本研究では、イアン·グッドフェローに授与ウェルカムトラストシニア·フェローシップによって資金を供給し、マリーキュリーイントラヨーロッパのフェローシップ（FP7欧州研究評議会）は、アルマンド·アリアスに授与されました。私達は私達に彼らのAgilentのbioanalyserを使用する許可を与え、RNAサンプルを実行すると助けるためにイオ香港チョン博士レベッカロービーとDrマイクスキナーに感謝します。

<ol>
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Gen. Virol. 73, 2125-2128 (1992).</li><li>Shuman, S., Hurwitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+mRNA+capping+by+vaccinia+virus+guanylyltransferase:+characterization+of+an+enzyme--guanylate+intermediate.">Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme--guanylate intermediate.</a> Proc. Natl. Acad. Sci. U.S.A. 78, 187-191 (1981).</li><li>Jang, S. K., Pestova, T. V., Hellen, C. U., Witherell, G. W., Wimmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-independent+translation+of+picornavirus+RNAs:+structure+and+function+of+the+internal+ribosomal+entry+site.">Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site.</a> Enzyme. 44, 292-309 (1990).</li><li>Chaudhry, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caliciviruses+differ+in+their+functional+requirements+for+eIF4F+components.">Caliciviruses differ in their functional requirements for eIF4F components.</a> J. Biol. Chem. 281, 25315-25325 (2006).</li><li>Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calicivirus+translation+initiation+requires+an+interaction+between+VPg+and+eIF+4.">Calicivirus translation initiation requires an interaction between VPg and eIF 4.</a> E. EMBO Rep. 6, 968-972 (2005).</li><li>Herbert, T. P., Brierley, I., Brown, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+protein+linked+to+the+genomic+and+subgenomic+mRNAs+of+feline+calicivirus+and+its+role+in+translation.">Identification of a protein linked to the genomic and subgenomic mRNAs of feline calicivirus and its role in translation.</a> J. Gen. Virol. 78 ( Pt5 ), 1033-1040 (1997).</li><li>Chaudhry, Y., Skinner, M. A., Goodfellow, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+genetically+defined+murine+norovirus+in+tissue+culture+by+using+a+fowlpox+virus+expressing+T7+RNA+polymerase.">Recovery of genetically defined murine norovirus in tissue culture by using a fowlpox virus expressing T7 RNA polymerase.</a> J. Gen. Virol. 88, 2091-20100 (2007).</li><li>Ward, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+infectious+murine+norovirus+using+pol+II-driven+expression+of+full-length+cDNA.">Recovery of infectious murine norovirus using pol II-driven expression of full-length cDNA.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 11050-11055 (2007).</li><li>Yunus, M. A., Chung, L. M., Chaudhry, Y., Bailey, D., Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+optimized+RNA-based+murine+norovirus+reverse+genetics+system.">Development of an optimized RNA-based murine norovirus reverse genetics system.</a> J. Virol. Methods. 169, 112-118 (2010).</li><li>Arias, A., Perales, C., Escarmis, C., Domingo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+mutants+of+VPg+reveal+new+cytopathology+determinants+in+a+picornavirus.">Deletion mutants of VPg reveal new cytopathology determinants in a picornavirus.</a> PLoS One. 5, e10735 (2010).</li><li>Werf, S. v. a. n. d. e. r., Bradley, J., Wimmer, E., Studier, F. W., Dunn, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+infectious+poliovirus+RNA+by+purified+T7+RNA+polymerase.">Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 2330-2334 (1986).</li><li>Bailey, D., Thackray, L. B., Goodfellow, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+amino+acid+substitution+in+the+murine+norovirus+capsid+protein+is+sufficient+for+attenuation+in+vivo.">A single amino acid substitution in the murine norovirus capsid protein is sufficient for attenuation in vivo.</a> J. Virol. 82, 7725-7728 (2008).</li><li>Bailey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+RNA+structures+present+at+the+3'+extremity+of+the+murine+norovirus+genome:+the+variable+polypyrimidine+tract+plays+a+role+in+viral+virulence.">Functional analysis of RNA structures present at the 3' extremity of the murine norovirus genome: the variable polypyrimidine tract plays a role in viral virulence.</a> J. Virol. 84, 2859-2870 (2010).</li></ol>

