The authors would like to acknowledge the Utah Science Technology and Research fund for support of YML and the Korea National Research Foundation grants (2009-0069679 and 2010-0010154) for support of SIY. This research was supported by the Utah Agricultural Experiment Station, Utah State University, and approved as journal paper number UAES #8753. Also, the authors thank Dr. Deborah McClellan for editorial assistance.

The authors have declared that they have no competing financial interests.

目前的协议已被成功地用来产生全长感染性cDNA克隆的两种不同菌株（CNU / LP2 27和SA 14 -14-2 28）JEV，黄病毒，其功能性的cDNA已被证明是本来就很难构建和由于传播宿主细胞毒性和克隆基因的遗传不稳定性8,74-76该协议包括三个主要部分组成:第一，采用高保真逆转录酶最大化的病毒RNA的忠实cDNA拷贝合成/放大/ DNA聚合酶;第二，克隆含有毒序列（未公布的数据）的病毒的prM-E的编码区74,77,78在从最初的cDNA亚克隆到最终的全长cDNA组装步骤非常低拷贝数载体的BAC;第三，利用克隆载体的BAC，可容纳外来DNA与120-350 kb的，19-21的平均尺寸，这显然容忍较大的DNA樱雪RTS比其他克隆载体。这种克隆方法将是普遍适用于许多其他正链RNA病毒，特别是那些具有约10〜32 kb的大的RNA基因组。的感染性cDNA克隆的产生是在发展为RNA病毒反向遗传学系统的一个关键步骤，特别是对正链RNA病毒，因为它的基因组作为病毒mRNA是受宿主细胞的核糖体翻译成蛋白质。因此，病毒复制可以通过导入的cDNA衍生的基因组长度RNA分子进入易感宿主细胞启动。感染性JEV cDNA克隆的可用性，当用重组DNA技术结合起来，还增加了我们的病毒生命周期的各个方面的理解，在分子水平上，如基因表达73,79和基因组的复制。63,64，一个全长的JEV cDNA克隆已被证明是用于抗病毒疫苗28和基因递送载体的发展的有价值的工具。&lt;SUP&gt; 80,81 如同所有的正链RNA病毒中，有在构建可靠官能的cDNA JEV从中高度传染性的RNA 可以在体外进行合成的多个关键步骤。理想的情况下，从全长cDNA的克隆转录合成RNA的顺序应该是相同的，所述病毒基因组RNA，特别是所需要的病毒RNA复制的起始的5&#39;-和3&#39;-端序列的60-62在当前的协议，正宗5&#39;-和3&#39;-末端确保通过将SP6启动子序列的病毒基因组的第一个腺嘌呤核苷酸的上游和定位一个独特人工的XbaⅠ限制性位点的最后的下游病毒基因组的胸腺嘧啶核苷酸，分别。封端的合成的RNA与由一个Xba I位线性化和MBN处理的cDNA德的径流转录被生产正宗5&#39;和3&#39;末端mplate使用SP6 RNA聚合酶底漆与M 7 G（5&#39;），PPP（5&#39;）的盖模拟。