

<ol>
	<li>Centers for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+of+deaths+associated+with+seasonal+influenza+---+United+States,+1976-2007.">Estimates of deaths associated with seasonal influenza --- United States, 1976-2007.</a> MMWR. Morbidity and mortality weekly report. 59 (33), 1057-1062 (2010).</li><li>Neumann, G., Noda, T., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+and+pandemic+potential+of+swine-origin+H1N1+influenza+virus.">Emergence and pandemic potential of swine-origin H1N1 influenza virus.</a> Nature. 459 (7249), 931-939 (2009).</li><li>Tumpey, T. M., Garcia-Sastre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenicity+of+Influenza+Viruses+with+Genes+from+the+1918+Pandemic+Virus:+Functional+Roles+of+Alveolar+Macrophages+and+Neutrophils+in+Limiting+Virus+Replication+and+Mortality+in+Mice.">Pathogenicity of Influenza Viruses with Genes from the 1918 Pandemic Virus: Functional Roles of Alveolar Macrophages and Neutrophils in Limiting Virus Replication and Mortality in Mice.</a> J Virol. 79 (23), 14933-14944 (2005).</li><li>Doherty, P. C., Turner, S. J., Webby, R. G., Thomas, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+and+the+challenge+for+immunology.">Influenza and the challenge for immunology.</a> Nat Immu. 7 (5), 449-455 (2006).</li><li>Webster, R. G., Govorkova, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuing+challenges+in+influenza.">Continuing challenges in influenza.</a> Ann N Y Acad Sci. 1323 (1), 115-139 (2014).</li><li>Szretter, K. J., Balish, A. L., Katz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unit+15G.1+Influenza:+propagation,+quantification,+and+storage.+.">Unit 15G.1 Influenza: propagation, quantification, and storage. .</a> Current protocols in microbiology. , (2006).</li><li>Short, K. R., Kroeze, E. J. B. V., Fouchier, R. A. M., Kuiken, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+influenza-induced+acute+respiratory+distress+syndrome.">Pathogenesis of influenza-induced acute respiratory distress syndrome.</a> Lancet Infect Dis. 14 (4), 57-69 (2014).</li><li>Ito, T., Suzuki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+sialic+acid-galactose+linkages+in+the+chicken+egg+amnion+and+allantois+influence+human+influenza+virus+receptor+specificity+and+variant+selection.">Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.</a> J Virol. 71 (4), 3357-3362 (1997).</li><li>Robertson, J. S., Nicolson, C., Major, D., Robertson, E. W., Wood, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+amniotic+passage+in+the+egg-adaptation+of+human+influenza+virus+is+revealed+by+haemagglutinin+sequence+analyses.">The role of amniotic passage in the egg-adaptation of human influenza virus is revealed by haemagglutinin sequence analyses.</a> J Gen Virol. 74 (Pt 10), 2047-2051 (1993).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells).">Replication and plaque assay of influenza virus in an established line of canine kidney cells).</a> Appl Microbiol. 16 (4), 588-594 (1968).</li></ol>

The authors have nothing to disclose.

インフルエンザは、疾患の大規模なグローバル負担が発生し、作業が肺損傷7の病因を理解することに継続する。この致命的な疾患で容易に研究し、種々の方法は、インフルエンザウイルス6を伝播するために開発されている。ここでは、鶏卵中でインフルエンザウイルスを製造する技術が記載されている。この方法の利点は、それが高度に再現可能であることであり、多くの<...

インフルエンザAは、ヒトの健康に対する大きな脅威であり続けている。これは、大規模なグローバルな負担が世界中で毎年150万の死亡までの原因と潜在的に壊滅的な呼吸器疾患である。インフルエンザウイルスは、オルトミクソウイルス科にあり、それらのゲノム1,2- 8ネガティブセンス一本鎖RNAを運ぶ。ウイルスゲノムの高い可変性（ すなわち 、抗原性「ドリフト」）は、長期的な免疫を防ぐことができます。また、インフルエンザは、抗ウイルス薬3にますます抵抗性がある。 2009年新型インフルエンザのパンデミックは、インフルエンザ疾患（パンデミック株、抗ウイルス耐性、遅延ワクチン製造）に関連するすべての困難な問題を強調した。ワクチン製剤は最も可能性の高い病原性インフルエンザ株4（H1N1、H3N2およびB型インフルエンザ用に1つずつ）のために世界保健機関が毎年決定 ​​されます。この方法は、予測されたインフルエンザ株に基づいているのでsは、病原性株は時折間違って識別され、ワクチンの有効性は劇的に低下する。また、新型インフルエンザ株の出現はパンデミック病気2を引き起こすこれらの予防プログラムを回避することができます。 ユニバーサルインフルエンザワクチンの作成 ​​は5とらえどころのないされている。そのため、研究は肺損傷の病因の理解に続けなければならない。施設研究室の研究のために、いくつかの方法は、ウイルスの単離および増殖6のために開発されている。ヒトインフルエンザウイルスは、哺乳動物細胞、種々の基材に増幅することができる。しかし、大量に発生する高力価のウイルスは、最高の孵化卵内で達成される。以下のプロトコルは、インフルエンザのために既存のウイルスストックからのウイルスの伝播とストレージを技術が記載されている。