我々は、開示することは何もありません。

ここでは、細胞培養における感染MNVの回復を可能にする2つの異なった逆遺伝学的アプローチを説明しました。両方を効果的に近づくし、細胞リボソームによって認識されるキャップMNVの転写産物の生成を介したウイルスRNAゲノムの5 &#39;末端にVPGの共有結合のための絶対的な要件をバイパスします。in vitro転写酵素キャッピング続いて、より効率的です。転写物はFPVキャッピング酵素によってキャップされるかもしれないT7 RNAポリメラーゼを発現する細胞に感染するプラスミドの転写より感染MNVの回復。これらの逆遺伝学システムで回収し、ウイルス力価は、感染した細胞培養液17（ 図5）から精製したウイルスVPG-結合型RNAのトランスフェクションによって得られたこれらに類似しています。寛容なRAW264.7細胞に覆われたMNV RNAのトランスフェクションは、transfectioを含む実験より1つだけログ低いウイルス力価をレンダリングn個のウイルスVPG-結合型RNA（ 図5）を含む感染細胞からのトータルRNA。この事実は、これらのシステムによって生成された転写産物の5 &#39;末端にVPG分子の添加はVPGに関連付けられている細胞にMNV感染性の根底にある機能的側面を明らかにすることが増加したウイルスの収率につながるかどうかを判断するためにさらなる調査を奨励しています。他のRNAウイルスに匹敵する高効率のいずれかの遺伝システムは現在、in vitro転写RNA のウイルス粒子19、20と実際の感染症に比べて唯一の10から100まで低価の回復を可能にするに使用される逆にそれにもかかわらず、我々は、このリバースジェネティクス系を考えています。 全体として、現在の方法論は、ノロウイルスの分子生物学の分野で大きな前進を構成し、ノロウイルスのゲノム中のタンパク質と保存されたRNAモチーフの機能的役割を調べるためのツールを提供してくれる。これらのアプローチは、既にCと組み合わされています10日未満4、21 -マウスurrentマウスモデル使用可能で、MNVが感染性cDNAから回収されたことが示されているが&gt; 80％STAT1-/の致死的感染を引き起こすことができます。我々はキャプシドタンパク質のおよび in vivo 21、22 でやや減衰の表現型を示すさまざまな宿主因子（PTBとPCBP）の結合に関与するポリピリミジン管の生存マウスノロウイルスの変異体を回復したこのシステムを活用した。さらに、我々は最近、ORF4からVF1タンパク質を発現する能力を欠いているウイルスが効率的に細胞培養で複製が再びWT MNV 8に対してマウスにおける病原性が低下していることを明らかにした。これらの研究は、私たちは潜在的なワクチン候補として検討することができMNVの研究に基づいて人間のノロウイルスの弱毒化のバージョンを設計することをお勧めします。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>塩化リチウム沈殿ソリューション</td><td> Ambionの</td><td> AM9480 </td></tr><tr><td> RNA·ストレージ·ソリューション</td><td> Ambionの</td><td> AM7000 </td></tr><tr><td> MEGAscript T7スクリプト</td><td> Ambionの</td><td> AM1333 </td></tr><tr><td> ScriptCap m7Gキャッピングシステム</td><td> EPICENTREバイオテクノロジー</td><td> SCCE0610 </td></tr><tr><td>ネオントランスフェクションシステム</td><td>インビトロジェン</td><td> MPK5000 </td></tr><tr><td>ネオントランスフェクション·システム·キット</td><td>インビトロジェン</td><td> MPK1025 </td></tr><tr><td>のOpti-MEM I </td><td>インビトロジェン</td><td> 31985070 </td></tr><tr><td>リポフェクトアミン2000トランスフェクション試薬</td><td>インビトロジェン</td><td> 11668-027 </td></tr><tr><td>アジレント·RNA 600ナノキット</td><td>アジレント</td><td> 5067-1511 </td></tr><tr><td> Agilent 2100バイオアナライザの双方向oanalyzer </td><td>アジレント</td><td> G2939AA </td></tr><tr><td> Illustra GFX PCR DNAとジェルバンド精製キット</td><td> GEヘルスケア</td><td> 28-9034-70 </td></tr><tr><td> RiboMAX大規模RNA生産システム-T7 </td><td>プロメガ</td><td> P1300 </td></tr></tbody></table>

