This study is supported by funding from Taipei Medical University Hospital (102TMU-TMUH-19) and the Ministry of Science and Technology of Taiwan (MOST103-2320-B-038-031-MY3).

注：細胞培養​​およびウイルス感染に関連するすべての手続きが処理されたサンプルのバイオセーフティーレベルに適した認定バイオセーフティフードに行われていることを確認してください。プロトコルを説明する目的のために、ガウシアルシフェラーゼレポータータグ付きHCVモデルウイルス32として使用されます。代表的な結果との関連では、化合物chebulagic酸（CHLA）とpunicalagin（PUG）を早期にウ ​​イルスの侵入31ステップの間に細胞表面グ ​​リコサミノグリカンとウイルス糖タンパク質相互作用を標的とする候補抗ウイルス剤として使用されています。多くのウイルス30,31,33,34の進入を妨害することが知られているヘパリンは、このような状況における陽性対照処理として使用されます。基本的なウイルス学技術の背景、ウイルスの増殖、ウイルス力価の決意、そしてプラーク形成単位で感染用量（PFU）の発現のために、形成単位（FFU）、または感染（MOI）を多数集中、読者は再です35を参照するように移し。前の例と代表的な結果に示されているウイルスを使用する最適化された条件のために、読者は 、表1、図1A、図2Aに記載されている参考文献30-32,36-39などの詳細に言及されます。 1.細胞培養、化合物の調製、および化合物の細胞毒性<ol><li>分析されるウイルス感染システム（ 表1）のためのそれぞれの細胞株を成長させます。 HCVについては、ダルベッコ改変イーグル培地（DMEM）、10％ウシ胎児血清（FBS）を補充した、200 U / mlのペニシリンG、200 / mlのストレプトマイシン、および0.5 / mlの中のHuh-7.5細胞は、アムホテリシンBを成長します</li><li>それぞれの溶媒を用いて、試験化合物およびコントロールを準備する：例えば、ジメチルスルホキシド（DMSO）にCHLAとPUGを溶解します。無菌二重蒸留水にヘパリンを準備します。後続のすべての希釈のために、培養培地を使用しています。 注：最後のコンセント試験化合物治療におけるDMSOの比は、実験には1％未満です。 1％のDMSOを比較するためのアッセイで陰性対照処理として含まれています。 </li><li>そのようなXTT（2,3-ビス[2-メトキシ-4-ニトロ-5-スルホフェニル]などの試薬 ​​を決定する細胞生存率を使用して、ウイルス感染細胞に対する試験化合物（例えば、CHLAとPUG）の細胞毒性を決定します-5-フェニルアミノ）カルボニル] -2H-テトラゾリウムヒドロキシド）。 <ol><li> HCVについては、96ウェルプレート（ウェル当たり1×10 4細胞 ）中のHuh-7.5細胞を播種し、単層を得るために、5％CO 2インキュベータO / N中で37℃でインキュベートします。 </li><li>三連で培養ウェルにDMSOコントロール（1％）または試験化合物のCHLAとPUGの濃度の増加（例：0、10、50、100、および500μM）を適用します。 </li><li>その後、72時間37℃でインキュベートし、プレート中の培地を廃棄し、二回リン酸緩衝生理食塩水を200μl（PBS）で細胞を洗浄します。 </li><li> assayin100μlのを追加Gの各ウェルにXTTベースのインビトロ毒性アッセイキットからの溶液とXTTをホルマザンの生産を可能にするために、別の3時間37℃で培養します。 </li><li> 492 nmでのテスト波長でマイクロプレートリーダーおよび690nmでの参照波長で吸光度を決定します。 </li><li>ここで、「時」と試験化合物と溶媒対照（元の吸光度を参照してください」と」、細胞生存率（％）=×100％として、/で1％のDMSO：次の式を用いて細胞の生存率を計算します。それぞれ）の治療、。製造業者のプロトコルに従って、このようなグラフパッドプリズムなどの分析ソフトウェアから試験化合物の50％細胞傷害（CC 50）の濃度を決定します。 </li></ol></li></ol>ウイルス感染の2読み出し注意：ウイルス感染の読み出しが用いられるウイルスのシステムに依存し、そのようなプラークアッセイやMEAなどの方法を含むことができますレポータータグ付きウイルスからのレポーターシグナルをジューリング。ルシフェラーゼレポーター活性に基づくレポーターHCV感染を検出するための方法について説明します。 <ol><li>感染したウェルからの上清を収集し、4℃で5分間、微量で17000×gで明らかにする。 </li><li> ガウルシフェラーゼアッセイキットからのルシフェラーゼ基質の50μlにテスト20μlの上清を混合し、直接製造元の指示に従って、ルミノメーターで測定します。 </li><li>ウイルス阻害（％）を決定するための相対的光単位（RLU）のlog 10として HCV感染を発現し、そして製造業者のプロトコルに従ってグラフパッドプリズムソフトウェアアルゴリズムを用いてHCV感染に対する試験化合物の50％有効濃度（EC 50）を計算します 。 </li></ol> 3.ウイルス不活性化アッセイ注：さまざまなウイルスの潜伏期間およびウイルス用量の例としてA図1Aに記載されている再。ウイルスのより高い濃度もMOI / PFUを増加させることによって試験することができます。 <ol><li>シードホ- 7.5 96ウェルプレート中の細胞（ウェル当たり1×10 4細胞 ）と単層を得るために、5％のCO 2インキュベーター中、37℃でO / Nインキュベートします。 </li><li>試験化合物またはコントロールインキュベート（最終濃度は以下のとおりです。