この作品は、アイスランドの研究基金とアイスランド研究基金の大学によって資金を供給された。

1。トランスフェクション分子クローンは、それぞれ12 kbと4.5kbの二つのプラスミド、p8XSp5 - EGFPとp67r、に含まれています。9,10 <ol><li>二つのプラスミド、すなわちp8XSp5 - EGFPの4.4μgのとトランスフェクションの前にXbaIおよびライゲーションとp67rの1.6μgの等モル量をカット。 </li><li>ライゲーションの場合は、16℃で一晩50μlの反応とインキュベートでリガーゼの1ワイスのユニットを使用してください。 </li><li>ライゲーションが完了すると、° Cリガーゼの活性化のための15分間65℃ライゲーションミックスをインキュベートする。このステップを省略してもよい。 </li><li>二日前にトランスフェクション、シード10 6 SCPのT25組織培養フラスコ中の細胞、およびコンフルエント90％の単層に抗生物質なしで5ミリリットルDMEM + 10％子羊血清および2mMのグルタミンで2日間細胞を育てる。細胞が二日後にコンフルエント90％に達していない場合は、1つの追加の日成長する細胞を残す。 </li></ol>注：子羊の血清はウシ胎児血清（FBS）で置換することができます。 MVVの一部の菌株は、FBSにより阻害されるので、我々は日常的に、子羊血清を使用して、このMVV株は、しかし、FBSによって阻害されていません。 <ol start="5"><li>細胞はトランスフェクションのための準備ができたら、抗生物質なしで1％の血清（子羊血清またはFBS）を含むDMEMに培地を変更してください。 我々は、トランスフェクションのためにLipofectamin 2000（Invitrogen）を使用してください。 </li><li> 500μLのOpti - MEM培地中でDNAを希釈する。 </li><li> 500μLのOpti - MEM培地で20μlのLipofectamin 2000を希釈し、5分間室温（RT）で残す。 </li><li> 5分のインキュベーション後、希釈したリポフェクタミン2000（合計量= 1000μL）で希釈したDNAを組み合わせる。穏やかに混合し、室温で20〜30分間インキュベートする。 </li><li> SCPの細胞と培地（5 ml）を含むT25フラスコにトランスフェクション混合物（1ml）を追加します。静かに揺らして混合します。 </li><li> 37 ° C、5％CO一夜2（18 - 24時間）。 </li><li> 1％の血清、2mMグルタミン、100IUペニシリンと100IUストレプトマイシンを含むDMEMに培地は、次の日を変更します。 </li><li>倒立蛍光顕微鏡によるSCPの細胞でGFPの生産を監視します。 15％ - 低いトランスフェクション効率は、通常5これらの初代培養細胞、と期待できる。 </li><li> 37さらにインキュベート℃、5％フラスコ中でウイルスの拡散を可能にするために数日間のためのCO 2。それは通常、完全な感染の7〜14日かかります。 </li><li>エッペンドルフチュー​​ブに上清を小分けしてウイルスを回収する。細胞破片を除去するために5分間3000rpmで遠心でスピン。新しいチューブにウイルスを含む上清を移す。 -80℃で保存する</li></ol> 2。ウイルスの滴定<ol><li> 96ウェルプレートにウェルあたり、10％血清、2mMグルタミン、100IU/mlペニシリンおよび100IU/mlのストレプトマイシンを添加した100μlのDMEM培地中でのシード3 × 10 4 SCPの細胞を。 37℃、5％CO 2。 </li><li>細胞が次の日にコンフルエントな場合は、1％血清、2mMグルタミン、100IU/mlペニシリンおよび100IU/mlのストレプトマイシン、各ウェルに100μlのDMEMに培地を変更してください。 </li><li>次の方法でウイルスを希釈する。 <ol><li> 96ウェル丸底またはV底プレートに1％の血清、2mMグルタミン、100IU/mlペニシリンおよび100IU/mlストレプトマイシンウェルあたりに4 × 10ウェルを180μlのDMEMを追加。 </li><li> 4部倍希釈系列の場合は、プレートの最初の列に4wellsのそれぞれにウイルス20μlを追加し、4チャンネルのピペットを使用して、サンプルが十分に混合されることを保証するために上下にピペッティング。ピペットチップを変更し、というように、次の4ウェルに20μlずつ移す。ネガティブコントロール用ウイルスなしで最後の井戸にしておきます。 </li></ol></li><li> SCPの細胞と培地にウイルス希釈液100μLを加える。 </li><li> 37℃および5％CO 2をインキュベートする。二週間のためにGFP蛍光と細胞変性効果を観察する。 細胞変性効果が丸いと多核出現細胞と細胞死で終わる、伸ばしプロセスとの正常な細胞よりも透明化細胞で構成されています。 </li><li>リードMuench法11を用いてウイルス力価（TCID50）を計算する。 </li></ol> 3。感染した細胞のFACS分析注：ウイルスの複製の急速な監視は、感染した細胞をフローサイトメトリーで調べることができます。 <ol><li>感染細胞を（約10 5）トリプシン、500μLPBSでPBSに懸濁すると1Xを洗う。 </li><li>室温で30分間インキュベート500μlの4％パラホルムアルデヒド溶液（最終濃度2％）を、追加。 </li><li> 5分間3000rpmで遠心で細胞をペレット化する。 </li><li> PBS 2X5分で洗い、FACスキャン分析装置で分析する。 10,000のイベントを数える。 </li></ol> 4。代表的な結果： 10 7 TCID 50 / mlの-このウイルスは完全に感染し、10 6の力価に複製する必要があります。感染細胞は蛍光性であるとDETすることができますフローサイトメトリーや蛍光顕微鏡法によるected。一般的に、GFPは、フローサイトメトリー（図1）を用いて感染細胞培養中の細胞の60％以上で検出することができます。同様に、蛍光顕微鏡で観察したとき、細胞の大多数は（図2AおよびB）蛍光灯です。 <img src="/files/ftp_upload/3483/3483fig1.jpg" alt="図1" /> 感染の7日後にKV1772 - EGFPウイルスに感染したSCPの細胞の図1。FACスキャン分析。 <img src="/files/ftp_upload/3483/3483fig2.jpg" alt="図2" /> インキュベーションの7日間（）の後にKV1772 - EGFPを感染させたSCPの細胞の図2。位相差顕微鏡。同じ細胞は蛍光顕微鏡（B）により可視化。

