We thank F. Chisari for providing Huh-7.5.1 cell line. We would like to thank Justine Ho for editing the manuscript. This work was supported by Cedars-Sinai Medical Center Institutional Programmatic Research Award and National Center for Advancing Translational Sciences, Grant UL1TR000124 to V.A.

プロトコルの概要を図1に示されている。 1。細胞<ol><li> 10〜15％ウシ胎児血清（FBS）、10 mMの非必​​須アミノ酸、10mMのHepes、ペニシリン（100単位/ ml）、ストレプトマイシン（100 mg / ml）を、および2mM L-グルタミンを含有する完全増殖培地を調製する。 </li><li> C型肝炎ウイルスの複製サイクルのインビトロ分析のための上記のサプリメントを含む完全な増殖培地中でホ- 7.5.1細胞の13を維持する。 </li><li> 5％のCO 2、37℃で指定された補足の増殖培地とホ- 7.5.1細胞で培養したウイルス株。 </li></ol> 2ウイルスやプラスミド構築<ol><li> HCV RNAの相補DNA（cDNA）の形態を生成し、遺伝子操作を容易にするためのプラスミドベクターにクローニングする。注：日本の劇症肝炎1の発見は、JFH-1（遺伝子型2aのHCV）、HCVの研究のための批判的とされている単離主にHCVの複製サイクル11から13を研究するために使用。 JFH-1のHCVに基づく間およびintragenotypicキメラウイルスは、一般的に、研究14〜18で使用されています。合成intragenotype 2aのキメラウイルス、FNX-HCV、およびモノシストロンキメラレポーター株の構築は、19と建設の詳細は、このプロトコルの範囲を超えて先に説明した。 </li><li>それは（ヌクレオチド1から2878）J6CF親株（NCBIアクセッション番号AF177036）の、よう5&#39;NTR、構造領域とP7とNS2非構造領域の一部が含まれてpFNX抗HCV intragenotype 2aのウイルスを使用して、よくJFH-1ウイルス株（NCBIアクセッション番号AB047639）の非構造部材として。注：FNX-HCVは、以前に報告された15 Jc1にintragenotype 2aのキメラHCVの配列に基づいて合成した。 </li><li> Rを挿入することで（ステップ2.2以上）pFNX-HCV株に基づいて、モノシストロニックキメラレポーターウイルス構築、pFNX-Rlucをを生成（NT 388と389の間）5 &#39;NTRとコア遺伝子との間enillaルシフェラーゼ遺伝子。続いて、切断シグナルとして働くだろう口蹄疫ウイルス2A（F2A）ペプチド配列を用いて、ルシフェラーゼ遺伝子およびその構築物のコア遺伝子を接続する。 </li><li> pFNX-HCVまたはpFNX-Rlucをウイルス背景のいずれかを使用して、RNAポリメラーゼ-ヌル（ポル·）ウイルス構築エンジニア。 、AAGのアミノ酸残基とこれらのウイルスの主鎖のいずれかの、NS5Bポリメラーゼ触媒残基、GDD（8615から8623をntのAA 2759年から2761年）を交換します。 </li></ol> 3 インビトロのHCV RNA転写<ol><li> intragenotype 2aのキメラウイルスFNX-HCVおよびFNX-HCVルポールヌルウイルス複製（ 図2A）の評価のための（ポル·）は、HCVを使用してください。 </li><li> XbaI制限酵素でウイルスプラスミドを直線化して、平滑末端を生成するために、緑豆ヌクレアーゼで処理する。陰イオン交換クロマトグラフィーによって消化プラスミドを精製する。 oを整合性を検証fは、ゲル電気泳動（ 図2B）をアガロースにDNAを施すことにより、プラスミドを線状化。 </li><li> T7-RNAポリメラーゼを用いて線形化されたウイルスのプラスミドを転写。 </li><li> RNA精製キットを用いて新たに合成されたDNase処理RNAを精製する。 </li><li>アガロースゲル電気泳動（ 図2C）により、RNAの生産を確認してください。 </li><li>分光測光法によりRNAを定量化する。 </li><li> 10μgの作業アリコートで-80℃で生成したRNAを保管してください。注：同じ時に各ウイルスのサンプルおよびコントロールのためのすべてのRNAを転写することにより、各実験計画の中にRNAの品質のばらつきを最小限に抑えます。 </li></ol> 4。のHCV RNAのトランスフェクションおよび検体採取<ol><li>トリプシンを用いたHuh-7.5.1細胞を採取する。 </li><li>細胞、遠心分離機をすすぎ、冷たい低血清培地で二回、サスペンションを再懸濁し。 ml当たり1×10 7個の細胞で低血清培地で細胞を再懸濁します。 </li><li> 10＆＃合計を混ぜる956; 0.4 cmのエレクトロポレーションキュベット中に再懸濁細胞（4×10 6細胞）を400μlの転写されたウイルスRNAのG。 、270 Vでエレクトロポレーションを介して細胞をトランスフェクト100Ω、および950μF。 </li><li> 15％FBSと10ミリリットル完全増殖培地中でエレクトロポレーションした細胞を再懸濁。注：許-7.5.1細胞は15％ウシ胎児血清中で培養したときにエレクトロポレーションした細胞生存性の増大が見られる。 </li><li> 4、48及び96時間：両方のT-25フラスコ（フラスコあたり〜1.2×10 6細胞）を、48ウェルプレートに以下の時点の各々について（ウェル当たり1×10 4細胞）で細胞をプレート。 </li><li>培養されたフラスコおよびプレートから死細胞破片を除去し、10％FBSを新鮮な増補増殖培地で4-8時間のトランスフェクション後にメディアを交換してください。 </li><li>収穫細胞培養上清48、96時間の時点で、4℃で10分間、1500rpmで細胞を遠心分離によって収集したサンプルから細胞破片を除去 ºCの</li><li> -80ºCで無細胞上清を保存する溶解ウエスタンブロットによるタンパク質およびRNA分析のために、示された時点で転写定量的PCRリバース細胞。 </li></ol> 5。HCVゲノムコピーを評価するための転写定量PCR（RT-qPCRの）をリバース<ol><li> HCVゲノムRNAコピー数を決定するために、ツーステップRT-定量PCRを行う。 </li><li>またはハウスキーピング遺伝子のペプチジルイソメラーゼG（PPIG R特異的なプライマー：逆、逆転写酵素と5&#39;NTR（5&#39;CCTATCAGGCAGTACCACA-3 &#39;JFH RTQ R）に結合し、HCVのセンス鎖特異的なプライマーを用いて、全細胞RNAの1μgのを転写：5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3 &#39;）。また、HCVのセンス鎖プライマーを用いて、既知のゲノムコピー（1月 10 日から9月 10 日までの標準）の（ステップ3で生成された）FNX-HCV RNAを逆転写。 </li><li> （HCV JFH RT特異プライマーを用いて得られた転写されたcDNA 50ngのを用いて定量PCRを行うことQF：5&#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R：5&#39;CCTATCAGGCAGTACCACA-3 &#39;）およびDNAの定量PCRスーパーミックスを含むグリーン色素を結合。だけでなく、ハウスキーピング遺伝子PPIGための定量PCRを行う（プライマーPPIG F：5&#39;-GAAGAGTGCGATCAAGAACCCATGAC-3 &#39;; PPIG R：5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#39;）注：サンプル間で正確な細胞内のHCV RNA量を計算するため、細胞のハウスキーピング遺伝子、PPIGを使用正規化のため、発現量（Ctの周期）。コピー数の決定のための標準としてFNX-HCVゲノムのコピー数を使用してください。 </li><li> 15秒95ºCと60：のHCV RNAコピー数を決定するために、定量PCRを実行するときに、以下の条件を使用してください&#160;リアルタイムPCRシステムを用いて30秒（40サイクル）ºC。 </li><li>ゲノム複製の結果を得るために、図3Aと図3Bを参照してください。 </li></ol> 6。ウェスタンブロッティング分析HCVタンパク質の発現（図3C）を検出する<ol><li>細胞溶解物のFRを使用して、OMウイルスRNAは、タンパク質ウェスタンブロッティング解析のための96時間後、トランスフェクションでトランスフェクトした。 </li><li> SDS-PAGE及びポリビニリデンジフルオリド（PVDF）膜に転写を用いて細胞溶解物を解決する。 </li><li> 5％スキムミルク、リン酸緩衝生理食塩水（PBS）中の0.2％Tween 20を含むブロッキング溶液を用いて膜をブロックする。 </li><li>一次マウスモノクローナル抗体のNS3（1千倍希釈）およびβ-アクチン（1 5000倍希釈）を有する膜をインキュベートする。 </li><li>西洋ワサビペルオキシダーゼ（1 5000倍希釈）に結合したヤギ抗マウスIgGを加え、化学発光によって検出します。 </li></ol> 7。免疫蛍光アッセイ（IFA） <ol><li>免疫蛍光アッセイのため-20℃で30分間メタノールを使用してHCV感染およびトランスフェクトされた細胞を固定します。 </li><li> PBSで三回細胞を洗浄し、IFAはブロッキング緩衝液でブロックする。 </li><li>ウサギポリクローナル抗NS5Aは、主に抗体（親切に博士ダスグプタ、UCLAによって提供される）またはマウスモノクローナル抗DSR使用NA抗体1:200（1μg/ ml）を希釈でJ2と5時間に一晩4でインキュベート ºCの</li><li> 3回の一次抗体の後、PBSで細胞を洗浄。 </li><li>ヤギ抗ウサギIgG-488ポリクローナル二次抗体または1:1,000希釈（1μg/ ml）を少なくともヤギ抗マウスIgG-594ポリクローナル二次抗体を添加し、室温で1時間インキュベートする。 </li><li>蛍光顕微鏡（ 図3D）を使用して、ヘキスト染料とビューを用いてPBS 3回染色核で細胞を洗浄。 </li></ol> 8。測定ウイルス力価<ol><li>プレートナイーブホ-7.5.1細胞を、約3×10 3細胞/ウェルで96ウェルプレートを用いて。翌日、増殖培地を用いて、HCVのRNAトランスフェクトした細胞から採取し、無細胞培養上清の10倍段階希釈を実施したHuh-7.5.1細胞の上に三重で接種する。 </li><li> -20℃で30分間、メタノールを（使用して72時間の感染後、細胞を固定</suC）およびセクション7で述べたようなHCV NS5Aタンパク質のための免疫染色ºP&gt;。 </li><li> NS5A陽性病巣のためにカウントしミリリットル当たり焦点形成単位の平均数（FFU）を計算するために最高希釈を使用しています。 HCV力価については、図4を参照してください。 </li></ol>ウイルスゲノム複製および感染のため9。 ウミルシフェラーゼレポーターアッセイ<ol><li>ウイルスゲノムの複製、48ウェルプレート中で三連でプレートHCVのRNA電気穿孔たHuh-7.5.1細胞（1×10 4細胞/ウェル）を評価する。 </li><li>溶解6時間での受動溶解緩衝液75μlを、48時間、および96時間後にトランスフェクションした細胞。 </li><li> -80ºCで室温店で15分間穏やかプレートを揺する</li><li> ウミシイタケルシフェラーゼ酵素活性を測定するために、室温にタンパク質溶解物およびウミシイタケルシフェラーゼアッセイ試薬を解凍する。 </li><li> 96-WELウェル当たりタンパク質溶解液20μlを追加<html> Lホワイト発光プレートルミノメーターでプレートを置き。 </li><li>ウェルあたり2秒先読み遅延の後にウミルシフェラーゼアッセイ試薬（セレンテラジン基質+バッファ）の100μLを分注する。 10秒間照射された光を統合する。 </li><li>生物学的な三連のルシフェラーゼ値から、各試料の平均値と標準偏差を計算し、統計的分析（ 図5）にデータを供する。 </li><li>感染性を評価するため、48ウェルプレートにナイーブホ - 7.5.1細胞の上に三重に指示された時点（48時間および96時間）のためのHCV RNAトランスフェクトした細胞から得られた無細胞上清500μlを接種。 </li><li> 6時間後に感染した後、ウェルあたりの新鮮な培地500μlのウイルス接種を交換してください。 </li><li>溶解ウミシイタケルシフェラーゼアッセイと48時間後に感染し、対象の細胞のステップ8.2から8.7（ 図5）で説明したように。 </li></ol>イトルを記入 &quot;&gt; 10。統計解析<ol><li>グラフ中のエラーバーは標準偏差（SD）を示す。 P値は、対応のないt検定により計算した。 </li></ol>

<ol>
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T., 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=Hepatitis+C+virus+p7+and+NS2+proteins+are+essential+for+production+of+infectious+virus.">Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus.</a> J Virol. 81 (16), 8374-8383 (2007).</li><li>Steinmann, E., 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=Hepatitis+C+virus+p7+protein+is+crucial+for+assembly+and+release+of+infectious+virions.">Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions.</a> PLoS Pathog. (7), (2007).</li></ol>

The authors have nothing to disclose.

この図は、C型肝炎ウイルス複製サイクルを分析するための方法を記載している。 HCVはヒトの病原体であり、所定の生物学的安全性プロトコルは、必ず守っていただきます。感染性HCV細胞培養系は、以前に11-13,16,17記載されている。示されたプロトコルを以下のときに我々が実装し、いくつかの重要なポイントがあります。まず、下流の研究のために無傷の完全長ウイルスゲノムRNAの?...

C型肝炎ウイルス（HCV）は、肝硬変および肝臓癌を引き起こす。それは35万人が毎年1-3死んで全世界1.7億人々に影響を与えます。 HCVは、9.6kbのゲノムサイズを有するプラス鎖RNAウイルスである。 HCVゲノムは、タンパク質分解的に10ポリペプチドに様々な細胞およびウイルスのプロテアーゼによって切断され〜3,000アミノ酸残基の単一ポリタンパク質として翻訳される。 HCVはヘパシウイルス属に原型ウイルスで、 フラビウイルス科 4に属しています。暴露されると、HCVは、個人の80％に慢性感染を確立します。感染は、診断が肝劣化を防止する治療的介入を可能にすることができる主に無症候性かつタイムリーである。現在の治療は、部分最適で、ワクチンは5,6ありません。 C型肝炎の病因は1989年7に記載した。HCVの複製を研究するC型肝炎ワクチンおよび治療 ​​の研究のために重要であるが、それがあった長い効率的なウイルス培養系の欠如によって妨げ。 HCVの分子クローンは、肝内接種8時チンパンジーにおける感染であることが示された。続いて、HCVサブゲノムレプリコンは、細胞培養系9,10におけるウイルスゲノムの複製段階を分析させ、これについて説明した。遺伝子型2aのHCVの発見は、JFH-1（日本劇症肝炎-1）を単離、細胞培養に感染することができる、HCV複製の研究11月13日のための新たな道を開いた。感染培養系をベース遺伝子型2a株JFH-1ベース間および内遺伝子型キメラウイルス遺伝子型1のHCVにも14〜18用意されています。 我々が正常タンパク質ドメインおよびシス作用RNAエレメント19,20の高分解能機能プロファイリングマップを得るために、JFH-1株およびHCV intragenotype 2aのキメラウイルスを使用している。これによれば、ここでは慣用的に使用可能にする効果的な培養システムを記載HCVの複製サイクルと宿主 - 病原体相互作用の様々な段階を研究。私たちは、ウイルスゲノムの複製とintragenotype 2aはHCVおよびウミシイタケルシフェラーゼに基づくレポーターHCVの新規の感染性を評価するために、ウイルス学的ア ​​ッセイを提示する。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Name of Material/ Equipment</td>
			<td style="width:267px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">10-017-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Non essential amino acid</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">MT25025CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">HEPES</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">15630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Glutamax</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Opti-MEM Reduced Serum Medium,no Phenol Red</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11058-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Huh-7.5.1</td>
			<td style="width:267px;">The Scripps Research Institute</td>
			<td style="width:119px;"></td>
			<td>The cell line was kindly provided by Dr. Francis Chisari to Dr. Arumugaswami under executed MTA between The Scripps Research Institute and Cedars-Sinai Medical Center</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:569px;">Plasmids (pFNX-HCV, pFNX-HCV Pol null, pFNX-Rluc, and pFNX-Rluc Pol null)&#160;</td>
			<td style="width:267px;">Cedars-Sinai Medical Center</td>
			<td style="width:119px;"></td>
			<td>The HCV plasmids were synthesized by Dr. Arumugaswami using overlapping oligo-nucleotides.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">XbaI</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">R0145S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Mung Bean Nuclease</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">M0250S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">T7 RiboMAX Express Large Scale RNA Production System</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">P1320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Rneasy Mini Kit</td>
			<td style="width:267px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Nanodrop 2000</td>
			<td style="width:267px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Electroporation Cuvette (4 mm)</td>
			<td style="width:267px;">Bioexpress</td>
			<td>E-5010-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Gene Pulser Xcell Total System</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">165-2660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-dsRNA antibody J2&#160;</td>
			<td>English &#38; Scientific Consulting Kft.</td>
			<td style="width:119px;">10010200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 488</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 594</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVDF membrane package</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">162-0263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Blotting Grade Blocker Non Fat Dry Milk</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6404XTU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Tween-20</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6531XTU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [8 G-2]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab65407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [H23]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab13830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td style="width:119px;">115-035-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham ECL Prime Western Blotting Detection Reagents&#160;</td>
			<td style="width:267px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SUPERSCRIPT III RT&#160;</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">18080085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SYBR QPCR SUPERMIX W/ROX</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11744500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ViiA 7 real-time PCR system</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Renilla Luciferase Assay System kit</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">E2810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">RNase-Free DNase</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">M6101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">GloMax-Multi Detection System (Luminometer)</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for analyzing hepatitis c virus replication