CHLAを= 50μM; PUG = 50μM;ヘパリン=千/ mlの; DMSO = 1％）、37℃（ 図1AでのHCV粒子と、「長期」 ）1：1の比率。例えば、1×10 4 FFUを含む100μlのウイルス接種物に、100μMのCHLA作業希釈液100μlを追加します。これは、50μMの最終濃度でCHLA治療をもたらします。 </li><li>試験化合物の「準治療」（無効）濃度のウイルス - 薬物混合物を希釈します。例えば、HCVに対するCHLAとPUGの非有効濃度は1μM31です。故にこれは、基本培地の9.8 mlの（2％FBSでの細胞培養培地）を用いて達成することができるウイルス - 薬物混合物の50倍希釈を必要とします。 注：サブ治療濃度に希釈が、試験化合物と宿主細胞表面との間に有意な相互作用を防止し、無細胞ビリオンに対する治療効果の検討を可能にします。この希釈は、特定のウイルス感染に対する試験化合物の抗ウイルス用量応答に依存して、この特定のアッセイ31を実行する前に決定されることに留意されたいです。 </li><li>比較のために、試験化合物とウイルスを混合し、直ちにサブ治療濃度感染前に（ 図1A、「短期」）に（何の潜伏期間）を希釈していません。 </li><li> （ウイルスの量は、1×10 2 FFUになりました;最終MOI = 0.01）ホ-7.5細胞単層上に希釈したHCV-薬物混合物の100μlを添加して、ウイルスを可能にするために、37℃で3時間インキュベート吸着。 </li><li>感染後、希釈された接種物を除去し、軽く二回200μlのPBSでウェルを洗浄します。 注：細胞を持ち上げ避けるために静かに洗浄液を実行します。 </li><li>各ウェルに基礎培地100μlを適用し、72時間、37℃でインキュベートします。 </li><li> 2 &#39;で説明したようにルシフェラーゼ活性のために上清をアッセイすることによって生じた感染を分析。ウイルス感染の読み出し」。 </li></ol> 4.ウイルスの付着アッセイ注：さまざまなウイルスの潜伏期間およびウイルス用量の例は、 図2Aに記載された「添付」されています。ウイルスのより高い濃度もMOI / PFUを増加させることによって試験することができます。 <ol><li>シードホ- 7.5 96ウェルプレート中の細胞（ウェル当たり1×10 4細胞 ）と単層を得るために、5％のCO 2インキュベーター中、37℃でO / Nインキュベートします。 </li><li> 4°C FOでプレート中の細胞単層を事前に冷やしますR 1時間。 </li><li> HCV接種（MOI = 0.01）を有する細胞と試験化合物またはコントロール共同治療（最終濃度は以下のとおりです。= 50μMCHLAを、PUG = 50μM;ヘパリン=1,000μgの/ mlの; DMSO = 1％）、4℃でのために3時間。例えば、1×10 2 FFUを含む90μlのウイルス接種物に、500μMのCHLA作業希釈液10μlを添加します。これは、50μMの最終濃度と細胞単層にMOI = 0.01でHCVによる感染でCHLA治療をもたらします。 注：これは、ウイルスの結合を可能にするが、最も効率的に、37℃で発生したエントリを排除するので、それは4℃で実験を行うことが重要です。温度を4℃に維持されることを保証するために氷と4℃の冷蔵庫で続くインキュベーションにウイルスおよび試験化合物の添加を行います。 </li><li>上清を除去し、優しく二回氷冷PBS200μlで細胞単層を洗浄します。 注：細胞を持ち上げ避けるために静かに洗浄液を実行します&lt;。/ LI&gt; <li>各ウェルに基礎培地100μlを適用し、72時間、37℃でインキュベートします。 </li><li> 2 &#39;で説明したようにルシフェラーゼ活性のために上清をアッセイすることによって生じた感染を分析。ウイルス感染の読み出し」。 </li></ol> 5.ウイルスの侵入/融合アッセイ注：インキュベーション期間および種々のウイルスのためのウイルスの投与量の例は、 図2（a）「侵入/融合」に記載されています。ウイルスのより高い濃度もMOI / PFUを増加させることによって試験することができます。 <ol><li>シードホ- 7.5 96ウェルプレート中の細胞（ウェル当たり1×10 4細胞 ）と単層を得るために、5％のCO 2インキュベーター中、37℃でO / Nインキュベートします。 </li><li> 1時間4℃でプレート中の細胞単層を事前に冷やし。 </li><li> 3時間、4℃で、HCVを有する細胞（MOI = 0.01）に感染。例えば、1×10 2 FFUを含む100μlのウイルス接種物を使用しています。 注意：ADDIを実行氷の上でウイルス接種し、ウイルス結合ではなく、エントリを許可し、4℃、温度を維持するために4℃の冷蔵庫でその後のインキュベーション化。 </li><li>上清を除去し、優しく二回氷冷PBS200μlで細胞単層を洗浄します。 注：細胞を持ち上げ避けるために静かに洗浄液を実行します。 </li><li> （最終濃度は以下のとおりです。PUG = 50μM;ヘパリン=千/ mlの、CHLA =50μMのDMSO = 1％）の試験化合物またはコントロールでウェルを治療し、3時間37℃でインキュベートします。たとえば、メディアの90μlに500μMのCHLAワーキング希釈液10μlを加え混ぜ、ウェルを扱います。これは、50μMの最終濃度でCHLA治療をもたらします。 注：37℃〜4℃からのシフトは現在、ウイルスの侵入/融合事象を容易にし、したがって、この特定のステップの試験化合物」効果の評価を可能にします。 </li><li>薬物含有上清を吸引除去し、非内在化を削除クエン酸緩衝液（50mMクエン酸ナトリウム、4mMの塩化カリウム、pHは3.0）または200μlのPBSのいずれかで洗浄することにより細胞外ウイルス。 72時間37℃でインキュベートする前に、基礎培地100μlを適用します。 </li><li> 2 &#39;で説明したようにルシフェラーゼ活性のために上清をアッセイすることによって生じた感染を分析。ウイルス感染の読み出し」。 </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+and+temperature+dependence+of+influenza+virus+membrane+fusion+at+neutral+pH.">Time and temperature dependence of influenza virus membrane fusion at neutral pH.</a> J Gen Virol. 67 (Pt 12), 2813-2817 (1986).</li><li>Haywood, A. M., Boyer, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sendai+virus+membrane+fusion:+time+course+and+effect+of+temperature,+pH,+calcium,+and+receptor+concentration).">Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration).</a> Biochemistry. 21, 6041-6046 (1982).</li><li>Wang, G., Hernandez, R., Weninger, K., Brown, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+cells+by+Sindbis+virus+at+low+temperature.">Infection of cells by Sindbis virus at low temperature.</a> Virology. 362, 461-467 (2007).</li><li>Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrolyzable+tannins+(chebulagic+acid+and+punicalagin)+target+viral+glycoprotein-glycosaminoglycan+interactions+to+inhibit+herpes+simplex+virus+1+entry+and+cell-to-cell+spread.">Hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread.</a> J Virol. 85, 4386-4398 (2011).</li><li>Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad-spectrum+antiviral+activity+of+chebulagic+acid+and+punicalagin+against+viruses+that+use+glycosaminoglycans+for+entry.">Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry.</a> BMC Microbiol. 13, 187 (2013).</li><li>Marukian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture-produced+hepatitis+C+virus+does+not+infect+peripheral+blood+mononuclear+cells.">Cell culture-produced hepatitis C virus does not infect peripheral blood mononuclear cells.</a> Hepatology. 48, 1843-1850 (2008).</li><li>Baba, M., Snoeck, R., Pauwels, R., de Clercq, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfated+polysaccharides+are+potent+and+selective+inhibitors+of+various+enveloped+viruses,+including+herpes+simplex+virus,+cytomegalovirus,+vesicular+stomatitis+virus,+and+human+immunodeficiency+virus.">Sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus.</a> Antimicrob Agents ChemotheR. 32, 1742-1745 (1988).</li><li>Barth, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+binding+of+hepatitis+C+virus+envelope+glycoprotein+E2+requires+cell+surface+heparan+sulfate.">Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.</a> J Biol CheM. 278, 41003-41012 (2003).</li><li>Flint, S. 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The authors declare that no competing interests exist.

In this report the methods to identify and evaluate antiviral compounds based on a mechanistic approach of dissecting the early viral entry events were described. Specifically, the assays allowed us to examine the effect of test compounds on free virus particles, viral attachment, and viral entry/fusion. Critical steps were implemented to distinctly evaluate the drug effect on the specific stage of early viral entry. For instance, in the &#8216;viral inactivation assay&#8217;, the dilution of the virus-drug mixture to sub-therapeutic concentration prevents significant interaction between the test compound and the host cell surface by &#8216;titrating out&#8217; the drug. This ensures that the inhibitory effect observed on the subsequent infection of the host cell is due to a direct interaction between the test compound and the cell-free virions, rather than an effect from the test compound on host cell membrane or membrane-associated molecules, including viral receptors30. Similarly, the shift in temperature between 4 &#176;C (which allows for virus binding but not entry) and 37 &#176;C (which facilitates virus entry/fusion) in the &#8216;viral attachment assay&#8217; and &#8216;viral entry/fusion assay&#8217; are crucial to determine the test compound&#8217;s effect on each of these specific events. This is feasible due to the temperature sensitivity of enveloped viruses during these steps in the infection24-29. It is therefore important that the assays are performed at the indicated temperature to ensure the accuracy of the results; for example, by carrying out the experiment on ice to maintain at 4 &#176;C and by placing the sample directly in a 37 &#176;C incubator for the temperature shift. In addition, the use of negative (ex. DMSO solvent for drug preparation) and positive (ex. heparin treatment) controls also help further establish the assays&#8217; accuracy. The utility and applicability of such methods have been demonstrated in many antiviral studies26,30,31,40,41. Note that while heparin is included as a control for all three assays in the context of the representative results, it typically blocks the initial virus binding rather than the ensuing fusion/entry step (as reflected by the data in Figure 2). Additional controls could also be used, such as neutralizing antibodies directed against the virus (for viral inactivation assay), antibodies that mask the cell surface receptors for the virus (for viral attachment assay), and membrane fusion inhibitors (for viral fusion/entry assay). 
The assays described in this report, which are specific to the early stages of the viral infection, are useful in terms of application as secondary tests to characterize the mechanism of action of candidate drugs from primary screens which typically target the viral infection more broadly. Alternatively, they could also be incorporated in primary screens if one is specifically looking for inhibitors of early viral entry, including virus inactivating agents, viral attachment antagonists, and inhibitors to viral entry/fusion. In this case, their use allows a more focused and precise screen analysis for the identification of mechanism-specific antiviral candidates, which, in turn, would expedite downstream drug development. 
The use of cell-based assays in identifying antiviral agents provides several important advantages compared to biochemical assays, including revealing potential off-target effects (such as cytotoxicity) and adding physiological relevance to the bioactivity of the test agents42. These issues are important considerations for deciding whether a candidate agent is of value for continuation in subsequent phases of drug development. Similarly, the early viral entry-specific assays described in this report allow examination of the drug effect on the distinct viral entry stage at the cellular level, and more specifically in the context of an authentic viral infection in vitro. The results obtained from such assays would therefore help better predict the antiviral efficacy of the test compounds and also identify potentially unwanted off-target effects against the host cell. One potential limitation though, is that an in vitro cell-based assay may not completely reflect the actual in vivo entry step in the context of a natural viral infection. Nonetheless, the assays presented in this protocol do serve as an analytical platform for mechanism-based identification and evaluation of novel antiviral agents.
The development of reporter viruses or reporter cell systems to quantitate the amount of viral infection has greatly facilitated cell-based screening and evaluation of antiviral compounds. Examples include the use of recombinant viruses carrying a reporter gene or by means of recombinant human cell lines containing a reporter gene driven by the specific virus promoter31,43. In this report, the infection from luciferase-tagged HCV can be easily monitored by quantitating the reporter signal, thus facilitating data analysis. By incorporating these useful reporter-based tools, the early viral entry assays described here can essentially be adapted into high-throughput format for mechanism-based screening of small molecule libraries.
In conclusion, a protocol was described for assays dissecting the early viral entry as a means of identifying and evaluating mechanism-specific antiviral compounds. Such assays would be useful for discovering novel antagonists/inhibitors to viral entry and help expand the scope of antiviral agents for development as prophylactic and/or therapeutic treatments.