该协议可以以多种方式进行修改。 体外转录，另一个噬菌体RNA聚合酶（例如，T3或T7）可以在与它的良好定义的启动子序列一起使用。27作为一个径流位点，不同的限制性位点可以使用，如果它不存在在病毒基因组，如果合成的RNA从线性的cDNA与正宗的3&#39;端结束。 3&#39;-端核苷酸序列的重要性已被证明在核糖核酸感染性〜10倍的下降，当一个合成的RNA包含三个或四个病毒无关的核苷酸在其3&#39;末端27在体外转录反应中，既第m 7 G（5&#39;），PPP（5&#39;）A和m 7 G（5&#39;），PPP（5&#39;）G帽类似物可同样用得好，病毒5后者虽然地方不相关的额外摹核苷酸上游&#39;末端，但是这此外不改变的感染性或合成的RNA复制。27此外，除去从RNA转录通过DNA酶I消化的cDNA模板是没有必要的核糖核酸的感染性的测试，因为与cDNA模板本身不是感染性的。27 上述BAC技术已被应用到构建感染性cDNA克隆为少数正链RNA病毒，即，二JEVs，CNU / LP2 27和SA 14 -14-2 28（基因组的大小，〜11 kb的）; 2登革热病毒，BR / 90 26和NGC 29（〜11 KB）;牛病毒性腹泻病毒，SD1（〜12 KB）; 25两种经典猪瘟病毒，C和帕德博恩（〜12 KB）; 24边界病病毒，吉夫霍恩（〜12 KB）; 24猪繁殖与呼吸综合征病毒，PL97-1 / LP1（〜15 KB）; 30传染性胃肠炎病毒，PUR46-MAD（〜29 KB）; 16猫传染性腹膜炎病毒，DF-2（〜29 KB）; 32严重急性呼吸系统综合症冠 ​​状病毒，乌尔巴尼（〜30 KB）; 9中东呼吸综合症，EMC / 2012（〜30 KB）; 17和人类冠状病毒，病毒OC43（〜 31 KB）31使用的BACs用于cDNA结构的主要优点是大的，1或2拷贝BAC质粒的高遗传稳定性。然而，其非常低拷贝数的固有性质是因为BAC DNA和随之减少在BAC DNA的纯度相对于宿主染色体DNA的非常低的产率的也是一个很大的缺点，。在当前的协议，BAC DNA的产率通过生长大肠杆菌最大化大肠杆菌 DH10B在营养丰富的介质，的2xYT转化的感染性BAC PBAC / SA 14 -14-2。尽管这样的努力，平均产量仅在从500毫升的2xYT肉汤BAC DNA的〜15微克。而且，BAC DNA的纯度最好使用氯化铯 - 溴化乙锭密度梯度离心进行纯化达到的，而不是通常使用的基于列的质粒分离。但是，要记住重要的是，BAC转化E.大肠杆菌不应过度生长，因为它可能会危及克隆的cDNA的遗传稳定性，和更高的增长并不必然导致更大的产率或更高纯度的BAC DNA。 这里描述的协议是一个优化的，高效和简化的遗传稳定的全长感染性cDNA克隆作为BAC为JEV的构造和繁殖方法中，一个过程曾经认为实际上不可能。此相同的克隆策略，​​也可以应用于其它许多正链RNA病毒。在一般情况下，感染性cDNA克隆使我们介绍了各种突变（例如，缺失，插入，和点突变）的成病毒RNA基因组来研究它们的生物学功能中的病毒复制和发病机理。该cDNA为基础的反向遗传学系统使得能够开发和TES吨疫苗和靶向感兴趣的特定正链RNA病毒的毒力因子（多个）治疗的候选人。此外，这种感染性cDNA技术也可以用来作为病毒载体，能够表达外来感兴趣的基因（多个）用于生物医学研究中的许多应用。

用于RNA病毒学家，重组DNA技术在20世纪70年代后期出现使得有可能转换的病毒RNA基因组成的cDNA克隆，其可随后被传播作为细菌的RNA病毒的基因操作的质粒。1所述的第一RNA病毒是分子克隆是噬菌体Qβ，一个正链RNA病毒，感染大肠杆菌 。一份载有Qβ基因组RNA的全长cDNA拷贝质粒引起感染Qβ噬菌体在引入E.大肠杆菌 。2之后不久，这种技术应用于脊髓灰质炎病毒，人类和动物的一个正链RNA病毒。质粒轴承脊髓灰质炎病毒基因组RNA的全长cDNA的感染性时转染到哺乳动物细胞和能够产生感染性病毒体3在这个"DNA的启动"的方法，将克隆的cDNA应在细胞内转录的启动病毒RNA的复制。然而，目前还不清楚如何转录启动以及如何转录物加工成正确的病毒序列。这种担忧导致了另一种"RNA推出"的发展方针，从而使病毒RNA基因组的完整基因副本是根据由E.识别的启动子克隆大肠杆菌或噬菌体RNA聚合酶，用于生产在体外合成的RNA具有确定的5&#39;和3&#39;末端，在导入宿主细胞经历完整的病毒复制循环的4,5的第一次成功，这种方法据报道为雀麦花叶病毒，6,7-植物的正链RNA病毒。此后，核糖核酸射方法已经被开发用于广泛的正链RNA病毒，包括杯状病毒，甲病毒，黄病毒，动脉炎病毒，和冠状病毒。1,4,5,8 在这两个DNA和RNA的启动反向遗传学系统，一个FUL的施工升的全长cDNA克隆的关键是产生感染性DNA或正链RNA病毒的RNA，但它成为相当大的技术挑战，作为病毒基因组的尺寸增加。9-17特别是，一个大的RNA基因组〜10 -32 kb的提出了三种主要障碍全长官能cDNA的克隆18的第一个困难是一个忠实的​​cDNA拷贝的合成，因为RT-PCR的保真度是成反比的病毒RNA的长度。第二关是有潜在毒性的序列的存在，因为长的RNA分子更可能含有能够使cDNA片段在质粒不稳定在大肠杆菌的意想不到的序列大肠杆菌 。第三也是最关键的问题是合适的载体的可用性，因为它是很难找到的克隆载体，可以容纳的&gt; 10kb的一个病毒cDNA插入物。在过去的三十年里，这些障碍已经被克服在酶学，方法论，是一些进步ðvectorology。1,4,5,8其中，最有前途的创新发展是大的正链RNA病毒传染性细菌人工染色体（BAC）的克隆。 BAC载体是一低拷贝质粒克隆（1-2个拷贝/细胞）的基础上E.大肠杆菌 F质粒，具有的平均DNA插入物大小:约120-350 kb的19-21的DNA片段被插入到以类似的方式将BAC载体以克隆到一般的克隆载体。所产生的BAC克隆是稳定的多代的E.大肠杆菌。22,23到目前为止，上述BAC技术已被用来创建感染性cDNA克隆的三个正链RNA病毒家族， 即 黄病毒科 ， 动脉炎 24-29，30和冠状的&gt; 10个成员。9,16,17 ，31,32 使用日本脑炎病毒（JEV）作为一个例子，目前的工作报告的详细过程，可以是用于构建遗传稳定的全长感染性BAC为各种正链RNA病毒。 JEV是一种人畜共患黄病毒33是在鸟类，猪和其他脊椎动物宿主之间性质通过蚊子载体传播。34,35在人类中，JEV感染可引起严重的常常是致命的神经系统疾病的日本脑炎（JE），36中发生的亚洲和西太平洋，37,38的部分地区有中〜50,000-175,000临床病例，估计每年的发病率。39,40乙脑病毒的基因组是〜11 KB，单股，正链RNA分子，由由两个非编码区（NCR的）在5&#39;和3&#39;侧翼的单个开放阅读框（ORF）结束。41,42的ORF编码是受宿主和病毒蛋白酶裂解，生成10个单独的蛋白质多蛋白，指定C，PRM，请E，NS1，NS2A，NS2B，NS3，NS4A，NS4B和NS5中的N至C端方向34,43,44此外，电子NS1（NS1&#39;）的xtended形式是由-1核糖体移密码子NS2A 8-9表示。45,46这些11种蛋白质中，有三种结构蛋白（C，的prM和E）是用于感染性的形成至关重要病毒粒子，47,48和其余八的非结构蛋白（NS1到NS5，和NS1&#39;）是病毒RNA复制，49-51颗粒组装，52-56和先天免疫逃避至关重要。57-59两者的5&#39;和3 &#39;NCR的含有保守的一级序列和形式的RNA二级/三级结构，60-62其中用于调节病毒RNA复制很重要。63,64 本协议描述的工具，方法和策略产生全长乙脑病毒感染性BAC SA 14 -14-2。28这种功能BAC克隆包含了乙脑病毒基因组RNA，它是通过一个启动子包含一个完整的基因副本，65对于SP6 RNA聚合酶上游的病毒的5&#39;末端和一个独特的XbaⅠ限制性病毒3&#39;末端的体外径流转录的下游位点。此BAC技术适用于构建一个功能完整的cDNA分子克隆用于正链RNA病毒的阵列。

