这项工作是由巴黎市，法国国家授予的ANR - 09 - JCJC - 0118 - 1，和紧急救济协调员开始格兰特RNAvirusPopDivNVax项目没有从医学和健康研究资助经费支持。 242719。

<ol>
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E., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quasispecies+diversity+determines+pathogenesis+through+cooperative+interactions+in+a+viral+population.">Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population.</a> Nature. 439, 344-348 (2006).</li><li>Vignuzzi, M., Wendt, E., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+attenuated+virus+vaccines+by+controlling+replication+fidelity.">Engineering attenuated virus vaccines by controlling replication fidelity.</a> Nat Med. 14, 154-161 (2008).</li><li>Crotty, S., Cameron, C., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribavirin's+antiviral+mechanism+of+action:+lethal+mutagenesis.">Ribavirin's antiviral mechanism of action: lethal mutagenesis.</a> J Mol Med. 80, 86-95 (2002).</li><li>Coffey, L. L., Vignuzzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+alternation+of+chikungunya+virus+increases+fitness+while+restricting+population+diversity+and+adaptability+to+novel+selective+pressures.">Host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures.</a> J Virol. 85, 1025-1035 (2011).</li><li>Ciota, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+mutant+spectrum+in+adaptation+and+replication+of+West+Nile+virus.">Role of the mutant spectrum in adaptation and replication of West Nile virus.</a> J Gen Virol. 88, 865-874 (2007).</li><li>Ibarra, K. D., Pfeiffer, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+ribavirin+antiviral+efficacy+via+nucleoside+transporter-mediated+drug+resistance.">Reduced ribavirin antiviral efficacy via nucleoside transporter-mediated drug resistance.</a> J Virol. 83, 4538-4547 (2009).</li><li>Levi, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fidelity+variants+of+RNA+dependent+RNA+polymerases+uncover+an+indirect,+mutagenic+activity+of+amiloride+compounds.">Fidelity variants of RNA dependent RNA polymerases uncover an indirect, mutagenic activity of amiloride compounds.</a> PLoS Pathog. 6, e1001163-e1001163 (2010).</li><li>Sierra, S., D&#225;vila, M., Lowenstein, P. R., Domingo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+foot-and-mouth+disease+virus+to+increased+mutagenesis:+influence+of+viral+load+and+fitness+in+loss+of+infectivity.">Response of foot-and-mouth disease virus to increased mutagenesis: influence of viral load and fitness in loss of infectivity.</a> J Virol. 74, 8316-8323 (2000).</li><li>Ruiz-Jarabo, C. M., Ly, C., Domingo, E., de la Torre, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+mutagenesis+of+the+prototypic+arenavirus+lymphocytic+choriomeningitis+virus+(LCMV).">Lethal mutagenesis of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV).</a> Virology. 308, 37-47 (2003).</li><li>Sierra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foot-and-mouth+disease+virus+mutant+with+decreased+sensitivity+to+ribavirin:+implications+for+error+catastrophe.">Foot-and-mouth disease virus mutant with decreased sensitivity to ribavirin: implications for error catastrophe.</a> J Virol. 81, 2012-2024 (2007).</li></ol>

