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 border="0" cellpadding="0" cellspacing="0" width="1054">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">1. Molecular Cloning</td>
			<td style="width:275px;"></td>
			<td style="width:243px;"></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">2xYT Broth</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">Y2377</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">[3H]UTP</td>
			<td style="width:275px;">PerkinElmer</td>
			<td style="width:243px;">NET380250UC</td>
			<td>Radioactive</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">50-mL Tube</td>
			<td style="width:275px;">Thermo Scientific (Nalgene)</td>
			<td style="width:243px;">3114-0050</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">250-mL Bottle</td>
			<td style="width:275px;">Beckman Coulter</td>
			<td style="width:243px;">356011</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Agarose</td>
			<td style="width:275px;">Lonza</td>
			<td style="width:243px;">50004</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Agarose (Low Melting Point)</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">16520-100</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">AvaI</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0152S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">AvrII</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0174S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">BamHI</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0136S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">BsiWI</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0553S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">BsrGI</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0575S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Butanol</td>
			<td style="width:275px;">Fisher Scientific</td>
			<td style="width:243px;">A399-1&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Cap Analog [m7G(5&#697;)ppp(5&#697;)A]</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">S1405S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Cesium Chloride</td>
			<td style="width:275px;">Fisher Scientific</td>
			<td style="width:243px;">BP1595-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Chloramphenicol</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">C0378</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Chloroform</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">C2432</td>
			<td>Carcinogenic</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">DE-81 Filter Paper</td>
			<td style="width:275px;">GE Healthcare Life Sciences</td>
			<td style="width:243px;">3658-023</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">dNTP mix</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">18427-088</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">E. coli DH10B&#160;</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">18297-010</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">EDTA</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">E5134</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Ethanol&#160;</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">E7023</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Ethidium Bromide</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">E7637</td>
			<td>Toxic and highly mutagenic</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Filter Column (Plasmid Maxiprep Kit)</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">K2100-26</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Glacial Acetic Acid</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">A6283</td>
			<td>Irritating</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Glucose</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">G5400</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Glycogen&#160;</td>
			<td style="width:275px;">Roche</td>
			<td style="width:243px;">10901393001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">High-Fidelity DNA Polymerase</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">M0491S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Isoamyl Alcohol</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">I9392</td>
			<td>Flammable</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Isopropanol&#160;</td>
			<td style="width:275px;">Amresco</td>
			<td style="width:243px;">0918</td>
			<td>Flammable</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">LB Broth</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">12795-027</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Lithium Chloride</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">L9650</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Lysozyme</td>
			<td style="width:275px;">Amresco</td>
			<td style="width:243px;">0663</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Magnesium Chloride</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">M8266</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">M-MLV Reverse Transcriptase</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">18080-044</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Mung Bean Nuclease</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">M0250S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Needle (18G, 20G)</td>
			<td style="width:275px;">BD</td>
			<td style="width:243px;">305196, 305175</td>
			<td>Biohazardous (Sharps waste)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">NotI</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0189S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Oligonucleotide</td>
			<td style="width:275px;">Integrated DNA Technologies</td>
			<td style="width:243px;">Custom Oligonucleotide Synthesis</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">PacI</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0547S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">pBeloBAC11</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">ER2420S (E4154S)</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Phenol (Buffer-Saturated)</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">15513-039</td>
			<td>Toxic and highly corrosive</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Phenol:Chloroform:Isoamyl Alcohol</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">15593-031</td>
			<td>Toxic and highly corrosive</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Phenol:Guanidine Isothiocyanate</td>
			<td style="width:275px;">Life Technologies (Ambion)</td>
			<td style="width:243px;">10296-010</td>
			<td>Toxic, corrosive, and irritating</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Pme I</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0560S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Potassium Acetate</td>
			<td style="width:275px;">Amresco</td>
			<td style="width:243px;">0698</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">RNase Inhibitor</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">10777-019</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">rNTP Set</td>