<table><tbody><tr><td>&lt;strong&gt;1. Molecular Cloning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2xYT Broth</td><td>Sigma-Aldrich</td><td>Y2377</td><td></td></tr><tr><td>[&lt;sup&gt;3&lt;/sup&gt;H]UTP</td><td>PerkinElmer</td><td>NET380250UC</td><td>Radioactive</td></tr><tr><td>50 ml Tube</td><td>Thermo Scientific (Nalgene)</td><td>3114-0050</td><td></td></tr><tr><td>250 ml Bottle</td><td>Beckman Coulter</td><td>356011</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>Agarose (Low Melting Point)</td><td>Life Technologies (Invitrogen)</td><td>16520-100</td><td></td></tr><tr><td>AvaI</td><td>New England BioLabs</td><td>R0152S</td><td></td></tr><tr><td>AvrII</td><td>New England BioLabs</td><td>R0174S</td><td></td></tr><tr><td>BamHI</td><td>New England BioLabs</td><td>R0136S</td><td></td></tr><tr><td>BsiWI</td><td>New England BioLabs</td><td>R0553S</td><td></td></tr><tr><td>BsrGI</td><td>New England BioLabs</td><td>R0575S</td><td></td></tr><tr><td>Butanol</td><td>Fisher Scientific</td><td>A399-1&amp;nbsp;</td><td></td></tr><tr><td>Cesium Chloride</td><td>Fisher Scientific</td><td>BP1595-1</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C0378</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>C2432</td><td>Carcinogenic</td></tr><tr><td>DE-81 Filter Paper</td><td>GE Healthcare Life Sciences</td><td>3658-023</td><td></td></tr><tr><td>dNTP mix</td><td>Life Technologies (Invitrogen)</td><td>18427-088</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; DH10B&amp;nbsp;</td><td>Life Technologies (Invitrogen)</td><td>18297-010</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Ethidium Bromide</td><td>Sigma-Aldrich</td><td>E7637</td><td>Toxic and highly mutagenic</td></tr><tr><td>Filter Column (Plasmid Maxiprep Kit)</td><td>Life Technologies (Invitrogen)</td><td>K2100-26</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma-Aldrich</td><td>A6283</td><td>Irritating</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G5400</td><td></td></tr><tr><td>Glycogen&amp;nbsp;</td><td>Roche</td><td>10901393001</td><td></td></tr><tr><td>High-fidelity DNA Polymerase</td><td>New England BioLabs</td><td>M0491S</td><td></td></tr><tr><td>Isoamyl Alcohol</td><td>Sigma-Aldrich</td><td>I9392</td><td>Flammable</td></tr><tr><td>Isopropanol&amp;nbsp;</td><td>Amresco</td><td>0918</td><td>Flammable</td></tr><tr><td>LB Broth</td><td>Life Technologies (Invitrogen)</td><td>12795-027</td><td></td></tr><tr><td>Lithium Chloride</td><td>Sigma-Aldrich</td><td>L9650</td><td></td></tr><tr><td>Lysozyme</td><td>Amresco</td><td>0663</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>M-MLV Reverse Transcriptase</td><td>Life Technologies (Invitrogen)</td><td>18080-044</td><td></td></tr><tr><td>Mung Bean Nuclease</td><td>New England BioLabs</td><td>M0250S</td><td></td></tr><tr><td>Needle (18G, 20G)</td><td>BD</td><td>305196, 305175</td><td>Biohazardous (Sharps waste)</td></tr><tr><td>NotI</td><td>New England BioLabs</td><td>R0189S</td><td></td></tr><tr><td>Oligonucleotide</td><td>Integrated DNA Technologies</td><td>Custom Oligonucleotide Synthesis</td><td></td></tr><tr><td>PacI</td><td>New England BioLabs</td><td>R0547S</td><td></td></tr><tr><td>pBeloBAC11</td><td>New England BioLabs</td><td>ER2420S (E4154S)</td><td></td></tr><tr><td>Phenol (Buffer-Saturated)</td><td>Life Technologies (Invitrogen)</td><td>15513-039</td><td>Toxic and highly corrosive</td></tr><tr><td>Phenol:Chloroform:Isoamyl Alcohol</td><td>Life Technologies (Invitrogen)</td><td>15593-031</td><td>Toxic and highly corrosive</td></tr><tr><td>Phenol:Guanidine Isothiocyanate</td><td>Life Technologies (Ambion)</td><td>10296-010</td><td>Toxic, corrosive, and