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Reagent/ Equipment</td>
			<td style="width:225px;">Company</td>
			<td style="width:128px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Phosphate buffered saline (PBS)</td>
			<td style="width:225px;">Cellgro&#160;</td>
			<td style="width:128px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serum pathogen-free (SPF) fertilized chicken eggs&#160;</td>
			<td>VALO BioMedia</td>
			<td style="width:128px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">humidified egg incubator&#160;</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 2100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">automatic egg turner</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 3200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">egg candler</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 3300&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1 ml syringe</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">18G needle</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">20G / 22G needle</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">305176 / 305156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">HEPES</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:128px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ethanol, &#62;99.5%</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:128px;">459844</td>
			<td>diluted to 70% using water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">glue gun &#34;Ad tech Hi Temp Project Pro&#34;</td>
			<td style="width:225px;">Ad tech, Adhesive Technologies, Inc.</td>
			<td>Model 1105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">glue &#34;Ad tech MINI SIZE, Multi Temp&#34;</td>
			<td style="width:225px;">Ad tech, Adhesive Technologies, Inc.</td>
			<td>220-34ZIP30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">MDCK cells</td>
			<td style="width:225px;">ATCC</td>
			<td>CRL-2935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Washed Pooled Turkey Red Blood Cells, 10%</td>
			<td style="width:225px;">Lampire Biological Laboratories</td>
			<td>724908</td>
			<td></td>
		</tr>
	</tbody>
</table>

influenza virus propagation in embryonated chicken eggs

インフルエンザ感染は約36000人が死亡、米国では20万人以上の入院毎年と関連している。突然変異再類別による新しいインフルエンザウイルス株の連続的な出現は、ウイルスの制御を複雑にし、新規薬剤およびワクチンの永久的な開発を必要とする。インフルエンザの実験室ベースの研究では、ウイルスの増殖のための信頼性とコスト効果的な方法が必要です。ここで、総合的なプロトコルは、一貫して高い力価のウイルスストックをもたらす肥沃鶏卵におけるインフルエンザAウイルスの増殖のために提供される。 11日目 - 要するに、血清病原体フリー（SPF）ニワトリ受精卵を37℃、10 55〜60％の湿度でインキュベートする。この期間、胚発生を容易に卵キャンドラーを用いてモニターすることができる。ウイルス接種針を用いて、尿膜腔へのウイルスストックを注入することにより行われる。 37℃でのインキュベーションの2日後、卵は4℃で少なくとも4時間冷却した。気嚢と漿尿膜上卵殻を注意深く開かれ、ウイルスを含む尿膜液を採取する。流体は、遠心分離によってデブリからクリア等分し、長期保存のために-80°Cに転送される。大量（卵あたりウイルス液5-10）に溶解し、通常、このプロトコルを用いて達成される、高ウイルス力価は、ウイルス調製物のために大量に必要とするin vitro試験のために、特に我々の有利な方法は、卵の使用を行っているウイルスの同じウイルスストックの高用量が必要とされている。

複数の方法は、インフルエンザウイルスを滴定するために開発されている。適切な方法を選択する一つの考慮事項は、他の人が実行可能なビリオンを評価する感染性、（ 例えば 、プラークアッセイ）6に基づいているのに対し、いくつかは関係なく実行可能性（ 例えば 、赤血球凝集素アッセイ）の合計粒子を決定することである。ここでは、マウス適合インフルエンザ...

Watch this Scientific Journal Video about Influenza Virus Propagation in Embryonated Chicken Eggs at JoVE.com

Influenza Virus Propagation in Embryonated Chicken Eggs

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 ...

influenza-virus-propagation-in-embryonated-chicken-eggs

Research

JoVE Journal

Immunology and Infection

42.6K ビュー.  Cedars-Sinai Medical Center. 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.

ビデオ: Influenza Virus Propagation in Embryonated Chicken Eggs

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, ...

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

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

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 ...

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce&#39;s disease (PD) of grapevine. Pierce&#8217;s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

の伝播<em&gt; Homalodisca coagulataウイルス01</em&gt;経由<em&gt; Homalodisca vitripennis</em&gt;細胞培養

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

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 ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

フローサイトメトリーによって、A549細胞におけるインフルエンザAウイルスの添付ファイルと内在化を測定

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

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.