			<td style="width:275px;">GE Healthcare Life Sciences</td>
			<td style="width:243px;">27-2025-01</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Sealable Polypropylene Tube (16 &#215; 76 mm)&#160;</td>
			<td style="width:275px;">Beckman Coulter</td>
			<td style="width:243px;">342413</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">SfiI</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0123S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Sma I</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0141S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Sodium Acetate</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">S2889</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Sodium Dodecyl Sulfate</td>
			<td style="width:275px;">Amresco</td>
			<td style="width:243px;">0227</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Sodium Hydroxide</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">S5881</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">SP6 RNA Polymerase</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">M0207S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Spin Column (Plasmid Miniprep Kit)</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">K2100-11</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Syringe</td>
			<td style="width:275px;">HSW NORM-JECT</td>
			<td style="width:243px;">4200.000V0</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">T4 DNA Ligase</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Tris</td>
			<td style="width:275px;">Amresco</td>
			<td style="width:243px;">0826</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">tRNA (yeast)</td>
			<td style="width:275px;">Life Technologies (Invitrogen)</td>
			<td style="width:243px;">15401-011</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">XbaI&#160;</td>
			<td style="width:275px;">New England BioLabs</td>
			<td style="width:243px;">R0145S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Name</td>
			<td style="width:275px;">Company</td>
			<td style="width:243px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">2. Cell Culture</td>
			<td style="width:275px;"></td>
			<td style="width:243px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Alpha Minimal Essential Medium</td>
			<td style="width:275px;">Life Technologies (Gibco)</td>
			<td style="width:243px;">12561-049</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Conical Tube (50 mL)</td>
			<td style="width:275px;">VWR</td>
			<td style="width:243px;">21008-242</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Crystal Violet&#160;</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">C0775</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Culture Dish (150 mm)&#160;</td>
			<td style="width:275px;">TPP</td>
			<td style="width:243px;">93150&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Cuvette (2-mm Gap)</td>
			<td style="width:275px;">Harvard Apparatus</td>
			<td style="width:243px;">450125&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Fetal Bovine Serum&#160;</td>
			<td style="width:275px;">Life Technologies (Gibco)</td>
			<td style="width:243px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Formaldehyde</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">F1635</td>
			<td>Toxic and carcinogenic</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Glutamine</td>
			<td style="width:275px;">Life Technologies (Gibco)</td>
			<td style="width:243px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Minimal Essential Medium</td>
			<td style="width:275px;">Life Technologies (Gibco)</td>
			<td style="width:243px;">61100-061</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Penicillin/Streptomycin</td>
			<td style="width:275px;">Life Technologies (Gibco)</td>
			<td style="width:243px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Potassium Chloride</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">P3911</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Potassium Phosphate Monobasic</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">P9791</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Six-Well Plate</td>
			<td style="width:275px;">TPP</td>
			<td style="width:243px;">92006</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Sodium Chloride</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">S3014</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Sodium Phosphate Dibasic</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">S3264</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:275px;">Life Technologies (Gibco)</td>
			<td style="width:243px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Vitamins</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:243px;">M6895</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Name</td>
			<td style="width:275px;">Company</td>
			<td style="width:243px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">3. Equipment</td>
			<td style="width:275px;"></td>
			<td style="width:243px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Agarose Gel Electrophoresis System</td>
			<td style="width:275px;">Mupid</td>
			<td style="width:243px;">MPDEXU-01</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">CO2 Incubator</td>
			<td style="width:275px;">Thermo Scientific</td>
			<td style="width:243px;">Heracell 150i</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Desktop Centrifuge&#160;</td>
			<td style="width:275px;">Thermo Scientific</td>
			<td style="width:243px;">ST16R</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Electroporator</td>
			<td style="width:275px;">Harvard Apparatus</td>
			<td style="width:243px;">ECM 830</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Longwave Ultraviolet Lamps (Handheld)</td>
			<td style="width:275px;">UVP</td>
			<td style="width:243px;">UVGL-58</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Tabletop Centrifuge</td>
			<td style="width:275px;">Beckman Coulter</td>
			<td style="width:243px;">368826</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Thermocycler</td>
			<td style="width:275px;">Life Technologies (Applied Biosystems)</td>
			<td style="width:243px;">GeneAmp PCR System 9700</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Vortexer</td>
			<td style="width:275px;">Scientific Industries</td>
			<td style="width:243px;">G-560</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:304px;">Water Bath</td>
			<td style="width:275px;">Jeio Tech</td>
			<td style="width:243px;">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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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

Research

JoVE Journal

Immunology and Infection

21.2K 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

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

科研

免疫与感染

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

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 ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...