irritating</td></tr><tr><td>Pme I</td><td>New England BioLabs</td><td>R0560S</td><td></td></tr><tr><td>Potassium Acetate</td><td>Amresco</td><td>0698</td><td></td></tr><tr><td>RNase Inhibitor</td><td>Life Technologies (Invitrogen)</td><td>10777-019</td><td></td></tr><tr><td>rNTP Set</td><td>GE Healthcare Life Sciences</td><td>27-2025-01</td><td></td></tr><tr><td>Sealable Polypropylene Tube (16 &amp;times; 76 mm)&amp;nbsp;</td><td>Beckman Coulter</td><td>342413</td><td></td></tr><tr><td>SfiI</td><td>New England BioLabs</td><td>R0123S</td><td></td></tr><tr><td>Sma I</td><td>New England BioLabs</td><td>R0141S</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Amresco</td><td>0227</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S5881</td><td></td></tr><tr><td>SP6 RNA Polymerase</td><td>New England BioLabs</td><td>M0207S</td><td></td></tr><tr><td>Spin Column (Plasmid Miniprep Kit)</td><td>Life Technologies (Invitrogen)</td><td>K2100-11</td><td></td></tr><tr><td>Syringe</td><td>HSW NORM-JECT</td><td>4200.000V0</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England BioLabs</td><td>M0202S</td><td></td></tr><tr><td>Tris</td><td>Amresco</td><td>0826</td><td></td></tr><tr><td>tRNA (yeast)</td><td>Life Technologies (Invitrogen)</td><td>15401-011</td><td></td></tr><tr><td>XbaI&amp;nbsp;</td><td>New England BioLabs</td><td>R0145S</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;2. Cell Culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alpha Minimal Essential Medium</td><td>Life Technologies (Gibco)</td><td>12561-049</td><td></td></tr><tr><td>Conical Tube (50 mL)</td><td>VWR</td><td>21008-242</td><td></td></tr><tr><td>Crystal Violet&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C0775</td><td></td></tr><tr><td>Culture Dish (150 mm)&amp;nbsp;</td><td>TPP</td><td>93150&amp;nbsp;</td><td></td></tr><tr><td>Cuvette (2-mm Gap)</td><td>Harvard Apparatus</td><td>450125&amp;nbsp;</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Life Technologies (Gibco)</td><td>16000-044</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F1635</td><td>Toxic and carcinogenic</td></tr><tr><td>Glutamine</td><td>Life Technologies (Gibco)</td><td>25030-081</td><td></td></tr><tr><td>Minimal Essential Medium</td><td>Life Technologies (Gibco)</td><td>61100-061</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies (Gibco)</td><td>15070-063</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Sigma-Aldrich</td><td>P9791</td><td></td></tr><tr><td>Six-Well Plate</td><td>TPP</td><td>92006</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma-Aldrich</td><td>S3264</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Life Technologies (Gibco)</td><td>25200-056</td><td></td></tr><tr><td>Vitamins</td><td>Sigma-Aldrich</td><td>M6895</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;3. Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose Gel Electrophoresis System</td><td>Mupid</td><td>MPDEXU-01</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Scientific</td><td>Heracell 150i</td><td></td></tr><tr><td>Desktop Centrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td>ST16R</td><td></td></tr><tr><td>Electroporator</td><td>Harvard Apparatus</td><td>ECM 830</td><td></td></tr><tr><td>Longwave Ultraviolet Lamps (Handheld)</td><td>UVP</td><td>UVGL-58</td><td></td></tr><tr><td>Tabletop Centrifuge</td><td>Beckman Coulter</td><td>368826</td><td></td></tr><tr><td>Thermocycler</td><td>Life Technologies (Applied Biosystems)</td><td>GeneAmp PCR System 9700</td><td></td></tr><tr><td>Vortexer</td><td>Scientific Industries</td><td>G-560</td><td></td></tr><tr><td>Water Bath</td><td>Jeio Tech</td><td>WB-10E</td><td></td></tr></tbody></table>