reverse genetics mediated recovery of infectious murine norovirus

人間のノロウイルスはヒトの胃腸炎（GE）は、世界中のほとんどの責任があると近い人から人への接触が1、2を回避することはできません環境での再発問題である。ここ数年の間に病院内で感染の発生率の増加は、その運用能力だけでなく、大規模な経済的損失に大きな混乱を引き起こして、報告されている。新たな抗ウイルスアプローチの識別は、細胞培養3で生産的な感染症を完了するには、人間のノロウイルスの無力のために限られていました。密接に人間のノロウイルス4に関連したマウスノロウイルス（MNV）、最近の分離がどのセル5に伝播することができますが、これらの病原体6,7の調査のための新しい道を開いた。 新しい正のセンスゲノムおよびサブゲノムRNA分子の合成MNVの複製の結果は、後者の最後のTHIに対応ウイルスゲノムのRD（ 図1）。 MNVは、ORF1は、ゲノムの大部分を占有し、ポリプロテイン前駆体から放出される7つの非構造タンパク質（NS1-7）をコードする4つの異なるオープンリーディングフレーム（ORF）が含まれています。 ORF2とORF3はサブゲノムRNA領域内に含まれており、キャプシドタンパク質（それぞれVP1およびVP2）（ 図1）エンコードされています。最近、我々はORF2が重複し、追加ORF4を同定したが、別のリーディングフレームで機能的であり、ミトコンドリアのローカライズされた病原性因子（VF1）8にエンコードしています。 ノロウイルスを含む正センスRNAウイルス、の複製は、新しい上限​​なしRNAゲノムの合成で得られる細胞質内で行われます。ウイルスの翻訳を促進するために、ウイルスが細胞のタンパク質合成機械9-11募集を目的としたさまざまな戦略を利用する。興味深いことに、ノロウイルスの変換は多機能ウイルスのタンパク質プライマーVPGによって駆動され共有ゲノムおよびサブゲノムRNAを12から14の両方の5 &#39;末端にリンクされています。翻訳のこの洗練されたメカニズムは、従来の逆遺伝学的アプローチによるウイルス回収の限られた効率の主要な要因となる可能性があります。 ここでは、5 &#39;末端にキャップされたマウスノロウイルス-1（同封MNVと呼ばれる）の転写産物の生成に基づいて2つの異なる戦略を報告します。 2番目のアプローチは、T7 RNAポリメラーゼを発現する細胞のcDNA MNVの転写を伴うのに対し、いずれかの方法では、in vitroでの合成およびウイルスRNAのキャップの両方が含まれます。 MNVと小動物モデルの研究のためのこれらの逆遺伝学·システムの可用性は、レプリケーションと病因15から17にウイルス配列の役割を分析するための前例のない能力を提供しています。

Watch this Scientific Journal Video about Reverse Genetics Mediated Recovery of Infectious Murine Norovirus at JoVE.com

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

ノロウイルスは胃腸炎の主要原因である、まだそれらの特性評価のための分子技術はまだ比較的新しいです。ここでは、マウスノロウイルス（MNV）、細胞培養で増殖させることができるこの属の唯一のメンバーを効率的に回収するための2つの異なる逆遺伝学のアプローチを報告します。

感染マウスノロウイルスのリバースジェネティクスを介したリカバリ

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

reverse-genetics-mediated-recovery-of-infectious-murine-norovirus

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

16.5K ビュー.  Imperial College London. ノロウイルスは胃腸炎の主要原因である、まだそれらの特性評価のための分子技術はまだ比較的新しいです。ここでは、マウスノロウイルス（MNV）、細胞培養で増殖させることができるこの属の唯一のメンバーを効率的に回収するための2つの異なる逆遺伝学のアプローチを報告します。

ビデオ: Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

エンテロウイルスと文化およびRT-qPCRにより水中のノロウイルス発生のEPAメソッド1615測定。パートIII。 RT-qPCRによりウイルス検出

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

環境学

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

のTaqManワンステップRT-PCR法によるキッズスツールからノロウイルスの検出とGenogrouping

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

医学

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

リフトバレー熱ウイルスワクチンの安全性および有効性を改善するためのMP - 12株のNSS遺伝子を操作する逆遺伝学を使用して

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

免疫・感染学

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

マウスノロウイルスのプラークアッセイ

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

FDAの承認を受けたVero細胞におけるワクチン開発のための組換えアレナウイルスの生成

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

cDNAからの組換えニューカッスル病ウイルスのレスキュー

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

ポジティブセンスRNAウイルス変異体を改変するためのリバースジェネティクス

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA ...

遺伝学

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

代替体外ウイルスのカプシド構造健全性の定量方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

世代、増幅、および組換え呼吸器合胞体ウイルスの滴定

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

レポータータンパク質を発現する組換えロタウイルスを回収する簡略逆遺伝学法

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian ...