这项研究是由威康信托高级奖学金颁发给伊恩·古德费洛，1居里夫人内欧洲奖学金（欧盟第七框架计划的欧洲研究理事会）授予阿曼多·阿里亚斯。我们要感谢给我们权限，使用安捷伦bioanalyser，帮助运行的RNA样品木卫一香港昌博士丽贝卡罗比和迈克·斯金纳博士。

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outbreaks+of+gastroenteritis+associated+with+noroviruses+on+cruise+ships--United+States.">Outbreaks of gastroenteritis associated with noroviruses on cruise ships--United States.</a> MMWR Morb. Mortal. Wkly. Rep. 51, 1112-1115 (2002).</li><li>Lopman, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+cost+of+nosocomial+gastroenteritis,+Avon,+England,+2002-2003.">Epidemiology and cost of nosocomial gastroenteritis, Avon, England, 2002-2003.</a> Emerg. Infect. Dis. 10, 2002-2003 (2004).</li><li>Duizer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+efforts+to+cultivate+noroviruses.">Laboratory efforts to cultivate noroviruses.</a> J. Gen. Virol. 85, 79-87 (2004).</li><li>Karst, S. M., Wobus, C. E., Lay, M., Davidson, J., Virgin, H. W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT1-dependent+innate+immunity+to+a+Norwalk-like+virus.">STAT1-dependent innate immunity to a Norwalk-like virus.</a> Science. 299, 1575-1575 (2003).</li><li>Wobus, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+Norovirus+in+cell+culture+reveals+a+tropism+for+dendritic+cells+and+macrophages.">Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.</a> PLoS Biol. 2, e432 (2004).</li><li>Bok, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+norovirus+replication+by+morpholino+oligomers+targeting+the+5'-end+of+the+genome.">Inhibition of norovirus replication by morpholino oligomers targeting the 5'-end of the genome.</a> Virology. 380, 328-337 (2008).</li><li>Kim, Y., Thapa, M., Hua, D. H., Chang, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+nanogels+for+oral+delivery+of+interferon+for+norovirus+infection.">Biodegradable nanogels for oral delivery of interferon for norovirus infection.</a> Antiviral Res. 89, 165-173 (2011).</li><li>McFadden, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+Regulation+of+the+Innate+Immune+Response+and+Apoptosis+Occurs+via+the+Product+of+the+Alternative+Open+Reading+Frame+4.">Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4.</a> PLoS Pathog. 7, e1002413 (2011).</li><li>Kormelink, R., van Poelwijk, F., Peters, D., Goldbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-viral+heterogeneous+sequences+at+the+5'+ends+of+tomato+spotted+wilt+virus+mRNAs.">Non-viral heterogeneous sequences at the 5' ends of tomato spotted wilt virus mRNAs.</a> J. 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>

我们什么都没有透露。

在这里，我们已经说明了两个不同的反向遗传学的方法，允许在细胞培养感染MNV恢复。这两种方法都有效地绕过为VPG通过代细胞的核糖体，然后由公认的皑皑MNV成绩单病毒RNA基因的5&#39;端共价联动的绝对要求。是更有效地在体外转录酶封顶传染病MNV转录比传染病质粒在细胞表达T7 RNA聚合酶的成绩单，其中可上限由鸡痘病毒封盖酶恢复。与这些反向遗传系统恢复病毒滴度类似VPG联病毒感染的细胞培养17（ 图5）纯化的RNA转染获得这些。皑皑MNV RNA进入许可Raw264.7细胞的转染呈现比实验涉及的transfectio的病毒滴度较低只有1日志N含有病毒的VPG联的RNA（ 图5）感染的细胞的总RNA。这实际上鼓励了进一步的调查，以确定是否可能导致病毒产量的增加，这可能揭示VPG相关的细胞的基本功能方面MNV传染性的VPG分子除了5&#39;这些系统所产生的最终成绩单。尽管如此，我们认为这种反向遗传学系统作为一种高效，与其他RNA病毒反向遗传学系统目前使用的体外转录RNA滴度只有真正感染病毒颗粒19，20比10-100允许恢复。 总体而言，目前的方法构成了诺罗病毒分子生物学领域的重要一步和提供的工具，我们调查的职能作用的蛋白质和诺罗病毒基因组保守的RNA图案。这些方法已经与C的结合urrent小鼠模型，并已显示出MNV传染性cDNA的恢复是能够引起致命感染- &gt; 80％STAT1的/小鼠在不到10天4，21。利用这一点，我们已经恢复了可行小鼠诺沃克病毒突变的衣壳蛋白和参与结合不同的宿主因素（PTB和选票详情规例）显示，在体内 21，22有点减毒表型的polypyrimidine道系统。此外，我们最近已表明，病毒缺乏表达能力从ORF4 VF1蛋白在细胞培养中有效地复制，但再次减少为WT MNV 8小鼠的毒性。这些研究，鼓励我们设计人类诺如病毒的减毒MNV可作为潜在的候选疫苗调查研究的基础版本。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>氯化锂沉淀解决方案</td><td> Ambion公司</td><td> AM9480 </td></tr><tr><td> RNA的存储解决方案</td><td> Ambion公司</td><td> AM7000 </td></tr><tr><td> MEGAscript T7启动脚本</td><td> Ambion公司</td><td> AM1333 </td></tr><tr><td> ScriptCap m7G旋盖系统</td><td>震中生物技术</td><td> SCCE0610 </td></tr><tr><td>霓虹灯转染系统</td><td> Invitrogen公司</td><td> MPK5000 </td></tr><tr><td>霓虹灯转染系统套件</td><td> Invitrogen公司</td><td> MPK1025 </td></tr><tr><td> OPTI-MEM我</td><td> Invitrogen公司</td><td> 31985070 </td></tr><tr><td>脂质体2000转染试剂</td><td> Invitrogen公司</td><td> 11668-027 </td></tr><tr><td>安捷伦RNA的600纳米套件</td><td>安捷伦</td><td> 5067-1511 </td></tr><tr><td>安捷伦2100 BIoanalyzer </td><td>安捷伦</td><td> G2939AA </td></tr><tr><td>插图GFX总DNA和凝胶带纯化试剂盒</td><td> GE医疗集团</td><td> 28-9034-70 </td></tr><tr><td> RiboMAX大规模RNA生产系统-T7 </td><td> Promega公司</td><td> P1300 </td></tr></tbody></table>

reverse genetics mediated recovery of infectious murine norovirus

人类诺如病毒是人类胃肠炎（GE）全球大多数情况下，负责，并在那里亲近的人对人的接触是无法避免的，1，2的环境中经常出现的问题。在医院暴发的发病率增加，在过去几年中已有报道，造成重大影响自身的业务能力以及巨大的经济损失。由于人类诺如病毒无法完成生产感染的细胞培养3，确定新的抗病毒方法已经被限制。小鼠诺沃克病毒（MNV）密切相关的人类诺罗病毒，但最近的隔离可在细胞5传播开辟了调查这些病原体6，7的新途径。 在新的积极意义的基因组和亚基因组RNA分子的合成的MNV复制结果，其中后者对应到最后THI病毒基因组的路（ 图1）。 MNV包含四个不同的开放阅读框（ORF），其中ORF1基因占据大部分的基因组，编码7个非结构蛋白（NS1蛋白-7）释放由聚易制毒化学。载的ORF2和ORF3内的亚基因组RNA地区和编码衣壳蛋白（VP1和VP2），分别为（ 图1）。最近，我们已经确定了额外的ORF4重叠的ORF2，但在不同的阅读框的功能和线粒体的本地化的致病因子（VF1）8编码。 积极意义的RNA病毒，包括诺如病毒，复制发生在细胞质中造成新的不封顶的RNA基因组的合成。促进翻译病毒，病毒利用不同的策略，旨在招聘细胞的蛋白质合成机器9-11。有趣的是，诺罗病毒的翻译是由多功能的病毒蛋白底漆VPG共价连接到5月底的两个基因组和亚基因组RNA 12-14。这种翻译复杂的机制很可能是在有限的病毒恢复传统的反向遗传学方法效率的主要因素。 在这里，我们报告两个不同的策略，基于上一代小鼠诺罗-1（简称MNV谨）在5&#39;端上限的成绩单。的方法之一，涉及体外病毒RNA的合成和封盖，而第二种方法需要的MNV细胞表达T7 RNA聚合酶基因的转录。这些反向遗传学的的MNV研究和小动物模型系统的可用性提供了一个前所未有的能力，解剖病毒复制和发病机制15-17序列的作用。

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

诺如病毒肠胃炎尚未分子技术是表征的主要原因仍然是相对较新的。在这里，我们报告两个不同的反向遗传学方法对小鼠诺沃克病毒（MNV），本属的唯一成员，可在细胞培养繁殖的高效回收。

反向遗传学感染小鼠诺沃克病毒介导的恢复

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

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Immunology and Infection

16.4K 次观看.  Imperial College London. 诺如病毒肠胃炎尚未分子技术是表征的主要原因仍然是相对较新的。在这里，我们报告两个不同的反向遗传学方法对小鼠诺沃克病毒（MNV），本属的唯一成员，可在细胞培养繁殖的高效回收。