Viral infections are a constant threat to the public health and a significant cause of epidemic diseases, morbidity, and deaths worldwide. Specific modes of control against viral infections include vaccine development and antiviral therapies. While vaccine efforts have proven successful in immunizing against several viruses, many viral pathogens remain without a protective vaccine including dengue virus (DENV), human cytomegalovirus (HCMV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV)1-5. Antivirals, on the other hand, play an important role for the management of these viral infections when prophylactic vaccines are unavailable. However, to date, only few licensed and cost-effective antiviral drugs are available compared to the number of viral pathogens that threatens the public health. More importantly, due to an increase in global travel and rapid urbanization, the situation is aggravated by risks of epidemic outbreaks from emerging/re-emerging viral infections that are being introduced into non-indigenous areas6. Recent outbreaks caused by severe acute respiratory syndrome (SARS) virus, influenza viruses (H1N1, H5N1, H3N2, and H7N9), DENV, West Nile virus (WNV), measles virus (MV), Middle East Respiratory Syndrome (MERS) virus, and Ebola virus6-12 are among the examples reflecting the need for antivirals development when immunization and/or therapeutics are unavailable. In addition, there is always a potential risk of generating drug-resistant infections with currently used antivirals. Thus, the continuous development and expansion of the scope of antivirals to these emerging/re-emerging viral infections are necessary to provide better management strategies and safeguard the public health.
Most antiviral therapies consist of direct acting antivirals (DAAs) which target a specific viral protein or cofactor that mediates important steps in the viral life cycle. For example, the nucleoside analogue Acyclovir inhibits herpesvirus DNA polymerase, protease inhibitors Boceprevir and Telaprevir antagonize the HCV NS3, and Oseltamivir and Zanamivir are neuraminidase inhibitors that block the release of influenza virus particles from infected cells13-15. There are however very few licensed viral entry inhibitors including Enfuvirtide, which targets HIV gp41 to prevent fusion, and Maraviroc, which blocks the HIV co-receptor CCR5, thereby preventing viral entry16. Exploring novel antagonists/inhibitors to viral entry could help provide additional therapeutics for prophylactic or therapeutic use, such as in combination with other antivirals with a different mechanism of action to better manage viral infections17-19. 
Identification of antivirals can involve structure-guided drug design and candidate drug screening-based strategy. Methods for assessing antiviral activity of test agents include biochemical assays of enzymatic activity and evaluation by cell-based systems20-23. In cell-based systems, the viral life cycle can be dissected into distinct stages of infection, such as entry events (attachment, fusion, uncoating), the replication phase (viral genome replication and protein translation), and virion egress (assembly, maturation, and release). Since the assays can be adapted to investigate each specific stage using various tools and methods, this approach allows identification/examination of potential candidate antivirals with a specific mechanism of action targeting the distinct stage analyzed. For instance, to analyze drug effect specifically on the free virus particles prior to binding to the host cell, a &#8216;viral inactivation assay&#8217; can be performed. In this assay, the virus is allowed to incubate with the test drug and then diluted to titrate out the drug before infecting a cell monolayer. Additional steps such as viral attachment and entry/fusion stages can also be analyzed individually, by shifting the temperature during the infection. For many enveloped viruses, viral entry/fusion at the host cell membrane is greatly facilitated at 37 &#176;C, but is typically precluded at 4 &#176;C which does not affect virus binding24-29. Finally, the use of reporter viruses or cell systems could facilitate these studies and permit a high-throughput analysis.
We previously employed the cell-based approach and dissected the early entry of various enveloped viruses for the examination of antiviral compounds that potentially antagonize viral entry30,31. Herein, the various methods used, including viral inactivation, viral attachment, and viral entry/fusion assays, are described.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>GIBCO</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>In vitro toxicology assay kit, XTT-based</td>
			<td>Sigma</td>
			<td>TOX2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4&#160;</td>
			<td>GIBCO</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Bio-Tek Instrument, Inc.</td>
			<td>ELx800&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002420</td>
			<td></td>
		</tr>
		<tr>
			<td>BioLux Gaussia luciferase assay kit</td>
			<td>New England Biolabs</td>
			<td>E3300L&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax-20/20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate, dihydrate</td>
			<td>Sigma</td>
			<td>71402</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
	</tbody>
</table>