C型肝炎ウイルス（HCV）は、世界の人口の3％に影響を及ぼし、慢性肝炎、肝硬変、および肝細胞癌を含む重篤な肝疾患を引き起こす。 HCVは、 フラビウイルス科に属するエンベロープRNAウイルスである。現在の治療は、十分に有効ではなく、有害な副作用を引き起こす。利用可能なHCVワクチンはありません。このように、継続的な努力は、ワクチン、より良い治療法を開発するために必要です。 HCV細胞培養系は、ウイルスの侵入、ゲノム複製、パッケージング、および出口を含むHCV増殖の様々な段階を研究するために重要である。提示現在の手順では、野生型intragenotype 2aのキメラウイルス、FNX-HCV、およびウイルス複製を研究するために、 ウミシイタケルシフェラーゼレポーター遺伝子を担持する組換えFNX-Rlucをウイルスを使用した。ヒト肝癌細胞株（ホ-7ベースの） インビトロでのトランスフェクションのために使用したのHCVゲノムRNAを転写した。無細胞培養上清を、タンパク質溶解物およびtotal RNAは、HCVの増殖を評価するために、トランスフェクション後様々な時点で採取した。 HCVゲノムの複製の状態は、定量的RT-PCRおよびHCVの存在を可視化する二本鎖RNAにより評価した。 HCVタンパク質の発現は、HCV NS3およびNS5Aタンパク質に特異的な抗体を用いてウェスタンブロットおよび免疫蛍光アッセイによって確認した。培養上清およびウイルス力価に感染性HCV粒子を放出するRNAトランスフェクトされた細胞を測定した。ルシフェラーゼレポーターアッセイはHCVの複製レベルと感染性を評価するために利用した。結論として、我々は、HCVの複製サイクルの異なる段階を特徴付けるための種々のウイルス学的アッセイ法を提示する。

C型肝炎ウイルスはRNAウイルスである。このように遺伝子操作の目的のために、HCVゲノムcDNAを、細菌プラスミドベクターにクローニングされている。 T7 RNAポリメラーゼプロモーター配列は、HCVゲノムの5 &#39;末端の直前に導入した。 HCV分析ワークフローの概要を図1に示す。正確な3 &#39;末端とHCVゲノムRNAを生成するために、プラスミドを含むHCVゲノムは、XbaI制限酵素で切?...

Watch this Scientific Journal Video about A Protocol for Analyzing Hepatitis C Virus Replication at JoVE.com

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

C型肝炎ウイルスの複製を分析するためのプロトコル

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

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JoVE Journal

Immunology and Infection

23.9K ビュー.  Cedars-Sinai Medical Center. Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle. 

ビデオ: A Protocol for Analyzing Hepatitis C Virus Replication

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

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

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

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

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

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

免疫・感染学

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

モデルヘルペスウイルスの溶菌レプリケーションにおける解剖ホストウイルス作用

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

単純ヘルペスウイルスの待ち時間と再活性化の研究の主ニューロン培養システム

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

表現型のハイスループットアッセイ<em&gt;サルモネラ菌</em&gt;ネズミチフス菌協会、侵略、およびマクロファージにおける複製

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

C型肝炎ウイルスに対するヒトラクトフェリンの細胞取り込みおよびそれに関連する抗ウイルス活性を監視するために、免疫蛍光

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

インフルエンザA型ウイルスのヘマグルチニンの結合力および特異性を評価するための小型糖鎖マイクロアレイアッセイ

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

複製サイクルの初期段階を監視するためにB型肝炎ウイルスのレポーターシステムの開発

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

新世界Zikaウイルス感染クローンからの組換えウイルスの救出とキャラクタリゼーション

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

初代肝細胞とクッパー細胞 B 型肝炎ウイルス感染の「肝臓-オン-チップ」文化

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

全血豚で表されるコーディングおよび非コーディングの RNA クラスの識別

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...