bacterial artificial chromosomes: a functional genomics tool for the study of positive-strand rna viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

对于所有的正链RNA病毒，反向遗传学系统的可靠性和效率取决于一个克隆的全长cDNA的遗传稳定性，其序列相当于共识的病毒基因组RNA序列。27 图1示出五步骤策略的全长感染性cDNA的构建作为BAC为JEV的SA 14 -14-2 28:第1步，病毒RNA从JEV感染的BHK-21细胞（图1A）的细胞培养物上清液纯化;步骤2，合成四个重叠的cDNA的扩增子（F1至F4）跨越整个病毒基因组（图1B）;步骤3中，亚克隆的四个邻接的cDNA片段的插入BAC载体，创建PBAC / F1至PBAC / F4（图1C）;步骤4中，将克隆的cDNA 用于体外径流用SP6 RNA聚合酶， 即转录的变形例中，放置一个SP6启动子序列紧接病毒的5&#39;末端（PBAC / F1的SP6）的上游侧，通过引入沉默点突变消除预先存在的内部XbaI位点在核苷酸9131，阿9134→T（PBAC / F3 正），并插入一个新的人工的XbaⅠ径流现场立即病毒3&#39;-端（PBAC / F4 RO）（图1D）的下游;和第5步，一个全长的组件SA 14 -14-2的cDNA的BAC，PBAC / SA 14 -14-2（图1E）。 表1列出了在此克隆程序中使用的寡核苷酸。28 用于功能JEV cDNA的结构中，第一重要的步骤是使用纯化病毒RNA作为用于RT-PCR的模板的四个重叠cDNA片段的合成。 图2提供了一个代表性的结果为四RT-PCR产物是分别电泳在0.8％琼脂糖凝胶。这种凝胶清楚显示的全长cDNA的JEV被放大成四个重叠cDNA片段。偶尔，RT-PCR反应可能产生，因为引物时cDNA合成/扩增非特异性退火的一个或多个附加的病毒特异性或非特异性的产品，大多是比预期的产物更小，。另一方面，很少或没有预期的，因为在病毒RNA分离或不当的RT-PCR性能意外RNase污染的RT-PCR产物将被扩增。 下一关键步骤是一个partial-或全长的cDNA JEV在BAC，它是一种使用标准重组DNA技术相对简单的过程的克隆和修饰。69 图3给出了为 BAC克隆的含有纯化的代表性结果JEV SA 14 -14-2的通过捆在氯化铯-溴化乙锭梯度的全长cDNA。在该实验中，离心分离16小时，401700×g下后，两个不同的频带， 即 ， 大肠杆菌大肠杆菌染色体DNA的上方和超螺旋的BAC质粒DNA的下面，是在管的下长波紫外光中间可见。一个最小的体积（〜400微升）作为低级BAC DNA带的小心地戳一个孔与在管侧的注射器收集。接着，溴化乙锭是从BAC DNA由丁醇萃取萃取，将乙锭 - 自由BAC的DNA通过乙醇沉淀浓缩。 最后一步是从核糖核酸转染到允许细胞（图4）之后的全长的SA 14 -14-2 BAC（PBAC / SA 14 -14-2） 体外转录的合成RNA的特异性感染性的测定。该步骤包括三个顺序步骤:在3&#39;步骤1，全长的线性化的SA 14 -14-2的cDNA病毒基因组（图4A）的末端;步骤2，从生产线性的cDNA合成的RNA通过径流转录（图4B）的;和第3步中，重组病毒在BHK-21转染的细胞合成的RNA（图4C）的救援。实验上，PBAC / SA 14 -14-2的两个独立的克隆，线性化用 XbaⅠ消化并用MBN处理以除去由XbaI位消化产生的四碱基的5&#39;突出端。将线性化的BAC进行清理通过苯酚 - 氯仿提取，接着乙醇沉淀。两个纯化的BAC的线性化证实在0.8％琼脂糖凝胶（图5A）。酚 - 氯仿提取必须小心，以确保线性化BAC的无RNA酶的。每两个线性化的BAC的担任用于使用SP6 RNA聚合酶在米7 G（5&#39;）ppp的存在下径流转录的cDNA模板（5&#39;）的盖模拟。合成RNA的完整性被证明通过在0.6％琼脂糖凝胶上运行两个转录反应混合物的等分试样，以及一个参考1 kb的DNA梯（ 图 5B）。在这个简单的测定法中，主要的突出的RNA带总是迁移刚下面的3 kb的DNA参考乐队，似乎是尖锐的。然而，降解的RNA会对同一凝胶的拖尾外观。 感染性中心试验则是金标准，用于确定合成的RNA的特异性感染性。该测定是通过电穿孔的BHK-21细胞的RNA样品，播种的10倍系列稀释的电穿孔的细胞的等份在6孔板含幼稚BHK-21细胞（3×10 5细胞/孔 ），并覆盖琼脂糖进行到细胞单层。培育4天后，存活细胞用甲醛固定并用结晶紫小号olution量化感染性中心（斑块）的数量，其对应于递送到细胞（图6A）的感染性RNA分子的数目。由于用于体外转录的cDNA模板已被证明是无感染性的，直接用于电穿孔的转录反应混合物的27等分试样。电穿孔是用于RNA转染的优选方法;可选地，分子RNA可以通过使用DEAE-葡聚糖和阳离子脂质体的其它方法来进行转染。核糖核酸电穿孔是非常有效的，但"电弧"电脉冲的发生很少如果盐存在的电穿孔反应或如果电穿孔杯中被重用。病毒蛋白的在RNA转染的细胞中的表达进行了检查，使用抗NS1兔抗血清（图6B）免疫荧光实验。在RNA转染的细胞的上清液积累病毒颗粒的生产是一个通过空斑测定（图6C）alyzed。这些实验的结果清楚地表明，该cDNA衍生的合成的RNA是在感染性许可BHK-21细胞，产生重组病毒的高滴度。 <table fo:keep-together.within-page="1"><tbody><tr><td> 寡核苷酸 </td><td> 序列一个 （5&#39;至3&#39;） </td><td> 位置 b </td><td> 极性 </td></tr><tr><td> 1RT </td><td> TAGGGATCTGGGCGTTTCTG GCAAAT </td><td> 2578年至2603年</td><td>反义</td></tr><tr><td> 1F </td><td> aatcccgggAGAAGTTTATC TGTGTGAACTT </td><td> 1-22 </td><td>感</td></tr><tr><td> 1R </td><td> attgcggccgcCCACGTCGT TGTGCACGAAGAT </td><td> 2532年至2553年</td><td>反义</td></tr><tr><td> 2RT </td><td> TTCTGCCTACTCTGCCCCTC CGTTGA </td><td> 5975-6000 </td><td>蚂蚁ISENSE </td></tr><tr><td> 2F </td><td> aatcccgggTCAAGCTCAGT