细胞株的选择。诱变作为RNA碱基类似物的疗效相关，他们通过不同类型的细胞11的相对摄取。通常病毒通过使用的细胞系，如果被证明是难治性诱变剂吸收或过于敏感（高细胞毒性），它可能需要使用另一个细胞系，以满足这些要求，仍然是宽松的病毒复制。一旦诱变阻力变异是孤立的，其余的表征，可以在原有的首选细胞株。 HeLa细胞在我们的经验，很容易采取诱变; BHK细胞需要多达10倍浓度较高和Vero细胞难治性诱变剂吸收。 选择在试图孤立高保真变种诱变处理，成功的可能性增加，如果使用一个以上的诱变诱变剂。基地模拟诱变不同的结构，在复制过程中的基因组中被错误地将主要在一个特定子集，在随后的复制周期的基因突变诱导的结果:利巴韦林治疗有利于 GtoA和CtoU过渡突变12; 5 -氮胞苷也有类似的偏见，与此外CtoG和GtoC颠13; 5 -氟尿嘧啶优先诱导AtoG和UtoC转换14。另外，较高浓度的Mg 2 +的或Mn 2 +可以补充介质，以提高整体RNA病毒的基因突变频率无上述 12所描述的偏见。根据病毒的密码子序列，密码子的变化，需要生成一个高保真的变种，这些条件将有利于比别人出现这种变异。对于更高的保真度的脊髓灰质炎病毒G64S和柯萨奇病毒A372V，利巴韦林治疗最容易选择的变种，因为在密码子网站的要求AtoG过渡与主要由这三氮唑核苷产生的突变。 教学语言与人口规模 ，病毒学，组织培养感染的协议特别注意感染复数（MOI），以避免缺陷干扰颗粒（低MOI）的积累，促进病毒之间的重组（高MOI），例子。要选择通过串口传代出现的事件，它也是重要的考虑病毒的人口规模。由于最初在低频存在耐药突变，最好是从一个通道传输尽可能大的人口规模（10 5 -10 6病毒，例如），以避免失去在每个通过这些新兴的变种。扩大井或烧瓶的大小（感染细胞的数量），可能有助于减少在教学语言的增加，如果这种担心。另一方面，低MOI感染病毒的敏感性诱变正在测试的实验，是为了增加在实验中发生的复制周期和更高的健身基因组，以避免救援诱变基因组，通过互补，在共同感染的细胞。这是很重要的，因为第一轮的复制过程中产生的后代基因组的突变不会立即检测。这些诱变的RNA大多数仍然会被包装成病毒颗粒。这是目前在这些基因组的致死突变会导致中止复制周期，减少病毒滴度在感染下一轮。这可能是必要允许致死突变的一个显着的效果观察前几轮积累的突变。最后，如​​果在通过一系列的诱变剂存在，病毒滴度继续下降，直至灭绝，那么研究者应​​尽量传代病毒在逐渐增加（从一个非常低的的浓度开始）诱变的金额。 分离和RNA的RNA的抗诱变人口的抗诱变剂从克隆的一代。RNA的诱变引入多个随机突变每个基因组，但电阻的选择只会丰富（和修复共识序列）抗突变。为了确定这种基因突变，我们序列的诱变抗性种群人口的共识，而不是个别的病毒。因此，单一的随机突变，诱变创建序列中没有检测到，只有在达成共识的变化，下面的选择，结果发现突变。根据我们的经验，我们只能识别一个或两个这样的共识序列的变化。一旦获得诱变抗性种群和抗突变是确定的，这是必要的，产生这种变异的更纯粹的股票。上面，我们描述了一个空斑纯化过程。另外，如果感兴趣的病毒不容易识别斑块，所需的变异可能purifIED通过有限稀释。这种做法实质上是一种TCID 50，低于50％的水井受到感染该病毒的股票是稀释在96孔板格式。使用这种稀释，采取上述相同的方法是，在隔离多达10个人的变种，并确认它们的DNA序列。如前所述，在最好的情况下，一种传染性病毒株的cDNA克隆可用。变种的隔离，因此没有必要。在我们的经验，保真度的变异是单一的氨基酸替代的结果，因此可以使用简单的商业化的突变试剂盒，如Quikchange（安捷伦）。一个次要的选项，使用密切相关的病毒株的cDNA克隆。但是，如果使用的是一个相关的应变，我们强烈建议使用这种方法和病毒分离（如斑块净化），因为我们发现，同样的保真度改变两个密切相关的病毒突变不一定会具有相同的效果。 富达和复制。RNA的抗诱变剂变种的选择，导致类似野生型对口 4,12,15与生长特性，在更高和更低的保真度变种的隔离。目前尚不完全清楚，聚合酶的活性率和保真度之间的联系。在体外用纯化的RNA聚合酶的生化研究表明，保真度更高的变种处理速度较慢，而较低的保真度变种往往有更快的处理1-3,12。在组织文化中，这些差异通常并不明显，这表明，资源的可用性，而不是内在的聚合酶的活性动力学，是限速步骤。如果从野生型没有显着不同的动力学复制的保真度变异，然后比较其基因突变频率可以直接作出。如果复制过程中的动力学的存在非常显着的变化，那么数据应该归帐户动力学差异，例如，通过比较，都发生了相同数量的病毒复制周期。在我们的经验，观察野生型和高逼真度的变体之间虽然在一步生长动力学无显着差异，我们观察到，保真度更高的变种一贯滴度较高（1日志内）与野生型相比略少，但他们核糖核酸（内在同一量级），进一步表明，他们生产的基因组包含更少的突变，因而，更具传染性。 样品制备和测序。对于在这些协议中的所有步骤，当务之急是高保真，校对酶用于PCR和RT - PCR检测，以限制引入额外的突变，因为他们不能从生物学相关的基因突变杰出。这是至关重要的病毒相比人口已准备在相同的条件下（通过历史，组织培养基，温度，RNA提取方法，RT - PCR检测等）也很重要，以确保足够的起始原料获得RNA的提取等，通过RT - PCR产生强烈的乐队是。一个1 / 100稀释的RNA样品也应给予检测RT - PCR检测带，表明样品中含有足够数量的RNA分子，以避免代表性偏差（反复放大相同的基因组）。由于基因突变频率分布，人们所期望的，将获得类似的价值观无论人口规模，提供上述偏见是没有发生。如图6所示，10 5倍稀释病毒存量给人一种不从父母的股票明显不同的基因突变频率。 直到发现TopoTA克隆的最佳条件，确认后，在场的插入，蓝/白筛选殖民地之前测序的PCR。作为一个突变噪声控制（RT - PCR和测序引入突变），从质粒在体外转录RNA病毒基因组相对应的轴承相同的病毒序列和/或克隆和序列RT - PCR产物的克隆PCR产物（BE知道，在体外转录酶的不同有不同的错误率，可能不会给的有用信息的真实背景，在你的程序错误）。一些病毒序列可能有毒细菌，所以重要的是要验证，才决定在地区的病毒基因组测序基因突变频率。在分析所TopoTA获得的序列，请注意，每个克隆应该只包含一个插入/序列。如果双峰观察，表明混合的人口，这是可能的，被选中两个相邻的细菌菌落。它也是可能的，虽然不大可能在细菌复制的突变频率低，在细菌培养的突变质粒的扩增期间推出。在斑块p双峰urified人口，可能代表重叠的斑块，或病毒，即获得一个新的突变或恢复斑块的发展过程中的突变。一致，并决定是否进行计数或不能指望这些突变。 最后，请记住，这里所用的突变频率是相对值。他们是有效的，只有在比较，在相同条件下生长的病毒人口，并在同一区域的测序！他们不应该被视为突变率，或整个基因组的突变频率的绝对值。然而，当条件控制，他们允许重现性好，在突变的分布和频率差异的定量比较。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>病毒唑</td><td>西格玛</td><td> R9644 - 10MG </td><td></td></tr><tr><td> 5 - 氟尿嘧啶</td><td>西格玛</td><td> F6627 - 1G </td><td></td></tr><tr><td> 5 - 氮胞苷</td><td>西格玛</td><td> A2385 - 100MG </td><td></td></tr><tr><td> MgCl 2的 </td><td>西格玛</td><td>由M1028 - 100ML </td><td></td></tr><tr><td> MnCl 2 </td><td>西格玛</td><td> M1787 </td><td></td></tr><tr><td>台盼蓝</td><td>西格玛</td><td> T8154 - 20ML </td><td></td></tr><tr><td> TopoTA克隆试剂盒</td><td> Invitrogen公司</td><td> 10351021 </td><td></td></tr><tr><td> Quikchange突变试剂盒</td><td>安捷伦</td><td> 200516 </td><td>如果感染性克隆的cDNA是可用</td></tr><tr><td> 96以及小量制备试剂盒</td><td>马歇雷- NAGEL </td><td> 740625 </td><td></td></tr><tr><td> Lasergene，Sequencher </td><td> DNASTAR，基因编码公司</td><td> <a href="www.dnastar.com">www.dnastar.com</a> <a href="www.genecodes.com">www.genecodes.com</a> </td><td>或其他定位软件</td></tr></tbody></table>