early viral entry assays for the identification and evaluation of antiviral compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

図1では 、「ウイルス不活性化アッセイは、「2つの特定の天然化合物CHLAとPUGは、細胞のない状態で別のエンベロープウイルスを不活性化し、その後の感染を防止することができるかどうかを調べるために行きました。細胞毒性化合物及びこれらの化合物の抗ウイルス用量応答は、従来の機構的研究31を実行することに決定しました。ウイルスは、試験化合物で前処理し、その後、ウイル​​ス薬の混合物は、各ウイルスシステムのそれぞれの細胞単層に接種する前に、副治療濃度に希釈しました。 図1に示すように 、CHLAとPUG両方がその後の感染から細胞単層を保護不可逆的効果をもたらす、無細胞ビリオンと相互作用するように思われました。 80％ブロックMV及びRSVに対して観測された - 60に対し2つの試験化合物は、HCMV、HCV、およびDENV-2に対してほぼ100％の阻害を達成しました。これらの結果はsuggEST CHLAとPUGは、それらを不活性化し、その感染性を中和することにより、これらの無料のウイルス粒子に直接的な影響を持っていること。 図2では 、添付ファイル、および侵入/融合アッセイは、HCMV、HCV、DENV-2、MV、およびRSVからこれらの初期のウイルスの侵入関連イベントに対してCHLAとPUGの影響を調査するために実施しました。 CHLAとPUGの両方が効果的に得られたウイルス感染の阻害（：灰色のバーを点灯図2、「添付ファイル」）によって示されるように、それぞれの宿主細胞に調査し、ウイルスの結合を防ぎます。 100％ -両方の化合物によるウイルスの添付ファイルに対する抑制効果は90に至るまで、HCMV（ 図2B）、HCV（図2C）、DENV-2（図2D）、およびRSV（ 図2F）に対して同様でした。一方、PUGはTWの阻害率と、（ 図2E）結合MV CHLAに対してより効果的であると思われました80％ - O化合物は、50の間で変化します。多くのウイルスの侵入を阻止することが知られている制御処理ヘパリンもまた、阻害さHCMVの付着、DENV-2、RSV、広告MVが、HCVに対してあまり効率的でした。その後の「ウイルスの侵入/融合アッセイは、「CHLAとPUGは、ウイルスの侵入/融合相中にその活性を保持しているか否かを調べた（ 図2、「侵入/融合」：暗い灰色のバー）。それぞれの細胞単層の90％保護効果- 50を得、 -ここでも、CHLAとPUGの両方を効果的に（F図2B）を調べ、ウイルスのウイルスの侵入/融合ステップを損なうことが観察されました。 DENV-2のヘパリンも強力に阻害侵入/融合およびRSV感染症が、HCMV、HCV、及びMV（平均の&lt;40％阻害）に対してあまり有効でした。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>このウイルス</td><td>細胞型</td></tr><tr><td> HCMV </td> &lt;TD&gt; HEL </td></tr><tr><td> HCV </td><td>ホ - 7.5 </td></tr><tr><td> DENV-2 </td><td>ベロ</td></tr><tr><td> MV </td><td> CHO-SLAM </td></tr><tr><td> RSV </td><td> HEp-2 </td></tr></tbody></table> 表1：ウイルス感染のための宿主細胞型の代表的な結果で説明した各ウイルス感染システムに用いられる細胞型が示されています。細胞に関するさらなる詳細は、参考文献31に見出すことができます。 <img alt="図1" src="/files/ftp_upload/53124/53124fig1.jpg" /> 図1.試験化合物のCHLAとPUGによるウイルス感染の不活性化の異なるウイルスが長期間にわたって試験化合物で処理した。（1.5インキュベート-滴定前に3時間、ライトグレーの棒グラフ）または短期間（すぐに希釈し、濃い灰色サブ治療concentraに希釈前の37℃でのバー）ると、各宿主細胞に感染のその後の分析最終ウイルス濃度（左図）実験（A）回路図（PFU /ウェルまたはMOI）、長期ウイルス薬のインキュベーション期間（I）および（ii）その後のインキュベーション時間は、右側のテーブル内の各ウイルスのために示しました。 （B）HCMV、（C）HCVの分析は、（D）DENV-2、（E）MV、及び（F）RSVは、各追加のパネルに示されています。結果は、3回の独立した実験からの平均（SEM）の標準誤差±手段は、ウイルス感染のDMSO陰性対照処理と示されたデータに対してプロットされています。この図は、参照31から変更されている。 <a href="https://www.jove.com/files/ftp_upload/53124/53124fig1highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/53124/53124fig2.jpg" /> 図2.ウイルスの付着および侵入/融合に対する試験化合物のCHLAとPUGの抗ウイルス活性の評価。（A）実験手順、ウイルス濃度（PFU /ウェルまたはMOI）、および試験化合物との付加及び処理の時間（I、II、III）は回路図および関連する表に各ウイルスのために提示されています。ウイルス付着分析（薄い灰色の棒）において、異なる細胞型の単層を、4℃でそれぞれのウイルスおよび試験化合物と同時処理し、その後、1時間4℃で予め冷却した（1.5 - 3時間; I）その後のインキュベーションのために接種し、および試験化合物を洗浄する前に、（37℃; ii）及びウイルス感染の検査。ウイルスの侵入/融合分析（濃い灰色の棒）で、シードされた細胞の単層は、1時間4℃で予備冷却し、次いで1.5 4℃でそれぞれのウイルスでチャレンジ - 3時間（I）。次いで、細胞を洗浄し、温度がウイルス侵入/融合事象を促進するために37°Cに移行した時に追加のインキュベーション期間のための試験化合物（II）で処理しました。インキュベーションの終わりに、細胞外のウイルスは、いずれのクエン酸緩衝液（pH 3.0）またはPBS洗浄によって除去し、細胞をさらにウイルス感染の分析のために（III）でインキュベートしました。 （B）HCMV、（C）HCVについての結果は、（D）DENV-2、（E）MV、及び（F）RSVは、各追加のパネルに示されています。データは、ウイルス感染のDMSO陰性対照処理に対してプロットされており、3つの独立した実験からの平均±SEMとして示されます。この図は、参照31から変更されている。 <a href="https://www.jove.com/files/ftp_upload/53124/53124fig2highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds at JoVE.com

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

抗ウイルス化合物の同定および評価のための早期のウイルス侵入アッセイ

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

early-viral-entry-assays-for-identification-evaluation-antiviral

Research

JoVE Journal

Immunology and Infection

30.0K ビュー.  Taipei Medical University Hospital. Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

ビデオ: Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

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

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

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

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

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

免疫・感染学

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

標準的な臨床アッセイによる検出限界以下のウイルス量とHIV感染個体におけるHIV - 1 RNAを増幅して定量化

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

タングウイルスに対する新規抗ウイルス剤の同定のためのアッセイ

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

プラークアッセイを通じてウイルス濃度の測定：伝統と新しいオーバーレイシステムの使用

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

ハイスループット定量的リアルタイムRT-PCRアッセイのタイプIおよびIIIインターフェロンサブタイプの発現プロファイルを決定するための

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性的および定量的検出のためのアッセイおよびタンパク質の抗菌薬のキャラクタリゼーション

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

の検出のための凍結乾燥LAMP法試薬の開発<em&gt; Q熱リケッチア</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

表面上のヒトノロウイルスの検出のためのスワブサンプリング方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...