GATGTTAACAT </td><td> 1800年至1821年</td><td>感</td></tr><tr><td> 2R </td><td> attgcggccgcGATGGGTTT CCGAGGATGACTC </td><td> 5929-5950 </td><td>反义</td></tr><tr><td> 3RT </td><td> ACGGTCTTTCCTTCTGCTGC AGGTCT </td><td> 9426-9451 </td><td>反义</td></tr><tr><td> 3F </td><td> aatcccgggGAGGATACATT GCTACCAAGGT </td><td> 5500-5521 </td><td>感</td></tr><tr><td> 3R </td><td> attgcggccgcGTAAGTCAG TTCAATTATGGCT </td><td> 9380-9401 </td><td>反义</td></tr><tr><td> 4RT </td><td> AGATCCTGTGTTCTTCCTCA CCACCA </td><td> 10952-10977 </td><td>反义</td></tr><tr><td> 4F </td><td> aatcccgggAGTGGAAGGCT CAGGCGTCCAA </td><td> 9200-9221 </td><td>感</td></tr><tr><td> 4R </td><td> attgcggccgcAGATCCTGT GTTCTTCCTCACC </td><td> 10956-10977 </td><td>反义</td></tr><tr><td> SP6F </td><td> cataccccgcgtattcccac TA </td><td></td><td>感</td></tr><tr><td> SP6R </td><td> ACAGATAAACTTCTctatag tgtcccctaaa </td><td> 1-14 </td><td>反义</td></tr><tr><td> F1F </td><td> aggggacactatagAGAAGT TTATCTGTGTG </td><td> 1-17 </td><td>感</td></tr><tr><td> F1R </td><td> TGGATCATTGCCCATGGTAA GCTTA </td><td> 638-662 </td><td>反义</td></tr><tr><td> X1F </td><td> CGAATGGATCGCACAGTGTG GAGAG </td><td> 8403-8427 </td><td>感</td></tr><tr><td> X1R </td><td> AAAGCTTCAAACTCAAGATA CCGTGCTCC </td><td> 9120-9148 </td><td>反义</td></tr><tr><td> X2F </td><td> GGAGCACGGTATCTTGAGTT TGAAGCTTT </td><td> 9120-9148 </td><td>感</td></tr><tr><td> X2R </td><td> cacgtggacgagggcatgcc tgcag </td><td></td><td>反义</td></tr><tr><td> ROF </td><td> CCAGGAGGACTGGGTTACCA AAGCC </td><td> 10670-10694 </td><td>感</td></tr><tr><td> ROR </td><td> agggcggccgctctagAGAT CCTGTGTTCTTCCTCACCAC </td><td> 10954-10977 </td><td>反义</td></tr><tr><td colspan="4"> 一个乙脑病毒序列用大写字母显示，和BAC序列表示小写字母。 b核苷酸位置是指JEV SA 14 -14-2（Genbank登录号JN604986）的完整基因组序列。 </td></tr></tbody></table> 表1: 用于cDNA合成，PCR扩增，和BAC诱变寡核苷酸。 <img alt="图1" src="/files/ftp_upload/53164/53164fig1.jpg" /> 图1。0;战略乙脑病毒SA 14 -14-2的全长cDNA 为BAC建设的病毒RNA从JEV颗粒（A）隔离。示出的是JEV的SA 14 -14-2的基因组RNA的示意图。 （B）的4重叠的cDNA片段合成（F1至F4）覆盖整个病毒基因组。 （ 三）亚克隆四个重叠的cDNA片段插入BAC载体，创造PBAC / F1至PBAC / F4。 （D）的变形例的克隆的cDNA 用于体外径流转录。 PBAC / F1的SP6是PBAC / F1的包含病毒的5&#39;末端上游的SP6启动子序列的衍生物。 PBAC / F3 KO是PBAC / F3的衍生物，它包含一个无声的点突变（A→9134 T，星号）。 PBAC / F4 RO是PBAC / F4的衍生物包含病毒3&#39;末端的下游人造的XbaⅠ决胜点。 （E </<html> STRONG&gt;）全长SA 14 -14-2 BAC组装（PBAC / SA 14 -14-2）。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53164/53164fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53164/53164fig2.jpg" /> 图2.合成四个重叠cDNA片段（F1至F4）跨越的JEV的SA 14 -14-2 的全长基因组RNA。四个RT-PCR产物通过电泳在0.8％琼脂糖凝胶评价。男，1 kb的DNA阶梯。四cDNA片段的预期大小显示在凝胶图像的底部。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53164/53164fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> /ftp_upload/53164/53164fig3.jpg"/&gt; 图3.含纯化乙脑病毒的全长cDNA SA 14 -14-2 的BAC中 的 BAC质粒从大肠杆菌中分离大肠杆菌 DH10B由SDS-碱裂解法和通过捆在氯化铯-溴化乙锭梯度进一步纯化。介绍的是使用16×76毫米密封聚丙烯管的氯化铯，溴化乙锭梯度的一个例子。