<ol>
	<li>Sigurdsson, B., Palsson, P. A., Grimsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visna,+a+demyelinating+transmissable+disease+of+sheep.">Visna, a demyelinating transmissable disease of sheep.</a> J. Neuropathol. Exp. Neurol. 14, 389-403 (1957).</li><li>Gorrell, M. D., Brandon, M. R., Sheffer, D., Adams, R. J., Narayan, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovine+lentivirus+is+macrophagetropic+and+does+not+replicate+productively+in+T+lymphocytes.">Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes.</a> J. 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A., Petursson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+viral+antigens+in+the+central+nervous+system+of+visna-infected+sheep:+an+immunohistochemical+study+on+experimental+visna+induced+by+virus+strains+of+increased+neurovirulence.">Expression of viral antigens in the central nervous system of visna-infected sheep: an immunohistochemical study on experimental visna induced by virus strains of increased neurovirulence.</a> Acta Neuropathol. 77, 299-306 (1989).</li><li>Turelli, P., Guiguen, F., Mornex, J. F., Vigne, R., Querat, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dUTPase-minus+caprine+arthritis-encephalitis+virus+is+attenuated+for+pathogenesis+and+accumulates+G-to-A+substitutions.">dUTPase-minus caprine arthritis-encephalitis virus is attenuated for pathogenesis and accumulates G-to-A substitutions.</a> J. Virol. 71, 4522-4530 (1997).</li><li>Petursson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visna+virus+dUTPase+is+dispensable+for+neuropathogenicity.">Visna virus dUTPase is dispensable for neuropathogenicity.</a> J. Virol. 72, 1657-1661 (1998).</li><li>Sigurdsson, B., Thormar, H., Palsson, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+visna+virus+in+tissue+culture.">Cultivation of visna virus in tissue culture.</a> Arch Gesamte Virusforsch. 10, 368-380 (1960).</li><li>Gudmundsdottir, H. S., Olafsdottir, K., Franzdottir, S. R., Andresdottir, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+an+infectious+molecular+clone+of+maedi-visna+virus+that+expresses+green+fluorescent+protein.">Construction and characterization of an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein.</a> J. Virol. Methods. 168, 98-102 (2010).</li><li>Andresson, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+and+biological+properties+of+a+pathogenic+proviral+molecular+clone+of+neurovirulent+visna+virus.">Nucleotide sequence and biological properties of a pathogenic proviral molecular clone of neurovirulent visna virus.</a> Virology. 193, 89-105 (1993).</li><li>Skraban, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+mutations+within+39+amino+acids+in+the+envelope+glycoprotein+of+maedi-visna+virus+alter+the+neutralization+phenotype.">Naturally occurring mutations within 39 amino acids in the envelope glycoprotein of maedi-visna virus alter the neutralization phenotype.</a> J. Virol. 73, 8064-8072 (1999).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> American Journal of Hygiene. 27, 493-497 (1938).</li><li>Berkowitz, R. D., Ilves, H., Plavec, I., Veres, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+systems+derived+from+Visna+virus:+analysis+of+virus+production+and+infectivity.">Gene transfer systems derived from Visna virus: analysis of virus production and infectivity.</a> Virology. 279, 116-129 (2001).</li></ol>

利害の衝突は宣言されません。

ここで提示MVVの分子クローンを表現するGFPは、宿主細胞の相互作用とin vitroおよびin vivoの両方MVVの病原性を解析するための有用なはずです。複製の7〜14日後に得られたウイルスは間違いなく、逆転写酵素の不正確さに起因するいくつかの変異を取得しています。しかし、単一サイクルのベクトルとしてこのクローンの使用に制限があります。一つは、クローンの使用は羊とヤギ由来の細胞に?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 10938025 </td></tr><tr><td>のOpti - MEM </td><td> GIBCO </td><td> 51985018 </td></tr><tr><td>リポフェクタミン2000 </td><td>インビトロジェン</td><td> 11668019 </td></tr></tbody></table>

propagating and detecting an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein

Maedi -ビスナウイルス（MVV）は徐々に進行性間質性肺炎や脳炎1を引き起こして、羊のレンチです。 in vivoでのMVVの主要な標的細胞は単球系2のものであると考えています。 MVVの特定の菌株は、しかし、3,4、他の細胞型で複製することができます。緑色蛍光タンパク質は、生きた細胞内でレンチウイルスを研究するための一般的に使用されるマーカーです。我々は、MVVのdUTPaseのための遺伝子にEGFP遺伝子を挿入した。 dUTPaseの遺伝子はよく羊とヤギの大部分のレンチウイルス株で保存されておりCAEV 5の複製に重要であることが示されている。しかし、dUTPaseは、in vitroおよびin vivo 6の両方本研究で使用したMVVの分子クローンの複製のためのなくても済むことが示されている。 MVVの複製は、厳密に羊やヤギ由来の細胞に限られている。我々は、トランスフェクションとウイルス7の増殖のための羊の脈絡叢（SCPの細胞）からの主な細胞株を使用してください。蛍光MVVは、完全に感染性であり、EGFPの発現は、少なくとも6通路8上安定しています。 TCID 50とEGFPの測定値の間に良好な相関関係がある。このウイルスは、従って、in vitroおよびin vivo 8の細胞指向性や病原性の研究における感染細胞の迅速な検出に有用でなければなりません。

Watch this Scientific Journal Video about Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein at JoVE.com

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

我々はGFPを発現し、完全に伝染性であるmaedi -ビスナウイルスの分子クローンを説明します。このウイルスの複製は、蛍光顕微鏡やフローサイトメトリーを用いて検出することができます。

伝播と緑色蛍光タンパク質を発現Maedi -ビスナウイルスの感染分子クローンの検出

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

propagating-detecting-an-infectious-molecular-clone-maedi-visna-virus

Research

JoVE Journal

Immunology and Infection

9.8K ビュー.  University of Iceland. 我々はGFPを発現し、完全に伝染性であるmaedi -ビスナウイルスの分子クローンを説明します。このウイルスの複製は、蛍光顕微鏡やフローサイトメトリーを用いて検出することができます。

ビデオ: Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

緑色蛍光タンパク質を発現するリビジョンに依存するレンチウイルスベクターを用いたHIV - 1陽性細胞の特定のマーキング

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

免疫・感染学

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Vero細胞培養アッセイによるフィールドコレクト蚊から感染性ウイルスの検出

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

のAlexa Fluor ®標識を通じてデング熱ウイルスの可視化

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Alphaherpesウイルス順行性輸送とスプレッドの生細胞イメージング

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Tetracistronicミニゲノムを含むウイルス様粒子とバイオセーフティレベル2の条件の下ではエボラウイルスのライフサイクルのモデル化

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

調節するc-ジ-GMPシグナリングで、小さな分子をスクリーニングするためのハイスループットセットアップの確立<em&gt;緑膿菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

ジカウイルス感染細胞培養系と<em&gt;インビトロ</emインターフェロンの&gt;予防効果

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

アン<em&gt;インビトロ</em&gt;結核病変における薬物侵入を予測するカゼウム結合アッセイ

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

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 ...