isolation of fidelity variants of rna viruses and characterization of virus mutation frequency

RNA病毒的RNA依赖的RNA聚合酶复制其基因组。这些酶的本质差错率高是一个大的贡献极端种群的多样性，有利于病毒的适应和进化的一代。越来越多的证据表明，内在的错误率，以及由此产生的RNA病毒的突变频率，可调制微妙的病毒聚合酶的氨基酸变化。虽然生化分析存在一些病毒的RNA聚合酶，允许注册成立的保真度的定量测量，在这里我们描述了一个简单的方法测量的RNA病毒，已被证明是确定高保真改变突变的生化方法的准确突变频率。该方法使用传统的病毒学和测序技术可以在大多数生物实验室进行。我们与许多不同的病毒经验的基础上，我们已经确定，必须进行优化，以增加隔离高保真的变种，并产生统计意义的数据的可能性的关键步骤。高保真改变突变的分离和表征聚合酶的结构和功能1-3可以提供新的见解。此外，这些高保真的变种可以成为有用的工具表征病毒的适应和演变 4-7机制。

Watch this Scientific Journal Video about Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency at JoVE.com

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

本文介绍了分离和鉴定RNA聚合酶的保真度变体的RNA病毒，以及如何利用基因突变频率的数据，以确认在​​组织培养中的保真度的变化所需的步骤。

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 ...

isolation-fidelity-variants-rna-viruses-characterization-virus

Research

JoVE Journal

Immunology and Infection

29.5K 次观看.  Institut Pasteur. 本文介绍了分离和鉴定RNA聚合酶的保真度变体的RNA病毒，以及如何利用基因突变频率的数据，以确认在​​组织培养中的保真度的变化所需的步骤。

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

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

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 ...

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 ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

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.

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 ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

中性粒细胞衍生微粒的分离与鉴定功能研究

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.