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53164/53164fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53164/53164fig4.jpg" /> 图4.概述传染性病毒的全长乙脑病毒SA 14 -14-2的cDNA组装在BAC 复苏 的 cDNA模板（一）线性化。全长的JEV BAC被切断与XbaⅠ和治疗MBN。 （二）合成的RNA转录物。将线性化的cDNA通过SP6 RNA聚合酶在米7 G（5&#39;）PPP（5&#39;）甲帽类似物的存在下转录。 （ 三）恢复合成JEVs的。 体外转录的RNA转染BHK-21细胞，通过电产生合成病毒的高滴度。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53164/53164fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53164/53164fig5.jpg" /> 图5.合成的RNA的体外 使用全长BAC JEV作为cDNA模板转录。线性化的全长的JEV BAC，PBAC / SA 14 -14-2的（A）的生成。 PBAC的两个独立克隆/ SA 14 -14-2（CL.1和Cl.2）通过消化XbaⅠ和后续治疗MBN线性化。将线性化的BAC通过电泳在0.8％琼脂糖凝胶检查。 （ 二）通过径流转录生产合成RNA的。每两个线性化的BAC的用作SP6 RNA聚合酶径流转录的模板。两个转录反应的等分试样上的0.6％琼脂糖凝胶上运行。男，1 kb的DNA阶梯。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53164/53164fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53164/53164fig6.jpg" /> 从全长JEV BAC和合成病毒的恢复转录合成RNA的图6.具体感染性。BHK-21细胞是模拟电穿孔（模拟），或电穿孔而得fr处的RNA转录嗡每个全长的JEV BAC（CL.1和Cl.2）的两个独立的克隆。 （A）RNA感染力。将细胞覆盖有琼脂糖和，以4天转染后以结晶紫染色。核糖核酸感染是由感染性中心试验测定来估算的感染性RNA的电穿孔进入细胞（左面板）的量。另外，感染性中心的有代表性图像示（右图）。 （二）蛋白的表达。细胞在4孔室载玻片中培养。在20小时后转染（HPT）中的RNA电穿孔的细胞的病毒蛋白的表达是​​通过使用一个初级抗NS1兔抗血清和次级的Cy3缀合的山羊抗兔IgG（红色）免疫荧光分析进行分析。细胞核被counterstained用4&#39;，6-二脒基-2-苯基（蓝色）。免疫荧光图像被叠加在它们相应的微分干涉对比图像。 （C）病毒的产量。将细胞培养于150毫米培养皿。在22和40 HPT积累了RNA电穿孔细胞的培养上清液感染性病毒粒子的生产是由斑块检测检查。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53164/53164fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses at JoVE.com

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

细菌人工染色体:对于正链RNA病毒研究的功能基因组学工具

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

bacterial-artificial-chromosomes-functional-genomics-tool-for-study

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

21.3K 次观看.  Utah State University. Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

视频: Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

简单而强大在体内和在体外方法研究病毒大会

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

科研

免疫与感染

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

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.

反向遗传学设计正义RNA病毒变异株

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 ...

遗传学

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

呼吸系统同步病毒病毒特定核胶囊的生成和组装

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

生物化学

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.

造型埃博拉病毒的生命周期在生物安全二级条件下的病毒样颗粒包含Tetracistronic小基因组

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 ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

粗病毒提取中 DNA 的组合分析及对感染叶 RNA 的分析发现新的病毒基因组

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

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.

重组呼吸道合胞病毒的生成、扩增和滴定

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

Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial&#160;propagation.

The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter.

从细菌人工染色体cDNA克隆中拯救重组寨卡病毒

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines ...